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Sample records for acid column chromatography

  1. Column Liquid Chromatography.

    ERIC Educational Resources Information Center

    Majors, Ronald E.; And Others

    1984-01-01

    Reviews literature covering developments of column liquid chromatography during 1982-83. Areas considered include: books and reviews; general theory; columns; instrumentation; detectors; automation and data handling; multidimensional chromatographic and column switching techniques; liquid-solid chromatography; normal bonded-phase, reversed-phase,…

  2. Post-column labeling techniques in amino acid analysis by liquid chromatography.

    PubMed

    Rigas, Pantelis G

    2013-10-01

    Amino acid analysis (AAA) has always presented an analytical challenge in terms of sample preparation, separation, and detection. Because of the vast number of amino acids, various separation methods have been applied taking into consideration the large differences in their chemical structures, which span from nonpolar to highly polar side chains. Numerous separation methods have been developed in the past 60 years, and impressive achievements have been made in the fields of separation, derivatization, and detection of amino acids (AAs). Among the separation methods, liquid chromatography (LC) prevailed in the AAA field using either pre-column or post-column labeling techniques in order to improve either separation of AAs or selectivity and sensitivity of AAA. Of the two approaches, the post-column technique is a more rugged and reproducible method and provides excellent AAs separation relatively free from interferences. This review considers current separations combined with post-column labeling techniques for AAA, comparison with the pre-column methods, and the strategies used to develop effective post-column methodology. The focus of the article is on LC methods coupled with post-column labeling techniques and studying the reactions to achieve optimum post-column derivatization (PCD) conditions in order to increase sensitivity and selectivity using various types of detectors (UV-Vis, fluorescence, electrochemical etc.) and illustrating the versatility of the PCD methods for practical analysis.

  3. 5-O-caffeoylshikimic acid from Solanum somalense leaves: advantage of centrifugal partition chromatography over conventional column chromatography.

    PubMed

    Chideh, Saïda; Pilard, Serge; Attoumbré, Jacques; Saguez, Robert; Hassan-Abdallah, Alshaimaa; Cailleu, Dominique; Wadouachi, Anne; Baltora-Rosset, Sylvie

    2014-09-01

    Solanum somalense leaves, used in Djibouti for their medicinal properties, were extracted by MeOH. Because of the high polyphenol and flavonoid contents of the extract, respectively, determined at 80.80 ± 2.13 mg gallic acid equivalent/g dry weight and 24.4 ± 1.01 mg quercetin equivalent/g dry weight, the isolation and purification of the main polyphenols were carried out by silica gel column chromatography and centrifugal partition chromatography. Column chromatography led to 11 enriched fractions requiring further purification, while centrifugal partition chromatography allowed the easy recovery of the main compound of the extract. In a solvent system composed of CHCl3/MeOH/H2O (9.5:10:5), 21.8 mg of this compound at 97% purity was obtained leading to a yield of 2.63%. Its structure was established as 5-O-caffeoylshikimic acid by mass spectrometry and NMR spectroscopy. This work shows that S. somalense leaves contain very high level of 5-O-caffeoylshikimic acid (0.74% dry weight), making it a potential source of production of this secondary metabolite that is not commonly found in nature but could be partly responsible of the medicinal properties of S. somalense leaves.

  4. Column switching liquid chromatography and post-column photochemically fluorescence detection to determine imidacloprid and 6-chloronicotinic acid in honeybees.

    PubMed

    García, M D Gil; Galera, M Martínez; Valverde, R Santiago; Galanti, A; Girotti, S

    2007-04-13

    The determination of imidacloprid and its main metabolite (6-chloronicotinic acid) in honeybees was performed by liquid chromatography with post-column photochemical derivatisation in alkaline medium and fluorescence detection. The compounds were extracted from honeybees with acetone under ultrasound conditions prior to liquid-liquid partition with dichloromethane. The separation of extract components was performed using a 50 mm x 4.6 mm i.d. short column packed with 5 microm Aquasil C(18) using an acetonitrile:water gradient program as mobile phase. Injection of samples in 0.1 mol L(-1) H(3)PO(4)/KH(2)PO(4) buffer solution (pH 3) improved the chromatographic separation between the most polar components of matrix and the 6-chloronicotinic acid. Matrix components were removed to waste using an on-line clean-up method previously to post-column reaction. Limits of quantification were 0.3 and 5.0 microg L(-1) (corresponding to 1.2 and 20.0 microg kg(-1) in the honeybee sample) for imidacloprid and 6-chloronicotinic acid, respectively. The recovery was ranged from 80.2 to 91.7% with a relative standard deviation lower than 9.0%.

  5. Application of Pre-Column Labeling Liquid Chromatography for Canine Plasma-Free Amino Acid Analysis

    PubMed Central

    Azuma, Kazuo; Hirao, Yoshiko; Hayakawa, Yoshihiro; Murahata, Yusuke; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Okamoto, Yoshiharu; Ito, Norihiko

    2016-01-01

    Plasma-free amino acid (PFAA) levels are a useful metric for diagnosing cancer and providing a prognosis. However, the use of analysis of PFAA levels has been limited in the veterinary medicine field. We addressed the application of liquid chromatography (LC) using a pre-column labeling technique for analysis of canine PFAA levels. This method significantly shortened the analysis time relative to conventional methods. No diurnal fluctuations were detected at 9:00 AM in most PFAA levels, and food intake increased the levels of some PFAAs, including valine, leucine, tyrosine, phenylalanine, and proline. These results indicate that LC with pre-column labeling is useful for measuring canine PFAA levels, for which time of day and interval after food intake must be taken into consideration. PMID:26771650

  6. Separation of tissue and serum acid phosphatase isoenzymes by ion-exchange column chromatography.

    PubMed

    Mercer, D W

    1977-01-01

    I describe a simple, rapid ion-exchange column-chromatographic technique for separating the acid phosphatase (EC 3.1.3.2) isoenzymes in human serum and tissue. Extracts of platelets, spleen, liver, erythrocytes, and prostate were used to determine optimum conditions for separating these isoenzymes. Samples layered on mini-colunms of DEAE-Sephadex A-50 were eluted stepwise with sodium chloride (100, 200, and 300 mmol/liter, buffered with tris (hydroxymethyl)aminomethane). Activity in column effluents was measured with p-nitrophenol phosphate as substrate, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Comparision of activity patterns so derived for various tissues revealed prostatic tissue to be a rich source of acid phosphatase isoenzyme 2 activity. Evaluation of sera from six patients with prostatic cancer revealed isoenzyme patterns with prominent amount of isoenzyme 2 (3.8 to 27.6 U/liter). sera from 10 healthy laboratory technicians contained isoenzyme 2 in the range of 0.3-0.5 U/liter. Samples from two patients with abnormally high activity owing to nonprostatic conditions (Gaucher's disease and carcinoma of lung) exhibited less than 2 U of isoenzyme 2 per liter and acid phosphatase isoenzymes 3-5 that were 50- to 100-fold the normal range. Quantification of isoenzyme 2 by DEAE-Sephadex column chromatography as described appears to provide a more sensitive and specific approach to diagnosis of prostatic cancer.

  7. An Undergraduate Column Chromatography Experiment.

    ERIC Educational Resources Information Center

    Danot, M.; And Others

    1984-01-01

    Background information, list of materials needed, and procedures used are provided for an experiment designed to introduce undergraduate students to the theoretical and technical aspects of column chromatography. The experiment can also be shortened to serve as a demonstration of the column chromatography technique. (JN)

  8. Separation of aliphatic carboxylic acids and benzenecarboxylic acids by ion-exclusion chromatography with various cation-exchange resin columns and sulfuric acid as eluent.

    PubMed

    Ohta, Kazutoku; Ohashi, Masayoshi; Jin, Ji-Ye; Takeuchi, Toyohide; Fujimoto, Chuzo; Choi, Seong-Ho; Ryoo, Jae-Jeong; Lee, Kwang-Pill

    2003-05-16

    The application of various hydrophilic cation-exchange resins for high-performance liquid chromatography (sulfonated silica gel: TSKgel SP-2SW, carboxylated silica gel: TSKgel CM-2SW, sulfonated polymethacrylate resin: TSKgel SP-5PW, carboxylated polymethacrylate resins: TSKgel CM-5PW and TSKgel OA-Pak A) as stationary phases in ion-exclusion chromatography for C1-C7 aliphatic carboxylic acids (formic, acetic, propionic, butyric, isovaleric, valeric, isocaproic, caproic, 2-methylhexanoic and heptanoic acids) and benzenecarboxylic acids (pyromellitic, trimellitic, hemimellitic, o-phthalic, m-phthalic, p-phthalic, benzoic, salicylic acids and phenol) was carried out using diluted sulfuric acid as the eluent. Silica-based cation-exchange resins (TSKgel SP-2SW and TSKgel CM-2SW) were very suitable for the ion-exclusion chromatographic separation of these benzenecarboxylic acids. Excellent simultaneous separation of these benzenecarboxylic acids was achieved on a TSKgel SP-2SW column (150 x 6 mm I.D.) in 17 min using a 2.5 mM sulfuric acid at pH 2.4 as the eluent. Polymethacrylate-based cation-exchange resins (TSKgel SP-5PW, TSKgel CM-5PW and TSKgel OA-Pak A) acted as advanced stationary phases for the ion-exclusion chromatographic separation of these C1-C7 aliphatic carboxylic acids. Excellent simultaneous separation of these C1-C7 acids was achieved on a TSKgel CM-5PW column (150 x 6 mm I.D.) in 32 min using a 0.05 mM sulfuric acid at pH 4.0 as the eluent.

  9. Determination of some aliphatic carboxylic acids in anaerobic digestion process waters by ion-exclusion chromatography with conductimetric detection on a weakly acidic cation-exchange resin column.

    PubMed

    Ito, Kazuaki; Takayama, Yohichi; Ikedo, Mikaru; Mori, Masanobu; Taoda, Hiroshi; Xu, Qun; Hu, Wenzhi; Sunahara, Hiroshi; Hayashi, Tsuneo; Sato, Shinji; Hirokawa, Takeshi; Tanaka, Kazuhiko

    2004-06-11

    The determination of seven aliphatic carboxylic acids, formic, acetic, propionic, isobutyric, n-butyric, isovaleric and n-valeric acids in anaerobic digestion process waters was examined using ion-exclusion chromatography with conductimetric detection. The analysis of these biologically important carboxylic acids is necessary as a measure for evaluating and controlling the process. The ion-exclusion chromatography system employed consisted of polymethacrylate-based weakly acidic cation-exchange resin columns (TSKgel OApak-A or TSKgel Super IC-A/C). weakly acidic eluent (benzoic acid), and conductimetric detection. Particle size and cation-exchange capacity were 5 microm and 0.1 meq./ml for TSKgel OApak-A and 3 microm and 0.2 meq./ml for TSKgel Super IC-A/C, respectively. A dilute eluent (1.0-2.0 mM) of benzoic acid was effective for the high resolution and highly conductimetric detection of the carboxylic acids. The good separation of isobutyric and n-butyric acids was performed using the TSKgel Super IC-A/C column (150 mm x 6.0 mm i.d. x 2). The simple and good chromatograms were obtained by the optimized ion-exclusion chromatography conditions for real samples from mesophilic anaerobic digestors, thus the aliphatic carboxylic acids were successfully determined without any interferences.

  10. Analysis of trace inorganic anions in weak acid salts by single pump cycling-column-switching ion chromatography.

    PubMed

    Huang, Zhongping; Ni, Chengzhu; Zhu, Zhuyi; Pan, Zaifa; Wang, Lili; Zhu, Yan

    2015-05-01

    The application of ion chromatography with the single pump cycling-column-switching technique was described for the analysis of trace inorganic anions in weak acid salts within a single run. Due to the hydrogen ions provided by an anion suppressor electrolyzing water, weak acid anions could be transformed into weak acids, existing as molecules, after passing through the suppressor. Therefore, an anion suppressor and ion-exclusion column were adopted to achieve on-line matrix elimination of weak acid anions with high concentration for the analysis of trace inorganic anions in weak acid salts. A series of standard solutions consisting of target anions of various concentrations from 0.005 to 10 mg/L were analyzed, with correlation coefficients r ≥ 0.9990. The limits of detection were in the range of 0.67 to 1.51 μg/L, based on the signal-to-noise ratio of 3 and a 25 μL injection volume. Relative standard deviations for retention time, peak area, and peak height were all less than 2.01%. A spiking study was performed with satisfactory recoveries between 90.3 and 104.4% for all anions. The chromatographic system was successfully applied to the analysis of trace inorganic anions in five weak acid salts.

  11. Self-regenerating column chromatography

    DOEpatents

    Park, W.K.

    1995-05-30

    The present invention provides a process for treating both cations and anions by using a self-regenerating, multi-ionic exchange resin column system which requires no separate regeneration steps. The process involves alternating ion-exchange chromatography for cations and anions in a multi-ionic exchange column packed with a mixture of cation and anion exchange resins. The multi-ionic mixed-charge resin column works as a multi-function column, capable of independently processing either cationic or anionic exchange, or simultaneously processing both cationic and anionic exchanges. The major advantage offered by the alternating multi-function ion exchange process is the self-regeneration of the resins.

  12. Dry-Column Flash Chromatography

    NASA Astrophysics Data System (ADS)

    Shusterman, Alan J.; McDougal, Patrick G.; Glasfeld, Arthur

    1997-10-01

    Dry-column flash chromatography is a safe, powerful, yet easily learned preparative chromatography technique. It has proven useful in research, and an adaptation of the technique for use in large teaching laboratories (general chemistry, organic chemistry) is described here. The student version is similar to vacuum filtration, uses the same compact, readily available glassware, and inexpensive and safe solvents (ethyl acetate and hexane) and adsorbent (Merck grade 60 silica gel). The technique is sufficiently simple and powerful that a beginning student can successfully resolve diastereomers on sample scales ranging from 100 mg to >1 g.

  13. Simultaneous determination of acetylsalicylic acid and salicylic acid in human plasma by isocratic high-pressure liquid chromatography with post-column hydrolysis and fluorescence detection.

    PubMed

    Hobl, Eva-Luise; Jilma, Bernd; Ebner, Josef; Schmid, Rainer W

    2013-06-01

    A selective, sensitive and rapid high-performance liquid chromatography method with post-column hydrolysis and fluorescence detection was developed for the simultaneous quantification of acetylsalicylic acid and its metabolite salicylic acid in human plasma. Following the addition of 2-hydroxy-3-methoxybenzoic acid as internal standard and simple protein precipitation with acetonitrile, the analytes were separated on a ProntoSIL 120 C18 ace-EPS column (150 × 2 mm, 3 µm) protected by a C8 guard column (5 µm). The mobile phase, 10 mm formic acid in water (pH 2.9) and acetonitrile (70:30, v/v), was used at a flow rate of 0.35 mL/min. After on-line post-column hydrolysis of acetylsalicylic acid (ASA) to salicylic acid (SA) by addition of alkaline solution, the analytes were measured at 290 nm (λex ) and 400 nm (λem ). The method was linear in the concentration ranges between 0.05 and 20 ng/μL for both ASA and SA with a lower limit of quantification of 25 pg/μL for SA and 50 pg/μL for ASA. The limit of detection was 15 pg/μL for SA and 32.5 pg/μL for ASA. The analysis of ASA and SA can be carried out within 8 min; therefore this method is suitable for measuring plasma concentrations of salicylates in clinical routine.

  14. RAPID ANALYSIS OF CYNANURIC ACID IN SWIMMING POOL WATERS BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING POROUS GRAPHITIC CARBON COLUMN

    EPA Science Inventory

    An innovative approach is presented for reducing analysis times of cyanuric acid in swimming pool waters by high performance liquid chromatography (HPLC). The HPLC method exploits the unique selectivity of porous graphitic carbon (PGC) to fully resolve cyanuric acid from other p...

  15. Determination of trans-10-hydroxy-2-decenoic acid content in pure royal jelly and royal jelly products by column liquid chromatography.

    PubMed

    Genç, M; Aslan, A

    1999-04-16

    In this research, several royal jellies and commercial products containing royal jelly were analysed for their trans-10-hydroxy-2-decenoic acid (10-HDA) content by using a column liquid chromatography technique. Ten samples claimed to be pure royal jelly, containing 10-HDA between 0.75 and 2.54%. Seven samples claimed to contain royal jelly as an ingredient which ranged from non-detectable to 0.054%. The technique was found to be rapid with high recovery.

  16. Fast comprehensive two-dimensional gas chromatography method for fatty acid methyl ester separation and quantification using dual ionic liquid columns.

    PubMed

    Nosheen, Asia; Mitrevski, Blagoj; Bano, Asghari; Marriott, Philip J

    2013-10-18

    Safflower oil is a complex mixture of C18 saturated and unsaturated fatty acids amongst other fatty acids, and achieving separation between these similar structure components using one dimensional gas chromatography (GC) may be difficult. This investigation aims to obtain improved separation of fatty acid methyl esters in safflower oil, and their quantification using comprehensive two-dimensional GC (GC×GC). Here, GC×GC separation is accomplished by the coupling of two ionic liquid (IL) column phases: the combination of SLB-IL111 with IL59 column phases was finally selected since it provided excellent separation of a FAME standard mixture, as well as fatty acids in safflower and linseed oil, compared to other tested column sets. Safflower oil FAME were well separated in a short run of 16min. FAME validation was demonstrated by method reproducibility, linearity over a range up to 500mgL(-1), and limits of detection which ranged from 1.9mgL(-1) to 5.2mgL(-1) at a split ratio of 20:1. Quantification was carried out using two dilution levels of 200-fold for major components and 20-fold for trace components. The fatty acids C15:0 and C17:0 were not reported previously in safflower oil. The SLB-IL111/IL59 column set proved to be an effective and novel configuration for separation and quantification of vegetable and animal oil fatty acids.

  17. Temperature programmable microfabricated gas chromatography column

    DOEpatents

    Manginell, Ronald P.; Frye-Mason, Gregory C.

    2003-12-23

    A temperature programmable microfabricated gas chromatography column enables more efficient chemical separation of chemical analytes in a gas mixture by the integration of a resistive heating element and temperature sensing on the microfabricated column. Additionally, means are provided to thermally isolate the heated column from their surroundings. The small heat capacity and thermal isolation of the microfabricated column improves the thermal time response and power consumption, both important factors for portable microanalytical systems.

  18. Use of a polystyrene-divinylbenzene-based weakly acidic cation-exchange resin column and propionic acid as an eluent in ion-exclusion/adsorption chromatography of aliphatic carboxylic acids and ethanol in food samples.

    PubMed

    Mori, Masanobu; Hironaga, Takahiro; Kajiwara, Hiroe; Nakatani, Nobutake; Kozaki, Daisuke; Itabashi, Hideyuki; Tanaka, Kazuhiko

    2011-01-01

    We developed an ion-exclusion/adsorption chromatography (IEAC) method employing a polystyrene-divinylbenzene-based weakly acidic cation-exchange resin (PS-WCX) column with propionic acid as the eluent for the simultaneous determination of multivalent aliphatic carboxylic acids and ethanol in food samples. The PS-WCX column well resolved mono-, di-, and trivalent carboxylic acids in the acidic eluent. Propionic acid as the eluent gave a higher signal-to-noise ratio, and enabled sensitive conductimetric detection of analyte acids. We found the optimal separation condition to be the combination of a PS-WCX column and 20-mM propionic acid. Practical applicability of the developed method was confirmed by using a short precolumn with a strongly acidic cation-exchange resin in the H(+)-form connected before the separation column; this was to remove cations from food samples by converting them to hydrogen ions. Consequently, common carboxylic acids and ethanol in beer, wine, and soy sauce were successfully separated by the developed method.

  19. Caprylic acid-induced impurity precipitation from protein A capture column elution pool to enable a two-chromatography-step process for monoclonal antibody purification.

    PubMed

    Zheng, Ji; Wang, Lu; Twarowska, Barbara; Laino, Sarah; Sparks, Colleen; Smith, Timothy; Russell, Reb; Wang, Michelle

    2015-01-01

    This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography.

  20. A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers.

    PubMed

    Hou, Hongwei; Xiong, Wei; Gao, Na; Zhang, Xiaotao; Tang, Gangling; Hu, Qingyuan

    2012-11-01

    The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.

  1. Determination of Phenolic Acids and Flavonoids in Taraxacum formosanum Kitam by Liquid Chromatography-Tandem Mass Spectrometry Coupled with a Post-Column Derivatization Technique

    PubMed Central

    Chen, Hung-Ju; Inbaraj, Baskaran Stephen; Chen, Bing-Huei

    2012-01-01

    A liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed for the determination of phenolic acids and flavonoids in a medicinal Chinese herb Taraxacum formosanum Kitam. Initially, both phenolic acids and flavonoids were extracted with 50% ethanol in a water-bath at 60 °C for 3 h and eventually separated into acidic fraction and neutral fraction by using a C18 cartridge. A total of 29 compounds were separated within 68 min by employing a Gemini C18 column and a gradient solvent system of 0.1% formic acid and acetonitrile at a flow rate of 1.0 mL/min. Based on the retention behavior as well as absorption and mass spectra, 19 phenolic acids and 10 flavonoids were identified and quantified in T. formosanum, with the former ranging from 14.1 μg/g to 10,870.4 μg/g, and the latter from 9.9 μg/g to 325.8 μg/g. For further identification of flavonoids, a post-column derivatization method involving shift reagents such as sodium acetate or aluminum chloride was used and the absorption spectral characteristics without or with shift reagents were compared. An internal standard syringic acid was used for quantitation of phenolic acids, whereas (±) naringenin was found suitable for quantitation of flavonoids. The developed LC-MS/MS method showed high reproducibility, as evident from the relative standard deviation (RSD) values for intra-day and inter-day variability being 1.0–6.8% and 2.0–7.7% for phenolic acids and 3.7–7.4% and 1.5–8.1% for flavonoids, respectively, and thus may be applied for simultaneous determination of phenolic acids and flavonoids in Chinese herb and nutraceuticals. PMID:22312251

  2. [Determination of twenty free amino acids in flue-cured tobacco leaves using ultra performance liquid chromatography-single quadruple mass spectrometry and pre-column derivatization].

    PubMed

    Li, Haoli; Zhao, Chunxia; Zhang, Junjie; Fu, Jiajun; Wang, Ying; Lu, Xin; Xu, Guowang

    2013-12-01

    Free amino acids in flue-cured tobacco leaves were investigated using the ultra performance liquid chromatography-single quadruple mass spectrometry detection and pre-column derivatization method. The validation results showed that the method could meet the analytical requirements. A total of 138 tobacco leaf samples were collected from 14 provinces in China in 2011 in which the free amino acids were determined. The relative standard deviations (RSDs) of the contents of free amino acids in different growing regions ranged from 28.50%-94.20%, and those of asparagine and glutamine were over 80%. The RSDs of the contents of free amino acids in full aroma tobacco leaves were larger than those in fresh aroma and medium aroma tobacco leaves. The principal component analysis (PCA) and non-parameter Mann-Whitney U test were used for data analysis. The free amino acids of the same aroma type grown in different regions or different aroma types in the same province showed great variation. The contents of free amino acids of full aroma tobacco grown in Southeast region were much lower than those in Huanghuai region. The contents of free amino acids in Hunan province were much lower than the average contents. The results showed that free amino acids in flue-cured tobacco leaves were affected by the growing region.

  3. "Dry-column" chromatography of plant pigments

    NASA Technical Reports Server (NTRS)

    Woeller, F. H.; Lehwalt, M. F.; Oyama, V. I.

    1973-01-01

    Separation of plant pigments which can be accomplished on thin-layer silica plates with mixture of petroleum ether, halocarbon, acetone, and polar solvent can be readily translated into dry-column technique that yields reproducible chromatograms after elution in fashion of liquid chromatography with fluorimeter as detector. Best solvent system was found to be mixture of petroleum ether, dichloromethane, acetone, and ethyl acetate.

  4. Non-planar microfabricated gas chromatography column

    DOEpatents

    Lewis, Patrick R.; Wheeler, David R.

    2007-09-25

    A non-planar microfabricated gas chromatography column comprises a planar substrate having a plurality of through holes, a top lid and a bottom lid bonded to opposite surfaces of the planar substrate, and inlet and outlet ports for injection of a sample gas and elution of separated analytes. A plurality of such planar substrates can be aligned and stacked to provide a longer column length having a small footprint. Furthermore, two or more separate channels can enable multi-channel or multi-dimensional gas chromatography. The through holes preferably have a circular cross section and can be coated with a stationary phase material or packed with a porous packing material. Importantly, uniform stationary phase coatings can be obtained and band broadening can be minimized with the circular channels. A heating or cooling element can be disposed on at least one of the lids to enable temperature programming of the column.

  5. An ultra-sensitive method for the analysis of perfluorinated alkyl acids in drinking water using a column switching high-performance liquid chromatography tandem mass spectrometry.

    PubMed

    Dasu, Kavitha; Nakayama, Shoji F; Yoshikane, Mitsuha; Mills, Marc A; Wright, J Michael; Ehrlich, Shelley

    2017-04-21

    In epidemiological research, it has become increasingly important to assess subjects' exposure to different classes of chemicals in multiple environmental media. It is a common practice to aliquot limited volumes of samples into smaller quantities for specific trace level chemical analyses. A novel method was developed for the determination of 14 perfluorinated alkyl acids (PFAAs) in small volumes (10mL) of drinking water using off-line solid phase extraction (SPE) pre-treatment followed by on-line pre-concentration on a WAX column before analysis on column-switching high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). In general, large volumes (100-1000mL) have been used for the analysis of PFAAs in drinking water. The current method requires approximately 10mL of drinking water concentrated by using an SPE cartridge and eluted with methanol. A large volume injection of the extract was introduced on to a column-switching HPLC-MS/MS using a mix-mode SPE column for the trace level analysis of PFAAs in water. The recoveries for most of the analytes in the fortified laboratory blanks ranged from 73±14% to 128±5%. The lowest concentration minimum reporting levels (LCMRL) for the 14 PFAAs ranged from 0.59 to 3.4ng/L. The optimized method was applied to a pilot-scale analysis of a subset of drinking water samples from an epidemiological study. These samples were collected directly from the taps in the households of Ohio and Northern Kentucky, United States and the sources of drinking water samples are both surface water and ground water, and supplied by different water distribution facilities. Only five PFAAs, perfluoro-1-butanesulfonic acid (PFBS), perfluoro-1- -hexanesulfonic acid (PFHxS), perfluoro-1-octanesulfonic acid (PFOS), perfluoro-n-heptanoic acid (PFHpA) and perfluoro-n-octanoic acid (PFOA) are detected above the LCMRL values. The median concentrations of these five PFAAs detected in the samples was ≤4.1ng/L with PFOS at 7.6ng

  6. Correlations of beta-aminoisobutyric acid in 8 hour and 24 hour urinary samples determined by dual column gas chromatography.

    PubMed

    Sjölin, K E; Nyholm, K K

    1980-05-01

    The correlations of beta-aminoisobutyric acid values in 8 hour and 24 hour urinary samples from 23 healthy persons were determined. beta-AIB in the 8 hour urinary samples was measured by gas chromatography and the 24 hour excretion was calculated from the results of three 8 hour determinations. Simultaneous determinations of urinary creatinine were performed by Jaffe's reaction. Based on the 8 hour values of urinary beta-AIB the results demonstrated a constant excretion of beta-aminoisobutyric acid within the 24 hour periods in both low and high excretors. The precision in distinguishing low and high 24 hour excretors of beta-AIB by using 8 hour values was 91%. If 8 hour values of beta-AIB were related to creatinine the same precision for this calculated ratio was 96.5%. However, for high excretors of beta-AIB, failures were 24.5% by using the 8 hour excretion of beta-AIB as indicator, but only 6.5% by using the ratio.

  7. On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

    PubMed

    Anguizola, Jeanethe; Bi, Cong; Koke, Michelle; Jackson, Abby; Hage, David S

    2016-08-01

    An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0 cm × 2.1 mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

  8. Spectrofluorometry, thin layer chromatography, and column high-performance liquid chromatography determination of rabeprazole sodium in the presence of its acidic and oxidized degradation products.

    PubMed

    Osman, Afaf Osman; Osman, Afaf; Osman, Mohamed

    2009-01-01

    The objective of this study is to develop validated stability-indicating spectrofluorometric, TLC-densitometric, and HPLC methods for the determination of rabeprazole sodium and its degradation products. The first method was based on measuring the fluorescence intensity of the drug at 416 and 311 nm for the emission and at 320 and 274 nm for the excitation for acid and oxidized solutions, respectively. The second method was based on the separation of the drug from its acidic and oxidized degradation products followed by densitometric measurement of the intact drug spot at 284 nm. The separation was carried out on Fluka TLC sheets of silica gel 60 F254 using isopropyl alcohol--30% ammonia (80 + 2, v/v) mobile phase. The third method was based on HPLC separation of rabeprazole sodium from its acidic and oxidized degradation products on a reversed-phase Waters Nova-Pak C18 column using 0.05 M potassium dihydrogen phosphate-methanol-acetonitrile (5 + 3 + 2, v/v/v) pH 7 +/- 0.2 mobile phase. The proposed procedures were successfully applied for the determination of rabeprazole sodium in pure form, laboratory-prepared mixtures, tablet, and expired batch. The obtained results were statistically compared with those of a reported method and validated according to United States Pharmacopeia guidelines. Two main acidic degradation products of the drug were separated and subjected to IR spectrometry and MS to confirm their structures, and the schemes for their formation were elucidated.

  9. Fluorescence determination of N-acetylaspartic acid in the rat cerebrum homogenate using high-performance liquid chromatography with pre-column fluorescence derivatization.

    PubMed

    Fukushima, Takeshi; Arai, Kotaro; Tomiya, Masayuki; Mitsuhashi, Shogo; Sasaki, Tsukasa; Santa, Tomofumi; Imai, Kazuhiro; Toyo'oka, Toshimasa

    2008-01-01

    N-acetyl-L-aspartic acid (NAA) is an endogenous compound, and its brain concentration is suggested to be altered in neurological disorders. In the present study, a fluorescence determination method for NAA was developed by employing reversed-phase high-performance liquid chromatography (HPLC) with pre-column fluorescence derivatization using 4-N,N-dimethylaminosulfonyl-7-N-(2-aminoethyl)amino-2,1,3-benzoxadiazole (DBD-ED). Using methylsuccinic acid as the internal standard, a linear calibration curve for NAA was constructed in the range 125-1000 microM (n=3). The detection limit on the column was approximately 5.0 fmol (signal-to-noise ratio 3). The proposed HPLC method was applied to determine NAA in the rat cerebrum homogenate. Cerebrum NAA was successfully determined using 10 microL of the homogenate, and the validation data for the proposed HPLC method demonstrated satisfactory results. Intra- and inter-day precision and accuracy were within 1.1-7.0 and -8.1-6.3%, respectively. The concentration of NAA in the male rat cerebrum (13 weeks old) was 84 +/-4.6 nmol/mg protein (n = 3) [corrected].

  10. Purification of matrix metalloproteinases by column chromatography.

    PubMed

    Imai, Kazushi; Okada, Yasunori

    2008-01-01

    Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin-Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes approximately 7-10 d.

  11. Low-capacity cation-exchange chromatography of amino acids using a novel sulfoacylated macroreticular polystyrene-divinylbenzene column with binary gradient elution.

    PubMed

    Yokoyama, Yukio; Wakabayashi, Natsuko; Furugaki, Yuki; Sato, Hisakuni

    2004-08-01

    This paper describes a versatile technique for amino-acid separation using a novel low-capacity sulfoacylated macroreticular polystyrene-divinylbenzene cation-exchange column with a simple binary high-pressure pH gradient elution. Proteinic 16 amino acids were well separated within 50 min using a H3PO4/Na2HPO4-CH3CN eluent system, and the cycle time was about 70 min. The chromatography with postcolumn OPA fluorescent detection was reproducible with RSDs less than 1% for retention times, and was quantitative with RSDs less than 5% for area responses. A linear regression line with an r2 value above 0.9990 was obtained for each analyte in concentration from 0.1 to 10 microM by 20 microL injection. The method was applicable to the separation and detection of urinary diagnostic amino acid due to inborn errors of metabolism, such as phenylketonuria. The analytical costs would be decreased by using the proposed method.

  12. Simultaneous Determination of Essential Oil Components and Fatty Acids in Fennel using Gas Chromatography with a Polar Capillary Column.

    PubMed

    Najdoska-Bogdanov, Menče; Bogdanov, Jane B; Stefova, Marina

    2015-09-01

    Cultivated and wild growing samples of fennel (Foeniculum vulgare Mill., Apiaceae) from R. Macedonia were studied for their volatiles and fatty acid composition. The main essential oil components isolated via hydrodistillation were: trans-anethole (>80%), estragole (< 6%), limonene (< 6%), anisaldehyde (< 1%) and 0.5 % fenchone. An alternative method for characterization of both the non-polar volatile and non volatile fractions was developed using n-hexane and dichloromethane (3:1, v/v) in a Soxhlet extraction followed by transesterification. The obtained extracts were then characterized and the dominant fatty acid was 18:1 (petroselinic and oleic acid) 75.0-82.8%, followed by 18:2 (linoleic acid) 10.8-16.2% and other fatty acids: palmitic (4.3-6.9%), stearic (1.2-1.7%) and myristic (0-2.9%). The results for the volatile fraction after Soxhlet extraction and transesterification did not significantly differ from results obtained after hydrodistillation, especially for the main components (trans-anethole, estragole, fenchone and limonene), implying that the developed method can be used for simultaneous determination of volatiles and fatty acids.

  13. Preparation and evaluation of poly(alkyl methacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithic columns for separating polar small molecules by capillary liquid chromatography.

    PubMed

    Lin, Shu-Ling; Wu, Yu-Ru; Lin, Tzuen-Yeuan; Fuh, Ming-Ren

    2015-04-29

    In this study, methacrylic acid (MAA) was incorporated with alkyl methacrylates to increase the hydrophilicity of the synthesized ethylene dimethacrylate-based (EDMA-based) monoliths for separating polar small molecules by capillary LC analysis. Different alkyl methacrylate-MAA ratios were investigated to prepare a series of 30% alkyl methacrylate-MAA-EDMA monoliths in fused-silica capillaries (250-μm i.d.). The porosity, permeability, and column efficiency of the synthesized MAA-incorporated monolithic columns were characterized. A mixture of phenol derivatives is employed to evaluate the applicability of using the prepared monolithic columns for separating small molecules. Fast separation of six phenol derivatives was achieved in 5 min with gradient elution using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. In addition, the effect of acetonitrile content in mobile phase on retention factor and plate height as well as the plate height-flow velocity curves were also investigated to further examine the performance of the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. Moreover, the applicability of prepared polymer-based monolithic column for potential food safety applications was also demonstrated by analyzing five aflatoxins and three phenicol antibiotics using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column.

  14. Rapid determination of caffeoylquinic acid derivatives in Cynara scolymus L. by ultra-fast liquid chromatography/tandem mass spectrometry based on a fused core C18 column.

    PubMed

    Shen, Qing; Dai, Zhiyuan; Lu, Yanbin

    2010-10-01

    An ultra-fast high-performance LC-ESI-MS/MS method was developed for the analysis and quantification of caffeoylquinic acid derivatives, including chlorogenic acid, 1,3-di-O-caffeoylquinic acid (cynarin) and 1,5-di-O-caffeoylquinic acid, in artichoke (Cynara scolymus L.) heads and leaves. The rapid separation (less than 4  min) was achieved based on a Halo fused core C18-silica column (50  mm × 2.1  mm id, 2.7  μm). The target compounds were detected and quantified by a triple-quadrupole mass spectrometer in multiple-reaction monitoring mode. The calibration function is linear from 0.06 to 2800  ng/mL for chlorogenic acid, 0.3-3000  ng/mL for cynarin and 0.24-4800  ng/mL for 1,5-di-O-caffeoylquinic acid, respectively. The average recoveries ranged from 92.1 to 113.2% with RSDs ≤6.5%. Moreover, four batches of artichoke head and leaf extracts were analyzed using the established method. The results indicated that the Halo fused core column provided much faster separations and higher sample throughput without sacrificing column ruggedness and reliability, and triple-quadrupole MS provided extraordinarily lower LOQs for most of the target analytes. Comparing to conventional quantitative approaches, the established method was fast, sensitive and reliable for the determination of caffeoylquinic acid derivatives in artichoke.

  15. Enantioseparation of N-derivatized amino acids by micro-liquid chromatography/laser induced fluorescence detection using quinidine-based monolithic columns.

    PubMed

    Wu, Huihui; Wang, Qiqin; Ruan, Meng; Peng, Kun; Zhu, Peijie; Crommen, Jacques; Han, Hai; Jiang, Zhengjin

    2016-03-20

    A novel carbamoylated quinidine based monolith, namely poly(O-9-[2-(methacryloyloxy)-ethylcarbamoyl]-10,11-dihydroquinidine-co-ethylene dimethacrylate (poly(MQD-co-EDMA)), was prepared for the micro-LC enantioseparation of N-derivatized amino acids. The influence of the mobile phase composition, including the organic modifier proportion, the apparent pH and the buffer concentration, on the enantioresolution of N-derivatized amino acids was systematically investigated. Satisfactory column performance in terms of permeability, efficiency and reproducibility was obtained in most cases. The majority of the enantiomers of the tested N-protected amino acids, including 3,5-DNB, 3,5-DClB, FMOC, 3,5-DMB, p-NB, m-ClB, p-ClB and B derivatives, could be baseline separated on the poly(MQD-co-EDMA) monolithic column within 25min. A self-assembled laser induced fluorescence (LIF) detector was employed to improve sensitivity when analyzing 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives of amino acids. Ten NBD-derivatized amino acids, including arginine and histidine whose enantioseparation on quinidine carbamate based CSPs has not been reported so far, were enantioresolved on the poly(MQD-co-EDMA) monolith column. It is worth noting that the d-enantiomers of NBD-derivatized amino acids eluted first, except in the case of glutamic acid. The LOD values obtained with the LIF detector were comparable to those reported using conventional LC-FL methods. The prepared poly(MQD-co-EDMA) monolithic column coupled with the LIF detector opens up interesting perspectives to the determination of trace D-amino acids in biological samples.

  16. Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

    NASA Technical Reports Server (NTRS)

    Rounds, M. A.; Nielsen, S. S.; Mitchell, C. A. (Principal Investigator)

    1993-01-01

    The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.

  17. Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates.

    PubMed

    Rounds, M A; Nielsen, S S

    1993-10-29

    The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.

  18. Using a box instead of a column for process chromatography.

    PubMed

    Ghosh, Raja

    2016-10-14

    Columns with relatively short bed-height to diameter ratios are frequently used for process-scale chromatography applications such as biopharmaceutical purification. Non-uniform flow distribution within such columns could result in broad and poorly resolved eluted peaks, which could in turn affect purity, recovery and productivity of the process. Different strategies centered on improved column header design have been proposed for addressing this problem. This paper describes a radically different approach, i.e. the use of a chromatography box (or chromato-box) instead of a column, for addressing the challenges posed by flow mal-distribution in process-scale, packed-bed chromatography devices. The design of the chromatography box devices used in this study is based on a laterally-fed membrane chromatography (or LFMC) device, that has been described and discussed in several recent papers. The performances of two chromatography box devices were compared with their equivalent columns in terms of sharpness and asymmetry of flow-through and eluted peaks, number of theoretical plates per metre, and peak resolution in binary and ternary protein separations. In each of the above comparisons, the chromatography box devices performed better than their equivalent columns, clearly indicating their potential as an alternative in process-scale chromatography applications.

  19. Differential coulometric oxidation following post column-switching high pressure liquid chromatography for fluorescence measurement of unmetabolized folic acid in human plasma.

    PubMed

    Bailey, Steven W; Ayling, June E

    2013-11-08

    Although many countries have fortified their grain supplies with folic acid (FA) to decrease the incidence of neural tube defects, others have not due to concerns that this synthetic folate might have some adverse effects. Persistent unmetabolized FA has been found even in plasma from fasted subjects. To facilitate measurement of low levels of folic acid in human plasma, post-column coulometric oxidative cleavage was used to convert poorly fluorescent FA into a highly fluorescent compound determined to be 6-formyl-pterin. To minimize sample work-up and maximize recovery, column-switching HPLC transferred a window of eluate containing the FA from the first column (C8) onto a second column (phenyl-hexyl). The pH of two mobile phases were adjusted to be above and then below a pK of the FA α-carboxyl group, thus promoting separation from compounds coeluting from the C8-column. This permitted sample preparation using only a simple high recovery protein precipitation. Definitive identification of FA in human plasma was accomplished by duplicate injections of sample with the electrochemical voltage set above and below its half-potential. The LOD (S/N=3) was 0.10 nM. The intra- and inter-assay CV's were 2.3% and 5%, respectively. Comparison of these results with those obtained by HPLC/MS/MS with stable isotope internal standard showed a slope of 1.00 ± 0.019. This simple, sensitive, and repeatable assay facilitates a more thorough investigation of the response of various human populations to folic acid intake. Post-column differential coulometric electrochemistry can expand the variety of compounds amenable to fluorescence detection.

  20. Direct probing of chromatography columns by laser-induced fluorescence

    NASA Astrophysics Data System (ADS)

    McGuffin, V. L.

    1992-12-01

    This report summarizes the progress and accomplishments of this research project from 1 Sep. 1989 to 28 Feb. 1993. During this period, we have accomplished all of the primary scientific objectives of the research proposal: (1) constructed and evaluated a laser-induced fluorescence detection system that allows direct examination of the chromatographic column, (2) examined nonequilibrium processes that occur upon solute injection and elution, (3) examined solute retention in liquid chromatography as a function of temperature and pressure, (4) examined solute zone dispersion in liquid chromatography as a function of temperature and pressure, and (5) developed appropriate theoretical models to describe these phenomena. In each of these studies, substantial knowledge has been gained of the fundamental processes that are responsible for chromatographic separations. In addition to these primary research objectives, we have made significant progress in three related areas: (1) examined pyrene as a fluorescent polarity probe in supercritical fluids and liquids as a function of temperature and pressure, (2) developed methods for the class-selective identification of polynuclear aromatic hydrocarbons in coal-derived fluids by microcolumn liquid chromatography with fluorescence quenching detection, and (3) developed methods for the determination of saturated and unsaturated (including omega-3) fatty acids in fish oil extracts by microcolumn liquid chromatography with laser-induced fluorescence detection. In these studies, the advanced separation and detection techniques developed in our laboratory are applied to practical problems of environmental and biomedical significance.

  1. Stationary phases for packed-column supercritical fluid chromatography.

    PubMed

    Poole, Colin F

    2012-08-10

    The properties of silica-based, chemically bonded, packed column stationary phases used in supercritical fluid chromatography are described with a focus on column design and retention mechanisms. Supercritical fluid chromatography has benefited substantially from innovations in column design for liquid chromatography even if the separation conditions employed are generally quite different. The mobile phase composition and column operating conditions play an interactive role in modifying selectivity in supercritical fluid chromatography by altering analyte solubility in the mobile phase and through selective solvation of the stationary phase resulting in a wider range and intensity of intermolecular interactions with the analyte. The solvation parameter model is used to identify the main parameters that affect retention in supercritical fluid chromatography using carbon dioxide-methanol as a mobile phase and as a basis for column characterization to facilitate the identification of stationary phases with different separation characteristics for method development. As a caution it is pointed out that these column characterization methods are possibly a product of both the stationary phase chemistry and the column operating conditions and are suitable for use only when columns of similar design and with similar operating conditions are used.

  2. Ion Exchange and Liquid Column Chromatography.

    ERIC Educational Resources Information Center

    Walton, Harold F.

    1980-01-01

    Emphasizes recent advances in principles and methodology in ion exchange and chromatography. Two tables list representative examples for inorganic ions and organic compounds. Cites 544 references. (CS)

  3. Simultaneous determination of neutral and acidic pharmaceuticals in wastewater by high-performance liquid chromatography-post-column photochemically induced fluorimetry.

    PubMed

    González-Barreiro, C; Lores, M; Casais, M C; Cela, R

    2003-04-18

    An analytical method for the simultaneous determination of acidic and neutral pharmaceutical active compound (PhACs) residues in wastewater has been developed based on the combination of high-performance liquid chromatography (HPLC) and photochemically induced fluorimetry. The photoderivatization conditions for each particular PhAC have been assessed. Off-line optimization of the HPLC separation for both neutral and acidic compounds has been utilised and evaluated. Detection limits in the low ng/ml range have been achieved without sample pretreatment. By applying the developed analytical method combined with solid-phase extraction to real wastewater samples an enrichment factor of approximately two orders of magnitude can be obtained.

  4. Methodology for optimally sized centrifugal partition chromatography columns.

    PubMed

    Chollet, Sébastien; Marchal, Luc; Jérémy Meucci; Renault, Jean-Hugues; Legrand, Jack; Foucault, Alain

    2015-04-03

    Centrifugal Partition Chromatography (CPC) is a separation process based on the partitioning of solutes between two partially miscible liquid phases. There is no solid support for the stationary phase. The centrifugal acceleration is responsible for both stationary phase retention and mobile phase dispersion. CPC is thus a process based on liquid-liquid mass transfer. The separation efficiency is mainly influenced by the hydrodynamics of the phases in each cell of the column. Thanks to a visualization system, called "Visual CPC", it was observed that the mobile phase can flow through the stationary phase as a sheet, or a spray. Hydrodynamics, which directly governs the instrument efficiency, is directly affected during scale changes, and non-linear phenomena prevent the successful achievement of mastered geometrical scale changes. In this work, a methodology for CPC column sizing is proposed, based on the characterization of the efficiency of advanced cell shapes, taking into account the hydrodynamics. Knowledge about relationship between stationary phase volume, cell efficiency and separation resolution in CPC allowed calculating the optimum cell number for laboratory and industrial scale CPC application. The methodology is highlighted with results on five different geometries from 25 to 5000 mL, for two applications: the separation of alkylbenzene by partitioning with heptane/methanol/water biphasic system; and the separation of peptides by partitioning with n-butanol/acetic acid/water (4/1/5) biphasic system. With this approach, it is possible to predict the optimal CPC column length leading to highest productivity.

  5. A Better Method for Filling Pasteur Pipet Chromatography Columns

    ERIC Educational Resources Information Center

    Ruekberg, Ben

    2006-01-01

    An alternative method for the preparation of Pasteur pipet chromatography columns is presented that allows the column to be filled with solvent without bubbles and allows greater control of fluid flow while the materials to be separated are added. Students are required to wear gloves and goggles and caution should be used while handling glass…

  6. Combining micro dry column chromatography and mass spectrometry

    NASA Technical Reports Server (NTRS)

    Bauman, A. J.

    1970-01-01

    Dry column chromatography principles applied in microscale produce technique to minimize time in preparing and analyzing colorless constituents of soluble mixtures. Glass pipette microcolumns filled with finely sieved adsorbents permit capillary attraction and separation in 3 to 15 minutes. Technique is adaptable to gas chromatography.

  7. Column precipitation chromatography: an approach to quantitative analysis of eigencolloids.

    PubMed

    Breynaert, E; Maes, A

    2005-08-01

    A new column precipitation chromatography (CPC) technique, capable of quantitatively measuring technetium eigencolloids in aqueous solutions, is presented. The CPC technique is based on the destabilization and precipitation of eigencolloids by polycations in a confined matrix. Tc(IV) colloids can be quantitatively determined from their precipitation onto the CPC column (separation step) and their subsequent elution upon oxidation to pertechnetate by peroxide (elution step). A clean-bed particle removal model was used to explain the experimental results.

  8. Effect of column dimension on observed column efficiency in very high pressure liquid chromatography.

    PubMed

    Wu, Naijun; Bradley, Ashley C

    2012-10-26

    The effect of extra-column volume on observed linear velocity was investigated for columns of various internal diameters in very high pressure liquid chromatography. The results showed that the observed linear velocities were approximately 4.5, 9.5, 16.8, and 39.5% lower than the linear velocities corrected for the extra-column volume contribution for 4.6, 3.0, 2.1, and 1.0mm internal diameter columns, respectively. An empirical relationship between extra-column band broadening and extra-column volume was obtained using 50 cm long tubings of various internal diameters. The peak variance from the extra-column volume is near linearly proportional to the square of the extra-column volume for tubings with 0.0635-0.178 mm (0.025-0.07 in.) i.d. using a 50/50 acetonitrile/water mobile phase at flow rates greater than 0.3 mL/min. The effect of column internal diameter and column length on observed efficiency was studied using 50mm columns with four different column internal diameters and 2.1mm i.d columns with three different lengths. The results showed that the observed column efficiencies for 3.0, 2.1, and 1.0mm internal diameter columns were 18, 33, and 73% lower than that for a 4.6mm internal diameter column for benzophenone (k=5.5), respectively. An approximate 20% decrease in theoretical plate number was observed for propiophenone (k=3.3) using a 50 mm × 2.1 mm column packed with 1.7 μm particles compared to a 150 mm × 2.1 mm column packed with 5.0 μm particles, while the former column provided 9 fold faster separation. It is the column to extra column volume ratio instead of absolute extra-column volume that determines the degree of extra-column band-broadening in VHPLC.

  9. Design, testing, and simulation of microscale gas chromatography columns

    SciTech Connect

    Hudson, M.L.; Kottenstette, R.; Matzke, C.M.; Frye-Mason, G.C.; Shollenberger, K.A.; Adkins, D.R.; Wong, C.C.

    1998-08-01

    A microscale gas chromatography column is one component in a microscale chemistry laboratory for detecting chemical agents. Several columns were fabricated using the Bosch etch process which allows deep, high aspect ratio channels of rectangular cross-section. A design tool, based on analytical models, was developed to evaluate the effects of operating conditions and column specifications on separation resolution and time. The effects of slip flow, channel configuration, and cross-sectional shape were included to evaluate the differences between conventional round, straight columns and the microscale rectangular, spiral columns. Experimental data were obtained and compared with the predicted flowrates and theoretical number of plates. The design tool was then employed to select more optimum channel dimensions and operating conditions for high resolution separations.

  10. Column Chromatography To Obtain Organic Cation Sorption Isotherms.

    PubMed

    Jolin, William C; Sullivan, James; Vasudevan, Dharni; MacKay, Allison A

    2016-08-02

    Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation.

  11. A hybrid fluorous monolithic capillary column with integrated nanoelectrospray ionization emitter for determination of perfluoroalkyl acids by nano-liquid chromatography-nanoelectrospray ionization-mass spectrometry/mass spectrometry.

    PubMed

    Zhang, Haiyang; Ou, Junjie; Wei, Yinmao; Wang, Hongwei; Liu, Zhongshan; Zou, Hanfa

    2016-04-01

    A hybrid fluorous monolithic column was simply prepared via photo-initiated free radical polymerization of an acrylopropyl polyhedral oligomeric silsesquioxane (acryl-POSS) and a perfluorous monomer (2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate) in UV-transparent fused-silica capillaries within 5min. The physical characterization of hybrid fluorous monolith, including scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, mercury intrusion porosimetry (MIP) and nitrogen adsorption/desorption measurement was performed. Chromatographic performance was also evaluated by capillary liquid chromatography (cLC). Due to the fluorous-fluorous interaction between fluorous monolith and analytes, fluorobenzenes could well be separated, and the column efficiencies reached 86,600-92,500plates/m at the velocity of 0.87mm/s for alkylbenzenes and 51,900-76,000plates/m at the velocity of 1.10mm/s for fluorobenzenes. Meanwhile, an approach to integrate nanoelectrospray ionization (ESI) emitter with hybrid fluorous monolithic column was developed for quantitative determination of perfluoroalkyl acids by nanoHPLC-ESI-MS/MS. The integration design could minimize extracolumn volume, thus excluding undesirable peak broadening and improving separation performance.

  12. Analyzing insulin samples by size-exclusion chromatography: a column degradation study.

    PubMed

    Teska, Brandon M; Kumar, Amit; Carpenter, John F; Wempe, Michael F

    2015-04-01

    Investigating insulin analogs and probing their intrinsic stability at physiological temperature, we observed significant degradation in the size-exclusion chromatography (SEC) signal over a moderate number of insulin sample injections, which generated concerns about the quality of the separations. Therefore, our research goal was to identify the cause(s) for the observed signal degradation and attempt to mitigate the degradation in order to extend SEC column lifespan. In these studies, we used multiangle light scattering, nuclear magnetic resonance, and gas chromatography-mass spectrometry methods to evaluate column degradation. The results from these studies illustrate: (1) that zinc ions introduced by the insulin product produced the observed column performance issues; and (2) that including ethylenediaminetetraacetic acid, a zinc chelator, in the mobile phase helped to maintain column performance.

  13. Estimation of alkane-water logP for neutral, acidic, and basic compounds using an alkylated polystyrene-divinylbenzene high-performance liquid chromatography column.

    PubMed

    Jensen, Derek A; Gary, Ronald K

    2015-10-23

    Reliable HPLC methods are available to estimate octanol-water partition coefficients, but there is no comparable method for alkane-water partition coefficients that is accurate and applicable across a broad span of logP(alk). This study describes a high-throughput method for determining HPLC-logP(alk), a chromatographic parameter closely related to logP(alk), using an alkylated polystyrene-divinylbenzene column and fast acetonitrile gradient. A structurally diverse set of neutral, acidic, and basic compounds was analyzed under ionization-suppressing pH conditions. In this chromatographic system, the relationship between gradient retention time and isocratic logk was essentially linear. Thus, gradient retention time could be used as the sole input needed to determine an apparent logP(alk)by HPLC. HPLC-logP(alk) showed linear correlation (R(2)>0.96, n=59) with reference logP(alk) values from shake-flask measurements over 8 orders of magnitude, ranging from -2.3 to +5.7. Linear solvation energy relationship (LSER) analysis revealed that the relative contributions of intermolecular forces effecting retention in the fast gradient system or its corresponding isocratic variant were highly similar to those governing partition in bulk alkane-water.

  14. Separation of enantiomers of chiral weak acids with polysaccharide-based chiral columns and aqueous-organic mobile phases in high-performance liquid chromatography: Typical reversed-phase behavior?

    PubMed

    Matarashvili, Iza; Ghughunishvili, Darejan; Chankvetadze, Lali; Takaishvili, Nino; Khatiashvili, Tamar; Tsintsadze, Maia; Farkas, Tivadar; Chankvetadze, Bezhan

    2017-02-03

    When polysaccharide-based chiral columns are used in combination with aqueous-organic mobile phases for the separation of enantiomers in high-performance liquid chromatography the separation mode is commonly called "reversed-phase" in analogy to achiral separations. In several earlier and recent studies on neutral and basic chiral analytes it was shown by our and other groups that due to multiple type of interactions involved in selector-selectand binding and enantioselective recognition with polysaccharide derivatives, the above mentioned separation system may not always behave like a reversed-phase system. In the present study additional examples of non-reversed-phase behavior are described for the first time for weak acidic chiral analytes. In addition, the reversal of enantiomer elution order was observed again for the first time for several analytes based on water-content in the mobile phase.

  15. Evaluating two process scale chromatography column header designs using CFD.

    PubMed

    Johnson, Chris; Natarajan, Venkatesh; Antoniou, Chris

    2014-01-01

    Chromatography is an indispensable unit operation in the downstream processing of biomolecules. Scaling of chromatographic operations typically involves a significant increase in the column diameter. At this scale, the flow distribution within a packed bed could be severely affected by the distributor design in process scale columns. Different vendors offer process scale columns with varying design features. The effect of these design features on the flow distribution in packed beds and the resultant effect on column efficiency and cleanability needs to be properly understood in order to prevent unpleasant surprises on scale-up. Computational Fluid Dynamics (CFD) provides a cost-effective means to explore the effect of various distributor designs on process scale performance. In this work, we present a CFD tool that was developed and validated against experimental dye traces and tracer injections. Subsequently, the tool was employed to compare and contrast two commercially available header designs.

  16. Novel Design for Centrifugal Countercurrent Chromatography: I. Zigzag Toroidal Column

    PubMed Central

    Yang, Yi; Aisa, Haji Akber; Ito, Yoichiro

    2009-01-01

    The toroidal coil using an equilateral triangular core has improved both retention of the stationary phase and peak resolution of the conventional toroidal coil in centrifugal countercurrent chromatography. To further improve the retention of stationary phase and peak resolution, a novel zigzag toroidal coil was designed and the performance of the system was evaluated at various flow rates. The results indicated that both retention of stationary phase and peak resolution were improved as the flow rate was decreased. Modification of the tubing by pressing at given intervals with a pair of pliers improved the peak resolution without increasing the column pressure. All these separations were performed under low column pressure indicating the separation can be further improved by increasing the column length and/or revolution speed without damaging the separation column. PMID:20046954

  17. A simple parallel gas chromatography column screening system.

    PubMed

    Schafer, Wes; Hamilton, Simon E; Pirzada, Zainab; Welch, Christopher J

    2012-01-01

    A simple approach to the automated screening of four different columns on a single gas chromatography (GC) instrument is used for rapid chiral GC method development. Configuration of a conventional GC instrument with a second autosampler and several inexpensive Y-splitters enables simultaneous evaluation of two different columns, allowing a total of four different columns to be evaluated in two automated back to back runs. The resulting system affords a simple and effective approach to chiral GC method development that speeds analysis while eliminating the need for slow and tedious manual interchange of columns. An example of developing a rapid isothermal GC method from the screening results obtained by the instrument is also shown.

  18. Analysis of Cordyceps by multi-column liquid chromatography.

    PubMed

    Qian, Zhengming; Li, Shaoping

    2017-03-01

    Cordyceps is a famous traditional Chinese medicine (TCM) that has been used in China for hundreds of years. In the present study a multi-column liquid chromatography (MC-LC) system was developed for the qualitative analysis of macromolecules and micromolecules in Cordyceps. The MC-LC system includes a size exclusion pre-column, a size exclusion column (SEC) and a reversed phase column (RP) which were controlled by column-switching valves. The sample was separated by the size exclusion pre-column into two fractions (macromolecules and micromolecules). These fractions were further separated on SEC and RP columns, respectively. A diode array detector (DAD) and a mass spectrometer (MS) were used to detect the components. This MC-LC method was utilized for analysis of Cordyceps samples. Two macromolecular peaks and 15 micromolecular peaks were found in Cordyceps, and 11 of the micromolecular peaks were identified as adenosine-5'-monophosphate (AMP), phenylalanine, uridine, hypoxanthine, inosine, guanine, guanosine, deoxyadenosine-5'-monophosphate (dAMP), adenosine, adenine and cordycepin (or its isomer). This method is useful for quality control of Cordyceps.

  19. Rapid column heating method for subcritical water chromatography.

    PubMed

    Fogwill, Michael O; Thurbide, Kevin B

    2007-01-19

    A novel resistive heating method is presented for subcritical water chromatography (SWC) that provides higher column heating rates than those conventionally obtained from temperature-programmed gas chromatography (GC) convection ovens. Since the polarity of water reduces dramatically with increasing temperature, SWC employs column heating to achieve gradient elution. As such, the rate at which the mobile phase is heated directly impacts the magnitude of such gradients applied in SWC. Data from the current study demonstrate that the maximum column heating rate attainable in a typical SWC apparatus (i.e. using a GC convection oven) is around 10 degrees C/min, even at instrument oven settings of over three times this value. Conversely, by wrapping the separation column with ceramic insulation and a resistively heated wire, the column heating rates are increased five-fold. As a result, elution times can be greatly decreased in SWC employing gradients. Separations of standard alcohol test mixtures demonstrate that the retention time of the latest eluting component decreases by 35 to 50% using the prototype method. Additionally, solute retention times in this mode deviate by less than 1% RSD over several trials, which compares very well to those obtained using a conventional GC convection oven. Results suggest that the developed method can be a useful alternative heating technique in SWC.

  20. Chemometrics applications in biotechnology processes: predicting column integrity and impurity clearance during reuse of chromatography resin.

    PubMed

    Rathore, Anurag S; Mittal, Shachi; Lute, Scott; Brorson, Kurt

    2012-01-01

    Separation media, in particular chromatography media, is typically one of the major contributors to the cost of goods for production of a biotechnology therapeutic. To be cost-effective, it is industry practice that media be reused over several cycles before being discarded. The traditional approach for estimating the number of cycles a particular media can be reused for involves performing laboratory scale experiments that monitor column performance and carryover. This dataset is then used to predict the number of cycles the media can be used at manufacturing scale (concurrent validation). Although, well accepted and widely practiced, there are challenges associated with extrapolating the laboratory scale data to manufacturing scale due to differences that may exist across scales. Factors that may be different include: level of impurities in the feed material, lot to lot variability in feedstock impurities, design of the column housing unit with respect to cleanability, and homogeneity of the column packing. In view of these challenges, there is a need for approaches that may be able to predict column underperformance at the manufacturing scale over the product lifecycle. In case such an underperformance is predicted, the operators can unpack and repack the chromatography column beforehand and thus avoid batch loss. Chemometrics offers one such solution. In this article, we present an application of chemometrics toward the analysis of a set of chromatography profiles with the intention of predicting the various events of column underperformance including the backpressure buildup and inefficient deoxyribonucleic acid clearance.

  1. A versatile noninvasive method for adsorber quantification in batch and column chromatography based on the ionic capacity.

    PubMed

    Huuk, Thiemo C; Briskot, Till; Hahn, Tobias; Hubbuch, Jürgen

    2016-05-01

    Within the Quality by Design (QbD) framework proposed by the International Conference on Harmonisation (ICH), high-throughput process development (HTPD) and mechanistic modeling are of outstanding importance for future biopharmaceutical chromatography process development. In order to compare the data derived from different column scales or batch chromatographies, the amount of adsorber has to be quantified with the same noninvasive method. Similarly, an important requirement for the implementation of mechanistic modeling is the reliable determination of column characteristics such as the ionic capacity Λ for ion-exchange chromatography with the same method at all scales and formats. We developed a method to determine the ionic capacity in column and batch chromatography, based on the adsorption/desorption of the natural, uv-detectable amino acid histidine. In column chromatography, this method produces results comparable to those of classical acid-base titration. In contrast to acid-base titration, this method can be adapted to robotic batch chromatographic experiments. We are able to convert the adsorber volumes in batch chromatography to the equivalent volume of a compressed column. In a case study, we demonstrate that this method increases the quality of SMA parameters fitted to batch adsorption isotherms, and the capability to predict column breakthrough experiments. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:666-677, 2016.

  2. Glycolipid class profiling by packed-column subcritical fluid chromatography.

    PubMed

    Deschamps, Frantz S; Lesellier, Eric; Bleton, Jean; Baillet, Arlette; Tchapla, Alain; Chaminade, Pierre

    2004-06-18

    The potential of packed-column subcritical fluid chromatography (SubFC) for the separation of lipid classes has been assessed in this study. Three polar stationary phases were checked: silica, diol, and poly(vinyl alcohol). Carbon dioxide (CO2) with methanol as modifier was used as mobile phase and detection performed by evaporative light scattering detection. The influence of methanol content, temperature, and pressure on the chromatographic behavior of sphingolipids and glycolipids were investigated. A complete separation of lipid classes from a crude wheat lipid extract was achieved using a modifier gradient from 10 to 40% methanol in carbon dioxide. Solute selectivity was improved using coupled silica and diol columns in series. Because the variation of eluotropic strength depending on the fluid density changes, a normalized separation factor product (NSP) was used to select the nature, the number and the order of the columns to reach the optimum glycolipid separation.

  3. Comparison of IMAC and MOAC for phosphopeptide enrichment by column chromatography.

    PubMed

    Negroni, Luc; Claverol, Stephane; Rosenbaum, Jean; Chevet, Eric; Bonneu, Marc; Schmitter, Jean-Marie

    2012-04-01

    Automated phosphopeptide enrichment prior to MS analysis by means of Immobilized Metal Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC) has been probed with packed columns. We compared POROS-Fe³⁺ and TiO₂ (respectively IMAC and MOAC media), using a simple mixture of peptides from casein-albumin and a complex mixture of peptides isolated from mouse liver. With theses samples, selectivity of POROS-Fe³⁺ and TiO₂ were pH dependant. In the case of liver extract, selectivity increased from 12-18% to 58-60% when loading buffer contained 0.1 M acetic acid or 0.1 M trifluoroacetic acid, respectively. However, with POROS-Fe³⁺ column, the number of identifications decreased from 356 phosphopeptides with 0.1 M acetic acid to 119 phosphopeptides with 0.1 M TFA. This decrease of binding capacity of POROS-Fe³⁺ was associated with strong Fe³⁺ leaching. Furthermore, repetitive use of IMAC-Fe³⁺ with the 0.5 M NH₄OH solution required for phosphopeptide elution induced Fe₂O₃ accumulation in the column. By comparison, MOAC columns packed with TiO₂ support do not present any problem of stability in the same conditions and provide a reliable solution for packed column phosphopeptide enrichment.

  4. Generator for ionic gallium-68 based on column chromatography

    DOEpatents

    Neirinckx, Rudi D.; Davis, Michael A.

    1981-01-01

    A physiologically acceptable solution of gallium-68 fluorides, having an activity of 0.1 to 50 millicuries per milliliter of solution is provided. The solution is obtained from a generator comprising germanium-68 hexafluoride bound to a column of an anion exchange resin which forms gallium-68 in situ by eluting the column with an acid solution to form a solution containing .sup.68 Ga-fluorides. The solution then is neutralized prior to administration.

  5. Comparison of automated pre-column and post-column analysis of amino acid oligomers

    NASA Technical Reports Server (NTRS)

    Chow, J.; Orenberg, J. B.; Nugent, K. D.

    1987-01-01

    It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers (n = 2-6). The investigations of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that the pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.

  6. Analysis of biomass sugars and galacturonic acid by gradient anion exchange chromatography and pulsed amperometric detection without post-column addition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While the most accurate method for analysis of sugars in biomass is based on gas chromatography of trimethylsilane or alditol acetate derivatives of sugars, the derivation method is time consuming and laborious. In comparison, sample preparation for sugar analysis of hydrolyzed biomass samples using...

  7. Column selectivity in reversed-phase liquid chromatography. IV. Type-B alkyl-silica columns.

    PubMed

    Gilroy, Jonathan J; Dolan, John W; Snyder, Lloyd R

    2003-06-06

    Columns for reversed-phase HPLC (RP-LC) can be characterized by five, retention-related parameters: H (hydrophobicity), S (steric selectivity), A (hydrogen-bond acidity), B (hydrogen-bond basicity), and C (cation-exchange behavior). In the present study, values of the latter parameters have been measured for 92 type-B (low metals content)alkyl-silica columns and compared to column properties such as ligand length,ligand concentration, pore diameter, and the presence or absence of end-capping. With the exception of five columns of unusual design, retention factors, k, for 16 representative test compounds were correlated with values of H, S, etc., within an average +/- 1.2% (1 standard deviation, SD), suggesting that all significant solute-column interactions are recognized by these five column parameters. A single-valued function F(s) is proposed to measure differences in selectivity for any two RP-LC columns whose values of H, S, etc., are known. This allows the easy selection of columns whose selectivity is desired to be either similar to or different from a starting column, for application in either routine analysis or method development.

  8. Hydrothermal preparation of hybrid carbon/silica monolithic capillary column for liquid chromatography.

    PubMed

    Yang, Peiling; Wang, Wentao; Xiao, Xing; Jia, Li

    2014-08-01

    A simple, easy and economical approach for the preparation of a hybrid carbon/silica monolithic capillary column was described for the first time by using silica monolith as framework in combination with hydrothermal carbonization at 180°C. During the preparation process, formamide was introduced to the reaction solutions to reduce the dissolution rate of monolithic silica skeleton and its optimal concentration was 1.5 M. Fourier transform infrared spectrometry, scanning electron microscopy, energy dispersive X-ray spectrometry, and inverse size exclusion chromatography were carried out to characterize the as-prepared column. The results demonstrated that carbon spheres ranging from 150 to 1000 nm were successfully attached to the surface of silica skeleton. The prepared hybrid carbon/silica column had a permeability of 4.4 × 10(-14) m(2). Chromatographic performance of the column was evaluated by separation of various compounds including alkylbenzenes, nucleosides and bases, and aromatic acids. The column exhibited an efficiency of 75,000 plates/m for butylbenzene at the optimal linear velocity of 0.23 mm/s. The successful separation of these compounds and the study on mechanism indicated that the column can be applied in mixed-mode chromatography.

  9. Comparison of the kinetic performance of different columns for fast liquid chromatography, emphasizing the contributions of column end structure.

    PubMed

    Lambert, Nándor; Miyazaki, Shota; Ohira, Masayoshi; Tanaka, Nobuo; Felinger, Attila

    2016-11-18

    The kinetic performance of five chromatographic columns designed for fast liquid chromatography with different column packing materials - including fully porous (2.0 and 1.9μm particles), core-shell (2.6μm particles) or monolithic packings - with identical column dimensions (2.1×50mm) was tested. Since the tested monolithic column showed systematically better efficiency for early eluting compounds than the packed columns, an additional band broadening effect was suspected for the packed columns. The effects of the presence of the frits and the bed heterogeneity of the columns near the frits were characterized by a column-reversal method. It has been shown that significant differences - even 20-25% difference in efficiency - can exist between the two ends of the packed columns, while the monolithic column shows rather similar performance at either column end.

  10. Determination of S-carboxymethylcysteine in serum by reversed-phase ion-pair liquid chromatography with column switching following pre-column derivatization with o-phthalaldehyde.

    PubMed

    De Schutter, J A; Van der Weken, G; Van den Bossche, W; de Moerloose, P

    1988-07-15

    A method is described for the determination of S-(carboxymethyl)-L-cysteine in serum. After addition of S-(carboxyethyl)-L-cysteine as internal standard, both compounds are extracted into methanol, converted into fluorescent derivatives with o-phthalaldehyde and quantitatively determined by reversed-phase high-performance liquid chromatography. Chromatography of unwanted amino acid derivatives is avoided by column switching, thereby shortening analysis time and increasing column lifetime. The technique was applied in a study of the bioavailability of S-(carboxymethyl)-L-cysteine after oral administration to humans. The concentration-response curve was linear from 2 to 16 micrograms/ml; mean serum concentrations are reported.

  11. If You Were a Molecule in a Chromatography Column, What Would You See?

    ERIC Educational Resources Information Center

    Mattice, John

    2008-01-01

    To visualize what takes place in a chromatography column, enlarge the molecules to human size and expand the columns to keep the ratio of size of molecule to size of column the same. If we were molecules, what would the columns be like? A typical gas chromatography (GC) capillary column would be 50 x 10 [superscript 6] 6 km (31 million mi) long,…

  12. Selective extraction and analysis of catecholamines in rat blood microdialysate by polymeric ionic liquid-diphenylboric acid-packed capillary column and fast separation in high-performance liquid chromatography-electrochemical detector.

    PubMed

    Zhou, Xinguang; Zhu, Anwei; Shi, Guoyue

    2015-08-28

    Concentration of blood catecholamines (CAs) is linked to a host of cardiovascular diseases, including hypertension and stenocardia. The matrix interferences and low concentration require tedious sample pretreatment methods before quantitative analysis by the gold standard method of high-performance liquid chromatography-electrochemical detector (HPLC-ECD). Solid phase extraction (SPE) has been widely used as the pretreatment technique. Here, a facile polymeric ionic liquid (PIL)-diphenylboric acid (DPBA)-packed capillary column was prepared to selectively extract dopamine (DA), noradrenaline (NE) and epinephrine (E) prior to their quantitative analysis by a fast separation in HPLC-ECD method, while microdialysis sampling method was applied to get the analysis sample. Parameters that influenced desorption efficiency, such as pH, salt concentration, acetonitrile content and wash time, were examined and optimized. The proposed method, combining microdialysis sampling technique, SPE and HPLC-ECD system, was successfully applied to detect CAs in rat blood microdialysate with high sensitivity and selectivity in small sample volumes (5-40μl) and a short analysis time (8min), yielding good reproducibility (RSD 6.5-7.7%) and spiked recovery (91-104.4%).

  13. Selective isolation of β-glucan from corn pericarp hemicelluloses by affinity chromatography on cellulose column.

    PubMed

    Yoshida, Tomoki; Honda, Yoichi; Tsujimoto, Takashi; Uyama, Hiroshi; Azuma, Jun-ichi

    2014-10-13

    A combination of anion-exchange chromatography and affinity chromatography on a cellulose column was found to be effective for the isolation of β-(1,3;1,4)-glucan (BG) from corn pericarp hemicelluloses (CPHs). CPHs containing 6.6% BG were extracted from corn pericarp with 6M urea-2 wt% NaOH solution and initially fractionated into neutral and acidic parts by anion exchange chromatography to remove acidic arabinoxylan consisting of arabinose (35.6%) and xylose (50.9%). The neutral fraction (yield; 10.1% on the basis of CPHs) consisting of 1.0% arabinose, 10.1% xylose and 80.3% glucose containing 28.4% BG was then applied to a cellulose column of Whatman CF-11. BG could be recovered from the adsorbed fraction on the cellulose column by elution with 2% NaOH in a yield of 2.6% on the basis of CPHs with a purity of 84.7%. The chemical structure of the isolated corn pericarp BG was confirmed by (13)C NMR spectroscopic, methylation and lichenase treatment analyses. The results indicate that the ratios of (1,4)/(1,3) linkage and cellotriosyl/cellotetraosyl segments of the BG were 2.60 and 2.5, respectively.

  14. Group type analysis of asphalt by column liquid chromatography

    SciTech Connect

    Zhang, C.; Yang, J.; Xue, Y.; Li, Y.

    2008-07-01

    An improved analysis method for characterization of asphalt was established. The method is based on column chromatography technique. The asphalts were separated into four groups: saturates, aromatics, resins, and asphaltenes, quantitatively. About 0.1 g of sample was required in each analysis. About 20 mL of n-heptanes was used to separate out saturates first. Then about 35 mL of n-heptanes/dichloromethane (.5, v/v) mixture was used to separate out aromatics. About 30 mL of dichloromethane/tetrahydrofuran (1/3, v/v) mixture was used to separate out resin. The quality of the separation was confirmed by infrared spectra (IR) and {sup 1}H NMR analysis. The model compounds, tetracosan for saturates, dibenz(o)anthracen for aromatics, and acetanilide for resins were used for verification. The IR and {sup 1}H NMR analysis of the prepared fractions from the column liquid chromatography were in good agreement that of pure reagents.

  15. ANALYSIS OF PERFLUORINATED CARBOXYLIC ACIDS IN SOILS II: OPTIMIZATION OF CHROMATOGRAPHY AND EXTRACTION

    EPA Science Inventory

    With the objective of detecting and quantitating low concentrations of perfluorinated carboxylic acids (PFCAs), including perfluorinated octanoic acid (PFOA), in soils, we compared the analytical suitability of liquid chromatography columns containing three different stationary p...

  16. Column selectivity in reversed-phase liquid chromatography. VI. Columns with embedded or end-capping polar groups.

    PubMed

    Wilson, N S; Gilroy, J; Dolan, J W; Snyder, L R

    2004-02-13

    A previous model of column selectivity for reversed-phase liquid chromatography (RP-LC) has been applied to an additional 21 columns with embedded or end-capping polar groups (EPGs). Embedded-polar-group columns exhibit a significantly different selectivity vs. non-EPG, type-B columns, generally showing preferential retention of hydrogen-bond donors, as well as decreased retention for hydrogen-bond acceptors or ionized bases. EPG-columns are also generally less hydrophobic (more polar) than are non-EPG-columns. Interestingly, columns with polar end-capping tend to more closely resemble non-EPG columns, suggesting that the polar group has less effect on column selectivity when used to end-cap the column versus the case of an embedded polar group. Column selectivity data reported here for EPG-columns can be combined with previously reported values for non-EPG columns to provide a database of 154 different columns. This enables a comparison of any two of these columns in terms of selectivity. However, comparisons that involve EPG columns are more approximate.

  17. Optimization and simulation of tandem column supercritical fluid chromatography separations using column back pressure as a unique parameter.

    PubMed

    Wang, Chunlei; Tymiak, Adrienne A; Zhang, Yingru

    2014-04-15

    Tandem column supercritical fluid chromatography (SFC) has demonstrated to be a useful technique to resolve complex mixtures by serially coupling two columns of different selectivity. The overall selectivity of a tandem column separation is the retention time weighted average of selectivity from each coupled column. Currently, the method development merely relies on extensive screenings and is often a hit-or-miss process. No attention is paid to independently adjust retention and selectivity contributions from individual columns. In this study, we show how tandem column SFC selectivity can be optimized by changing relative dimensions (length or inner diameter) of the coupled columns. Moreover, we apply column back pressure as a unique parameter for SFC optimization. Continuous tuning of tandem column SFC selectivity is illustrated through column back pressure adjustments of the upstream column, for the first time. In addition, we show how and why changing coupling order of the columns can produce dramatically different separations. Using the empirical mathematical equation derived in our previous study, we also demonstrate a simulation of tandem column separations based on a single retention time measurement on each column. The simulation compares well with experimental results and correctly predicts column order and back pressure effects on the separations. Finally, considerations on instrument and column hardware requirements are discussed.

  18. Novel design for centrifugal counter-current chromatography: VI. Ellipsoid column.

    PubMed

    Gu, Dongyu; Yang, Yi; Xin, Xuelei; Aisa, Haji Akber; Ito, Yoichiro

    2015-01-01

    A novel ellipsoid column was designed for centrifugal counter-current chromatography. Performance of the ellipsoid column with a capacity of 3.4 mL was examined with three different solvent systems composed of 1-butanol-acetic acid-water (4:1:5, v/v) (BAW), hexane-ethyl acetate-methanol-0.1 M HCl (1:1:1:1, v/v) (HEMH), and 12.5% (w/w) PEG1000 and 12.5% (w/w) dibasic potassium phosphate in water (PEG-DPP) each with suitable test samples. In dipeptide separation with BAW system, both stationary phase retention (Sf) and peak resolution (Rs) of the ellipsoid column were much higher at 0° column angle (column axis parallel to the centrifugal force) than at 90° column angle (column axis perpendicular to the centrifugal force), where elution with the lower phase at a low flow rate produced the best separation yielding Rs at 2.02 with 27.8% Sf at a flow rate of 0.07 ml/min. In the DNP-amino acid separation with HEMW system, the best results were obtained at a flow rate of 0.05 ml/min with 31.6% Sf yielding high Rs values at 2.16 between DNP-DL-glu and DNP-β-ala peaks and 1.81 between DNP-β-ala and DNP-L-ala peaks. In protein separation with PEG-DPP system, lysozyme and myolobin were resolved at Rs of 1.08 at a flow rate of 0.03 ml/min with 38.9% Sf. Most of those Rs values exceed those obtained from the figure-8 column under similar experimental conditions previously reported.

  19. Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography

    ERIC Educational Resources Information Center

    Davies, Don R.; Johnson, Todd M.

    2007-01-01

    A simple experiment for undergraduate organic chemistry students to separate a colorless mixture using column chromatography and then monitor the outcome of the separation using thin-layer chromatography (TLC) and infrared spectroscopy(IR) is described. The experiment teaches students the principle and techniques of column and thin-layer…

  20. Preparation and evaluation of molecularly imprinted polymer liquid chromatography column for the separation of Cathine enantiomers

    PubMed Central

    Balamurugan, Krishnamoorthy; Gokulakrishnan, Kannan; Prakasam, Tangirala

    2011-01-01

    In this study molecular imprinting technology was employed to prepare a specific affinity sorbent for the resolution of Cathine, a chiral drug product. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting with either (+) or (−)-Cathine (threo-2-amino-1-hydroxy-1-phenyl propane; norpseudoephedrine) as the template. Methacrylic acid and ethylene glycol di-methacrylate were copolymerized in the presence of the template molecule. The bulk polymerization was carried out in chloroform with 2,2′-azobisisobutyronitrile as the initiator, at 5 °C and under UV radiation. The resulting MIP was ground into powders, which were slurry packed into analytical columns. After removal of template molecules, the MIP-packed columns were found to be effective for the resolution of (±)-Cathine racemates. The separation factor for the enantiomers ranged between 1.5 and 2.4 when the column was packed with MIP prepared with (+)-Cathine as the template. A separation factor ranging from 1.6 to 2.9 could be achieved from the column packed with MIP, prepared with (−)-Cathine as the template. Although the separation factor was higher with that previously obtained from reversed-phase column chromatography following derivatization with a chiral agent, elution peaks were broader due to the heterogeneity of binding sites on MIP particles and the possible non-specific interaction. PMID:23960776

  1. Capillary monolithic titania column for miniaturized liquid chromatography and extraction of organo-phosphorous compounds.

    PubMed

    Abi Jaoudé, Maguy; Randon, Jérôme

    2011-05-01

    A new sol-gel protocol was designed and optimized to produce titanium-dioxide-based columns within confined geometries such as monolithic capillary columns and porous-layer open-tubular columns. A surface pre-treatment of the capillary enabled an efficient anchorage of the monolith to the silica capillary wall during the synthesis. The monolith was further synthesized from a solution containing titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly(ethylene oxide) as pore template. The chromatographic application of capillary titania-based columns was demonstrated with the separation of a set of phosphorylated nucleotides as probe molecules using aqueous normal-phase liquid chromatography conditions. Capillary titania monoliths offered a compromise between the high permeability and the important loading capacity needed to potentially achieve miniaturized sample preparations. The specificity of the miniaturized titania monolithic support is illustrated with the specific enrichment of 5'-adenosine mono-phosphate. The monolithic column offered a ten times higher loading capacity of 5'-adenosine mono-phosphate compared with that of the capillary titania porous-layer open-tubular geometry.

  2. Analytical and preparative enantioseparation of DL-penicillamine and DL-cysteine by high-performance liquid chromatography on alpha-acid glycoprotein and beta-cyclodextrin columns using ninhydrin as a reversible tagging reagent.

    PubMed

    Bhushan, Ravi; Kumar, Rajender

    2009-04-10

    Two sulfur-containing amino acids, DL-cysteine (Cys) and DL-penicillamine (PenA), were condensed with ninhydrin to form their spirothiazolidine derivatives. These were separated by HPLC using alpha-acid glycoprotein (AGP) and beta-cyclodextrin (beta-CD) columns. The resolution conditions were optimized and the results were compared. Since the method provided resolution greater than 2 it was also applied to preparative separation. After separation, each of them was detagged using Zn dust and 10% aqueous trifluoroacetic acid. For analytical purposes dinitrophenyl (DNP) derivatives of DL-Cys and DL-PenA were also prepared and were resolved on both the columns. The detection was carried out using photodiode array detection system at 231 nm. The limits of detection were found to be 0.01% and 0.004% for spirothiazolidine carboxylic acid and DNP derivatives, respectively.

  3. Ionic liquid-based zwitterionic organic polymer monolithic column for capillary hydrophilic interaction chromatography.

    PubMed

    Wang, Tingting; Chen, Yihui; Ma, Junfeng; Zhang, Xiaodan; Zhang, Lihua; Zhang, Yukui

    2015-08-21

    In the current study, a novel ionic liquid-based zwitterionic organic polymer monolithic column was developed by copolymerizing 1-vinyl-3-(butyl-4-sulfonate) imidazolium, acrylamide and N,N'-methylenebisacrylamide in a quaternary porogenic solvent consisting of formamide, dimethyl sulphoxide, polyethylene glycol 8000 and polyethylene glycol 10,000 for capillary hydrophilic interaction chromatography. The monolithic stationary phase was optimized by adjusting the amount of monomer in the polymerization solution along with the composition of porogenic solvent. The optimized monolith exhibited excellent selectivity and favorable retention for nucleosides and benzoic acid derivatives. The primary factors affecting the separation efficiency of the monolithic column (including acetonitrile content, pH, and buffer salt concentration in the mobile phase) have been thoroughly evaluated. Excellent reproducibility of the retention times for five nucleosides was achieved, with relative standard deviations of run-to-run (n = 3), column-to-column (n = 3) and batch-to-batch (n = 3) in the range of 0.18-0.48%, 2.33-4.20% and 3.07-6.50%, respectively.

  4. Advances in silver ion chromatography for the analysis of fatty acids and triacylglycerols-2001 to 2011.

    PubMed

    Momchilova, Svetlana M; Nikolova-Damyanova, Boryana M

    2012-01-01

    An effort is made to critically present the achievements in silver ion chromatography during the last decade. Novelties in columns, mobile-phase compositions and detectors are described. Recent applications of silver ion chromatography in the analysis of fatty acids and triacylglycerols are presented while stressing novel analytical strategies or new objects. The tendencies in the application of the method in complementary ways with reversed-phase chromatography, chiral chromatography and, especially, mass detection are outlined.

  5. Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogenous peptides in multidimensional liquid chromatography. Structure and function of strong cation-exchange restricted access materials.

    PubMed

    Machtejevas, E; Denoyel, R; Meneses, J M; Kudirkaite, V; Grimes, B A; Lubda, D; Unger, K K

    2006-08-04

    In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 degrees C, intrusion-extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08 mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (approximately 150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

  6. Halo columns: new generation technology for high speed liquid chromatography.

    PubMed

    Ali, Imran; Gaitonde, Vinay D; Grahn, Anders

    2010-01-01

    Fast speed and high sample loading and the pressing demands of industries and researchers are compelling scientists and manufacturers to explore the new horizons in column technology. Recently, superficially porous silica particle columns are manufactured with some salient features such as super fast speed, sharp peaks, good sample loading, and low backpressure. The commercially available columns are Halo (Advanced Material Technology, Wilmington, DE), Express (Supelco, Bellefonte, PA), and Proshell 120 (Agilent, Santa Clara, CA). Halo columns are of C(8), C(18), RP Amide, and HILIC types with 2.7 microm over all diameters, 0.5 microm porous thick layers containing 90 A as pore diameter, and 150 m(2)/g surface area. These columns have been used for fast separation of low molecular weight compounds with some exception for large molecules such as protein, peptides, and DNA. The present article describes the importance of these state-of-the-art superficially porous silica particles based columns with special emphasis on Halo columns. The different aspects of these columns such as structures, mechanism of separations, applications, and comparison, with conventional columns have been discussed.

  7. Isolation of human beta-interferon receptor by wheat germ lectin affinity and immunosorbent column chromatographies

    SciTech Connect

    Zhang, Z.Q.; Fournier, A.; Tan, Y.H.

    1986-06-15

    Radioiodinated human beta-interferon-Ser 17 (Betaseron) was reversibly cross-linked to Daudi cells by dithiobis(succinimidylpropionate). The radioactive ligand was cross-linked to three macromolecules forming labeled complexes of apparent Mr values of 130,000, 220,000, and 320,000. Betaseron, human alpha-interferon, human interleukin 2 but not recombinant human gamma-interferon competed with the labeled ligand for binding to these putative receptor(s). Human leukocyte-produced gamma-interferon competed weakly with /sup 125/I-Betaseron for binding to Daudi cells. The Betaseron-receptor complex(es) was purified by passage through a wheat germ lectin column followed by chromatography on an anti-interferon immunosorbent column and semipreparative gel electrophoresis. The cross-linked ligand-receptor complex was shown to be highly purified by sodium dodecyl sulfate and acetic acid:urea:Triton X-100 polyacrylamide gel electrophoresis. It can be dissociated into the labeled Betaseron (Mr = 17,000) ligand and a receptor moiety which has an apparent molecular weight of 110,000. The chromatographic behavior of the ligand-receptor complex on wheat germ lectin column suggests that the receptor is a glycoprotein. The described procedure yielded about 1 microgram of Betaseron receptor from 10(10) Daudi cells, estimated to contain a maximum of about 15 micrograms of the receptor.

  8. Planar gas chromatography column on aluminum plate with multi-walled carbon nanotubes as stationary phase

    NASA Astrophysics Data System (ADS)

    Platonov, I. A.; Platonov, V. I.; Pavelyev, V. S.

    2016-04-01

    The high selectivity of the adsorption layer for low-boiling alkanes is shown, the separation factor (α) couple iso-butane / butane is 1.9 at a column temperature of 50 °C.The paper presents sorption and selective properties of planar gas chromatography column on aluminum plate with multi-walled carbon nanotubes as the stationary phase.

  9. Colorful Column Chromatography: A Classroom Demonstration of a Three-Component Separation

    ERIC Educational Resources Information Center

    Heumann, Lars V.

    2008-01-01

    A classroom demonstration detailing the procedure for the separation of a ternary mixture consisting of intensely colored compounds using silica gel column chromatography is described. The audience can follow the compounds during their passage through the column as individual, colored bands while learning about different tools and techniques used…

  10. Silica-based monolithic capillary columns modified by liposomes for characterization of analyte-liposome interactions by capillary liquid chromatography.

    PubMed

    Moravcová, Dana; Planeta, Josef; Wiedmer, Susanne K

    2013-11-22

    This study introduces a silica-based monolith in a capillary format (0.1 mm × 100 mm) as a support for immobilization of liposomes and its characterization in immobilized liposome chromatography. Silica-based monolithic capillary columns prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea were modified by (3-aminopropyl)trimethoxysilane, whereby amino groups were introduced to the monolithic surface. These groups undergo reaction with glutaraldehyde to form an iminoaldehyde, allowing covalent binding of pre-formed liposomes containing primary amino groups. Two types of phospholipid vesicles were used for column modification; these were 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidyl choline with and without 1,2-diacyl-sn-glycero-3-phospho-L-serine. The prepared columns were evaluated under isocratic separation conditions employing 20mM phosphate buffer at pH 7.4 as a mobile phase and a set of unrelated drugs as model analytes. The liposome layer on the synthesized columns significantly changed the column selectivity compared to the aminopropylsilylated monolithic stationary phase. Monolithic columns modified by liposomes were stable under the separation conditions, which proved the applicability of the suggested preparation procedure for the synthesis of capillary columns dedicated to study analyte-liposome interactions. The column efficiency originating from the silica monolith was preserved and reached, e.g., more than 120,000 theoretical plates/m for caffeine as a solute.

  11. At-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography for bioassay-guided separation of antioxidants from vine tea (Ampelopsis grossedentata).

    PubMed

    Ma, Ruyi; Zhou, Rongrong; Tong, Runna; Shi, Shuyun; Chen, Xiaoqing

    2017-01-01

    Vine tea (Ampelopsis grossedentata), a widely used healthy tea, beverage and herbal medicine, exhibited strong antioxidant activity. However, systematic purification of antioxidants, especially for those with similar structures or polarities, is a challenging work. Here, we present a novel at-line hyphenation of high-speed countercurrent chromatography with Sephadex LH-20 column chromatography (HSCCC-Sephadex LH-20 CC) for rapid and efficient separation of antioxidants from vine tea target-guided by 1,1-diphenyl-2-picryl-hydrazyl radical-high performance liquid chromatography (DPPH-HPLC) experiment. A makeup pump, a six-port switching valve and a trapping column were served as interface. The configuration had no operational time and mobile phase limitations between two dimensional chromatography and showed great flexibility without tedious sample-handling procedure. Seven targeted antioxidants were firstly separated by stepwise HSCCC using petroleum ether-ethyl acetate-methanol-water (4:9:4:9, v/v/v/v) and (4:9:5:8, v/v/v/v) as solvent systems, and then co-eluted antioxidants were on-line trapped, concentrated and desorbed to Sephadex LH-20 column for further off-line purification by methanol. It is noted that six elucidated antioxidants with purity over 95% exhibited stronger activity than ascorbic acid (VC). More importantly, this at-line hyphenated strategy could sever as a rapid and efficient pathway for systematic purification of bioactive components from complex matrix.

  12. Stationary phase modulation in liquid chromatography through the serial coupling of columns: A review.

    PubMed

    Alvarez-Segura, T; Torres-Lapasió, J R; Ortiz-Bolsico, C; García-Alvarez-Coque, M C

    2016-06-07

    Liquid chromatography with single columns often does not succeed in the analysis of complex samples, in terms of resolution and analysis time. A relatively simple solution to enhance chromatographic resolution is the modulation of the stationary phase through the serial coupling of columns. This can be implemented with any type of column using compatible elution conditions and conventional instruments. This review describes the key features of column coupling and published procedures, where two or more columns were coupled in series to solve separation problems. In all reports, the authors could not resolve their samples with single columns, whereas significant enhancement in chromatographic performance was obtained when the columns were combined. Particularly interesting is the reduction in the analysis time in the isocratic mode, which alleviates the "general elution problem" of liquid chromatography, and may represent a stimulus for the proposal of new procedures, especially in combination with mass spectrometric, electrochemical and refractometric detection. Developments proposed to make the serial coupling of columns useful in routine and research laboratories are outlined, including optimisation strategies that facilitate the selection of the appropriate column combination and elution conditions (solvent content, flow rate or temperature) in both isocratic and gradient modes. The availability of zero dead volume couplers, able to connect standard columns, and the commercialisation of short columns with multiple lengths, have expanded the possibilities of success.

  13. Investigation of a new core-shell particle column for ion-pair reversed-phase liquid chromatography analysis of oligonucleotides.

    PubMed

    Biba, Mirlinda; Welch, Christopher J; Foley, Joe P

    2014-08-05

    A new core-shell particle column showed excellent performance and durability for separation of short (∼21-mer) ribonucleic acid (RNA) oligonucleotides by ion-pair reversed-phase liquid chromatography (IP-RPLC). Previously investigated core-shell C18 columns showed excellent peak shapes and separations of closely eluting impurities by IP-RPLC. However, these columns showed only modest long-term stability at the neutral pH and elevated column temperatures of ≥60°C, typically used for IP-RPLC analysis of oligonucleotides. The newly introduced SunShell C18 column provided separations comparable to the previously evaluated core-shell columns, but with significantly improved long-term column stability when operated at neutral pH and elevated column temperature.

  14. SEPARATION OF T-MAZ ETHOXYLATED SORBITAN FATTY ACID ESTERS BY SUPERCRITICAL FLUID CHROMATOGRAPHY

    EPA Science Inventory

    The application of supercritical fluid chromatography (SFC) to the analysis of T-MAZ ethoxylated sorbitan fatty acid esters is described. FC separation methods utilize a density programming technique and a 50 um I.D. capillary column. his work demonstrates that capillary column S...

  15. Separation of donor and recipient bacteria by column chromatography.

    PubMed

    Zsigray, R M; Fulk, G E; Lawton, W D

    1970-08-01

    When donor and recipient strains of Escherichia coli were added to columns containing Cellex-P (a cation-exchange cellulose), more than 80% of the female cells passed through the column but only 11% or less of the male cells were eluted. However, when donor strains were blended before their addition to the column, the majority of these cells were eluted. These results indicated that the filamentous appendages termed F pili (which are removed by blending) were the structures responsible for the adherence of donor cells to the cellulose.

  16. Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.

    PubMed

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2014-06-20

    Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.

  17. Separation of Be and Al for AMS using single-step column chromatography

    NASA Astrophysics Data System (ADS)

    Binnie, Steven A.; Dunai, Tibor J.; Voronina, Elena; Goral, Tomasz; Heinze, Stefan; Dewald, Alfred

    2015-10-01

    With the aim of simplifying AMS target preparation procedures for TCN measurements we tested a new extraction chromatography approach which couples an anion exchange resin (WBEC) to a chelating resin (Beryllium resin) to separate Be and Al from dissolved quartz samples. Results show that WBEC-Beryllium resin stacks can be used to provide high purity Be and Al separations using a combination of hydrochloric/oxalic and nitric acid elutions. 10Be and 26Al concentrations from quartz samples prepared using more standard procedures are compared with results from replicate samples prepared using the coupled WBEC-Beryllium resin approach and show good agreement. The new column procedure is performed in a single step, reducing sample preparation times relative to more traditional methods of TCN target production.

  18. Preparation and Characterization of a Polymeric Monolithic Column for Use in High-Performance Liquid Chromatography (HPLC)

    ERIC Educational Resources Information Center

    Bindis, Michael P.; Bretz, Stacey Lowery; Danielson, Neil D.

    2011-01-01

    The high-performance liquid chromatography (HPLC) experiment, most often done in the undergraduate analytical instrumentation laboratory course, generally illustrates reversed-phase chromatography using a commercial C[subscript]18 silica column. To avoid the expense of periodic column replacement and introduce a choice of columns with different…

  19. Surface modification of polytetrafluoroethylene column for two-stationary phase separations by counter-current chromatography.

    PubMed

    Quan, Kai-jun; Huang, Xin-yi; Li, Xiao-ting; Wang, Gao-hong; Liu, Yan-juan; Duan, Wen-da; Di, Duo-long

    2015-11-27

    To improve the separation capability of CCC, a novel solid-liquid two-stationary phases CCC (ASP-CCC) column was prepared employing graphene oxide (GO) conjugated poly-dopamine (PD) coating (GO/PD) as auxiliary stationary phase (ASP). The results of Scanning electron microscopy (SEM), contact angle and X-ray photoelectron spectroscopy (XPS) indicated that nanostructured GO and PD were successfully grafted on the inner wall of the PTFE column. Three alkaloid compounds were selected as the target analytes to evaluate the performance of the novel column. Because of the intermolecular force (hydrogen bond, electrostatic interaction and π-π interaction) between the ASP and model compounds, three analytes were well separated with this novel ASP-CCC column. Additionally, the novel column exhibited higher stationary phase retention ratio, about 8%, than original column without changing the chromatographic condition. Furthermore, the eluotropic sequence of analytes on novel column was in accordance with that in the original column. This suggested that the novel column is a CCC column with auxiliary stationary phase (ASP) in its own right, and the present separation mode is the combination of partition chromatography and adsorption chromatography.

  20. Sum of ranking differences to rank stationary phases used in packed column supercritical fluid chromatography.

    PubMed

    West, Caroline; Khalikova, Maria A; Lesellier, Eric; Héberger, Károly

    2015-08-28

    The identification of a suitable stationary phase in supercritical fluid chromatography (SFC) is a major source of difficulty for those with little experience in this technique. Several protocols have been suggested for column classification in high-performance liquid chromatography (HPLC), gas chromatography (GC), and SFC. However, none of the proposed classification schemes received general acceptance. A fair way to compare columns was proposed with the sum of ranking differences (SRD). In this project, we used the retention data obtained for 86 test compounds with varied polarity and structure, analyzed on 71 different stationary phases encompassing the full range in polarity of commercial packed columns currently available to the SFC chromatographer, with a single set of mobile phase and operating conditions (carbon dioxide-methanol mobile phase, 25°C, 150bar outlet pressure, 3ml/min). First, a reference column was selected and the 70 remaining columns were ranked based on this reference column and the retention data obtained on the 86 analytes. As these analytes previously served for the calculation of linear solvation energy relationships (LSER) on the 71 columns, SRD ranks were compared to LSER methodology. Finally, an external comparison based on the analysis of 10 other analytes (UV filters) related the observed selectivity to SRD ranking. Comparison of elution orders of the UV filters to the SRD rankings is highly supportive of the adequacy of SRD methodology to select similar and dissimilar columns.

  1. Devising an adjustable splitter for dual-column gas chromatography.

    PubMed

    Wang, Chieh-Heng; Chang, Chih-Chung; Wang, Jia-Lin

    2007-09-07

    A flow controlled adjustable splitter was configured from a Deans switch and employed in an automated dual column gas chromatographic (GC) system for analyzing mono-aromatic compounds. Volatile organic compounds (VOCs), thermally desorbed from the sorbent trap, were split by the adjustable splitter onto two columns of different phases for separation and then detection by flame ionization detection (FID). Unlike regular splitters in which the split ratio is passively determined by the diameter and/or length of the connecting columns or tubing, the split ratio in our adjustable splitter is controlled by the auxiliary flow in the Deans switch. The auxiliary flow serves as a gas plug on either side of the column for decreasing the sample flow in one transfer line, but increasing the flow in the other. By adjusting the auxiliary flow and therefore the size of the gas plug, the split ratio can be easily varied and favorable to the side of no auxiliary gas. As an illustration, two columns, DB-1 and Cyclodex-B, were employed in this study for separating benzene, toluene, ethylbenzene, xylenes, denoted as BTEX, in particular the structural isomers of o-, m-, p-xylenes. This configuration demonstrates that BTEX cannot be fully separated with either column, but can be deconvoluted by simple algebra if dual columns are used with a splitter. The applicability of the proposed concept was tested by analyzing a gas standard containing BTEX at different split ratios and with various sample sizes, all leading to a constant ratio of m-xylene versus p-xylene.

  2. Orthogonal separation on one beta-cyclodextrin column by switching reversed-phase liquid chromatography and hydrophilic interaction chromatography.

    PubMed

    Feng, Jia-tao; Guo, Zhi-mou; Shi, Hui; Gu, Jiang-ping; Jin, Yu; Liang, Xin-miao

    2010-06-15

    A dual retention combined with reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) has been observed on beta-cyclodextrin (beta-CD) bonded stationary phase. A typical U-shaped retention curve was achieved owing to dual retention mechanism. Based on this observation, a beta-CD column can be operated under reversed-phase liquid chromatography (RP-LC) and hydrophilic interaction chromatography (HILIC) modes. Two-dimensional liquid chromatography (2D-LC) analysis can be realized on just a beta-CD column by switching these two different separation modes. In this study, off-line 2D-LC analysis for a natural product was carried out to prove the orthogonal separation between RP-LC and HILIC modes on a Click beta-CD column. Herba Hedyotis Diffusae, the whole grass of Hedyotis Diffusae wild was extracted with water, pretreated with macroporous resin and then first separated at RP-LC mode on the Click beta-CD column to obtain successive fractions, which were then reanalyzed at HILIC mode on the same Click beta-CD column. The result proved that both separation modes on the Click beta-CD column have good retention and peak shape, and these two separation modes have good orthogonality. 2D-LC analysis revealed abundant information in the natural product. Especially numerous minor components were enriched and separated. The mobile phase used in RP-LC and HILIC modes can be same and the switch between these two separation modes is easily realized by changing the ratio of the acetonitrile and water. Hence the mobile phase in this 2D-LC system is completely compatible. This advantage makes this combination is an appropriate 2D-LC method for the solutes having retention at both separation modes.

  3. Characterization by the solvation parameter model of the retention properties of commercial ionic liquid columns for gas chromatography.

    PubMed

    Rodríguez-Sánchez, S; Galindo-Iranzo, P; Soria, A C; Sanz, M L; Quintanilla-López, J E; Lebrón-Aguilar, R

    2014-01-24

    For the first time, four commercial ionic liquid columns (SLB-IL59, SLB-IL76, SLB-IL82 and SLB-IL100) for gas chromatography have been comprehensively evaluated in terms of efficiency, polarity and solvation properties. Grob tests and McReynolds constants showed that they were all high-efficiency columns of high polarity, but with low inertness to compounds with hydrogen bonding capabilities. The solvation parameter model was used to characterize the solvation interactions of the four columns in the 80-160°C temperature range. Results revealed that all the ionic liquids studied can be considered moderately hydrogen-bond acid and highly cohesive stationary phases, on which the dominant contributions to retention were the dipolar-type and hydrogen-bond base interactions, while π-π and n-π interactions were barely significant. The SLB-IL59 column provided the best separation of homologs, while the SLB-IL76 and SLB-IL100 columns had the most basic and the most acidic phases, respectively. A principal component analysis for the commonly used stationary phases in capillary GC showed that these commercial ionic liquid columns fill an empty area of the available selectivity space, which clearly enhances the separation capacity of this technique.

  4. A straightforward methodology for designing continuous monoclonal antibody capture multi-column chromatography processes.

    PubMed

    Gjoka, Xhorxhi; Rogler, Karl; Martino, Richard Alexander; Gantier, Rene; Schofield, Mark

    2015-10-16

    A simple process development strategy for continuous capture multi-column chromatography (MCC) is described. The approach involves a few single column breakthrough experiments, based on several simplifying observations that enable users to rapidly convert batch processes into well-designed multi-column processes. The method was validated using a BioSMB(®) (Pall Life Sciences) lab scale multi-column system and a mAb capture process employing Protein A resin. The approach enables users to optimize MCC processes based on their internal preferences and constraints without requiring any mathematical modeling expertise.

  5. Ion exclusion chromatography of aromatic acids.

    PubMed

    Mansour, Fotouh R; Kirkpatrick, Christine L; Danielson, Neil D

    2013-08-01

    The determination of aromatic acids by ion exclusion chromatography is challenging due to peak tailing and the long retention time of hydrophobic solutes. This review discusses the retention mechanisms and the factors affecting retention, eluents and detection methods used in ion exclusion chromatography of aromatic acids such as mono-, di-, tri- and tetra-carboxylic acids, amino acids, sulfonates and phenol. In addition, the different approaches used to improve the chromatographic separation of these compounds are also discussed. These approaches include introducing an internal gradient of the ionic strength, using vacancy ion exclusion chromatography, employing a hydrophilic cation exchange resin or adding a modifier such as heptanol to the dilute sulfuric acid mobile phase. The applications of these methods in the analysis of aromatic acids are provided with a table summarizing the stationary phases, the mobile phases and the detection methods.

  6. Multi-column chromatography of urinary steriods and adrenocortical dysfunction.

    PubMed

    Sayegh, J F; Vestergaard, P

    1978-01-01

    The potential of the multi-column assay for urinary neutral steroids in work with samples from patients with adrenocortical pathology is demonstrated through analyses performed on urine samples from Cushing and congenital adrenal hyperplasia cases, after modification of the routine methodology to include the quantitation of additional steroids of particular importance for pathological samples.

  7. Robust naphthyl methacrylate monolithic column for high performance liquid chromatography of a wide range of solutes.

    PubMed

    Jonnada, Murthy; El Rassi, Ziad

    2015-08-28

    An organic monolithic column based on the co-polymerization of 2-naphthyl methacrylate (NAPM) as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the crosslinker was introduced for high performance reversed-phase liquid chromatography (RPC). The co-polymerization was performed in situ in a stainless steel column of 4.6mm i.d. in the presence of a ternary porogen consisting of 1-dodecanol and cyclohexanol. This monolithic column (referred to as naphthyl methacrylate monolithic column or NMM column) showed high mechanical stability at relatively high mobile phase flow velocity indicating that the column has excellent hydrodynamic characteristics. To characterize the NMM column, different probe molecules including alkyl benzenes, and aniline, benzene, toluene and phenol derivatives were chromatographed on the column and the results in terms of k, selectivity and plate counts were compared to those obtained on an octadecyl silica (ODS) column in order to assess the presence of π-π and hydrophobic interactions on the NMM column under otherwise the same elution conditions. The NMM column offered additional π-π interactions with aromatic molecules in addition to hydrophobic interactions under RPC elution conditions. Run-to-run and column-to-column reproducibility of solute k values were evaluated, and percent relative standard deviation of <1% and ∼2-3.5%, respectively, were obtained. Six standard proteins were readily separated on the NMM column using shallow (30min at 1.0mL/min), steep (10min at 1.0mL/min) and ultra steep (1min at 3.0mL/min) linear gradient elution at increasing ACN concentration in the mobile phase using a 10cm×4.6mm i.d. column in case of shallow and steep linear gradients and a 3cm×4.6mm i.d. column for ultra steep linear gradient.

  8. How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases.

    PubMed

    Borges, Endler M

    2014-01-07

    Three RP-LC column characterization protocols [Tanaka et al. (1989), Snyder et al. (PQRI, 2002), and NIST SRM 870 (2000)] were evaluated using both Euclidian distance and Principal Components Analysis to evaluate effectiveness at identifying equivalent columns. These databases utilize specific chromatographic properties such as hydrophobicity, hydrogen bonding, shape/steric selectivity, and ion exchange capacity of stationary phases. The chromatographic parameters of each test were shown to be uncorrelated. Despite this, the three protocols were equally successful in identifying similar and/or dissimilar stationary phases. The veracity of the results has been supported by some real life pharmaceutical separations. The use of Principal Component Analysis to identify similar/dissimilar phases appears to have some limitations in terms of loss of information. In contrast, the use of Euclidian distances is a much more convenient and reliable approach. The use of auto scaled data is favoured over the use of weighted factors as the former data transformation is less affected by the addition or removal of columns from the database. The use of these free databases and their corresponding software tools shown to be valid for identifying similar columns with equivalent chromatographic selectivity and retention as a "backup column". In addition, dissimilar columns with complimentary chromatographic selectivity can be identified for method development screening strategies.

  9. Two-column sequential injection chromatography--new approach for fast and effective analysis and its comparison with gradient elution chromatography.

    PubMed

    Chocholous, Petr; Satínský, Dalibor; Sklenárová, Hana; Solich, Petr

    2010-05-23

    This work presents novel approach in low-pressure chromatography flow systems--two-column Sequential Injection Chromatography (2-C SIC) and its comparison with gradient elution chromatography on the same instrument. The system was equipped with two different chromatographic columns (connected to selection valve in parallel design) for isocratic separation and determination of all components in composed anti-inflammatory pharmaceutical preparation (tablets). The sample was first injected on the first column of length 30 mm where less retained analytes were separated and then the sample was injected on the second column of length 10 mm where more retained analytes were separated. The SIC system was based on a commercial SIChrom manifold (8-port high-pressure selection valve and medium-pressure syringe pump with 4 mL reservoir) (FIAlab, USA) with two commercially available monolithic columns the "first column" Chromolith Flash RP-18e (25 mm x 4.6 mm i.d. with guard column 5 mm x 4.6 mm i.d.) and the "second column" Chromolith RP-18e (10 mm x 4.6 mm i.d.) and CCD UV-vis detector USB 4000 with micro-volume 1.0 cm Z flow cell. Two mobile phases were used for analysis (one for each column). The mobile phase 1 used for elution of paracetamol, caffeine and salicylic acid (internal standard) was acetonitrile/water (10:90, v/v, the water part of pH 3.5 adjusted with acetic acid), flow rate was 0.9 mL min(-1) (volume 3.0 mL of mobile phase per analysis). The mobile phase 2 used for elution of propyphenazone was acetonitrile/water (30:70, v/v); flow rate was 1.2 mL min(-1) (volume 1.5 mL of mobile phase per analysis). Absorbance was monitored at 210 nm. Samples were prepared by dissolving of one tablet in 30% acetonitrile and 10 microL of filtered supernatant was injected on each column (2 x 10 microL). The chromatographic resolution between all compounds was >1.45 and analysis time was 5.5 min under the optimal conditions. Limits of detection were determined at 0.4 microg m

  10. Highly crosslinked silicon polymers for gas chromatography columns

    NASA Technical Reports Server (NTRS)

    Shen, Thomas C. (Inventor)

    1994-01-01

    A new highly crosslinked silicone polymer particle for gas chromatography application and a process for synthesizing such copolymer are described. The new copolymer comprises vinyltriethoxysilane and octadecyltrichlorosilane. The copolymer has a high degree of crosslinking and a cool balance of polar to nonpolar sites in the porous silicon polymer assuring fast separation of compounds of variable polarity.

  11. Developing Inquiry-Based Labs Using Micro-Column Chromatography

    ERIC Educational Resources Information Center

    Barden-Gabbei, Laura M.; Moffitt, Deborah L.

    2006-01-01

    Chromatography is a process by which mixtures can be separated or substances can be purified. Biological and chemical laboratories use many different types of chromatographic processes. For example, the pharmaceutical industry uses chromatographic techniques to purify drugs, medical labs use them to identify blood components such as cholesterol,…

  12. Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis.

    PubMed

    Hetzel, Terence; Loeker, Denise; Teutenberg, Thorsten; Schmidt, Torsten C

    2016-10-01

    The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 μm fully porous as well as the 2.7 μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 μm fully porous particle packed column for ultra-fast liquid chromatography.

  13. Resolution of diacylglycerol moieties of natural glycerophospholipids by gas-liquid chromatography on polar capillary columns.

    PubMed

    Myher, J J; Kuksis, A

    1982-06-01

    A rapid and practical method has been developed for the gas-liquid chromatographic determination of the sn-1,2-diacylglycerol moieties of natural glycerophospholipids using polar wall-coated open tubular columns. The method gives complete resolution and quantitative estimates for all species according to molecular weight and degree of unsaturation, including stearoyl docosahexaenoylglycerol and related polyunsaturates. For this purpose the sn-1,2-diacylglycerols are obtained from the glycerophospholipids by hydrolysis with phospholipase C and are converted into the trimethylsilyl or tertiary-butyldimethylsilyl ethers. The silyl ethers are separated by gas-liquid chromatography on the capillary glass columns coated with a polar cyanopropylsiloxane polymer, in the temperature range 175-250 degrees C, using hydrogen as the carrier gas. Practical applications of the method are illustrated by analyses of the sn-1,2-diacylglycerol moieties of the phosphatidylcholines of soybean phosphatides, egg yolk, and rat liver. The method of analysis is applicable to other classes of glycerophospholipids and the total time requirements for the analysis of any one phospholipid class are comparable to those for a fatty acid analysis.

  14. [High-performance liquid chromatography determination of histamine in nasal mucosa of guinea pig after post-column derivatization].

    PubMed

    Huang, X H; Chen, B M; Liang, S X; Deng, F L

    2000-06-28

    A rapid and simple method for determining histamine by post-column derivatization liquid chromatography with fluorescent detection was described. SCX weakly acidic cation exchange column was used as an analytical column. Histamine was eluted at 13 min 12 s by 40 mmol.L-1 of trisodium citrate (pH5.50) at flow rate of 1.0 ml.min-1. The recoveries of histamine ranged from 1.0 mumol.L-1 to 100 mumol.L-1 were above 92%. The detection limit for histamine was 50 nmol.L-1 and the linear range for histamine was 50 nmol.L(-1)-500 mumol.L-1.

  15. Comprehensive two-dimensional high performance liquid chromatography system with immobilized liposome chromatography column and monolithic column for separation of the traditional Chinese medicine Schisandra chinensis.

    PubMed

    Wang, Shuowen; Wang, Chen; Zhao, Xin; Mao, Shilong; Wu, Yutian; Fan, Guorong

    2012-02-03

    A comprehensive two-dimensional (2D) separation is one that employs two separation dimensions (columns) and draws on all of the available resolving power from each of the dimensions of separate the components in a sample. In this study, a comprehensive 2D chromatography approach was developed for the separation and identification of membrane permeable compounds in a famous traditional Chinese medicine of Schisandra chinensis. The first dimensional column was the immobilized liposome chromatography (ILC) column, which mimics the biological membranes and can be used to study drug-membrane interactions in liquid chromatography. Using an automatic ten-port switching valve equipped with two sample loops, the section of the first-dimension was introduced in the second-dimension consist of a silica monolithic column. More than 40 components in Schisandra chinensis were resolved by using the developed separation system and among them 14 compounds were identified interacting with the ILC column based on their retention action, UV and mass data. With this comprehensive 2D-HPLC system, the three-dimensional chromatographic fingerprints of Schisandra chinensis were preliminarily established and processed by using principal component analysis and hierarchical clustering analysis. The obtained information can distinguish the unacceptable samples of the quality control. The result demonstrated that the 2D biochromatography system has been demonstrated to have more advantages of finding strong binding bioactive components, providing an enhanced peak capacity, good sensitivity and powerful resolution biological fingerprinting analysis of complex TCMs, which was a useful means to control the quality of and to clarify the membrane permeability of the compounds in Schisandra chinensis.

  16. Evaluation of a silicon oxynitride hydrophilic interaction liquid chromatography column in saccharide and glycoside separations.

    PubMed

    Wan, Huihui; Sheng, Qianying; Zhong, Hongmin; Guo, Xiujie; Fu, Qing; Liu, Yanfang; Xue, Xingya; Liang, Xinmiao

    2015-05-01

    The retention characteristics of a silicon oxynitride stationary phase for carbohydrate separation were studied in hydrophilic interaction chromatography mode. Four saccharides including mono-, di-, and trisaccharides were employed to investigate the effects of water content and buffer concentration in the mobile phase on hydrophilic interaction liquid chromatography retention. For the tested saccharides, the silicon oxynitride column demonstrated excellent performance in terms of separation efficiency, hydrophilicity, and interesting separation selectivity for carbohydrates compared to the bare silica stationary phase. Finally, the silicon oxynitride hydrophilic interaction liquid chromatography column was employed in the separation of complex samples of fructooligosaccharides, saponins, and steviol glycoside from natural products. The resulting chromatograms demonstrated good separation efficiency and longer retention compared with silica, which further confirmed the advantages and potential application of silicon oxynitride stationary phase for hydrophilic interaction liquid chromatography separation.

  17. Twin-column CaptureSMB: a novel cyclic process for protein A affinity chromatography.

    PubMed

    Angarita, Monica; Müller-Späth, Thomas; Baur, Daniel; Lievrouw, Roel; Lissens, Geert; Morbidelli, Massimo

    2015-04-10

    A twin-column counter-current chromatography processes, CaptureSMB, was used for the protein A affinity capture of a monoclonal antibody (mAb). By means of sequential loading, the process improves the utilization of the stationary phase by achieving loadings much closer to the static binding capacity of the resin in comparison to batch chromatography. Using a mAb capture case study with protein A affinity chromatography, the performance and product quality obtained from CaptureSMB and batch processes were compared. The effect of the flow rate, column length and titer concentration on the process performance and product quality were evaluated. CaptureSMB showed superior performance compared to batch chromatography with respect to productivity, capacity utilization, product concentration and buffer consumption. A simplified economic evaluation showed that CaptureSMB could decrease resin costs of 10-30% depending on the manufacturing scenario.

  18. Characterization of peak capacity of microbore liquid chromatography columns using gradient kinetic plots.

    PubMed

    Hetzel, Terence; Blaesing, Christina; Jaeger, Martin; Teutenberg, Thorsten; Schmidt, Torsten C

    2017-02-17

    The performance of micro-liquid chromatography columns with an inner diameter of 0.3mm was investigated on a dedicated micro-LC system for gradient elution. Core-shell as well as fully porous particle packed columns were compared on the basis of peak capacity and gradient kinetic plot limits. The results for peak capacity showed the superior performance of columns packed with sub-2μm fully porous particles compared to 3.0μm fully porous and 2.7μm core-shell particles within a range of different gradient time to column void time ratios. For ultra-fast chromatography a maximum peak capacity of 16 can be obtained using a 30s gradient for the sub-2μm fully porous particle packed column. A maximum peak capacity of 121 can be achieved using a 5min gradient. In addition, the influence of an alternative detector cell on the basis of optical waveguide technology and contributing less to system variance was investigated showing an increased peak capacity for all applied gradient time/column void time ratios. Finally, the influence of pressure was evaluated indicating increased peak capacity for maximum performance whereas a limited benefit for ultra-fast chromatography with gradient times below 30s was observed.

  19. Improved micromachined column design and fluidic interconnects for programmed high-temperature gas chromatography separations.

    PubMed

    Gaddes, David; Westland, Jessica; Dorman, Frank L; Tadigadapa, Srinivas

    2014-07-04

    This work focuses on the development and experimental evaluation of micromachined chromatographic columns for use in a commercial gas chromatography (GC) system. A vespel/graphite ferrule based compression sealing technique is presented using which leak-proof fluidic interconnection between the inlet tubing and the microchannel was achieved. This sealing technique enabled separation at temperatures up to 350°C on a μGC column. This paper reports the first high-temperature separations in microfabricated chromatographic columns at these temperatures. A 2m microfabricated column using a double Archimedean spiral design with a square cross-section of 100μm×100μm has been developed using silicon microfabrication techniques. The microfabricated column was benchmarked against a 2m 100μm diameter commercial column and the performance between the two columns was evaluated in tests performed under identical conditions. High temperature separations of simulated distillation (ASTM2887) and polycyclic aromatic hydrocarbons (EPA8310) were performed using the μGC column in temperature programmed mode. The demonstrated μGC column along with the high temperature fixture offers one more solution toward potentially realizing a portable μGC device for the detection of semi-volatile environmental pollutants and explosives without the thermal limitations reported to date with μGC columns using epoxy based interconnect technology.

  20. Vacancy ion-exclusion chromatography of haloacetic acids on a weakly acidic cation-exchange resin.

    PubMed

    Helaleh, Murad I H; Tanaka, Kazuhiko; Mori, Masanobu; Xu, Qun; Taoda, Hiroshi; Ding, Ming-Yu; Hu, Wenzhi; Hasebe, Kiyoshi; Haddad, Paul R

    2003-05-16

    A new and simple approach is described for the determination of the haloacetic acids (such as mono-, di- and trichloroacetic acids) usually found in drinking water as chlorination by-products after disinfection processes and acetic acid. The new approach, termed vacancy ion-exclusion chromatography, is based on an ion-exclusion mechanism but using the sample solution as the mobile phase, pure water as the injected sample, and a weakly acidic cation-exchange resin column (TSKgel OApak-A) as the stationary phase. The addition of sulfuric acid to the mobile phase results in highly sensitive conductivity detection with sharp and well-shaped peaks, leading to excellent and efficient separations. The elution order was sulfuric acid, dichloroacetic acid, monochloroacetic acid, trichloroacetic acid, and acetic acid. The separation of these acids depends on their pKa values. Acids with lower pKa values were eluted earlier than those with higher pKa, except for trichloroacetic acid due to a hydrophobic-adsorption effect occurring as a side-effect of vacancy ion-exclusion chromatography. The detection limits of these acids in the present study with conductivity detection were 3.4 microM for monochloroacetic acid, 0.86 microM for dichloroacetic acid and 0.15 microM for trichloroacetic acid.

  1. Effect of extra-column volume on practical chromatographic parameters of sub-2-μm particle-packed columns in ultra-high pressure liquid chromatography.

    PubMed

    Wu, Naijun; Bradley, Ashley C; Welch, Christopher J; Zhang, Li

    2012-08-01

    Effects of extra-column volume on apparent separation parameters were studied in ultra-high pressure liquid chromatography with columns and inlet connection tubings of various internal diameters (id) using 50-mm long columns packed with 1.8-μm particles under isocratic conditions. The results showed that apparent retention factors were on average 5, 11, 18, and 41% lower than those corrected with extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns, respectively, when the extra-column volume (11.3 μL) was kept constant. Also, apparent pressures were 31, 16, 12, and 10% higher than those corrected with pressures from extra-column volumes for 4.6-, 3.0-, 2.1-, and 1.0-mm id columns at the respective optimum flow rate for a typical ultra-high pressure liquid chromatography system. The loss in apparent efficiency increased dramatically from 4.6- to 3.0- to 2.1- to 1.0-mm id columns, less significantly as retention factors increased. The column efficiency was significantly improved as the inlet tubing id was decreased for a given column. The results suggest that maximum ratio of extra-column volume to column void volume should be approximately 1:10 for column porosity more than 0.6 and a retention factor more than 5, where 80% or higher of theoretically predicted efficiency could be achieved.

  2. Control of column temperature in reversed-phase liquid chromatography.

    PubMed

    Wolcott, R G; Dolan, J W; Snyder, L R; Bakalyar, S R; Arnold, M A; Nichols, J A

    2000-02-11

    When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

  3. Dress-up chiral columns for the enantioseparation of amino acids based on fluorous separation.

    PubMed

    Todoroki, Kenichiro; Ishii, Yasuhiro; Toyoda, Koji; Ikawa, Takashi; Min, Jun Zhe; Inoue, Koichi; Akai, Shuji; Toyo'oka, Toshimasa

    2013-10-01

    In this paper, we report a new type of chiral high-performance liquid chromatography (HPLC) column--a so-called dress-up chiral column--featuring a chiral stationary phase adsorbed reversibly in a commercial fluorous HPLC column through fluorous interactions. We synthesized perfluroalkylated proline derivatives as chiral stationary phase compounds and then adsorbed them reversibly in the fluorous HPLC column through the pumping of their solutions. By using this dress-up chiral column and fluorophobic elution of an aqueous copper(II) sulfate/MeOH mixture, we could enantioseparate seven racemic amino acids within 40 min. When we washed the dress-up chiral column with fluorophilic tetrahydrofuran or MeOH, the adsorbed chiral stationary phase compounds desorbed from the column, completely destroying its enantioseparation ability. The relative standard deviation of the retention times, the number of theoretical plates, and the resolution for each of four preparations of the dress-up columns were all less than or equal to 9.53% in 20-times repeated analysis, and were all less than or equal to 18.7% in four different preparations, respectively.

  4. Polymer-based monolithic column with incorporated chiral metal-organic framework for enantioseparation of methyl phenyl sulfoxide using nano-liquid chromatography.

    PubMed

    Wang, Xin; Lamprou, Alexandros; Svec, Frantisek; Bai, Yu; Liu, Huwei

    2016-12-01

    A new approach to the preparation of enantioselective porous polymer monolithic columns with incorporated chiral metal-organic framework for nano-liquid chromatography has been developed. While no enantioseparation was achieved with monolithic poly(4-vinylpyridine-co-ethylene dimethacrylate) column, excellent separations of both enantiomers of (±)-methyl phenyl sulfoxide were achieved with its counterpart prepared after admixing metal-organic framework [Zn2 (benzene dicarboxylate)(l-lactic acid)(dmf)], which is synthesized from zinc nitrate, l-lactic acid, and benzene dicarboxylic acid in the polymerization mixture. These novel monolithic columns combined selectivity of the chiral framework with the excellent hydrodynamic properties of polymer monoliths, may provide a great impact on future studies in the field of chiral analysis by liquid chromatography.

  5. Sub-to super-ambient temperature programmable microfabricated gas chromatography column

    DOEpatents

    Robinson, Alex L.; Anderson, Lawrence F.

    2004-03-16

    A sub- to super-ambient temperature programmable microfabricated gas chromatography column enables more efficient chemical separation of chemical analytes in a gas mixture by combining a thermoelectric cooler and temperature sensing on the microfabricated column. Sub-ambient temperature programming enables the efficient separation of volatile organic compounds and super-ambient temperature programming enables the elution of less volatile analytes within a reasonable time. The small heat capacity and thermal isolation of the microfabricated column improves the thermal time response and power consumption, both important factors for portable microanalytical systems.

  6. Hydrophilic interaction chromatography of seized drugs and related compounds with sub 2 μm particle columns.

    PubMed

    Lurie, Ira S; Li, Li; Toske, Steven G

    2011-12-30

    The use of hydrophilic interaction chromatography (HILIC) with sub 2 μm particle columns for the analysis of drugs and related compounds of forensic interest is described. This technique uses a high organic/low aqueous buffered mobile phase with a polar stationary phase, and is excellent for the separation of many of the charged solutes that are found in forensic drug exhibits. In this study, HILIC is investigated for 11 solutes of forensic interest, including weak bases, weak acids, and a neutral solute. In addition, for columns containing either ethylene bridged hybrid particles with or without an amide bonded phase, the effects of acetonitrile concentration, buffer type, buffer concentration, linear velocity, and sample concentration were studied. Based on these studies, HILIC with sub 2 μm particle columns can offer highly efficient, selective, and rapid isocratic separations of drugs and related compounds of forensic interest, with excellent peak shapes and low back pressures. This is in contrast to reverse phase chromatography (RPLC), where gradient elution is usually required, which can result in extensive overlap between acidic, neutral, and basic solutes. In addition, since HILIC exhibits a much greater loading capacity than RPLC, it could be a preferred technique for drug profiling. Furthermore, because high organic content mobile phases are highly amenable to mass spectrometric detection, the use of HILIC with tandem mass spectrometric detection for the analysis of seized drugs is described.

  7. Methacrylate-bonded covalent-organic framework monolithic columns for high performance liquid chromatography.

    PubMed

    Liu, Li-Hua; Yang, Cheng-Xiong; Yan, Xiu-Ping

    2017-01-06

    Covalent-organic frameworks (COFs) are a newfangled class of intriguing microporous materials. Considering their unique properties, COFs should be promising as packing materials for high performance liquid chromatography (HPLC). However, the irregular shape and sub-micrometer size of COFs synthesized via the traditional methods render the main obstacles for the application of COFs in HPLC. Herein, we report the preparation of methacrylate-bonded COF monolithic columns for HPLC to overcome the above obstacles. The prepared COF bonded monolithic columns not only show good homogeneity and permeability, but also give high column efficiency, good resolution and precision for HPLC separation of small molecules including polycyclic aromatic hydrocarbons, phenols, anilines, nonsteroidal anti-inflammatory drugs and benzothiophenes. Compared with the bare polymer monolithic column, the COF bonded monolithic columns show enhanced hydrophobic, π-π and hydrogen bond interactions in reverse phase HPLC. The results reveal the great potential of COF bonded monoliths for HPLC and COFs in separation sciences.

  8. Modeling Transport in Gas Chromatography Columns for the Micro-ChemLab

    SciTech Connect

    ADKINS,DOUGLAS R.; FRYE-MASON,GREGORY CHARLES; HUDSON,MARY L.; KOTTENSTETTE,RICHARD; MATZKE,CAROLYN M.; SALINGER,ANDREW G.; SHADID,JOHN N.; WONG, CHUNGNIN CHANN

    1999-09-01

    The gas chromatography (GC) column is a critical component in the microsystem for chemical detection ({mu}ChemLab{trademark}) being developed at Sandia. The goal is to etch a meter-long GC column onto a 1-cm{sup 2} silicon chip while maintaining good chromatographic performance. Our design strategy is to use a modeling and simulation approach. We have developed an analytical tool that models the transport and surface interaction process to achieve an optimized design of the GC column. This analytical tool has a flow module and a separation module. The flow module considers both the compressibility and slip flow effects that may significantly influence the gas transport in a long and narrow column. The separation module models analyte transport and physico-chemical interaction with the coated surface in the GC column. It predicts the column efficiency and performance. Results of our analysis will be presented in this paper. In addition to the analytical tool, we have also developed a time-dependent adsorption/desorption model and incorporated this model into a computational fluid dynamics (CFD) code to simulate analyte transport and separation process in GC columns. CFD simulations can capture the complex three-dimensional flow and transport dynamics, whereas the analytical tool cannot. Different column geometries have been studied, and results will be presented in this paper. Overall we have demonstrated that the modeling and simulation approach can guide the design of the GC column and will reduce the number of iterations in the device development.

  9. Evaluation of column hardware on liquid chromatography-mass spectrometry of phosphorylated compounds.

    PubMed

    Sakamaki, Hiroshi; Uchida, Takeharu; Lim, Lee Wah; Takeuchi, Toyohide

    2015-02-13

    The influences of column hardware, such as chromatographic tubes and frits, on liquid chromatography-mass spectrometry (LC-MS) analysis of phosphorylated compounds were evaluated. The signal to noise ratio (S/N) and the intensity of flavin adenine dinucleotide (FAD) using a glass lined tube and polyethylene frit (GL-PE) column was approximately 170 and 90 times higher, respectively, than those using conventional stainless steel tube and stainless steel frit (S-S) column. In addition, the retention time of FAD using GL-PE column was the shortest compared to other columns. Interaction between phosphorylated compounds and metal ions in the flow path in the S-S column was stronger than that between them and the GL-PE column. Thus, the metal ions in the flow path in GL-PE column were low. Since the specific surface area of a pair of frits was 70 times larger than that of a chromatographic tube (150 mm×2.1 mm), the frits were found to have more effective improvement of the S/N as well as the intensity than the chromatographic tubes, when phosphorylated compounds were analyzed by LC-MS. When the evaluated phosphorylated compounds were analyzed by LC-MS(/MS) using a GL-PE column, the intensity and S/N were increased.

  10. Applications of monolithic columns in liquid chromatography-based clinical chemistry assays.

    PubMed

    Bunch, Dustin R; Wang, Sihe

    2011-08-01

    Monolithic columns have slowly been applied to HPLC methods for clinical chemistry testing in the last 10 years. The application areas include therapeutic drug monitoring, drugs of abuse, vitamins, porphyrins, and steroids. In comparison with conventional particulate columns, the monolithic columns may offer shorter chromatography time, more robustness, and better resolution for certain analytes. The potential drawback of large mobile phase consumption may be improved with smaller id columns, which are currently on the market. Methods covered in this review are those searchable in PubMed up to December 2010. This review highlights the emergence of monolithic column technology in HPLC methods used for clinical chemistry testing. The goals of this review are threefold: (i) To identify the areas of clinical chemistry that analytical monolithic columns have been used in HPLC methods. (ii) To demonstrate the application of analytical monolithic columns in HPLC methods using different detection systems. (iii) To discuss the advantages and limitations of the monolithic columns compared with particulate columns in the clinical chemistry applications.

  11. A Computer-Interfaced Drop Counter as an Inexpensive Fraction Collector for Column Chromatography

    ERIC Educational Resources Information Center

    Nash, Barbara T.

    2008-01-01

    A computer-interfaced drop counter is described that serves as an inexpensive alternative to a fraction collector for column chromatography experiments. Undergraduate biochemistry laboratories frequently do not have the budget to purchase fraction collectors. Protocols that call for the manual measurement of fraction volumes as well as the manual…

  12. Separation of the Carotenoid Bixin from Annatto Seeds Using Thin-Layer and Column Chromatography

    ERIC Educational Resources Information Center

    McCullagh, James V.; Ramos, Nicholas

    2008-01-01

    In this experiment the carotenoid bixin is isolated from annatto ("Bixa orellana") seeds using column chromatography. The experiment has several key advantages over previous pigment separation experiments. First, unlike other experiments significant quantities of the carotenoid (typically 20 to 25 mg) can be isolated from small quantities of plant…

  13. Investigation of Pinus mugo essential oil oxygenated fraction by combined use of gas chromatography and dry column chromatography.

    PubMed

    A, M B; Coran, S A; Giannellini, V; Vincieri, F F; Moneti, G

    1981-09-01

    The oxygenated compounds of Pinus mugo Turra essential oil were investigated by a combination of GC and dry column chromatography (DCC) coordinated by GC data processing. The collected data resulted in a bar graph ("normalized" gas chromatogram) giving the RRT's and relative amounts of 68 components; 38 of them were identified by MS and IR. The described procedure may be used for essential oil analysis in general.

  14. Simple column-switching ion chromatography method for determining eight monosaccharides and oligosaccharides in honeydew and nectar.

    PubMed

    Ni, Chengzhu; Zhu, Binhe; Wang, Nani; Wang, Muhua; Chen, Suqing; Zhang, Jiajie; Zhu, Yan

    2016-03-01

    Honeydew is excreted by aphids as a sweet waste and nectar is floral honey. Honeydew and nectar are complicated samples which consist of various sugars and amino acids. In this work, a simple ion chromatography with column-switching method was developed for the simultaneous analysis of 8 monosaccharides and oligosaccharides in honeydew and nectar. A reversed-phase column was used as a pretreatment column to eliminate organics on-line and sugars were eluted from a collection loop to analytical column by using column-switching technique. This method showed good linearity (r⩾0.9994) and afforded low limits of detection ranging from 1.55 to 10.17μgL(-1) for all the analytes. Recoveries ranged from 95% to 105% and repeatability results were acceptable with relative standard deviation of less than 3.21% (n=6). This method was successfully applied to quantification of these sugars in honeydew and nectar. These results showed honeydew had much more oligosaccharides than nectar.

  15. Multidimensional high-performance liquid chromatography on Pinkerton ISRP and RP18 columns: direct serum injection to quantify creatinine.

    PubMed

    Puhlmann, A; Dülffer, T; Kobold, U

    1992-10-02

    A two-dimensional high-performance liquid chromatographic method for the determination of creatinine with direct serum injection without sample pretreatment has been developed. The column-switching technique allowed a switch from columns packed with internal surface reversed-phase (ISRP) material to columns of almost any other material, even if the eluents necessary in a particular case do not appear to be directly compatible. A Pinkerton ISRP column, which stands out because of its very good stability when loaded with undiluted serum samples, was used as precolumn. The creatinine-containing fraction was switched to a reversed-phase Shandon RP18 column and was focused there by alteration of the eluent from pH 6.5 to phosphoric acid-ion-pair reagent. The separation occurs via a pH gradient, with ultraviolet detection at 234 nm. This method stands out particularly for its good long-term stability, simple sample handling without pretreatment, high selectivity, a broad linearity (0.3-30 mg/dl creatinine), good reproducibility (inter-assay coefficient of variation less than 3%) and high recovery (97-100%) relative to values obtained with gas chromatography-mass spectrometry.

  16. High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization.

    PubMed

    Saito, K; Horie, M; Nose, N; Nakagomi, K; Nakazawa, H

    1992-03-20

    An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.

  17. Standardized test mixture for the characterization of comprehensive two-dimensional gas chromatography columns: the Phillips mix.

    PubMed

    Dimandja, Jean-Marie D; Clouden, Garrick C; Colón, Ivelisse; Focant, Jean-François; Cabey, Whitney V; Parry, Ritchard C

    2003-11-26

    A novel column characterization test mixture is developed for use in comprehensive two-dimensional gas chromatography (GC x GC). This mixture has been named the "Phillips mix" in honor of the late professor John B. Phillips, the father of GC x GC. The mixture comprises a series of homologous compounds from structural groups that cover a volatility and polarity range that is similar to the Grob mix, and includes saturated hydrocarbons (alkanes), unsaturated hydrocarbons (alkenes and alkynes), carbonyls (ketones and aldehydes), primary alcohols, fatty acid methyl esters, alkyl ethers, carboxylic acids, aromatics, as well as other unique functional groups (such as amines, etc.). Similarly to the Grob mix in conventional one-dimensional GC, the Phillips mix can be used as a standardized test for performance characterization of GC x GC column sets. Unlike the Grob mix, however, the Phillips mix's most important use is as a practical guideline for column users. This paper addresses some qualitative aspects of the use of the Phillips mix through an investigation of the chromatographic fingerprints of two different GC x GC column combinations.

  18. Quantification of PSP toxins in toxic shellfish matrices using post-column oxidation liquid chromatography and pre-column oxidation liquid chromatography methods suggests post-column oxidation liquid chromatography as a good monitoring method of choice.

    PubMed

    Rey, Verónica; Botana, Ana M; Botana, Luis M

    2017-04-01

    Different shellfish samples were analyzed by Pre- and Post-Column Oxidation Liquid Chromatography to compare the toxins profiles and get information about the degree of accomplishment of both methods. Comparison of the results obtained, the linear correlation coefficient (r(2) = 0.94) and the paired t test (two tails, α = 0.05), indicated that there were not significant differences between both sets of data. Nevertheless, important differences related to toxins profiles were found: it was remarkable the difference in results for both Gonyautoxins 1 and 4 and Decarbamoylgonyautoxins 2 and 3, depending on the method of choice, due to an overestimation in the Pre-Column method. It was necessary to modify the elution conditions in the Post-Column method to avoid the interference of matrix peaks at retention times closer to the retention times of the calibrants, mostly when working with oyster and scallop matrices, although it is a good method to use routinely.

  19. Rapid determination of Papaver somniferum alkaloids in process streams using monolithic column high-performance liquid chromatography with chemiluminescence detection.

    PubMed

    Costin, Jason W; Lewis, Simon W; Purcell, Stuart D; Waddell, Lucy R; Francis, Paul S; Barnett, Neil W

    2007-07-30

    We have combined high-performance liquid chromatography (HPLC) separations using a monolithic column with acidic potassium permanganate and tris(2,2'-bipyridyl)ruthenium(II) chemiluminescence detection in a rapid and highly sensitive method to monitor the process of extracting opiate alkaloids from Papaver somniferum. Due to the high flow rates allowed with the monolithic column and the inherent selectivity of the chemiluminescence reactions, the four predominant alkaloids--morphine, codeine, oripavine and thebaine--were determined in less than 2 min. The results obtained with numerous process samples compared favourable with those of the standard HPLC methodology. Limits of detection were 1x10(-10) M, 5x10(-10) M, 5x10(-10) M and 1x10(-9) M, for morphine, codeine, oripavine and thebaine, respectively.

  20. Determination of the column hold-up volume in supercritical fluid chromatography using nitrous-oxide.

    PubMed

    Vajda, Péter; Guiochon, Georges

    2013-09-27

    This study introduces a new tracer that is useful for the determination of the hold-up time or volume of packed columns, particularly of those used in supercritical fluid chromatography. The thermodynamic void volume of three columns packed with different adsorbents were determined using the weight difference method. These void volumes were used as the reference point in the further discussion. The hold-up volumes of these columns were determined under dynamic conditions, using nitrous oxide dissolved in methanol as the hold-up time marker. Changes in the hold-up volumes of these columns were monitored during changes of the volumetric flow rate of pure supercritical carbon dioxide and of dilute mixtures of organic modifier and supercritical carbon dioxide. The results suggest significant methanol enrichment on the adsorbent surface.

  1. Tuning preparation conditions towards optimized separation performance of thermally polymerized organo-silica monolithic columns in capillary liquid chromatography.

    PubMed

    Gharbharan, Deepa; Britsch, Denae; Soto, Gabriela; Weed, Anna-Marie Karen; Svec, Frantisek; Zajickova, Zuzana

    2015-08-21

    Tuning of preparation conditions, such as variations in the amount of a porogen, concentration of an aqueous acid catalyst, and adjustment in polymerization temperature and time, towards optimized chromatographic performance of thermally polymerized monolithic capillaries prepared from 3-(methacryloyloxy)propyltrimethoxysilane has been carried out. Performance of capillary columns in reversed-phase liquid chromatography was assessed utilizing various sets of solutes. Results describing hydrophobicity, steric selectivity, and extent of hydrogen bonding enabled comparison of performance of hybrid monolithic columns prepared under thermal (TSG) and photopolymerized (PSG) conditions. Reduced amounts of porogen in the polymerization mixture, and prolonged reaction times were necessary for the preparation of monolithic columns with enhanced retention and column efficiency that reached to 111,000 plates/m for alkylbenzenes with shorter alkyl chains. Both increased concentration of catalyst and higher temperature resulted in faster polymerization but inevitably in insufficient time for pore formation. Thermally polymerized monoliths produced surfaces, which were slightly more hydrophobic (a methylene selectivity of 1.28±0.002 TSG vs 1.20±0.002 PSG), with reduced number of residual silanols (a caffeine/phenol selectivity of 0.13±0.001 TSG vs 0.17±0.003 PSG). However, steric selectivity of 1.70±0.01 was the same for both types of columns. The batch-to-batch repeatability was better using thermal initiation compared to monolithic columns prepared under photopolymerized conditions. RSD for retention factor of benzene was 3.7% for TSG capillaries (n=42) vs. 6.6% for PSG capillaries (n=18). A similar trend was observed for columns prepared within the same batch.

  2. Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column.

    PubMed

    Zemenova, Jana; Sykora, David; Adamkova, Hana; Maletinska, Lenka; Elbert, Tomas; Marek, Ales; Blechova, Miroslava

    2017-03-01

    In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography-mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme-linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80-120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1-250 μg/mL.

  3. Fabrication of electrolytic cell for online post-column electrochemical derivatization in ion chromatography.

    PubMed

    Wu, Shuchao; Xu, Wei; Yang, Bingcheng; Ye, Mingli; Zhang, Peimin; Shen-Tu, Chao; Zhu, Yan

    2012-07-20

    An electrolytic cell (EC), composed of two ruthenium-plated titanium electrodes separated by cation-exchange membranes, was fabricated and evaluated for online postcolumn derivatization in ion chromatography (IC). Folic acid (FA) and methotrexate (MTX) were preliminarily used as prototype analytes to test the performance of EC. After separation by an anion exchange column, FA and MTX, which emit very weak fluorescence when excited, were electrochemically oxidized online in the anode chamber of the EC. The compounds with strong fluorescence, which are oxidation products, were detected by the fluorescence detector. The phosphate buffer solution (100 mM KH(2)PO(4)) served as an optimal eluent for anion exchange chromatographic separation and a suitable supporting electrolyte for electro-oxidation, leading to ideal compatibility between IC separation and the postcolumn electrochemical derivatization. For the presently proposed method, the linear ranges were from 0.01 mg L(-1) to 5 mg L(-1) for both FA and MTX. The detection limits of FA and MTX were 1.8 and 2.1 μg L(-1), and the relative standard deviations (RSD, n=7) were 2.9% and 3.6%, respectively. The method was applied for the simultaneous determination of FA and MTX in the plasma of patients being treated for rheumatoid arthritis. The determination of MTX in the urine of the patients of diffuse large B cell lymphoma was also demonstrated.

  4. Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak's extracts

    NASA Astrophysics Data System (ADS)

    Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat, Suzery, Meiny

    2015-12-01

    Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak's extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r2=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak's extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

  5. Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak’s extracts

    SciTech Connect

    Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,; Suzery, Meiny

    2015-12-29

    Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak’s extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r{sup 2}=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak’s extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

  6. Preparation and characterization of alkyl methacrylate-based monolithic columns for capillary gas chromatography applications.

    PubMed

    Yusuf, Kareem; Aqel, Ahmad; A L Othman, Zeid; Badjah-Hadj-Ahmed, Ahmed Yacine

    2013-08-02

    Gas chromatography (GC) is considered the least common application of both polymer and silica-based monolithic columns. This study describes the fabrication of alkyl methacrylate monolithic materials for use as stationary phases in capillary gas chromatography. Following the deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (TMSM), the monoliths were formed by the co-polymerization of either hexyl methacrylate (HMA) or lauryl methacrylate (LMA) with different percentage of ethylene glycol dimethacrylate (EDMA) in presence of an initiator (azobisisobutyronitrile, AIBN) and a mixture of porogens include 1-propanol, 1,4-butanediol and water. The monoliths were prepared in 500mm length capillaries possessing inner diameters of 250μm. The efficiencies of the monolithic columns for low molecular weight compounds significantly improved as the percentage of crosslinker was increased, because of the greater proportion of pores less than 50nm. The columns containing lower percentages of crosslinker were able to rapidly separate a series of 8 alkane members in 0.7min, but the separation was less efficient for the light alkanes. Columns prepared with the lauryl methacrylate monomer yielded a different morphology for the monolith-interconnected channels. The channels were more branched, which increased the separation time, and unlike the other columns, allowed for temperature programming.

  7. Enantioseparation of methamphetamine by supercritical fluid chromatography with cellulose-based packed column.

    PubMed

    Segawa, Hiroki; Iwata, Yuko T; Yamamuro, Tadashi; Kuwayama, Kenji; Tsujikawa, Kenji; Kanamori, Tatsuyuki; Inoue, Hiroyuki

    2017-02-04

    The enantiomers of methamphetamine were differentiated by supercritical fluid chromatography (SFC) with an enantioselective cellulose-based packed column. The optimization of the chromatographic conditions was achieved by changing column temperature, co-solvent proportion, additive concentration, flow rate and back pressure. In particular, the additive concentration crucially changed the resolution between the enantiomers. After determining the optimized conditions, the enantiomers of methamphetamine were successfully separated. The analytical precision, accuracy and limit of detection were checked by using the authentic standard and seized real samples. We believe that chiral SFC is a promising method for enantioseparation of forensic samples.

  8. [Analysis of aliphatic carboxylic acids in anaerobic digestion process waters by ion-exclusion chromatography].

    PubMed

    Ito, Kazuaki; Sakamoto, Jun; Nagaoka, Kazuya; Takayama, Yohichi; Kanahori, Takashi; Sunahara, Hiroshi; Hayashi, Tsuneo; Sato, Shinji; Hirokawa, Takeshi; Tanaka, Kazuhiko

    2012-04-01

    The analysis of seven aliphatic carboxylic acids (formic, acetic, propionic, iso-butyric, n-butyric, iso-valeric and n-valeric acid) in anaerobic digestion process waters for biogas production was examined by ion-exclusion chromatography with dilute acidic eluents (benzoic acid, perfluorobutyric acid (PFBA) and sulfuric acid) and non-suppressed conductivity/ultraviolet (UV) detection. The columns used were a styrene/divinylbenzene-based strongly acidic cation-exchange resin column (TSKgel SCX) and a polymethacrylate-based weakly acidic cation-exchange resin column (TSKgel Super IC-A/C). Good separation was performed on the TSKgel SCX in shorter retention times. For the TSKgel Super IC-A/C, peak shape of the acids was sharp and symmetrical in spite of longer retention times. In addition, the mutual separation of the acids was good except for iso- and n-butyric acids. The better separation and good detection was achieved by using the two columns (TSKgel SCX and TSKgel Super IC-A/C connected in series), lower concentrations of PFBA and sulfuric acid as eluents, non-suppressed conductivity detection and UV detection at 210 nm. This analysis was applied to anaerobic digestion process waters. The chromatograms with conductivity detection were relatively simpler compared with those of UV detection. The use of two columns with different selectivities for the aliphatic carboxylic acids and the two detection modes was effective for the determination and identification of the analytes in anaerobic digestion process waters containing complex matrices.

  9. Metal-Organic Framework Thin Films as Stationary Phases in Microfabricated Gas-Chromatography Columns.

    SciTech Connect

    Read, Douglas; Sillerud, Colin Halliday

    2016-01-01

    The overarching goal of this project is to integrate Sandia's microfabricated gas-chromatography ( GC) columns with a stationary phase material that is capable of retaining high-volatility chemicals and permanent gases. The successful integration of such a material with GCs would dramatically expand the repertoire of detectable compounds for Sandia's various microanalysis systems. One such promising class of candidate materials is metal-organic frameworks (MOFs). In this report we detail our methods for controlled deposition of HKUST-1 MOF stationary phases within GC columns. We demonstrate: the chromatographic separation of natural gas; a method for determining MOF film thickness from chromatography alone; and the first-reported GC x GC separation of natural gas -- in general -- let alone for two disparate MOF stationary phases. In addition we determine the fundamental thermodynamic constant for mass sorption, the partition coefficient, for HKUST-1 and several light hydrocarbons and select toxic industrial chemicals.

  10. Optical isomer separation of single-chirality carbon nanotubes using gel column chromatography.

    PubMed

    Liu, Huaping; Tanaka, Takeshi; Kataura, Hiromichi

    2014-11-12

    We report a gel column chromatography method for easily separating the optical isomers (i.e., left- and right-handed structures) of single-chirality carbon nanotubes. This method uses the difference in the interactions of the two isomers of a chiral single-wall carbon nanotube (SWCNT) with an allyl dextran-based gel, which result from the selective interaction of the chiral moieties of the gel with the isomers. Using this technique, we sorted optical isomers of nine distinct (n, m) single-chirality species from HiPco SWCNTs, which is the maximum number of isolable species of SWCNTs reported to date. Because of its advantages of technical simplicity, low cost, and high efficiency, gel column chromatography allows researchers to prepare macroscopic ensembles of single-structure SWCNTs and enables the complete discovery of intrinsic properties of SWCNTs and advances their application.

  11. Size exclusion chromatography of synthetic polymers and biopolymers on common reversed phase and hydrophilic interaction chromatography columns.

    PubMed

    Caltabiano, Anna M; Foley, Joe P; Barth, Howard G

    2016-03-11

    This work describes the applicability of common reversed phase and HILIC columns for size exclusion chromatography of synthetic and natural polymers. Depending on the nature of the solute and column stationary phase, a "non-retention" condition must be created with the aid of the mobile phase to achieve a unique size-based separation in isocratic mode. The various bonded phases show remarkable differences in size separations that are controlled by mobile phase conditions. Polymer-mobile phase and column-mobile phase solvation interactions determine polymer hydrodynamic volume (or solute bulkiness) and polymer-column steric interaction. Solvation interactions in turn depend on polymer, mobile phase and stationary phase polarities. Column-mobile phase solvation interactions determine the structural order of the bonded ligands that can vary from ordered (extended, aligned away from the silica substrate) to disordered (folded, pointing toward the silica substrate). Chain order increases with increased solvent penetration into the bonded phase. Increased chain order reduces pore volume, and therefore decreases the size-separation efficiency of a column. Conversely, decreased chain order increases pore volume and therefore increases the size-separation efficiency. The thermodynamic quality of the mobile phase also plays a significant role in the separation of polymers. "Poor" solvents can significantly reduce the hydrodynamic diameter of a solute and thus change their retention behavior. Medium polarity stationary phases, such as fluoro-phenyl and cyano, exhibit a unique retention behavior. With an appropriate polarity mobile phase, polar and non-polar synthetic polymers of the same molecular masses can be eluted at the same retention volumes.

  12. Signal analysis of NEMS sensors at the output of a chromatography column

    SciTech Connect

    Bertholon, François; Harant, Olivier; Bourlon, Bertrand; Gerfault, Laurent; Grangeat, Pierre; Jutten, Christian

    2015-01-13

    This article introduces a joined Bayesian estimation of gas samples issued from a gas chromatography column (GC) coupled with a NEMS sensor based on Giddings Eyring microscopic molecular stochastic model. The posterior distribution is sampled using a Monte Carlo Markov Chain and Gibbs sampling. Parameters are estimated using the posterior mean. This estimation scheme is finally applied on simulated and real datasets using this molecular stochastic forward model.

  13. Evaluation of sequential injection chromatography for reversed phase separation of triazine herbicides exploiting monolithic and core-shell columns.

    PubMed

    de Prá Urio, Ricardo; Masini, Jorge C

    2015-01-01

    This paper describes the development of reversed phase sequential injection chromatography (SIC) methods for separation of simazine (SIM) and atrazine (AT), as well as their metabolites deethylatrazine (DEA), deisopropylatrazine (DIA) and hydroxyatrazine (HAT) exploiting silica based monolithic (50 × 4.6 mm) and core-shell (30 × 4.6 mm, 2.7 µm particles) columns. The separation was made by stepwise elution with two mobile phases: MP1 composed of 15:85 (v/v) acetonitrile: 2.5 mmol L(-1) acetic acid/ammonium acetate buffer (pH 4.2), and MP2, composed of 35:65 (v/v) acetonitrile: 2.5 mmol L(-1) acetic acid/ammonium acetate buffer (pH 4.2).The less hydrophobic compounds (DIA, HAT and DEA) eluted with MP1, whereas SIM and AT eluted with MP2. The method using core-shell column exhibited better chromatographic efficiency compared with monolithic column for separation of SIM and AT, but failed to provide base line separation of DIA and HAT. The proposed composition of mobile phases enabled the monolithic column to separate all the studied compounds with resolution >2.3 at flow rate of 35 µL s(-1) and sampling throughput of 8 analyses per hour, whereas in the core-shell the maximum flow rate allowed in the SIC system was 8 µL s(-1) (sampling throughput of 3 analyses per hour). The limits of detection were between 24 µg L(-1) (AT) and 40 µg L(-1) (DEA) using the monolithic column, and between 20 µg L(-1) (SIM) and 38 µg L(-1) (DEA) with the core-shell. Ultrasound-assisted extraction (80:20 v/v acetonitrile:water) of a soil sample enriched with the five triazines (250, 500 and 1000 µg kg(-1)) resulted recoveries between 51% and 121% of the spiked concentrations.

  14. High performance mini-gas chromatography-flame ionization detector system based on micro gas chromatography column

    NASA Astrophysics Data System (ADS)

    Zhu, Xiaofeng; Sun, Jianhai; Ning, Zhanwu; Zhang, Yanni; Liu, Jinhua

    2016-04-01

    Monitoring Volatile organic compounds (VOCs) was a very important measure for preventing environmental pollution, therefore, a mini gas chromatography (GC) flame ionization detector (FID) system integrated with a mini H2 generator and a micro GC column was developed for environmental VOC monitoring. In addition, the mini H2 generator was able to make the system explode from far away due to the abandoned use of a high pressure H2 source. The experimental result indicates that the fabricated mini GC FID system demonstrated high repeatability and very good linear response, and was able to rapidly monitor complicated environmental VOC samples.

  15. Flow rate dependent extra-column variance from injection in capillary liquid chromatography.

    PubMed

    Aggarwal, Pankaj; Liu, Kun; Sharma, Sonika; Lawson, John S; Dennis Tolley, H; Lee, Milton L

    2015-02-06

    Efficiency and resolution in capillary liquid chromatography (LC) can be significantly affected by extra-column band broadening, especially for isocratic separations. This is particularly a concern in evaluating column bed structure using non-retained test compounds. The band broadening due to an injector supplied with a commercially available capillary LC system was characterized from experimental measurements. The extra-column variance from the injection valve was found to have an extra-column contribution independent of the injection volume, showing an exponential dependence on flow rate. The overall extra-column variance from the injection valve was found to vary from 34 to 23 nL. A new mathematical model was derived that explains this exponential contribution of extra-column variance on chromatographic performance. The chromatographic efficiency was compromised by ∼130% for a non-retained analyte because of injection valve dead volume. The measured chromatographic efficiency was greatly improved when a new nano-flow pumping system with integrated injection valve was used.

  16. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-04

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column.

  17. Core-Shell Columns in High-Performance Liquid Chromatography: Food Analysis Applications

    PubMed Central

    Preti, Raffaella

    2016-01-01

    The increased separation efficiency provided by the new technology of column packed with core-shell particles in high-performance liquid chromatography (HPLC) has resulted in their widespread diffusion in several analytical fields: from pharmaceutical, biological, environmental, and toxicological. The present paper presents their most recent applications in food analysis. Their use has proved to be particularly advantageous for the determination of compounds at trace levels or when a large amount of samples must be analyzed fast using reliable and solvent-saving apparatus. The literature hereby described shows how the outstanding performances provided by core-shell particles column on a traditional HPLC instruments are comparable to those obtained with a costly UHPLC instrumentation, making this novel column a promising key tool in food analysis. PMID:27143972

  18. A micro gas chromatography column with a micro thermal conductivity detector for volatile organic compound analysis.

    PubMed

    Sun, J H; Cui, D F; Chen, X; Zhang, L L; Cai, H Y; Li, H

    2013-02-01

    In this paper, a micro gas chromatography (μGC) system contained a μGC column and a micro thermal conductivity detector (μTCD) was proposed. In order to reduce the volume of the system, some micro heaters were integrated on the surface and backside of the GC column, which could provide a robust temperature programming capability and rapidly increase the temperature of the μGC column. In addition, a silicon-glass μTCD with four-thermistor thermal conductivity cells that can offer significant advantages over previously reported designs including low dead volume, good thermal isolation, and elimination of the thermal noise was proposed in this paper. Experimental results have indicated that the μGC system with a detection limit of several ppm concentration levels separated and detected the benzene, toluene, and styrene in less than 3 min, and the μGC system also exhibited a good linear response in the test range.

  19. Parallel extraction columns and parallel analytical columns coupled with liquid chromatography/tandem mass spectrometry for on-line simultaneous quantification of a drug candidate and its six metabolites in dog plasma.

    PubMed

    Xia, Y Q; Hop, C E; Liu, D Q; Vincent, S H; Chiu, S H

    2001-01-01

    A method with parallel extraction columns and parallel analytical columns (PEC-PAC) for on-line high-flow liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated for simultaneous quantification of a drug candidate and its six metabolites in dog plasma. Two on-line extraction columns were used in parallel for sample extraction and two analytical columns were used in parallel for separation and analysis. The plasma samples, after addition of an internal standard solution, were directly injected onto the PEC-PAC system for purification and analysis. This method allowed the use of one of the extraction columns for analyte purification while the other was being equilibrated. Similarly, one of the analytical columns was employed to separate the analytes while the other was undergoing equilibration. Therefore, the time needed for re-conditioning both extraction and analytical columns was not added to the total analysis time, which resulted in a shorter run time and higher throughput. Moreover, the on-line column extraction LC/MS/MS method made it possible to extract and analyze all seven analytes simultaneously with good precision and accuracy despite their chemical class diversity that included primary, secondary and tertiary amines, an alcohol, an aldehyde and a carboxylic acid. The method was validated with the standard curve ranging from 5.00 to 5000 ng/mL. The intra- and inter-day precision was no more than 8% CV and the assay accuracy was between 95 and 107%.

  20. Pressure Tuning of First Dimension Columns in Comprehensive Two-Dimensional Gas Chromatography.

    PubMed

    Sharif, Khan M; Kulsing, Chadin; Marriott, Philip J

    2016-09-20

    The experimental approach and mechanism of pressure tuning (PT) are introduced for the first stage of a comprehensive two-dimensional gas chromatography (GC × GC) separation. The PT-GC × GC system incorporates a first dimension ((1)D) coupled column ensemble comprising a pair of (1)D columns ((1)D1 and (1)D2) connected via a microfluidic splitter device, allowing variable decompression of carrier gas across each (1)D column, and a conventional (2)D narrow bore column. By variation of junction pressure between the (1)D1 and (1)D2 columns, tunable total (1)D retentions of analytes are readily derived. Separations of a standard mixture comprising a number of different chemical classes (including alkanes, monoaromatics, alcohols, aldehydes, ketones, and esters) and Australian tea tree oil (TTO) were studied as practical examples of the PT-GC × GC system application. This illustrated the change of analyte retention time with experimental conditions depending on void time and retention on the different columns. In addition to void time change, variation of carrier gas relative decompression in the (1)D ensemble leads to tunable contribution of the (1)D1/(1)D2 columns that changes apparent polarity and selectivity of the ensemble. The resulting changes in (1)D elution order further altered elution temperature and thus retention of each analyte on the (2)D column in temperature-programmed GC × GC. 2D orthogonality measurements were then conducted to evaluate overall separation performance under application of different (1)D junction pressure. As a result, distribution and selectivity of particular target compounds, monoterpenes, sesquiterpenes, and oxygenated terpenes in 2D space, and thus orthogonality, could be adequately tuned. This indicates the potential of PT-GC × GC to be applicable for practical sample separation and provides a general approach to tune selectivity of target compounds.

  1. [Purification of Al13 species in polyaluminum chloride (PAC) by column chromatography and the character of the fractions].

    PubMed

    Chu, Yong-bao; Gao, Bao-yu; Yue, Qin-yan; Wang, Shu-guang; Wang, Yan

    2005-05-01

    The polynuclear keggin species, Al12AlO4(OH)24(H2O)12(7+) (Al13), was separated and purified from polyaluminum chloride (PAC) by column chromatography method. The three fractions were characterized by Al-Ferron timed complexation spectrophotometry, TEM and ZATESIZER tester. Jar tests were performed to test the coagulation efficiency of the three fractions in treating synthetic or actual water samples. The results show that the sample purified by the column chromatography method influenced by the size of the species. The bigger molecules were eluted first, then the smaller ones. Al13 would be obtained by taking out the middle fraction. Compared with the other two fractions, the third fraction gives the best results for turbidity, humic acid and color removal, and achieves the highest charge-neutralizing ability. Al13 species is a higher positive-charged species and can be applied in a wide pH range, and it is much more steady in hydrolyzing process and most effective polymeric Al species in water and wastewater treatment.

  2. Ultra high pressure liquid chromatography. Column permeability and changes of the eluent properties.

    PubMed

    Gritti, Fabrice; Guiochon, Georges

    2008-04-11

    The behavior of four similar liquid chromatography columns (2.1mm i.d. x 30, 50, 100, and 150 mm, all packed with fine particles, average d(p) approximately 1.7 microm, of bridged ethylsiloxane/silica hybrid-C(18), named BEH-C(18)) was studied in wide ranges of temperature and pressure. The pressure and the temperature dependencies of the viscosity and the density of the eluent (pure acetonitrile) along the columns were also derived, using the column permeabilities and applying the Kozeny-Carman and the heat balance equations. The heat lost through the external surface area of the chromatographic column was directly derived from the wall temperature of the stainless steel tube measured with a precision of +/-0.2 degrees C in still air and +/-0.1 degrees C in the oven compartment. The variations of the density and viscosity of pure acetonitrile as a function of the temperature and pressure was derived from empirical correlations based on precise experimental data acquired between 298 and 373 K and at pressures up to 1.5 kbar. The measurements were made with the Acquity UPLC chromatograph that can deliver a maximum flow rate of 2 mL/min and apply a maximum column inlet pressure of 1038 bar. The average Kozeny-Carman permeability constant of the columns was 144+/-3.5%. The temperature hence the viscosity and the density profiles of the eluent along the column deviate significantly from linear behavior under high-pressure gradients. For a 1000 bar pressure drop, we measured DeltaT=25-30 K, (Deltaeta/eta) approximately 100%, and (Deltarho/rho) approximately 10%. These results show that the radial temperature profiles are never fully developed within 1% for any of the columns, even under still-air conditions. This represents a practical advantage regarding the apparent column efficiency at high flow rates, since the impact of the differential analyte velocity between the column center and the column wall is not maximum. The interpretation of the peak profiles recorded in

  3. High-performance, static-coated silicon microfabricated columns for gas chromatography.

    PubMed

    Reidy, Shaelah; Lambertus, Gordon; Reece, Jennifer; Sacks, Richard

    2006-04-15

    A procedure is described for the preparation of high-performance etched silicon columns for gas chromatography. Rectangular channels, 150 mum wide by 240 mum deep are fabricated in silicon substrates by gas-phase reactive ion etching. A 0.1-0.2-mum-thick film of dimethyl polysiloxane stationary phase is deposited on the channel walls by filling the channel with a dilute solution in 1:1 n-pentane and dichloromethane and pumping away the solvent. A thermally activated cross-linking agent is used for in situ cross-linking. A 3-m-long microfabricated column generated approximately 12 500 theoretical plates at optimal operating conditions using air as carrier gas. A kinetic model for the efficiency of rectangular cross-section columns is used to evaluate column performance. Results indicate an additional source of gas-phase dispersion beyond longitudinal diffusion and nonequilibrium effects, probably resulting from numerous turns in the gas flow path through the channel. The columns are thermally stable to at least 180 degrees C using air carrier gas. Temperature programming is demonstrated for the boiling point range from n-C5 to n-C12. A 3.0-m-long column heated at 10 degrees C/min obtains a peak capacity of over 100 peaks with a resolution of 1.18 and a separation time of approximately 500 s. With a 0.25-m-long column heated at 30 degrees C/min, a peak capacity of 28 peaks is obtained with a separation time of 150 s. Applications are shown for the analysis of air-phase petroleum hydrocarbons and the high-speed analysis of chemical warfare agent and explosive markers.

  4. Overloading study of basic compounds with a positively charged C18 column in liquid chromatography.

    PubMed

    Wang, Chaoran; Guo, Zhimou; Long, Zhen; Zhang, Xiuli; Liang, Xinmiao

    2013-03-15

    While tailing and overloading of basic compounds remain problematic on most RP columns, a new kind of positively charged RP column named XCharge C18 was found to be superior good for the separation of alkaloids in our practical use. In this work, the surface charge property of the XCharge C18 column was evaluated by the retention of NO(3)(-) under different pH values and buffer concentrations. A considerable and pH-dependent positive charge was confirmed on the column. Then overloading behaviors of bases were systematically studied using amitriptyline as a basic probe. Good peak shapes (Tf<1.5) and extra high loadability with a C(0.5) of about 30,000 mg/L were observed on the column, with commonly used 0.1% formic acid as mobile phase additive. However, increasing the ionic strength of buffer with phosphates led to tailing peaks at high sample amount and sharp decline in loadability (C(0.5) of 2000-3000 mg/L), although it brought higher column efficiency at low sample amount. Higher pH also induced worse performance and lower loadability. The overall results demonstrated the importance of an appropriate level of ionic repulsion for the XCharge C18 column to achieve the good performance for bases, which could be explained by the multiple-site adsorption theory as ionic repulsion would shield the solute from occupying high-energy sites deeper in C18 layer.

  5. Automated hydrophobic interaction chromatography column selection for use in protein purification.

    PubMed

    Murphy, Patrick J M; Stone, Orrin J; Anderson, Michelle E

    2011-09-21

    In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein (1). The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification. HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH(4;))(2;)SO(4;)). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) (2). As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter (3). Automated column scouting allows for an efficient approach for determining

  6. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    PubMed Central

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-01-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan. PMID:27470880

  7. In-column ATR-FTIR spectroscopy to monitor affinity chromatography purification of monoclonal antibodies

    NASA Astrophysics Data System (ADS)

    Boulet-Audet, Maxime; Kazarian, Sergei G.; Byrne, Bernadette

    2016-07-01

    In recent years many monoclonal antibodies (mAb) have entered the biotherapeutics market, offering new treatments for chronic and life-threatening diseases. Protein A resin captures monoclonal antibody (mAb) effectively, but the binding capacity decays over repeated purification cycles. On an industrial scale, replacing fouled Protein A affinity chromatography resin accounts for a large proportion of the raw material cost. Cleaning-in-place (CIP) procedures were developed to extend Protein A resin lifespan, but chromatograms cannot reliably quantify any remaining contaminants over repeated cycles. To study resin fouling in situ, we coupled affinity chromatography and Fourier transform infrared (FTIR) spectroscopy for the first time, by embedding an attenuated total reflection (ATR) sensor inside a micro-scale column while measuring the UV 280 nm and conductivity. Our approach quantified the in-column protein concentration in the resin bed and determined protein conformation. Our results show that Protein A ligand leached during CIP. We also found that host cell proteins bound to the Protein A resin even more strongly than mAbs and that typical CIP conditions do not remove all fouling contaminants. The insights derived from in-column ATR-FTIR spectroscopic monitoring could contribute to mAb purification quality assurance as well as guide the development of more effective CIP conditions to optimise resin lifespan.

  8. High resolution capillary column development for selective separations in gas chromatography

    SciTech Connect

    Przybyciel, M.

    1985-01-01

    A review of techniques for the preparation of high resolution capillary columns for gas chromatography is presented. Surface roughing, surface deactivation, stationary phase coating, and stationary phase crosslinking are discussed. Criteria for the selection of GC stationary phases and procedures for column evaluation are presented. A method is proposed for the isolation and determination of crude oil contamination in tropical plants and sediments. The method uses Florisil (TM) chromatography for the simultaneous clean-up and fractionation of aliphatic and aromatic hydrocarbons. Crosslinked SE-54 fused silica capillary columns prepared in our laboratory were employed for all GC separations. Mass spectrometry was used to help locate and identify specific oil components despite the intense background of the chromatogram. Crude oil components were identified in extracts of mangrove plant samples collected from the Peck Slip oil spill site at Media Munda, Puerto Rico. Crude oil components were also identified in sediment samples from controlled oil spill of Prudhoe Bay oil at Laguna de Chiriqui, Panama.

  9. Simultaneous analysis of tea catechins, caffeine, gallic acid, theanine and ascorbic acid by micellar electrokinetic capillary chromatography.

    PubMed

    Aucamp, J P; Hara, Y; Apostolides, Z

    2000-04-21

    A micellar electrokinetic capillary chromatography (MEKC) method for the simultaneous analysis of five tea catechins, theanine, caffeine, gallic acid and ascorbic acid has been developed. The catechins are (-)-epicatechin, (+)-catechin, (-)-epigallocatechin, (-)-epicatechin gallate and (-)-epigallocatechin gallate. p-Nitrophenol serves as both reference and internal standard. All the components are separated within 13 min with a 57 cm uncoated fused-silica column. On-column detection was carried out at 200 nm. This method has been used to measure these compounds in fresh tea leaves and tea liquor. The limit of detection for all analytes ranged from 1 to 20 microg/ml.

  10. Rapid, economical qualitative method for separation of aflatoxins B-1, B-2 & G-1, G-2 by dry column chromatography.

    PubMed

    Megalla, S E

    1983-12-01

    A good correlation of four components of aflatoxins was accomplished by using the dry column chromatography method. The decolorization process of interfering substances, by 0.01 N KOH and defatting the extract with petroleum ether yields a clean residue for DCC separation. It is clear that the dry column chromatography is a very simple and time-saving procedure for separation of aflatoxins. DCC columns are more economical than precoated 'thick layer' preparative plates and, in DCC, no large developing tanks need to be used. Hazards associated with the use of large volumes of flammable solvents are greatly reduced.

  11. Electrochemically-modulated liquid chromatography (EMLC): Column design, retention processes, and applications

    SciTech Connect

    Ting, En -Yi

    1997-10-08

    This work describes the continued development of a new separation technique, electrochemically-modulated liquid chromatography (EMLC), from column design, retention mechanisms to pharmaceutical applications. The introduction section provides a literature review of the technique as well as a brief overview of the research in each of the chapters. This section is followed by four chapters which investigate the issues of EMLC column design, the retention mechanism of monosubstituted aromatic compounds, and the EMLC-based applications to two important classes of pharmaceutical compounds (i.e., corticosteroids and benzodiazepines). These four sections have been removed to process separately for inclusion on the database. The dissertation concludes with a general summary, a prospectus, and a list of references cited in the General Introduction. 32 refs.

  12. Determination of fungicide residues in fruits by coupled-column liquid chromatography.

    PubMed

    Zamora, Tatiana; Hidalgo, Carmen; López, Francisco J; Hernández, Félix

    2004-06-01

    Coupled-column liquid chromatography with fluorescence detection was applied to the determination of o-phenylphenol and bitertanol residues in orange and banana fruits. After extraction with a mixture of acetone, dichloromethane-petroleum ether, and ethyl acetate, an extract aliquot of 100 microL was injected directly without any additional clean-up into the chromatographic system using two reversed phase C18 coupled columns. The LC-LC approach allowed automated sample clean up of the vegetal extracts, leading to a simple and rapid analytical procedure, with limits of quantification between 0.01 and 0.05 mg kg(-1). Recovery experiments performed on orange and banana samples fortified at different concentrations (0.01 - 4 mg kg(-1)) gave average recoveries between 70 and 113% with relative standard deviations lower than 15%. The procedure developed was finally applied to orange and banana samples from different geographical locations and the results were confirmed by GC-MS.

  13. Kinetic efficiency of polar monolithic capillary columns in high-pressure gas chromatography.

    PubMed

    Kurganov, A A; Korolev, A A; Shiryaeva, V E; Popova, T P; Kanateva, A Yu

    2013-11-08

    Poppe plots were used for analysis of kinetic efficiency of monolithic sorbents synthesized in quartz capillaries for utilization in high-pressure gas chromatography. Values of theoretical plate time and maximum number of theoretical plates occurred to depend significantly on synthetic parameters such as relative amount of monomer in the initial polymerization mixture, temperature and polymerization time. Poppe plots let one to find synthesis conditions suitable either for high-speed separations or for maximal efficiency. It is shown that construction of kinetic Poppe curves using potential Van Deemter data demands compressibility of mobile phase to be taken into consideration in the case of gas chromatography. Model mixture of light hydrocarbons C1 to C4 was then used for investigation of influence of carrier gas nature on kinetic efficiency of polymeric monolithic columns. Minimal values of theoretical plate times were found for CO2 and N2O carrier gases.

  14. Impact of reversed phase column pairs in comprehensive two-dimensional liquid chromatography.

    PubMed

    Allen, Robert C; Barnes, Brian B; Haidar Ahmad, Imad A; Filgueira, Marcelo R; Carr, Peter W

    2014-09-26

    A major issue in optimizing the resolving power of two-dimensional chromatographic separations is the choice of the two phases so as to maximize the distribution of the analytes over the separation space. In this work, we studied the choice of appropriate reversed phases to use in on-line comprehensive two-dimensional liquid chromatography (LC×LC). A set of four chemically different conventional bonded reversed phases was used in the first dimension. The second dimension column was either a conventional bonded C18 phase or a carbon-clad phase (CCP). The LC×LC chromatograms and contour plots were all rather similar indicating that the selectivities of the two phases were also similar regardless of the reverse phase column used in the first dimension. Further, the spatial coverage seen with all four first dimension stationary phases when paired with a second dimension C18 phase were low and the retention times were strongly correlated. However, when the C18 column was replaced with the CCP column much improved separations were observed with higher spatial coverages, greater orthogonalities and significant increases in the number of observed peaks.

  15. Capture chromatography for Mo-99 recovery from uranyl sulfate solutions: minimum-column-volume design method.

    PubMed

    Ling, Lei; Chung, Pei-Lun; Youker, Amanda; Stepinski, Dominique C; Vandegrift, George F; Wang, Nien-Hwa Linda

    2013-09-27

    Molybdenum-99 (Mo-99), generated from the fission of Uranium-235 (U-235), is the radioactive parent of the most widely used medical isotope, technetium-99m (Tc-99m). An efficient, robust, low-pressure process is developed for recovering Mo-99 from uranyl sulfate solutions. The minimum column volume and the maximum column length for required yield, pressure limit, and loading time are determined using a new graphical method. The method is based on dimensionless groups and intrinsic adsorption and diffusion parameters, which are estimated using a small number of experiments and simulations. The design is tested with bench-scale experiments with titania columns. The results show a high capture yield and a high stripping yield (95±5%). The design can be adapted to changes in design constraints or the variations in feed concentration, feed volume, or material properties. The graph shows clearly how the column utilization is affected by the required yield, loading time, and pressure limit. The cost effectiveness of various sorbent candidates can be evaluated based on the intrinsic parameters. This method can be used more generally for designing other capture chromatography processes.

  16. Separation of nucleotides by hydrophilic interaction chromatography using the FRULIC-N column.

    PubMed

    Padivitage, Nilusha L T; Dissanayake, Milan K; Armstrong, Daniel W

    2013-11-01

    A stationary phase composed of silica-bonded cyclofructan 6 (FRULIC-N) was evaluated for the separation of four cyclic nucleotides, six nucleoside monophosphates, four nucleoside diphosphates, and five nucleoside triphosphates via hydrophilic interaction chromatography (HILIC) in both isocratic and gradient conditions. The gradient conditions gave significantly better separations by narrowing peak widths. Sixteen out of nineteen nucleotides were baseline separated on the FRULIC-N column in one run. Unlike other known HILIC stationary phases, there can be dual-retention mechanisms unique to this media. Traditional hydrogen bonding/dipolar interactions can be supplemented by dynamic ion interaction effects for anionic analytes. This occurs because the FRULIC-N stationary phase is able to bind certain buffer cations. The extent of the ion interaction is tunable, in comparison to stationary phases with embedded charged groups, where the inherent ionic properties are fixed. The best mobile phase conditions were determined by varying the organic modifier (acetonitrile) content, as well as salt type/concentration and electrolyte pH. The thermodynamic characteristic of the FRULIC-N column was investigated by evaluating the column temperature effect on retention and utilizing van't Hoff plots. This study shows that there is a greater entropic contribution for the retention of nucleotide di and triphosphates, whereas there is a greater enthalphic contribution for the cyclic nucleotides with the FRULIC-N column.

  17. Blind column selection protocol for two-dimensional high performance liquid chromatography.

    PubMed

    Burns, Niki K; Andrighetto, Luke M; Conlan, Xavier A; Purcell, Stuart D; Barnett, Neil W; Denning, Jacquie; Francis, Paul S; Stevenson, Paul G

    2016-07-01

    The selection of two orthogonal columns for two-dimensional high performance liquid chromatography (LC×LC) separation of natural product extracts can be a labour intensive and time consuming process and in many cases is an entirely trial-and-error approach. This paper introduces a blind optimisation method for column selection of a black box of constituent components. A data processing pipeline, created in the open source application OpenMS®, was developed to map the components within the mixture of equal mass across a library of HPLC columns; LC×LC separation space utilisation was compared by measuring the fractional surface coverage, fcoverage. It was found that for a test mixture from an opium poppy (Papaver somniferum) extract, the combination of diphenyl and C18 stationary phases provided a predicted fcoverage of 0.48 and was matched with an actual usage of 0.43. OpenMS®, in conjunction with algorithms designed in house, have allowed for a significantly quicker selection of two orthogonal columns, which have been optimised for a LC×LC separation of crude extractions of plant material.

  18. Simple, specific analysis of organophosphorus and carbamate pesticides in sediments using column extraction and gas chromatography

    USGS Publications Warehouse

    Belisle, A.A.; Swineford, D.M.

    1988-01-01

    A simple, specific procedure was developed for the analysis of organophosphorus and carbamate pesticides in sediment. The wet soil was mixed with anhydrous sodium sulfate to bind water and the residues were column extracted in acetone:methylene chloride (1:l,v/v). Coextracted water was removed by additional sodium sulfate packed below the sample mixture. The eluate was concentrated and analyzed directly by capillary gas chromatography using phosphorus and nitrogen specific detectors. Recoveries averaged 93 % for sediments extracted shortly after spiking, but decreased significantly as the samples aged.

  19. Inverse gas chromatography. V - Computer simulation of diffusion processes on the column

    NASA Technical Reports Server (NTRS)

    Hattam, Paul; Munk, Petr

    1988-01-01

    The elution behavior of low molecular weight probes on inverse gas chromatography (IGC) columns is simulated using a computer. The IGC model is based on a polymer stationary phase of uniform thickness with a nonnegligible resitance to probe penetration. Three characteristic numbers are found to determine the whole process: Z(p) characterizing the distribution of the probe between phases, Z(f) describing the diffusion in the polymer phase, and Z(g) related to diffusion in the gaseous phase. For situations when Z(p)/Z(f) is less than 2, the standard evaluation procedures are virtually useless. The actual behavior of such systems is described.

  20. Stationary phase optimized selectivity liquid chromatography: Basic possibilities of serially connected columns using the "PRISMA" principle.

    PubMed

    Nyiredy, Sz; Szucs, Zoltán; Szepesy, L

    2007-07-20

    A new procedure (stationary phase optimized selectivity liquid chromatography: SOS-LC) is described for the optimization of the HPLC stationary phase, using serially connected columns and the principle of the "PRISMA" model. The retention factors (k) of the analytes were determined on three different stationary phases. By use of these data the k values were predicted applying theoretically combined stationary phases. These predictions resulted in numerous intermediate theoretical separations from among which only the optimal one was assembled and tested. The overall selectivity of this separation was better than that of any individual base stationary phase. SOS-LC is independent of the mechanism and the scale of separation.

  1. Comprehensive two-dimensional liquid chromatography: ion chromatography × reversed-phase liquid chromatography for separation of low-molar-mass organic acids.

    PubMed

    Brudin, Stella S; Shellie, Robert A; Haddad, Paul R; Schoenmakers, Peter J

    2010-10-22

    In the work presented here a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Ion chromatography is chosen for the first-dimension separation and reversed-phase liquid chromatography is chosen for the second-dimension separation mode. The coupling of these modes is made possible by neutralising the first-dimension effluent, containing KOH, prior to transfer to the second-dimension reversed-phase column. A test mixture of 24 low-molar-mass organic acids is used for optimisation of the system. Three food and beverage samples were analysed in order to evaluate the developed methodology, the resulting two-dimensional separation is near-orthogonal, the set-up is simple and all instrumental components are available commercially. The method proved to be robust and suitable for the analysis of wine, orange juice and yogurt.

  2. Chemically modified polymeric resins for separation of cations, organic acids, and small polar moleculea by high performance liquid chromatography

    SciTech Connect

    Morris, John B.

    1993-07-01

    This thesis is divided into 4 parts: a review, ion chromatography of metal cations on carboxylic resins, separation of hydrophilic organic acids and small polar compounds on macroporous resin columns, and use of eluent modifiers for liquid chromatographic separation of carboxylic acids using conductivity detection.

  3. Sequential injection chromatography with post-column reaction/derivatization for the determination of transition metal cations in natural water samples.

    PubMed

    Horstkotte, Burkhard; Jarošová, Patrícia; Chocholouš, Petr; Sklenářová, Hana; Solich, Petr

    2015-05-01

    In this work, the applicability of Sequential Injection Chromatography for the determination of transition metals in water is evaluated for the separation of copper(II), zinc(II), and iron(II) cations. Separations were performed using a Dionex IonPAC™ guard column (50mm×2mm i.d., 9 µm). Mobile phase composition and post-column reaction were optimized by modified SIMPLEX method with subsequent study of the concentration of each component. The mobile phase consisted of 2,6-pyridinedicarboxylic acid as analyte-selective compound, sodium sulfate, and formic acid/sodium formate buffer. Post-column addition of 4-(2-pyridylazo)resorcinol was carried out for spectrophotometric detection of the analytes׳ complexes at 530nm. Approaches to achieve higher robustness, baseline stability, and detection sensitivity by on-column stacking of the analytes and initial gradient implementation as well as air-cushion pressure damping for post-column reagent addition were studied. The method allowed the rapid separation of copper(II), zinc(II), and iron(II) within 6.5min including pump refilling and aspiration of sample and 1mmol HNO3 for analyte stacking on the separation column. High sensitivity was achieved applying an injection volume of up to 90µL. A signal repeatability of<2% RSD of peak height was found. Analyte recovery evaluated by spiking of different natural water samples was well suited for routine analysis with sub-micromolar limits of detection.

  4. [Determination of 14 sulfonamide residues in shrimps by high performance liquid chromatography coupled with post-column derivatization].

    PubMed

    Huang, Dongmei; Huang, Xuanyun; Gu, Runrun; Hui, Yunhua; Tian, Liangliang; Feng, Bing; Zhang, Xuan; Yu, Huijuan

    2014-08-01

    A method for the determination of 14 sulfonamide residues in shrimps by high performance liquid chromatography coupled with post-column derivatization was established. The sulfonamide residues were extracted with ethyl acetate after adding sulfapyridine as internal standard. The extracts were vacuum-concentrated and reverse-extracted by 2 mol/L hydrochloric acid solution for clean-up, and then the hydrochloric acid solution was defatted with n-hex- ane. The solution after filtration was blended with a mixed solution of methanol, acetonitrile and 3. 5 mol/L sodium acetate solution (5:5:20, v/v/v). The sulfonamides were separated on a C18 column by RP-HPLC and on-line derivatized with a fluorescamine and detected with a fluorescence detector. The standard addition method was used for quantitative analysis. The parameters of post-column derivatization system, such as concentration of fluorescamine solution, velocity of reagent solution and reaction temperature, were optimized. The calibration curves of the method showed good linearity in the range of 5 - 200 μg/L. The limits of quantification (LOQ, S/N= 10) were 1.0-5.0 μg/kg for the 14 sulfonamides. The recoveries were 77.8%- 103. 6% in the spiked range of 1. 0-100.0 μg/kg in shrimps with the relative standard deviations of 2.9%-9.1% (n= 6). The results indicated that the method is sensitive, efficient and more accurate. It is suitable for the simultaneous determination of the 14 sulfonamide residues in shrimps.

  5. Chromatography column comparison and rapid pretreatment for the simultaneous analysis of amantadine, rimantadine, acyclovir, ribavirin, and moroxydine in chicken muscle by ultra high performance liquid chromatography and tandem mass spectrometry.

    PubMed

    Zhang, Qiaoyan; Xiao, Chaogeng; Wang, Wei; Qian, Mingrong; Xu, Jie; Yang, Hua

    2016-10-01

    In this work, a chromatography column comparison and rapid pretreatment development were carried out. A multi-class method was built based on the quick, easy, cheap, effective, rugged, and safe pretreatment method with hydrophilic interaction ultra high performance liquid chromatography and tandem mass spectrometry for the high-throughput analysis of five antivirals in chicken muscle. The HSS T3 column, BEH HILIC column and BEH Amide column were studied, and their chemical functionalities and chromatographic separation effectiveness were compared. The BEH Amide column was selected to perform the mass spectrometry analysis under the hydrophilic interaction chromatography mode. First, a different strategy without adding MgSO4 and NaCl into the muscle samples was considered. Then, different concentrations of formic acid, acetic acid, and ammonia in acetonitrile were compared for better extraction efficiency. Nine sorbents (C18 , PSA, NH2 , Florisil, Alumina-B, Alumina-N, PestiCarb, NANO, and NANO-NH2 ) were studied. The optimized procedure consisted of the use of 10% acetic acid in acetonitrile for the extraction solvent and NANO-NH2 for clean-up. NANO-NH2 had not been applied in other matrix and pollutants so far. The developed method provided favorable trueness, precision, and acceptable matrix effect. Meanwhile, the method was sensitive, the limits of detection of amantadine, rimantadine, acyclovir, ribavirin, and moroxydine achieved were 0.56, 0.50, 0.30, 2.22, and 0.51 μg/kg, respectively, and were successfully applied for the routine detection of antivirals in the chicken samples.

  6. A strategy for identification and structural characterization of compounds from Gardenia jasminoides by integrating macroporous resin column chromatography and liquid chromatography-tandem mass spectrometry combined with ion-mobility spectrometry.

    PubMed

    Wang, Lu; Liu, Shu; Zhang, Xueju; Xing, Junpeng; Liu, Zhiqiang; Song, Fengrui

    2016-06-24

    In this paper, an analysis strategy integrating macroporous resin (AB-8) column chromatography and high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) combined with ion mobility spectrometry (IMS) was proposed and applied for identification and structural characterization of compounds from the fruits of Gardenia jasminoides. The extracts of G. jasminoides were separated by AB-8 resin column chromatography combined with reversed phase liquid chromatography (C18 column) and detected by electrospray ionization tandem mass spectrometry. Additionally, ion mobility spectrometry (IMS) was employed as a supplementary separation technique to discover previously undetected isomers from the fruits of G. jasminoides. A total of 71 compounds, including iridoids, flavonoids, triterpenes, monoterpenoids, carotenoids and phenolic acids were identified by the characteristic high resolution mass spectrometry and the ESI-MS/MS fragmentations. In conclusion, the IMS-MS technique achieved the separation of isomers in crocin-3 and crocin-4 according to their acquired mobility drift times differing from classical analysis by mass spectrometry. The proposed strategy can be used as a highly sensitive and efficient procedure for identification and separation isomeric components in extracts of herbal medicines.

  7. Determination of lansoprazole enantiomers in dog plasma by column-switching liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

    PubMed

    Wang, Hao; Sun, Yantong; Meng, Xiangjun; Yang, Bo; Wang, Jian; Yang, Yan; Gu, Jingkai

    2015-09-01

    Lansoprazole, a selective proton pump inhibitor, has a chiral benzimidazole sulfoxide structure and is used for the treatment of gastric acid hypersecretory related diseases. To investigate its stereoselective pharmacokinetics, a column-switching liquid chromatography with tandem mass spectrometry method was developed for the determination of lansoprazole enantiomers in dog plasma using (+)-pantoprazole as an internal standard. After a simple protein precipitation procedure with acetonitrile, matrix components left behind after sample preparation were further eliminated from the sample by reversed-phase chromatography on a C18 column. The fluent was fed to a chiral column for the separation of lansoprazole enantiomers. Baseline separation of lansoprazole enantiomers was achieved on a Chiralcel OZ-RH column using acetonitrile/0.1% formic acid in water (35:65, v/v) as the mobile phase at 40°C. The linearity of the calibration curves ranged from 3 to 800 ng/mL for each enantiomer. Intra- and inter-day precisions ranged from 2.1 to 7.3% with an accuracy of ±1.7% for (+)-lansoprazole, and from 1.6 to 6.9% with an accuracy of ±3.5% for (-)-lansoprazole, respectively. The validated method was successfully applied for the stereoselective pharmacokinetic study of lansoprazole in beagle dog after intravenous infusion.

  8. Utilization of different crown ethers impregnated polymeric resin for treatment of low level liquid radioactive waste by column chromatography.

    PubMed

    Attallah, M F; Borai, E H; Hilal, M A; Shehata, F A; Abo-Aly, M M

    2011-11-15

    The main goal of this study was to find a novel impregnated resin as an alternative for the conventional resin (KY-2 and AN-31) used for low and intermediate level liquid radioactive waste treatment. Novel impregnated ion exchangers namely, poly (acrylamide-acrylic acid-acrylonitril)-N,N'-methylenedi-acrylamide-4,4'(5')di-t-butylbenzo 18 crown 6 [P(AM-AA-AN)-DAM/DtBB18C6], poly (acrylamide-acrylic acid-acrylonitril)-N,N'-methylenediacrylamide-dibenzo 18 crown 6 [P(AM-AA-AN)-DAM/DB18C6], and poly (acrylamide-acrylic acid-acrylonitril)-N,N'-methylenediacrylamide-18 crown 6 [P(AM-AA-AN)-DAM/18C6] were prepared and their removal efficiency of some radionuclides was investigated. Preliminary batch experiments were performed in order to study the influence of the different derivatives of 18 crown 6 on the characteristic removal performance. Separation of (134)Cs, (60)Co, (65)Zn and ((152+154))Eu radionuclides from low level liquid radioactive waste was investigated by using column chromatography with P(AM-AA-AN)-DAM/DtBB18C6 and metal salt solutions traced with the corresponding radionuclides. Breakthrough data was obtained in a fixed bed column at room temperature (298K) using different bed heights and flow rates. The breakthrough capacities were found to be 94.7, 83.3, 58.7, 43.1 (mg/g) for (60)Co, (65)Zn, (134)Cs, and ((152+154))Eu, respectively. Pre-concentration and separation of all radionuclides under study have been carried out using different concentration of nitric and/or oxalic acids.

  9. On-column micro gas chromatography detection with capillary-based optical ring resonators.

    PubMed

    Shopova, Siyka I; White, Ian M; Sun, Yuze; Zhu, Hongying; Fan, Xudong; Frye-Mason, Greg; Thompson, Aaron; Ja, Shiou-jyh

    2008-03-15

    We developed a novel on-column micro gas chromatography (microGC) detector using capillary based optical ring resonators (CBORRs). The CBORR is a thin-walled fused silica capillary with an inner diameter ranging from a few tens to a few hundreds of micrometers. The interior surface of the CBORR is coated with a layer of stationary phase for gas separation. The circular cross section of the CBORR forms a ring resonator and supports whispering gallery modes (WGMs) that circulate along the ring resonator circumference hundreds of times. The evanescent field extends into the core and is sensitive to the refractive index change induced by the interaction between the gas sample and the stationary phase. The WGM can be excited and monitored at any location along the CBORR by placing a tapered optical fiber against the CBORR, thus enabling on-column real-time detection. Rapid separation of both polar and nonpolar samples was demonstrated with subsecond detection speed. Theoretical work was also established to explain the CBORR detection mechanism. While low-nanogram detection limits are observed in these preliminary tests, many methods for improvements are under investigation. The CBORR is directly compatible with traditional capillary GC columns without any dead volumes. Therefore, the CBORR-based muGC is a very promising technology platform for rapid, sensitive, and portable analytical devices.

  10. Direct coupling of packed column supercritical fluid chromatography to continuous corona discharge ion mobility spectrometry.

    PubMed

    Rahmanian, A; Ghaziaskar, H S; Khayamian, T

    2013-01-11

    In this study, packed column supercritical fluid chromatography (SFC) was directly coupled to a continuous corona discharge (CD) ion mobility spectrometer (IMS) with several modifications. The main advantage of the developed detector is its capability to introduce full column effluent up to 2000 mL min(-1) CO(2) gas directly into the IMS cell relative to 40 mL min(-1) CO(2) gas as a maximum tolerance, reported for the previous IMS detectors. This achievement was made possible because of using corona discharge instead of (63)Ni as an ionization source and locating the inlet and outlet of the CO(2) gas in the counter electrode of the CD in opposite direction. In addition, a heated interface was placed between back pressure regulator (BPR) and the IMS cell to heat the output of the BPR for introducing sample as the gas phase into the IMS cell. Furthermore, a make-up methanol flow was introduced between the column outlet and BPR to provide a more uniform flow through the BPR and also to prevent freezing and deposition of the analytes in the BPR. The performance of the SFC-CD-IMS was evaluated by analysis of testosterone, medroxyprogesterone, caffeine, and theophylline as test compounds and figures of merit for these compounds have been calculated.

  11. Analysis of fifteen estrogen metabolites using packed column supercritical fluid chromatography-mass spectrometry.

    PubMed

    Xu, Xia; Roman, John M; Veenstra, Timothy D; Van Anda, Jennifer; Ziegler, Regina G; Issaq, Haleem J

    2006-03-01

    Packed column supercritical fluid chromatography with tandem mass spectrometry was used for the separation of estrone, estradiol, estriol, 16-epiestriol, 17-epiestriol, 16-ketoestradiol, 16alpha-hydroxyestrone, 2-methoxyestrone, 4-methoxyestrone, 2-hydroxyestrone-3-methyl ether, 2-methoxyestradiol, 4-methoxyestradiol, 2-hydroxyestrone, 4-hydroxyestrone, and 2-hydroxyestradiol. A gradient of methanol in carbon dioxide (0-30% methanol in 15 min, 2% change/min) at a flow rate of 2 mL/min and cyanopropyl silica column connected in series with a diol column, both 2.1 mm i.d. x 150 mm long, packed with 5-mum spherical silica-based particles, resulted in the separation and quantification of all 15 estrogens in less than 10 min. The limit of detection (LOD) and limit of quantitation (LOQ) of this pSFC MS/MS method was determined to be 0.5 (S/N = 3), and 5 pg, respectively. Compared with RP-HPLC MS analysis of the same mixture in terms of speed of analysis and sensitivity, pSFC MS is much faster, 10 versus 70 min, with comparable LOD and LOQ.

  12. Evaluation of steric exclusion chromatography on cryogel column for the separation of serum proteins.

    PubMed

    Wang, Chuan; Bai, Shu; Tao, Shi-Peng; Sun, Yan

    2014-03-14

    Steric exclusion chromatography (SXC) is a new mode of protein chromatography, in which large proteins are retained on hydrophilic stationary phase surface due to the steric exclusion of polyethylene glycol (PEG) in the mobile phase, and thereafter the retained proteins can be eluted by reducing PEG concentration. In this work, SXC was evaluated on a polyacrylamide cryogel monolith. Microscopic observation of γ-globulin precipitates on the gel surface in SXC was reported for the first time. Due to the compact packing of protein precipitates on the stationary phase surface, the dynamic retention capacity of the cryogel monolith for γ-globulin reached 20 mg/mL bed volume, much higher than those of cryogel beds in adsorption-based chromatography. The effect of molecular weight and concentration of PEG, solution pH and salt concentration on protein retention capacity was in agreement with the earlier work on SXC. Because the cryogel monoliths with interconnected macropores (10-100 μm) allow much easy flow-through of viscous PEG buffer, the SXC can be operated at low back pressure. Hence, the cryogel monoliths are more suitable for SXC than other monoliths of narrow pores reported previously. In the separation of bovine serum proteins, albumin was recovered in the breakthrough fraction with high purity, and globulin was over eight times concentrated in the elution pool. This work has, thus, demonstrated the rapid serum protein separation and concentration by SXC on the cryogel monolith columns.

  13. Monolithic stationary phases with incorporated fumed silica nanoparticles. Part I. Polymethacrylate-based monolithic column with incorporated bare fumed silica nanoparticles for hydrophilic interaction liquid chromatography.

    PubMed

    Aydoğan, Cemil; El Rassi, Ziad

    2016-05-06

    Fumed silica nanoparticles (FSNPs), were incorporated for the first time into a polymethacrylate monolithic column containing glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in order to develop a new monolithic column for hydrophilic interaction high performance liquid chromatography (HILIC). When compared to poly(GMM-EDMA) monolithic column without FSNPs, the same monolithic column with incorporated FSNPs yielded important effects on HILIC separations. The effects of monomers and FSNPs content of the polymerization mixture on the performance of the monolithic column were examined in details, and the optimized stationary phase was investigated over a wide range of mobile phase composition with polar acidic, weakly basic and neutral analytes including hydroxy benzoic acids, nucleotides, nucleosides, dimethylformamide, formamide and thiourea. The retention of these analytes was mainly controlled by hydrophilic interactions with the FSNPs and electrostatic repulsion from the negatively charged silica surface in the case of hydroxy benzoic acids and nucleotides. The electrostatic repulsion was minimized by decreasing the pH of the aqueous component of the mobile phase, which in turn enhanced the retention of acidic solutes. Nucleotides were best separated using step gradient elution at decreasing pH as well as ACN concentration in the mobile phase. Improved peak shape and faster analysis of nucleosides were attained by a fast linear gradient elution with a shallow decrease in the ACN content of the ACN-rich mobile phase. The run-to-run and column-to-column reproducibility were satisfactory. The percent relative standard deviations (%RSDs) for the retention times of tested solutes were lower than 2.5% under isocratic conditions and lower than 3.5 under gradient conditions.

  14. Displacement chromatography of proteins using a retained pH front in a hydrophobic charge induction chromatography column.

    PubMed

    Pinto, N D S; Frey, Douglas D

    2015-03-27

    The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.

  15. Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization.

    PubMed

    Koshiishi, I; Kobori, Y; Imanari, T

    1990-10-26

    A high-performance liquid chromatographic method was developed for the determination of citrulline and homocitrulline using a post-column colorimetric reaction with o-phthaladehyde and N-(1-naphthyl)-ethylenediamine. Citrulline and homocitrulline were determined with no interferences from protein amino acids. The results show that the level of citrulline in the plasma of patients with uremia on intermittent hemodialysis is higher than that in healthy human plasma, and that homocitrulline is excreted into the urine of healthy adults.

  16. Virus elimination during the purification of monoclonal antibodies by column chromatography and additional steps.

    PubMed

    Roberts, Peter L

    2014-01-01

    The theoretical potential for virus transmission by monoclonal antibody based therapeutic products has led to the inclusion of appropriate virus reduction steps. In this study, virus elimination by the chromatographic steps used during the purification process for two (IgG-1 & -3) monoclonal antibodies (MAbs) have been investigated. Both the Protein G (>7log) and ion-exchange (5 log) chromatography steps were very effective for eliminating both enveloped and non-enveloped viruses over the life-time of the chromatographic gel. However, the contribution made by the final gel filtration step was more limited, i.e., 3 log. Because these chromatographic columns were recycled between uses, the effectiveness of the column sanitization procedures (guanidinium chloride for protein G or NaOH for ion-exchange) were tested. By evaluating standard column runs immediately after each virus spiked run, it was possible to directly confirm that there was no cross contamination with virus between column runs (guanidinium chloride or NaOH). To further ensure the virus safety of the product, two specific virus elimination steps have also been included in the process. A solvent/detergent step based on 1% triton X-100 rapidly inactivating a range of enveloped viruses by >6 log inactivation within 1 min of a 60 min treatment time. Virus removal by virus filtration step was also confirmed to be effective for those viruses of about 50 nm or greater. In conclusion, the combination of these multiple steps ensures a high margin of virus safety for this purification process.

  17. [Online enrichment ability of restricted-access column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride].

    PubMed

    Zhang, Xiaohui; Wang, Rong; Xie, Hua; Yin, Qiang; Li, Xiaoyun; Jia, Zhengping; Wu, Xiaoyu; Zhang, Juanhong; Li, Wenbin

    2013-05-01

    The online enrichment ability of the restricted-access media (RAM) column coupled with high performance liquid chromatography by column switching technique for benazepril hydrochloride in plasma was studied. The RAM-HPLC system consisted of an RAM column as enrichment column and a C18 column as analytical column coupled via the column switching technique. The effects of the injection volume on the peak area and the systematic pressure were studied. When the injection volume was less than 100 microL, the peak area increased with the increase of the injection volume. However, when the injection volume was more than 80 microL, the pressure of whole system increased obviously. In order to protect the whole system, 80 microL was chosen as the maximum injection volume. The peak areas of ordinary injection and the large volume injection showed a good linear relationship. The enrichment ability of RAM-HPLC system was satisfactory. The system was successfully used for the separation and detection of the trace benazepril hydrochloride in rat plasma after its administration. The sensitivity of HPLC can be improved by RAM pre-enrichment. It is a simple and economic measurement method.

  18. Rapid tandem-column micro-gas chromatography based on optofluidic ring resonators with multi-point on-column detection.

    PubMed

    Sun, Yuze; Liu, Jing; Howard, Daniel J; Frye-Mason, Greg; Thompson, Aaron K; Ja, Shiou-jyh; Fan, Xudong

    2010-01-01

    We demonstrated a novel tandem-column micro-gas chromatography (microGC) based on optofluidic ring resonator (OFRR). The OFRR is a thin-walled fused silica capillary whose interior surface is coated with a polymeric stationary phase. The circular cross section of the OFRR forms the micro-ring resonator and supports whispering gallery modes (WGMs). Via tapered optical fibers in contact with the OFRR, the WGM can be excited externally at any positions along the OFRR capillary, thus enabling multi-point, on-column, real-time detection of vapor molecules flowing through the OFRR. In the present OFRR-based tandem-column-based microGC implementation, a 180 cm long conventional GC column coated with a nonpolar stationary phase was followed by a relatively short OFRR column coated with a polar phase. Two detection positions, one at the inlet of the OFRR and the other a few centimeters downstream, were used to monitor the separation achieved by the first and the second column, respectively. Owing to the multi-point on-column detection that provides complementary retention time information on each chemical compound, co-eluted analytes can be well separated and identified on at least one detection channel and no modulation is needed at the interface of tandem columns. Separation and detection of twelve analytes with various volatilities and polarities within four minutes were demonstrated. In addition, the chromatograms obtained from three different locations along the OFRR column demonstrated the system's capability of on-column monitoring of the separation process for the target analyte in a vapor mixture. Our results will lead to the development of a rapid, simple, and portable microGC system with significantly improved selectivity and chemical identification capabilities.

  19. Simultaneous determination of three chloroacetic acids, three herbicides, and 12 anions in water by ion chromatography.

    PubMed

    Luo, Ximing; Chen, Liang; Zhao, Yanqing

    2015-09-01

    An ion chromatography method was developed for the simultaneous detection of three soluble herbicides (glyphosate, bentazone and picloram), three chlorine disinfection byproducts (monochloroacetic acid, dichloroacetic acid and trichloroacetic acid) and 12 anions in water (Cl(-), Br(-), SO4(2-), CO3(2-), ClO3(-), ClO4(-), BrO3(-), PO4(3-), NO2(-), NO3(-), CH3COO(-) and COO(-)). High linearity (r(2) > 0.996) was observed for all target analytes for each respective concentration range. The limit of detection and limit of quantitation were between 0.21-0.85 and 0.06-25.46 μg/L, respectively. However, the interference effect of Cl(-), NO3(-) , SO4 (2-) and CO3(2-) on some target analytes must be considered during the analysis. Sample pre-treatment by a hydrogen column (H-column) required to reduce the negative effect of CO3(2-). Additionally, sample pre-treatment by a sliver-hydrogen column (Ag-H-column) is required when Cl(-) > 100 mg/L and SO4(2-) < 50 mg/L, and pre-treatment by both a barium column (Ba-column) and an H-column is required when Cl(-) > 100 mg/L and SO4(2-) > 50 mg/L. When Cl(-) > 100 mg/L, SO4(2-) > 50 mg/L and CO3(2-) > 20 mg/L, the sample pre-treatment by either an Ag-H-Ba-column or an Ag-H-column and Ba-column is required to minimize interference.

  20. Intrinsic advantages of packed capillaries over narrow-bore columns in very high-pressure gradient liquid chromatography.

    PubMed

    Gritti, Fabrice; McDonald, Thomas; Gilar, Martin

    2016-06-17

    250μm×100mm fused silica glass capillaries were packed with 1.8μm high-strength silica (HSS) fully porous particles. They were prepared without bulky stainless steel endfittings and metal frits, which both generate significant sample dispersion. The isocratic efficiencies and gradient peak capacities of these prototype capillary columns were measured for small molecules (n-alkanophenones) using a home-made ultra-low dispersive micro-HPLC instrument. Their resolution power was compared to that of standard 2.1mm×100mm very high-pressure liquid chromatography (vHPLC) narrow-bore columns packed with the same particles. The results show that, for the same column efficiency (25000 plates) and gradient steepness (0.04min(-1)), the peak capacity of the 250μm i.d. capillary columns is systematically 15-20% higher than that of the 2.1mm i.d. narrow-bore columns. A validated model of gradient chromatography enabled one to predict accurately the observed peak capacities of the capillary columns for non-linear solvation strength retention behavior and under isothermal conditions. Thermodynamics applied to the eluent quantified the temperature difference for the thermal gradients in both capillary and narrow-bore columns. Experimental data revealed that the gradient peak capacity is more affected by viscous heating than the column efficiency. Unlike across 2.1mm i.d. columns, the changes in eluent composition across the 250μm i.d. columns during the gradient is rapidly relaxed by transverse dispersion. The combination of (1) the absence of viscous heating and (2) the high uniformity of the eluent composition across the diameter of capillary columns explains the intrinsic advantage of capillary over narrow-bore columns in gradient vHPLC.

  1. Separation and quantitation of free fatty acids and fatty acid methyl esters by reverse phase high pressure liquid chromatography.

    PubMed

    Aveldano, M I; VanRollins, M; Horrocks, L A

    1983-01-01

    Reverse phase high pressure liquid chromatography (HPLC) on octadecylsilyl columns separates mixtures of either free fatty acids or fatty acid methyl esters prepared from mammalian tissue phospholipids. Acetonitrile-water mixtures are used for the elution of esters. Aqueous phosphoric acid is substituted for water for the separation of the free acids. Unsaturated compounds are detected and quantitated by their absorption at 192 nm. Saturates are detected better at 205 nm. The order of elution of fatty acids in complex mixtures varies as a function of acetonitrile concentration. At any given concentration, some compounds overlap. However, by varying the solvent strength, any fatty acid of interest can be resolved including many geometrical and positional isomers. Methyl esters prefractionated according to unsaturation by argentation thin-layer chromatography (TLC) are rapidly and completely separated by elution with CH3CN alone. Argentation TLC-reverse phase HPLC can be used as an analytical as well as a preparative procedure. Octylsilyl columns are used for rapid resolution and improved detection of minor or low ultraviolet-absorbing components in the fractions. For example, monoenoic fatty acids with up to 32 carbons have been detected in bovine brain glycerophospholipids. Specific radioactivities of 3H- and 14C-labeled fatty acids and the distribution of radioactivity among acyl groups from complex lipids are measured. The method is not recommended for complete compositional analysis, but is useful for determinations of specific radioactivities during studies on turnover and metabolic conversions of labeled fatty acids.

  2. Enantiomeric separation of asymmetric triacylglycerol by recycle high-performance liquid chromatography with chiral column.

    PubMed

    Nagai, Toshiharu; Mizobe, Hoyo; Otake, Ikuko; Ichioka, Kenji; Kojima, Koichi; Matsumoto, Yumiko; Gotoh, Naohiro; Kuroda, Ikuma; Wada, Shun

    2011-05-20

    In our previous studies, we employed recycle HPLC for the separation of triacylglycerol (TAG)-positional isomers (PIs). In this study, a recycle HPLC system equipped with a polysaccharide-based chiral column was applied to the enantiomeric separation of some asymmetric TAGs having straight-chain C16-C18 acyl residues. As a result, 1,2-dipalmitoyl-3-oleoyl-rac-glycerol (rac-PPO), 1,2-dioleoyl-3-palmitoyl-rac-glycerol (rac-OOP), and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol (rac-PPL) were resolved into their respective enantiomers. However, neither 1,2-dioleoyl-3-linoleoyl-rac-glycerol (rac-OOL), consisting of only unsaturated fatty acids, nor 1,2-dipalmitoyl-3-stearoyl-rac-glycerol (rac-PPS), consisting of only saturated fatty acids, was resolved. These results suggest that the asymmetric TAGs, used in this study, having both a palmitic acid moiety and an oleic acid (or a linoleic acid) moiety at the sn-1 or sn-3 positions are resolved by the chiral column. This new chiral separation method can be used in combination with atmospheric pressure chemical ionization mass spectrometry to determine the sn-OOP/sn-POO ratio in palm oil. This method is applicable for the chiral separation of asymmetric TAGs in palm oil.

  3. Online micro-solid-phase extraction based on boronate affinity monolithic column coupled with high-performance liquid chromatography for the determination of monoamine neurotransmitters in human urine.

    PubMed

    Yang, Xiaoting; Hu, Yufei; Li, Gongke

    2014-05-16

    Quantification of monoamine neurotransmitters is very important in diagnosing and monitoring of patients with neurological disorders. We developed an online analytical method to selectively determine urinary monoamine neurotransmitters, which coupled the boronate affinity monolithic column micro-solid-phase extraction with high-performance liquid chromatography (HPLC). The boronate affinity monolithic column was prepared by in situ polymerization of vinylphenylboronic acid (VPBA) and N,N'-methylenebisacrylamide (MBAA) in a stainless capillary column. The prepared monolithic column showed good permeability, high extraction selectivity and capacity. The column-to-column reproducibility was satisfactory and the enrichment factors were 17-243 for four monoamine neurotransmitters. Parameters that influence the online extraction efficiency, including pH of sample solution, flow rate of extraction and desorption, extraction volume and desorption volume were investigated. Under the optimized conditions, the developed method exhibited low limit of detection (0.06-0.80μg/L), good linearity (with R(2) between 0.9979 and 0.9993). The recoveries in urine samples were 81.0-105.5% for four monoamine neurotransmitters with intra- and inter-day RSDs of 2.1-8.2% and 3.7-10.6%, respectively. The online analytical method was sensitive, accurate, selective, reliable and applicable to analysis of trace monoamine neurotransmitters in human urine sample.

  4. Silica-based monolithic columns with mixed-mode reversed-phase/weak anion-exchange selectivity principle for high-performance liquid chromatography.

    PubMed

    Nogueira, Raquel; Lubda, Dieter; Leitner, Alexander; Bicker, Wolfgang; Maier, Norbert M; Lämmerhofer, Michael; Lindner, Wolfgang

    2006-05-01

    This article describes the synthesis, chromatographic characterization, and performance evaluation of analytical (100 x 4.6 mm id) and semipreparative (100 x 10 mm id) monolithic silica columns with mixed-mode RP/weak anion-exchange (RP/WAX) surface modification. The monolithic RP/WAX columns were obtained by immobilization of N-(10-undecenoyl)-3-aminoquinuclidine onto thiol-modified monolithic silica columns (Chromolith) by a radical addition reaction. Their chromatographic characterization by Engelhardt and Tanaka tests revealed slightly lower hydrophobic selectivities than C-8 phases, as well as higher polarity and also improved shape selectivity than RP-18e silica rods. The surface modification enabled separation by both RP and anion-exchange chromatography principles, and thus showed complementary selectivities to the RP-18e monoliths. The mixed-mode monoliths have been tested for the separation of peptides and turned out to be particularly useful for hydrophilic acidic peptides, which are usually insufficiently retained on RP-18e monolithic columns. Compared to a corresponding particulate RP/WAX column (5 microm, 10 nm pore diameter), the analytical RP/WAX monolith caused lower system pressure drops and showed, as expected, higher efficiency (e.g. by a factor of about 2.5 lower C-term for a tetrapeptide). The upscaling from the analytical to semipreparative column dimension was also successful.

  5. Temperature-assisted On-column Solute Focusing: A General Method to Reduce Pre-column Dispersion in Capillary High Performance Liquid Chromatography

    PubMed Central

    Groskreutz, Stephen R.; Weber, Stephen G.

    2014-01-01

    Solvent-based on-column focusing is a powerful and well known approach for reducingthe impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created thatlead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retentionTASF is used effectivelyto compress injection bands at the head of the column through the transient reduction in column temperature to 5 °C for a defined 7 mm segment of a 6 cm long 150 μm I.D. column. Following the 30 second focusing time, the column temperature is increased rapidly to the separation temperature of 60 °C releasing the focused band of analytes. We developed a model tosimulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance.All samples have solvent compositions matching the mobile phase. Over the 45 to 1050 nL injection volume range evaluated, TASF reducesthe peak width for all soluteswith k’ greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45 nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it canbe used where on-column focusing is required, but where implementation of solvent-based focusing is difficult. PMID:24973805

  6. Temperature-assisted on-column solute focusing: a general method to reduce pre-column dispersion in capillary high performance liquid chromatography.

    PubMed

    Groskreutz, Stephen R; Weber, Stephen G

    2014-08-08

    Solvent-based on-column focusing is a powerful and well known approach for reducing the impact of pre-column dispersion in liquid chromatography. Here we describe an orthogonal temperature-based approach to focusing called temperature-assisted on-column solute focusing (TASF). TASF is founded on the same principles as the more commonly used solvent-based method wherein transient conditions are created that lead to high solute retention at the column inlet. Combining the low thermal mass of capillary columns and the temperature dependence of solute retention TASF is used effectively to compress injection bands at the head of the column through the transient reduction in column temperature to 5°C for a defined 7mm segment of a 6cm long 150μm I.D. column. Following the 30s focusing time, the column temperature is increased rapidly to the separation temperature of 60°C releasing the focused band of analytes. We developed a model to simulate TASF separations based on solute retention enthalpies, focusing temperature, focusing time, and column parameters. This model guides the systematic study of the influence of sample injection volume on column performance. All samples have solvent compositions matching the mobile phase. Over the 45-1050nL injection volume range evaluated, TASF reduces the peak width for all solutes with k' greater than or equal to 2.5, relative to controls. Peak widths resulting from injection volumes up to 1.3 times the column fluid volume with TASF are less than 5% larger than peak widths from a 45nL injection without TASF (0.07 times the column liquid volume). The TASF approach reduced concentration detection limits by a factor of 12.5 relative to a small volume injection for low concentration samples. TASF is orthogonal to the solvent focusing method. Thus, it can be used where on-column focusing is required, but where implementation of solvent-based focusing is difficult.

  7. Capillary ion chromatography with on-column focusing for ultra-trace analysis of methanesulfonate and inorganic anions in limited volume Antarctic ice core samples.

    PubMed

    Rodriguez, Estrella Sanz; Poynter, Sam; Curran, Mark; Haddad, Paul R; Shellie, Robert A; Nesterenko, Pavel N; Paull, Brett

    2015-08-28

    Preservation of ionic species within Antarctic ice yields a unique proxy record of the Earth's climate history. Studies have been focused until now on two proxies: the ionic components of sea salt aerosol and methanesulfonic acid. Measurement of the all of the major ionic species in ice core samples is typically carried out by ion chromatography. Former methods, whilst providing suitable detection limits, have been based upon off-column preconcentration techniques, requiring larger sample volumes, with potential for sample contamination and/or carryover. Here, a new capillary ion chromatography based analytical method has been developed for quantitative analysis of limited volume Antarctic ice core samples. The developed analytical protocol applies capillary ion chromatography (with suppressed conductivity detection) and direct on-column sample injection and focusing, thus eliminating the requirement for off-column sample preconcentration. This limits the total sample volume needed to 300μL per analysis, allowing for triplicate sample analysis with <1mL of sample. This new approach provides a reliable and robust analytical method for the simultaneous determination of organic and inorganic anions, including fluoride, methanesulfonate, chloride, sulfate and nitrate anions. Application to composite ice-core samples is demonstrated, with coupling of the capillary ion chromatograph to high resolution mass spectrometry used to confirm the presence and purity of the observed methanesulfonate peak.

  8. Modeling on-column reduction of trisulfide bonds in monoclonal antibodies during protein A chromatography.

    PubMed

    Ghose, Sanchayita; Rajshekaran, Rupshika; Labanca, Marisa; Conley, Lynn

    2017-01-06

    Trisulfides can be a common post-translational modification in many recombinant monoclonal antibodies. These are a source of product heterogeneity that add to the complexity of product characterization and hence, need to be reduced for consistent product quality. Trisulfide bonds can be converted to the regular disulfide bonds by incorporating a novel cysteine wash step during Protein A affinity chromatography. An empirical model is developed for this on-column reduction reaction to compare the reaction rates as a function of typical operating parameters such as temperature, cysteine concentration, reaction time and starting level of trisulfides. The model presented here is anticipated to assist in the development of optimal wash conditions for the Protein A step to effectively reduce trisulfides to desired levels.

  9. Purification Or Organic Acids Using Anion Exchange Chromatography.

    DOEpatents

    Ponnampalam; Elankovan

    2001-09-04

    Disclosed is a cost-effective method for purifying and acidifying carboxylic acids, including organic acids and amino acids. The method involves removing impurities by allowing the anionic form of the carboxylic acid to bind to an anion exchange column and washing the column. The carboxylic anion is displaced as carboxylic acid by washing the resin with a strong inorganic anion. This method is effective in removing organic carboxylic acids and amino acids from a variety of industrial sources, including fermentation broths, hydrolysates, and waste streams.

  10. Effect of pre- and post-column band broadening on the performance of high-speed chromatography columns under isocratic and gradient conditions.

    PubMed

    Vanderlinden, Kim; Broeckhoven, Ken; Vanderheyden, Yoachim; Desmet, Gert

    2016-04-15

    We report on the results of an experimental and theoretical study of the effect of the extra-column band broadening (ECBB) on the performance of narrow-bore columns filled with the smallest particles that are currently commercially available. Emphasis is on the difference between the effect of ECBB under gradient and isocratic conditions, as well as on the ability to model and predict the ECBB effects using well-established band broadening expressions available from the theory of chromatography. The fine details and assumptions that need to be taken into account when using these expressions are discussed. The experiments showed that, the steeper the gradient, the more pronounced the extra-column band broadening losses become. Whereas the pre-column band broadening can in both isocratic and gradient elution be avoided by playing on the possibilities to focus the analytes on top of the column (e.g. by using the POISe injection method when running isocratic separations), the post-column extra-column band broadening is inescapable in both cases. Inducing extra-column band broadening by changing the inner diameter of the post-column tubing from 65 to 250 μm, we found that all peaks in the chromatogram are strongly affected (around a factor of 1.9 increase in relative peak width) when running steep gradients, while usually only the first eluting peak was affected in the isocratic mode or when running shallow gradients (factor 1.6-1.8 increase in relative peak width for the first eluting analyte).

  11. Determination of Sudan dyes in chili pepper powder by online solid-phase extraction with a butyl methacrylate monolithic column coupled to liquid chromatography with tandem mass spectrometry.

    PubMed

    Liu, Yao; Wang, Man-Man; Ai, Lian-Feng; Zhang, Chang-Kun; Li, Xin; Wang, Xue-Sheng

    2014-07-01

    A poly(butyl methacrylate-co-ethylene dimethacrylate) monolithic column was fabricated and used as a novel sorbent for online solid-phase extraction coupled to liquid chromatography with tandem mass spectrometry for the simultaneous determination of Sudan I-IV in chili pepper powder. The prepared columns were characterized by scanning electron microscopy, nitrogen adsorption-desorption, and pressure drop measurements. Online solid-phase extraction was performed on the synthesized monolithic column using 10 mM ammonium acetate solution as the loading solution with the aid of an online cleanup chromatography system. The desorption of Sudan I-IV was achieved with acetonitrile as the eluting solution at the flow rate of 0.5 mL/min. The extracted analytes were subsequently eluted into a C18 analytical column for chromatographic separation using a mixture of 10% acetonitrile/90% formic acid (0.5%) solution as the mobile phase. Under the optimized conditions, the developed method had linear range of 1.0-50 μg/kg, a detection limit of 0.3 μg/kg, and a quantification limit of 1.0 μg/kg for each analyte. The intraday and interday recoveries of Sudan I-IV in chili pepper powder samples ranged from 94.8 to 100.9% and 94.9 to 99.4%, respectively. The intraday and interday precision were between 3.37-7.01% and 5.01-7.68%, respectively.

  12. Determination of methandrostenolone and its metabolites in equine plasma and urine by coupled-column liquid chromatography with ultraviolet detection and confirmation by tandem mass spectrometry.

    PubMed

    Edlund, P O; Bowers, L; Henion, J

    1989-02-24

    Monitoring steroid use requires an understanding of the metabolism in the species in question and development of sensitive methods for screening of the steroid or its metabolites in urine. Qualitative information for confirmation of methandrostenolone and identification of its metabolites was primarily obtained by coupled-column high-performance liquid chromatography-tandem mass spectrometry. The steroids and a sulphuric acid conjugate were isolated and identified by their daughter ion mass spectra in the urine of both man and the horse following administration of methandrostenolone. Spontaneous hydrolysis of methandrostenolone sulphate gave 17-epimethandrostenolone and several dehydration products. This reaction had a half-life of 16 min in equine urine at 27 degrees C. Mono- and dihydroxylated metabolites were also identified. Several screening methods were evaluated for detection and confirmation of methandrostenolone use including thin-layer chromatography and high-performance liquid chromatography. Coupled-column liquid chromatography was used for automated clean-up of analytes difficult to isolate by manual methods. The recovery of methandrostenolone was 101 +/- 3.3% (mean +/- S.D.) at 6.5 ng/ml and both methandrostenolone and 17-epimethandrostenolone were quantified in urine by ultraviolet detection up to six days after a 250-mg intramuscular dose to a horse. The utility of on-line tandem mass spectrometry for confirmation of suspected metabolites is also shown.

  13. Streamlined pentafluorophenylpropyl column liquid chromatography-tandem quadrupole mass spectrometry and global (13)C-labeled internal standards improve performance for quantitative metabolomics in bacteria.

    PubMed

    Yang, Song; Sadilek, Martin; Lidstrom, Mary E

    2010-11-19

    Streamlined quantitative metabolomics in central metabolism of bacteria would be greatly facilitated by a high-efficiency liquid chromatography (LC) method in conjunction with accurate quantitation. To achieve this goal, a methodology for LC-tandem quadrupole mass spectrometry (LC-MS/MS) involving a pentafluorophenylpropyl (PFPP) column and culture-derived global (13)C-labeled internal standards (I.Ss.) has been developed and compared to hydrophilic interaction liquid chromatography (HILIC)-MS/MS and published combined two-dimensional gas chromatography and LC methods. All 50 tested metabolite standards from 5 classes (amino acids, carboxylic acids, nucleotides, acyl-CoAs and sugar phosphates) displayed good chromatographic separation and sensitivity on the PFPP column. In addition, many important critical pairs such as isomers/isobars (e.g. isoleucine/leucine, methylsuccinic acid/ethylmalonic acid and malonyl-CoA/3-hydroxybutyryl-CoA) and metabolites of similar structure (e.g. malate/fumarate) were resolved better on the PFPP than on the HILIC column. Compared to only one (13)C-labeled I.S., the addition of global (13)C-labeled I.Ss. improved quantitative linearity and accuracy. PFPP-MS/MS with global (13)C-labeled I.Ss. allowed the absolute quantitation of 42 metabolite pool sizes in Methylobacterium extorquens AM1. A comparison of metabolite level changes published previously for ethylamine (C2) versus succinate (C4) cultures of M. extorquens AM1 indicated a good consistency with the data obtained by PFPP-MS/MS, suggesting this single approach has the capability of providing comprehensive metabolite profiling similar to the combination of methods. The more accurate quantification obtained by this method forms a fundamental basis for flux measurements and can be used for metabolism modeling in bacteria in future studies.

  14. Concentration and fractionation of hydrophobic organic acid constituents from natural waters by liquid chromatography

    USGS Publications Warehouse

    Thurman, E.M.; Malcolm, R.L.

    1979-01-01

    A scheme is presented which used adsorption chromatography with pH gradient elution and size-exclusion chromatography to concentrate and separate hydrophobic organic acids from water. A review of chromatographic processes involved in the flow scheme is also presented. Organic analytes which appear in each aqueous fraction are quantified by dissolved organic carbon analysis. Hydrophobic organic acids in a water sample are concentrated on a porous acrylic resin. These acids usually constitute approximately 30-50 percent of the dissolved organic carbon in an unpolluted water sample and are eluted with an aqueous eluent (dilute base). The concentrate is then passed through a column of polyacryloylmorpholine gel, which separates the acids into high- and low-molecular-weight fractions. The high- and low-molecular-weight eluates are reconcentrated by adsorption chromatography, then are eluted with a pH gradient into strong acids (predominately carboxylic acids) and weak acids (predominately phenolic compounds). For standard compounds and samples of unpolluted waters, the scheme fractionates humic substances into strong and weak acid fractions that are separated from the low molecular weight acids. A new method utilizing conductivity is also presented to estimate the acidic components in the methanol fraction.

  15. Increased Yield of High Purity Recombinant Human Interferon-γ Utilizing Reversed Phase Column Chromatography

    PubMed Central

    Reddy, Praveen K.; Reddy, Srinivasa G.; Narala, Venkata R.; Majee, Sangita S.; Konda, Sudhakar; Gunwar, Sripad; Reddy, Raju C.

    2007-01-01

    Increasing therapeutic applications for recombinant human interferon-γ (rhIFN-γ), an antiviral proinflammatory cytokine, has broadened interest in optimizing methods for its production and purification. We describe a reversed phase chromatography (RPC) procedure using Source-30™ matrix in the purification of rhIFN-γ from Escherichia coli that results in a higher yield than previously reported. The purified rhIFN-γ monomer from the RPC column is refolded in Tris buffer. Optimal refolding occurs at protein concentrations between 50–100 μg/ml. This method yields greater than 90% of the dimer form with a yield of 40 mg g−1 cell mass. Greater than 99% purity is achieved with further purification over a Superdex G-75 column to obtain specific activities of from 2 to 4 × 107 IU/mg protein as determined via cytopathic antiviral assay. The improved yield of rhIFN-γ in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule. PMID:17049266

  16. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  17. Identification of hepatic copper-binding proteins from tilapia by column chromatography with proteomic approaches.

    PubMed

    Chen, Dongshi; Chan, King Ming

    2012-08-01

    Although copper is an essential element, it shows cytotoxic effects when present in excessive amounts with the production of hydroxyl radicals, which can damage phospholipids and enzymes. This necessitates a tight cellular control mechanism for copper homeostasis including its uptake and removal. The high copper contents in the liver of tilapia make this fish a suitable model for the study of copper binding proteins (CBPs). The liver was dissected from tilapia injected with Cu(2+) and cytosolic fractions were separated by using Superdex 75 column chromatography followed by atomic absorption spectrometry. Fractions in two major peaks containing CBPs were analyzed by using differential proteomic approaches, and loaded on a Cu chelating ion-immobilized affinity column (Cu-IMAC). Of the 113 differentially expressed proteins in these two peaks, 28 proteins were found to have copper binding ability, including well-characterized CBPs, such as copper transporter ATP7A and metallothionein. The networks of CBPs built up by Ingenuity Pathway Analysis (IPA) would help us to understand the transportation pathway and function of CBPs, which were related to free radical scavenging, cellular development and lipid metabolism. In addition, our results suggest that Cu(2+) would compete with Fe(2+) and Ca(2+) in binding with some target proteins, such as ferritin, transferrin, and calmodulin.

  18. Chromatographic performance of large-pore versus small-pore columns in micellar liquid chromatography.

    PubMed

    McCormick, Timothy J; Foley, Joe P; Lloyd, David K

    2003-02-25

    Micellar liquid chromatography (MLC) is useful in bioanalysis because proteinaceous biofluids can be directly injected onto the column. The technique has been limited in part because of the apparently weak eluting power of micellar mobile phases. It has recently been shown [Anal. Chem. 72 (2000) 294] that this may be overcome by the use of large pore size stationary phases. In this work, large-pore (1000 A) C(18) stationary phases were evaluated relative to conventional small-pore (100 A) C(18) stationary phases for the direct sample injection of drugs in plasma. Furthermore, the difference between the large and small pore phases in gradient elution separations of mixtures of widely varying hydrophobicities was investigated. Large-pore stationary phases were found to be very effective for eluting moderately to highly hydrophobic compounds such as ibuprofen, crotamiton, propranolol, and dodecanophenone, which were highly retained on the small-pore stationary phases typically used in MLC. The advantages of direct introduction of biological samples (drugs in plasma) and rapid column re-equilibration after gradient elution in MLC were maintained with large-pore phases. Finally, recoveries, precision, linearity, and detection limits for the determination of quinidine and DPC 961 in spiked bovine plasma were somewhat better using MLC with wide pore phases.

  19. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    PubMed

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.

  20. Rapid determination of beta-aminoisobutyric acid by reversed-phase high-performance liquid chromatography.

    PubMed

    Ladrón de Guevara, O; Cortinas de Nava, C; Padilla, P; Espinosa, J; Cebrian, M; García, L

    1990-06-08

    For the determination of beta-aminoisobutyric acid (BAIBA) in urine samples in which the beta-alanine concentrations are higher than those of BAIBA, the resolution between these two amino acids, separated by reversed-phase liquid chromatography on an octadecylsilane column, was optimized. The chromatographic analysis included precolumn derivatization of amino acids with o-phthalaldehyde, followed by a 15-min isocratic elution and detection at 340 nm. Because of its simplicity, this method should be useful for monitoring urinary excretion of BAIBA.

  1. Understanding and diminishing the extra-column band broadening effects in supercritical fluid chromatography.

    PubMed

    De Pauw, Ruben; Shoykhet Choikhet, Konstantin; Desmet, Gert; Broeckhoven, Ken

    2015-07-17

    Supercritical fluid chromatography, where a low-viscosity mobile phase such as carbon dioxide is used, proves to be an excellent technique for fast and efficient separations, especially when sub-2μm particles are used. However, to achieve high velocities when using these small particles, and in order to stay within the flow rate range of current SFC-instruments, narrow columns (e.g. 2.1mm ID) must be used. Unfortunately, state-of-the-art instrumentation is limiting the full separation power of these narrower columns due to significant extra-column band broadening effects. The present work identifies and quantifies the different contributions to extra-column band broadening in SFC such as the influence of the sample solvent, injection volume, extra-column volumes and detector cell volume/design. When matching the sample solvent to the mobile phase in terms of elution strength and polarity (e.g. using hexane/ethanol/isopropanol 85/10/5vol%) and lowering the injection volume to 0.4μL, the plate count can be increased from 7600 to 21,300 for a low-retaining compound (k'=2.3) on a 2.1mm×150mm column (packed with 1.8μm particles). The application of a water/acetonitrile mixture as sample solvent was also investigated. It was found that when the volumetric ratio of water/acetonitrile was optimized, only a slightly lower plate count was measured compared to the hexane-based solvent when minimizing injection and extra-column volume. This confirms earlier results that water/acetonitrile can be used if water-soluble samples are considered or when a less volatile solvent is preferred. Minimizing the ID of the connection capillaries from 250 to 65μm, however, gives no further improvement in obtained efficiency for early-eluting compounds when a standard system configuration with optimized sample solvent was used. When switching to a state-of-the-art detector design with reduced (dispersion) volume (1.7-0.6μL), an increase in plate count is observed (from 11,000 to 14

  2. Direct determination of acylcarnitines in amniotic fluid by column-switching liquid chromatography with electrospray tandem mass spectrometry.

    PubMed

    Cho, Sung-Hee; Lee, Jeongae; Lee, Won-Yong; Chung, Bong Chul

    2006-01-01

    A direct, simple, and simultaneous determination of acylcarnitines in amniotic fluid was developed using column-switching liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analytes can be assayed within 20 min without any sample preparation process, and we monitored separated acylcarnitines with positive electrospray ionization (ESI)-MS/MS. The calibration ranges of acylcarnitines were 1 to 100 nmol/L. The linearity of the method was 0.992 to 0.999, and the limits of detection at a signal-to-noise ratio of 3 were 1 nmol/L. The coefficients of variation were in the range of 5.2 to 13.3% for within-day variation and 6.7 to 11.9% for day-to-day, respectively. We detected acylcarnitines in the amniotic fluid of 22 women in the early stages of their pregnancies in the range of 2.2 to 17.2 nmol/L. The proposed method could be applied to diagnosis, monitoring, and biomedical investigations of inborn errors of the organic acid and fatty acid metabolism of the embryo.

  3. Dual-column cation-exchange chromatographic method for beta-aminoisobutyric acid and beta-alanine in biological samples.

    PubMed

    Kuo, K C; Cole, T F; Gehrke, C W; Waalkes, T P; Borek, E

    1978-08-01

    A rapid, automated chromatographic method has been developed for the quantitation of the nucleic acid catabolites beta-aminoisobutyric acid and beta-alanine in urine, serum, and other physiological fluids. The analyses were performed on a modified Beckman 121M amino acid analyzer with dual ion-exchange columns and the use of a single sodium citrate buffer (pH 4.38, 0.20 mol/liter). By carefully matching the elution pattern for the two ion-exchange columns and alternating use of these columns, analyses are completed every 40 min. The chromatography, regeneration, and equilibration of the two columns are precisely programmed, thus the detector sees only the elution of beta-aminoisobutyric acid and beta-alanine alternately from each column. Long-term precision and analytical recovery for the two metabolites in urine were 1.9 and 102%, and 3.3 and 101%, respectively. Their normal physiological values were determined in human serum and urine. Their excretion in the urine was also studied as a function of collection time, to validate a more convenient, less costly method of sampling. This study shows that randomly collected samples are acceptable when the concentration of the two metabolites are expressed in terms of creatinine excretion. In addition, the distribution of the free and conjugated forms of the two metabolites in urine and serum was studied. A preparative method was also developed for the quantitative isolation of beta-amino-isobutyric acid from urine samples. The alternating dual-column technique may be applied to any ion-exchange chromatographic method where many analyses must be performed. This method is currently used in our laboratories for measuring these beta-amino acids in urine and serum of patients with various types of cancers.

  4. Selection of column dimensions and gradient conditions to maximize the peak-production rate in comprehensive off-line two-dimensional liquid chromatography using monolithic columns.

    PubMed

    Eeltink, Sebastiaan; Dolman, Sebastiaan; Vivo-Truyols, Gabriel; Schoenmakers, Peter; Swart, Remco; Ursem, Mario; Desmet, Gert

    2010-08-15

    The peak-production rate (peak capacity per unit time) in comprehensive off-line two-dimensional liquid chromatography (LC/x/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A first-dimension ((1)D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the (1)D fractions on a monolithic column operating at a pH of 2. To obtain the highest peak-production rate, effects of column length, gradient duration, and sampling time were examined. RP/x/RP was performed at undersampling conditions using a short 10 min (1)D gradient. The peak-production rate was highest using a 50 mm long (2)D column applying an 8-10 min (2)D gradient time and was almost a factor of two higher than when a 250 mm monolithic column was used. The best way to obtain a higher peak-production rate in off-line LC/x/LC proved to be an increase in the number of (1)D fractions collected. Increasing the (2)D gradient time was less effective. The potential of the optimized RP/x/RP method is demonstrated by analyzing proteomics samples of various complexities. Finally, the trade-off between peak capacity and analysis time is discussed in quantitative terms for both one-dimensional RP gradient-elution chromatography and the off-line two-dimensional (RP/x/RP) approach. At the conditions applied, the RP/x/RP approach provided a higher peak-production rate than the (1)D-LC approach when collecting three (1)D fractions, which corresponds to a total analysis time of 60 min.

  5. Evaluation and application of static headspace-multicapillary column-gas chromatography-ion mobility spectrometry for complex sample analysis.

    PubMed

    Denawaka, Chamila J; Fowlis, Ian A; Dean, John R

    2014-04-18

    An evaluation of static headspace-multicapillary column-gas chromatography-ion mobility spectrometry (SHS-MCC-GC-IMS) has been undertaken to assess its applicability for the determination of 32 volatile compounds (VCs). The key experimental variables of sample incubation time and temperature have been evaluated alongside the MCC-GC variables of column polarity, syringe temperature, injection temperature, injection volume, column temperature and carrier gas flow rate coupled with the IMS variables of temperature and drift gas flow rate. This evaluation resulted in six sets of experimental variables being required to separate the 32 VCs. The optimum experimental variables for SHS-MCC-GC-IMS, the retention time and drift time operating parameters were determined; to normalise the operating parameters, the relative drift time and normalised reduced ion mobility for each VC were determined. In addition, a full theoretical explanation is provided on the formation of the monomer, dimer and trimer of a VC. The optimum operating condition for each VC calibration data was obtained alongside limit of detection (LOD) and limit of quantitation (LOQ) values. Typical detection limits ranged from 0.1ng bis(methylthio)methane, ethylbutanoate and (E)-2-nonenal to 472ng isovaleric acid with correlation coefficient (R(2)) data ranging from 0.9793 (for the dimer of octanal) through to 0.9990 (for isobutyric acid). Finally, the developed protocols were applied to the analysis of malodour in sock samples. Initial work involved spiking an inert matrix and sock samples with appropriate concentrations of eight VCs. The average recovery from the inert matrix was 101±18% (n=8), while recoveries from the sock samples were lower, that is, 54±30% (n=8) for sock type 1 and 78±24% (n=6) for sock type 2. Finally, SHS-MCC-GC-IMS was applied to sock malodour in a field trial based on 11 volunteers (mixed gender) over a 3-week period. By applying the SHS-MCC-GC-IMS database, four VCs were

  6. Using Artificial Soil and Dry-Column Flash Chromatography to Simulate Organic Substance Leaching Process: A Colorful Environmental Chemistry Experiment

    ERIC Educational Resources Information Center

    de Avellar, Isa G. J.; Cotta, Tais A. P. G.; Neder, Amarilis de V. Finageiv

    2012-01-01

    Soil is an important and complex environmental compartment and soil contamination contributes to the pollution of aquifers and other water basins. A simple and low-cost experiment is described in which the mobility of three organic compounds in an artificial soil is examined using dry-column flash chromatography. The compounds were applied on top…

  7. Reversed-phase high-performance liquid chromatography of tenuazonic acid and related tetramic acids.

    PubMed

    Shephard, G S; Thiel, P G; Sydenham, E W; Vleggaar, R; Marasas, W F

    1991-05-03

    A reversed-phase high-performance liquid chromatographic system for the determination of the fungal toxin, tenuazonic acid, (5S,8S)-3-acetyl-5-sec.-butyltetramic acid, is described. The system utilizes a column packed with deactivated end-capped C18 silica with a high carbon load to overcome the problem of poor chromatographic performance of this beta-diketone on reversed-phase liquid chromatography which previously necessitated the use of anion-exchange, ligand-exchange or ion-pairing methods. The reversed-phase system allows the separation of tenuazonic acid from its (5R,8S)-diastereomer, allo-tenuazonic acid and was applied to the detection of tenuazonic acid in cultures of Alternaria alternata and Phoma sorghina. By means of diode-array ultraviolet detection, (5S)-3-acetyl-5-isopropyltetramic acid was observed in extracts of culture material. This metabolite was purified using the analytical reversed-phase system and was identified by 1H and 13C nuclear magnetic resonance spectroscopy.

  8. Trend analysis of performance parameters of pre-packed columns for protein chromatography over a time span of ten years.

    PubMed

    Scharl, Theresa; Jungreuthmayer, Christian; Dürauer, Astrid; Schweiger, Susanne; Schröder, Tim; Jungbauer, Alois

    2016-09-23

    Pre-packed small scale chromatography columns are increasingly used for process development, for determination of design space in bioprocess development, and for post-licence process verifications. The packing quality of 30,000 pre-packed columns delivered to customers over a period 10 years has been analyzed by advanced statistical tools. First, the data were extracted and checked for inconsistencies, and then were tabulated and made ready for statistical processing using the programming language Perl (https://www.perl.org/) and the statistical computing environment R (https://www.r-project.org/). Reduced HETP and asymmetry were plotted over time to obtain a trend of packing quality over 10 years. The obtained data were used as a visualized coefficient of variation analysis (VCVA), a process that has often been applied in other industries such as semiconductor manufacturing. A typical fluctuation of reduced HETP was seen. A Tsunami effect in manufacturing, the effect of propagation of manufacturing deviations leading to out-of-specification products, was not observed with these pre-packed columns. Principal component analysis (PCA) showed that all packing materials cluster. Our data analysis showed that the current commercially available chromatography media used for biopharmaceutical manufacturing can be reproducibly and uniformly packed in polymer-based chromatography columns, which are designed for ready-to-use purposes. Although the number of packed columns has quadrupled over one decade the packing quality has remained stable.

  9. Refolding and simultaneous purification of recombinant human proinsulin from inclusion bodies on protein-folding liquid-chromatography columns.

    PubMed

    Yuan, Jie; Zhou, Huifang; Yang, Yicong; Li, Weimin; Wan, Yi; Wang, Lili

    2015-05-01

    Protein-folding liquid chromatography (PFLC) is an effective and scalable method for protein renaturation with simultaneous purification. However, it has been a challenge to fully refold inclusion bodies in a PFLC column. In this work, refolding with simultaneous purification of recombinant human proinsulin (rhPI) from inclusion bodies from Escherichia coli were investigated using the surface of stationary phases in immobilized metal ion affinity chromatography (IMAC) and high-performance size-exclusion chromatography (HPSEC). The results indicated that both the ligand structure on the surface of the stationary phase and the composition of the mobile phase (elution buffer) influenced refolding of rhPI. Under optimized chromatographic conditions, the mass recoveries of IMAC column and HPSEC column were 77.8 and 56.8% with purifies of 97.6 and 93.7%, respectively. These results also indicated that the IMAC column fails to refold rhPI, and the HPSEC column enables efficient refolding of rhPI with a low-urea gradient-elution method. The refolded rhPI was characterized by circular dichroism spectroscopy. The molecular weight of the converted human insulin was further confirmed with SDS-18% PAGE, Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and the biological activity assay by HP-RPLC.

  10. Optimal performance of single-column chromatography and simulated moving bed processes for the separation of optical isomers

    NASA Astrophysics Data System (ADS)

    Medi, Bijan; Kazi, Monzure-Khoda; Amanullah, Mohammad

    2013-06-01

    Chromatography has been established as the method of choice for the separation and purification of optically pure drugs which has a market size of about 250 billion USD. Single column chromatography (SCC) is commonly used in the development and testing phase of drug development while multi-column Simulated Moving Bed (SMB) chromatography is more suitable for large scale production due to its continuous nature. In this study, optimal performance of SCC and SMB processes for the separation of optical isomers under linear and overloaded separation conditions has been investigated. The performance indicators, namely productivity and desorbent requirement have been compared under geometric similarity for the separation of a mixture of guaifenesin, and Tröger's base enantiomers. SCC process has been analyzed under equilibrium assumption i.e., assuming infinite column efficiency, and zero dispersion, and its optimal performance parameters are compared with the optimal prediction of an SMB process by triangle theory. Simulation results obtained using actual experimental data indicate that SCC may compete with SMB in terms of productivity depending on the molecules to be separated. Besides, insights into the process performances in terms of degree of freedom and relationship between the optimal operating point and solubility limit of the optical isomers have been ascertained. This investigation enables appropriate selection of single or multi-column chromatographic processes based on column packing properties and isotherm parameters.

  11. Analysis of drugs in plasma samples from schizophrenic patients by column-switching liquid chromatography-tandem mass spectrometry with organic-inorganic hybrid cyanopropyl monolithic column.

    PubMed

    Domingues, Diego Soares; Souza, Israel Donizeti de; Queiroz, Maria Eugênia Costa

    2015-07-01

    This study reports on the development of a rapid, selective, and sensitive column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze sixteen drugs (antidepressants, anticonvulsants, anxiolytics, and antipsychotics) in plasma samples from schizophrenic patients. The developed organic-inorganic hybrid monolithic column with cyanopropyl groups was used for the first dimension of the column-switching arrangement. This arrangement enabled online pre-concentration of the drugs (monolithic column) and their subsequent analytical separation on an XSelect SCH C18 column. The drugs were detected on a triple quadrupole tandem mass spectrometer (multiple reactions monitoring mode) with an electrospray ionization source in the positive ion mode. The developed method afforded adequate linearity for the sixteen target drugs; the coefficients of determination (R(2)) lay above 0.9932, the interassay precision had coefficients of variation lower than 6.5%, and the relative standard error values of the accuracy ranged from -14.0 to 11.8%. The lower limits of quantification in plasma samples ranged from 63 to 1250pgmL(-1). The developed method successfully analyzed the target drugs in plasma samples from schizophrenic patients for therapeutic drug monitoring (TDM).

  12. Evaluation of reversible interconversion in comprehensive two-dimensional gas chromatography using enantioselective columns in first and second dimensions.

    PubMed

    Kröger, Sabrina; Wong, Yong Foo; Chin, Sung-Tong; Grant, Jacob; Lupton, David; Marriott, Philip J

    2015-07-24

    The reversible molecular interconversion behaviour of a synthesised oxime (2-phenylpropanaldehyde oxime; (C6H5)CH(CH3)CHN(OH)) was investigated by both, single dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Previous studies on small molecular weight oximes were extended to this larger aromatic oxime (molar mass 149.19gmol(-1)) with interest in the extent of interconversion, enantioselective resolution, and retention time. On a polyethylene glycol (PEG; wax-type) column, a characteristic interconversion zone between two antipodes of E and Z isomers was formed by molecules which have undergone isomerisation on the column (E⇌Z). The extent of interconversion was investigated by varying chromatographic conditions (oven temperature and carrier flow rate) to understand the nature of the behaviour observed. The extent of interconversion was negligible in both enantioselective and methyl-phenylpolysiloxane phase-columns, correlating with the low polarity of the stationary phase. In order to obtain isomerisation along with enantio-resolution, a wax-type and an enantioselective column were coupled in either enantioselective-wax or wax-enantioselective order. The most appropriate column arrangement was selected for study by using a GC×GC experiment with either a wax-phase or phenyl-methylpolysiloxane phase as (2)D column. In addition to evaluation of these fast elution columns, a long narrow-bore enantioselective column (10m) was introduced as (2)D, providing an enantioselective-PEG (coupled-column ensemble: (1)D1+(1)D2)×enantioselective ((2)D) column combination. In this instance, the (1)D1 enantioselective column provides enantiomeric separation of the corresponding enantiomers ((R) and (S)) of (E)- and (Z)-2-phenylpropanaldehyde oxime, followed by E/Z isomerisation in the coupled (1)D2 PEG (reactor) column. The resulting chromatographic interconversion region was modulated and separated into either E/Z isomers

  13. Hydrophilic interaction chromatography using a meter-scale monolithic silica capillary column for proteomics LC-MS.

    PubMed

    Horie, Kanta; Kamakura, Takeo; Ikegami, Tohru; Wakabayashi, Masaki; Kato, Takashi; Tanaka, Nobuo; Ishihama, Yasushi

    2014-04-15

    A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

  14. Simultaneous separation of inorganic anions and cations by using anion-exchange and cation-exchange columns connected in tandem in ion chromatography.

    PubMed

    Karim, Khairil Juhanni Binti Abd; Jin, Ji-Ye; Takeuchi, Toyohide

    2003-05-02

    Inorganic anions and cations in environmental waters were determined by ion chromatography. Stationary and mobile phases were examined for the simultaneous separation of both anions and cations. Cations detection by UV detection requires a mobile phase with a UV absorbing additive, which indirectly visualizes cations as negative peaks. Simultaneous separation of anions and cations were achieved when using an eluent that consists of inorganic acid with weak basic amino acid as additives. It was convenient to separate both anions and cations by coupling anion-exchange and cation-exchange columns in tandem. The order of the separation columns connected affected the elution profiles. When the eluent comprises of multiple anions and a single cation, the anion-exchange column should be connected in the upper stream, whereas when the eluent comprises multiple cations and a single anion, the cation-exchange column should be connected in the upper stream. Use of switching valves also allowed simultaneous separation of anions and cations in a single chromatographic run. In the present work, operating conditions were optimized for the simultaneous separation of anions and cations.

  15. Determination of biogenic amines in beer with pre-column derivatization by high performance liquid chromatography.

    PubMed

    Tang, Tao; Shi, Tianyu; Qian, Kun; Li, Pingliang; Li, Jianqiang; Cao, Yongsong

    2009-02-15

    Eighteen samples of commercially available Chinese beer were analyzed in order to determine the content of biogenic amines. The method involves pre-column derivatization of the amines with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF) and subsequent analysis by RP-HPLC (reversed phase-high performance liquid chromatography) with diode array detection. The labeled biogenic amines were separated on a Kromasil C18 column (250mmx4.6mm, 5microm) at room temperature and UV detection was applied at 254nm. The separation of seven labeled biogenic amines was achieved within 22min by elution acetonitrile and HAc-NaAc buffers. The method linearity, calculated for each biogenic amine, has a correlation coefficient higher than 0.9925, in concentrations ranging from 2.9micromolL(-1) to 565micromolL(-1). Detection limits of biogenic amines were 0.056-0.87micromolL(-1), at a signal-to-noise ratio of 3. The proposed method has been applied to the quantitative determination of spermine, phenethylamine, spermidine, histamine, tyramine, tryptamine and putrescine in beer with recoveries of 91.9-103.1% and R.S.D. of 2.86-5.63%. Quantitation is relative to external standards. The results showed that each kind of beer examined contained at least three biogenic amines. Putrescine, histamine and tyramine were detected in all samples. Spermidine was detected in 89% of the beers. Spermine, tryptamine and phenylethylamine occurred in 78%, 61% and 44% of the beers examined, respectively. These levels were below the level that may elicit direct adverse reactions for most consumers.

  16. Are molecular weights of proteins determined by superose 12 column chromatography correct?

    PubMed

    Lee, Shih-Chieh; Whitaker, John R

    2004-08-11

    Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.

  17. Sensitive and simple determination of bromate in foods disinfected with hypochlorite reagents using high performance liquid chromatography with post-column derivatization.

    PubMed

    Yokota, Azusa; Kubota, Hiroki; Komiya, Satomi; Sato, Kyoko; Akiyama, Hiroshi; Koshiishi, Ichiro

    2012-11-02

    A novel analytical method for the quantification of bromate in fresh foods using high performance liquid chromatography (HPLC) with a post-column reaction has been developed. The fresh food sample solutions were pretreated with homogenization, centrifugal ultrafiltration and subsequent solid phase extraction using a strong anion-exchange resin. After separation on a strong anion-exchange chromatography column using a highly concentrated NaCl solution (0.3M) as the eluent, the bromate was quantified by detection using a post-column reaction with a non-carcinogenic reagent (tetramethylbenzidine). The developed HPLC technique made it possible to quantify bromate in salt-rich fresh foods. The recoveries from fresh foods spiked with bromate at low levels (2 or 10 ng/g) satisfactorily ranged from 75.3 to 90.7%. The lowest quantification limit in fresh foods was estimated to be 0.6 ng/g as bromic acid. The method should be helpful for the quantification of bromate in fresh foods disinfected with hypochlorite solutions.

  18. [Determination of organic acids in fermentation broth of spiramycin by high performance liquid chromatography].

    PubMed

    Li, You-yuan; Chen, Chang-hua; Tao, Ping

    2002-01-01

    A method for determining organic acids in spiramycin fermentation broth by high performance liquid chromatography is described. The operating conditions were Zorbax 300-SB C18 column (5 microns, 4.6 mm i.d. x 15 cm) at 35 degrees C, 0.01 mol/L phosphoric acid buffer solution (pH 2.32) and methanol as mobile phase at a flow rate of 0.6 mL/min and UV detection at 210 nm. The relative standard deviations were 0.33%-0.10% and the recoveries were 99.95%-100.08%. It's a simple, rapid and accurate method.

  19. Separation of high-purity syringol and acetosyringone from rice straw-derived bio-oil by combining the basification-acidification process and column chromatography.

    PubMed

    Hao, Shilai; Chen, Kaifei; Cao, Leichang; Zhu, Xiangdong; Luo, Gang; Zhang, Shicheng; Chen, Jianmin

    2016-10-01

    Numerous technologies have been used to reclaim valuable chemicals from bio-oil. In this study, a combination of the basification-acidification process and column chromatography was employed for the separation of high-purity syringol and acetosyringone from rice straw-derived bio-oil. The optimal conditions for the basification-acidification process and the possible precipitation mechanism of the basification were explored. The results showed the following as the optimal conditions for the basification process: mass ratio of calcium hydroxide (Ca(OH)2 ) to bio-oil, 2.0; reaction temperature, 70°C; and reaction time, 30 min. The results also showed that 1.6 mol of hydrochloric acid (HCl) per gram of bio-oil was optimal for the acidification. The precipitation was found to proceed via a possible mechanism involving the reaction of the phenolic compounds in the bio-oil with Ca(OH)2 to produce a precipitate. After further separation by column chromatography, purities of 91.4 and 96.2% (from gas chromatography-mass spectrometry) were obtained for syringol and acetosyringone, respectively. Their recoveries for the whole process were 73.0 and 39.3%, respectively.

  20. Amino acids as chiral selectors in enantioresolution by liquid chromatography.

    PubMed

    Bhushan, Ravi; Dixit, Shuchi

    2012-08-01

    Amino acids are unique in terms of their structural features and multidimensional uses. With their simple structures and the ready availability of both enantiomers, amino acids not only serve as a chiral pool for synthesis but also provide an inexpensive pool for resolution studies. There has been no attempt to review the application of amino acids as chiral selectors for chromatographic enantioresolution of pharmaceuticals and other compounds. The present paper deals with application of l-amino acids and complexes of l-amino acids with a metal ion, particularly Cu(II), as an impregnating reagent in thin-layer chromatography or as a chiral ligand exchange reagent or a chiral mobile phase additive in both thin-layer chromatography and high-performance liquid chromatography. Enantiomeric resolution of β-blockers, nonsteroidal anti-inflammatories, amino acids (and their derivatives) and certain other compounds is discussed.

  1. Factors influencing the separation of oligonucleotides using reversed-phase/ion-exchange mixed-mode high performance liquid chromatography columns.

    PubMed

    Biba, Mirlinda; Jiang, Eileen; Mao, Bing; Zewge, Daniel; Foley, Joe P; Welch, Christopher J

    2013-08-23

    New mixed-mode columns consisting of reversed-phase and ion-exchange separation modes were evaluated for the analysis of short RNA oligonucleotides (∼20mers). Conventional analysis for these samples typically involves using two complementary methods: strong anion-exchange liquid chromatography (SAX-LC) for separation based on charge, and ion-pair reversed-phase liquid chromatography (IP-RPLC) for separation based on hydrophobicity. Recently introduced mixed-mode high performance liquid chromatography (HPLC) columns combine both reversed-phase and ion-exchange modes, potentially offering a simpler analysis by combining the benefits of both separation modes into a single method. Analysis of a variety of RNA oligonucleotide samples using three different mixed-mode stationary phases showed some distinct benefits for oligonucleotide separation and analysis. When using these mixed-mode columns with typical IP-RPLC mobile phase conditions, such as ammonium acetate or triethylammonium acetate as the primary ion-pair reagent, the separation was mainly based on the IP-RPLC mode. However, when changing the mobile phase conditions to those more typical for SAX-LC, such as salt gradients with NaCl or NaBr, very different separation patterns were observed due to mixed-mode interactions. In addition, the Scherzo SW-C18 and SM-C18 columns with sodium chloride or sodium bromide salt gradients also showed significant improvements in peak shape.

  2. A reduced order model for the study of asymmetries in linear gas chromatography for homogeneous tubular columns.

    SciTech Connect

    Whiting, Joshua J.; Romero, Louis Anthony; Parks, Michael L.

    2005-08-01

    In gas chromatography, a chemical sample separates into its constituent components as it travels along a long thin column. As the component chemicals exit the column they are detected and identified, allowing the chemical makeup of the sample to be determined. For correct identification of the component chemicals, the distribution of the concentration of each chemical along the length of the column must be nearly symmetric. The prediction and control of asymmetries in gas chromatography has been an active research area since the advent of the technique. In this paper, we develop from first principles a general model for isothermal linear chromatography. We use this model to develop closed-form expressions for terms related to the first, second, and third moments of the distribution of the concentration, which determines the velocity, diffusion rate, and asymmetry of the distribution. We show that for all practical experimental situations, only fronting peaks are predicted by this model, suggesting that a nonlinear chromatography model is required to predict tailing peaks. For situations where asymmetries arise, we analyze the rate at which the concentration distribution returns to a normal distribution. Numerical examples are also provided.

  3. Gas chromatographic separation of fatty acid esters of cholesterol and phytosterols on an ionic liquid capillary column.

    PubMed

    Hammann, Simon; Vetter, Walter

    2015-12-15

    Steryl esters are high molecular weight compounds (600-700g/mol) regularly present as a minor lipid class in animal and plant lipids. Different sterol backbones (e.g., cholesterol, β-sitosterol and brassicasterol) which can be esterified with various fatty acids can result in highly complex steryl ester patterns in food samples. The gas chromatographic (GC) analysis of intact steryl esters is challenging, since high elution temperatures are required for their elution. On nonpolar GC phases, steryl esters with fatty acids with differing degree of unsaturation (e.g., oleate and linoleate) cannot be separated and there are only few polar columns available with sufficient temperature stability. In this study, we used gas chromatography with mass spectrometry (GC/MS) and analyzed intact steryl esters on a commercial room temperature ionic liquid (RTIL) column which was shortened to a length of 12m. The column separated the steryl esters both by total carbon number and by degree of unsaturation of the fatty acid. For instance, cholesteryl esters with stearic acid (18:0), oleic acid (18:1n-9), linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) could be resolved (R≥1.3) from each other. By analysis of synthesized standard substances, the elution orders for different steryl backbones and different fatty acids on a given sterol backbone could be determined. Analysis of spreads and plant oils allowed to determine retention times for 37 steryl esters, although a few co-elutions were observed. The ionic liquid column proved to be well-suited for the analysis of intact steryl esters.

  4. High-Throughput Analysis of Sucrose Fatty Acid Esters by Supercritical Fluid Chromatography/Tandem Mass Spectrometry

    PubMed Central

    Hori, Katsuhito; Tsumura, Kazunobu; Fukusaki, Eiichiro; Bamba, Takeshi

    2014-01-01

    Supercritical fluid chromatography (SFC) coupled with triple quadrupole mass spectrometry was applied to the profiling of sucrose fatty acid esters (SEs). The SFC conditions (column and modifier gradient) were optimized for the effective separation of SEs. In the column test, a silica gel reversed-phase column was selected. Then, the method was used for the detailed characterization of commercial SEs and the successful analysis of SEs containing different fatty acids. The present method allowed for fast and high-resolution separation of monoesters to tetra-esters within a shorter time (15 min) as compared to the conventional high-performance liquid chromatography. The applicability of our method for the analysis of SEs was thus demonstrated. PMID:26819875

  5. Half-width plots, a simple tool to predict peak shape, reveal column kinetics and characterise chromatographic columns in liquid chromatography: state of the art and new results.

    PubMed

    Baeza-Baeza, J J; Ruiz-Ángel, M J; García-Álvarez-Coque, M C; Carda-Broch, S

    2013-11-01

    Peak profiles in chromatography are characterised by their height, position, width and asymmetry; the two latter depend on the values of the left and right peak half-widths. Simple correlations have been found between the peak half-widths and the retention times. The representation of such correlations has been called half-width plots. For isocratic elution, the plots are parabolic, although often, the parabolas can be approximated to straight-lines. The plots can be obtained with the half-widths/retention time data for a set of solutes experiencing the same kinetics, eluted with a mobile phase at fixed or varying composition. When the analysed solutes experience different resistance to mass transfer, the plots will be solute dependent, and should be obtained with the data for each solute eluted with mobile phases at varying composition. The half-width plots approach is a simple tool that facilitates the prediction of peak shape (width and asymmetry) with optimisation purposes, reveal the interaction kinetics of solutes in different columns, and characterise chromatographic columns. This work shows half-width plots for different situations in isocratic elution, including the use of different flows, the effect of temperature, the modification of the stationary phase surface by an additive, the existence of specific interactions within the column, and the comparison of columns. The adaptation to gradient elution is also described. Previous knowledge on half-width plots is structured and analysed, to which new results are added.

  6. Application of coupled-column liquid chromatography combined with post-column photochemically induced fluorimetry derivatization and fluorescence detection to the determination of pyrethroid insecticides in vegetable samples.

    PubMed

    Parrilla Vázquez, P; Gil García, M D; Barranco Martínez, D; Martínez Galera, M

    2005-03-01

    This study reports the first application of coupled-column liquid chromatography-photochemically induced fluorimetry-fluorescence detection (LC-LC-PIF-FD), demonstrating its potential for the quantitative and selective detection of seven pyrethroids in vegetable samples such as cucumber, green bean, tomato and aubergine. An internal surface reversed-phase (ISRP) column coupled to a C18 column for analyte clean-up and determination were used, respectively. In comparison with a C18 column, the ISRP substantially improved the separation between analytes and interferences from the vegetable matrix. The limits of detection ranged from 0.01 to 0.22 microg kg(-1) in the vegetable samples (equivalent to 0.01 and 0.13 microg L(-1) in the extract injected), and limits of determination ranged from 0.56 to 8.33 microg kg(-1) in the vegetable samples (equivalent to 0.34 and 5.00 microg L(-1) in the extract injected). Samples were extracted into dichloromethane to yield mean recoveries at two levels of concentration between 72.8 and 110.0% in all cases. Relative standard deviations were lower than 11%.

  7. CRC handbook of chromatography: Nucleic acids and related compounds

    SciTech Connect

    Krstulovic, A.M.

    1987-01-01

    This book's contents include: Structure Elucidation of Nucleic Acid Components; Fundamentals of HPLC; Analysis of Nucleic Acids and Oligonucleotides; Extraction of Nucleic Acids from Tissues; Gel Filtration Chromatography of RNAs and DNS Fragments; Separation of tRNAs and Oligonucleotides by Mixed Mode Chromatography; Anion-Exchange and Reversed-Phase HPLC of Synthetic Oligonucleotides; Nucleic Acid Components in Biological Fluids; RPLC Separation of RNA and DNA Hydrolysates; Nucleotides in Tissue Extracts; and Determination of Adenine Nucleotides and Creatine Phosphate in Various Mammalian Tissues.

  8. Greener liquid chromatography using a guard column with micellar mobile phase for separation of some pharmaceuticals and determination of parabens.

    PubMed

    Youngvises, Napaporn; Chaida, Thanatcha; Khonyoung, Supada; Kuppithayanant, Nattawan; Tiyapongpattana, Warawut; Itharat, Arunporn; Jakmunee, Jaroon

    2013-03-15

    In this research, a greener chromatography employing a short column, Zorbax SB C18 cartridge (12.5 × 4.6 mm, 5 μm) commonly used as a guard column in a reverse phase high performance liquid chromatography (RP-HPLC), was utilized as the analytical column in conjunction with a more eco-friendly micellar mobile phase of sodium dodecyl sulfate (SDS) for separation tertiary mixtures of local anesthetics and antihistamines; and binary mixture of colds drugs; and quaternary mixture of some parabens with different separation conditions. The chromatographic behavior of these analytes was studied to demonstrate separation efficiency of this guard column in a micellar mobile phase. Moreover, this column and SDS mobile phase was exploited for determination of parabens in 64 samples of cosmetic product, both those that were produced locally in the community and those that were commercially manufactured. Linear calibration graphs of the parabens as detected at 254 nm were obtained in the range of 1-100 μmol L(-1) with R(2)>0.9990. Percentage recoveries were 92.4-109.2 with %RSD<3, and the limit of detection and quantitation were 0.04-0.10 and 0.20-0.80 μmol L(-1), respectively. This analytical system is not only greener but also faster and employing simpler sample preparation than a conventional liquid chromatographic system.

  9. On-column refolding of denatured lysozyme by the conjoint chromatography composed of SEC and immobilized recombinant DsbA.

    PubMed

    Luo, Man; Guan, Yi-Xin; Yao, Shan-Jing

    2011-10-15

    DsbA (disulfide bond formation protein A) located in the periplasm of Escherichia coli is a disulfide isomerase, which is vital to disulfide bonds formation directly affecting the nascent peptides folding to the correct conformation. In this paper, recombinant DsbA was firstly immobilized onto NHS-activated Sepharose Fast Flow gel. Then Sephadex G-100 gel was sequentially packed on the top of recDsbA Sepharose Fast Flow, and a so-called conjoint chromatography column composed of SEC and immobilized recombinant DsbA was constructed. Denatured lysozyme was applied on the conjoint column. The effect of SEC volume, flow rate, loading amount and volume, pre-equilibrium mode and KCl concentration in the buffer on lysozyme refolding were investigated in detail and the stability of DsbA immobilization was evaluated. Finally the reusability of the conjoint refolding column was also tested. When loading 2.4 mg denatured lysozyme in 0.5 ml solution, the activity recovery reached 92.7% at optimized experimental conditions, and the conjoint column renaturation capacity decreased only 7.7% after six run reuse due to the use of SEC section in the chromatographic refolding process. The conjoint chromatography offers an efficient strategy to refold proteins in vitro with high productivity and column reusability.

  10. High-performance liquid chromatography separation of unsaturated organic compounds by a monolithic silica column embedded with silver nanoparticles.

    PubMed

    Zhu, Yang; Morisato, Kei; Hasegawa, George; Moitra, Nirmalya; Kiyomura, Tsutomu; Kurata, Hiroki; Kanamori, Kazuyoshi; Nakanishi, Kazuki

    2015-08-01

    The optimization of a porous structure to ensure good separation performances is always a significant issue in high-performance liquid chromatography column design. Recently we reported the homogeneous embedment of Ag nanoparticles in periodic mesoporous silica monolith and the application of such Ag nanoparticles embedded silica monolith for the high-performance liquid chromatography separation of polyaromatic hydrocarbons. However, the separation performance remains to be improved and the retention mechanism as compared with the Ag ion high-performance liquid chromatography technique still needs to be clarified. In this research, Ag nanoparticles were introduced into a macro/mesoporous silica monolith with optimized pore parameters for high-performance liquid chromatography separations. Baseline separation of benzene, naphthalene, anthracene, and pyrene was achieved with the theoretical plate number for analyte naphthalene as 36,000 m(-1). Its separation function was further extended to cis/trans isomers of aromatic compounds where cis/trans stilbenes were chosen as a benchmark. Good separation of cis/trans-stilbene with separation factor as 7 and theoretical plate number as 76,000 m(-1) for cis-stilbene was obtained. The trans isomer, however, is retained more strongly, which contradicts the long- established retention rule of Ag ion chromatography. Such behavior of Ag nanoparticles embedded in a silica column can be attributed to the differences in the molecular geometric configuration of cis/trans stilbenes.

  11. [Determination of histamine in canned fish by high performance liquid chromatography with pre-column derivatization].

    PubMed

    Jin, Gaowa; Cai, Youqiong; Yu, Huijuan; Qian, Beilei

    2010-11-01

    A pre-column derivatization-high performance liquid chromatographic (HPLC) method has been developed for the determination of histamine in canned fish. The homogenated samples were ultrasonically extracted with perchloric acid aqueous solution, derivatized with dansyl chloride and diluted with acetonitrile to a desired volume. The samples were determined by HPLC with ultraviolet detector and quantified by external standard method. Adopting a C18 column with 1.8 microm stationary phase particles, the analysis time for each sample was smaller than 5 min with the flow rate of 0.3 mL/min. It can decrease the consumption of the mobile phase and save the cost. The linear range was 0.08-8.00 mg/L for histamine. The correlation coefficient was 0.999 98. The average recoveries of histamine at different concentration levels in spiked samples were greater than 96% and the relative standard deviations (RSDs) were smaller than 2.5%. The quantitation limit was 5.00 mg/kg for histamine in canned fish by HPLC. The results indicated that this HPLC method is fast, sensitive, reproducible and practical for the routine analysis of histamine in canned fish.

  12. Application of gas-liquid chromatography and high-performance liquid chromatography to the analysis of trace amounts of salicylic acid, acetylsalicylic anhydride and acetylsalicylsalicylic acid in aspirin samples and aspirin formulations.

    PubMed

    Ali, S L

    1976-11-03

    The gas-liquid chromatographic (GLC) determination of salicylic acid (SA) in 12 commercial acetylsalicylic acid (aspirin, ASA) samples and 12 ASA formulations is reported. The GLC determination of SA as an impurity in ASA, utilising methylation with methyl iodide in the presence of potassium carbonate, requires a column chromatographic separation of SA prior to derivatization. Trace amounts of SA in ASA have also been determined by high-performance liquid chromatography (HPLC) on a Sil-X-I adsorption column using light petroleum-ethyl acetate-acetic acid as the mobile phase. Acetylsalicylic anhydride (ASN) and acetylsalicylsalicylic acid (ASSA) were determined by HPLC on a reversed-phase C18 column with water-methanol mixtures as the mobile phase. GLC was also applied to the determination of ASN as an impurity in ASA formulations.

  13. Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. I. Injection procedure and column stability.

    PubMed

    Mengerink, Y; Peters, R; Kerkhoff, M; Hellenbrand, J; Omloo, H; Andrien, J; Vestjens, M; van der Wal, S

    2000-04-21

    We report a method for reliable routine polymer sample introduction with minimal bias, a separation method of the first six linear and cyclic oligomers by liquid chromatography, quantification using group equivalents and long term method performance. Injecting a polymer sample in a mobile phase containing an aqueous non-solvent often results in blocked systems as the polymer precipitates in the connecting capillaries. In this first part we focus on a new injection technique, in which the dissolved polyamide is placed between two zones of formic acid, preventing the polymer to precipitate before it reaches the column. Development of this sandwich injection method makes direct injection of the polymer into an aqueous acetonitrile gradient feasible. The oligomeric polyamide recovery of this technique, extraction, dissolution/precipitation and direct injection on a hexafluoro-isopropanol (HFIP) gradient were compared. With the sandwich injection method the polymer remains on the column, slowly changing the stationary phase. The influence of this on resolution and retention was studied. Column stability allows sixty injections before cleaning or replacing the column is necessary.

  14. [Application of spiral disk column in high-speed counter-current chromatography for peptide and protein separation].

    PubMed

    Hu, Guanghui; Cao, Xueli

    2009-04-01

    In order to improve the stationary phase retention of polar solvent systems and aqueous two-phase systems (ATPSs), we designed a multiple spiral disk assembly for type-J high-speed counter-current chromatography (HSCCC). The stationary phase retention was studied under different elution modes by using two solvent systems that contained 1-butanol-acetic acid-water (4:1:5, V/V/V) and polyethylene glycol (PEG) 1000-K2HPO4-water (12.5:12.5:75, W/W/W). The best retention was obtained in L-I-T, U-O-H, L-I-H three modes by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a relatively high flow rate. Meanwhile, the relationship between retention percentage of the stationary phase (Sf) and various parameters such as flow-rate (F), rotation speed (w) and column temperature (T) was also studied. Sf increased with the increase of w and decreased with the increase of F. Regression analysis showed a linear relationship between Sf and F1/2/w. The influence of T on Sf was not obvious between 20 degrees C and 40 degrees C, lower temperature than 20 degrees C was not suitable for viscous ATPSs. Acceptable resolutions were achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9.0) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8.0) system.

  15. Study on the Alkaloids in Tibetan Medicine Aconitum pendulum Busch by HPLC-MSn Combined with Column Chromatography.

    PubMed

    Wang, Beibei; Dong, Jie; Ji, Jiaojiao; Yuan, Jiang; Wang, Jiali; Wu, Jiarui; Tan, Peng; Liu, Yonggang

    2016-01-01

    A rapid, convenient and effective identification method of alkaloids was established and an attempt on isolating and analyzing the alkaloids in Aconitum pendulum Busch was conducted successfully. In this article, four high-content components including deoxyaconitine, benzoylaconine, aconine and neoline were isolated by using column chromatography. HPLC-MS(n)was employed to deduce the regulations of fragmentation of diterpenoid alkaloids which displayed a characteristic behavior of loss of CO(28u), CH3COOH(60u), CH3OH(32u), H2O(18u) and C6H5COOH(122u). Then, according to fragmentation regulation of mass spectrometry, 42 alkaloids were found inA. pendulum Among them, 38 compounds were identified and 29 alkaloids were reported for the first time for this herb. Therefore, this means that HPLC-MS(n)combined with column chromatography could work as an effective and reliable tool for rapid identification of the chemical components of herbal medicine.

  16. [Determination of 2-methyl-3-nitrobenzoic acid through pretreatment with diazomethane by gas chromatography].

    PubMed

    Xue, Ke-She; Nan, Zhi-Xiang

    2002-09-01

    A method for the quantitative determination of 2-methyl-3-nitrobenzoic acid by gas chromatography is described. 2-Methyl-3-nitrobenzoic acid was esterified by pretreatment with diazomethane prior to analysis. A CP-Sil-43CB capillary column(25 m x 0.32 mm i.d. x 0.2 microm), a flame ionization detector and the area normalization method were used. The average recovery was 99.81%. The RSD was 0.08% and the detection limit was 3 x 10(-11) g. The results showed that the method is practical and reliable. It was realized that the higher purity and higher boiler matter was analyzed by gas chromatography. The method can be used to monitor the purity of this type of materials. analysis of research and production. It can be used in the development of new products and in the process.

  17. Determination of dissolved bromate in drinking water by ion chromatography and post column reaction: interlaboratory study.

    PubMed

    Cordeiro, Fernando; Robouch, Piotr; de la Calle, Maria Beatriz; Emteborg, Håkan; Charoud-Got, Jean; Schmitz, Franz

    2011-01-01

    A collaborative study, International Evaluation Measurement Programme-25a, was conducted in accordance with international protocols to determine the performance characteristics of an analytical method for the determination of dissolved bromate in drinking water. The method should fulfill the analytical requirements of Council Directive 98/83/EC (referred to in this work as the Drinking Water Directive; DWD). The new draft standard method under investigation is based on ion chromatography followed by post-column reaction and UV detection. The collaborating laboratories used the Draft International Organization for Standardization (ISO)/Draft International Standard (DIS) 11206 document. The existing standard method (ISO 15061:2001) is based on ion chromatography using suppressed conductivity detection, in which a preconcentration step may be required for the determination of bromate concentrations as low as 3 to 5 microg/L. The new method includes a dilution step that reduces the matrix effects, thus allowing the determination of bromate concentrations down to 0.5 microg/L. Furthermore, the method aims to minimize any potential interference of chlorite ions. The collaborative study investigated different types of drinking water, such as soft, hard, and mineral water. Other types of water, such as raw water (untreated), swimming pool water, a blank (named river water), and a bromate standard solution, were included as test samples. All test matrixes except the swimming pool water were spiked with high-purity potassium bromate to obtain bromate concentrations ranging from 1.67 to 10.0 microg/L. Swimming pool water was not spiked, as this water was incurred with bromate. Test samples were dispatched to 17 laboratories from nine different countries. Sixteen participants reported results. The repeatability RSD (RSD(r)) ranged from 1.2 to 4.1%, while the reproducibility RSD (RSDR) ranged from 2.3 to 5.9%. These precision characteristics compare favorably with those of ISO

  18. Performance of different C18 columns in reversed-phase liquid chromatography with hydro-organic and micellar-organic mobile phases.

    PubMed

    Ruiz-Angel, M J; Pous-Torres, S; Carda-Broch, S; García-Alvarez-Coque, M C

    2014-05-30

    Column selection in reversed-phase liquid chromatography (RPLC) can become a challenge if the target compounds interact with the silica-based packing. One of such interactions is the attraction of cationic solutes to the free silanols in silica-based columns, which is a slow sorption-desorption interaction process that gives rise to tailed and broad peaks. The effect of silanols is minimised by the addition of a competing agent in the mobile phase, such as the anionic surfactant sodium dodecyl sulphate (SDS). In micellar-organic RPLC, the adsorption of an approximately fixed amount of SDS monomers gives rise to a stable modified stationary phase, with properties remarkably different from those of the underlying bonded phase. The chromatographic behaviour (in terms of selectivity, analysis time and peak shape) of eight C18 columns in the analysis of weakly acidic phenols and basic β-blockers was examined with hydro-organic and micellar-organic mobile phases. The behaviour of the columns differed significantly when the cationic basic drugs were eluted with hydro-organic mobile phases. With micellar-organic mobile phases, the adsorption of surfactant, instead of making the columns similar, gave rise to a greater diversity of behaviours (especially in terms of selectivity and analysis time), for both groups of phenols and β-blockers, which should be explained by the residual effect of the underlying bonded stationary phase and the different amount of surfactant covering the packing. Therefore, the implementation of a micellar-organic procedure in RPLC will depend significantly on the selected type of C18 column.

  19. Separation and conductimetric detection of C1-C7 aliphatic monocarboxylic acids and C1-C7 aliphatic monoamines on unfunctionized polymethacrylate resin columns.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi; Takeuchi, Toyohide

    2004-06-11

    The application of unfunctionized polymethacrylate resin (TSKgel G3000PWXL) as a stationary phase in liquid chromatography with conductimetric detection for C1-C7 aliphatic monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, 3,3-dimethylbutyric acid, 4-methylvaleric acid, hexanoic acid, 2-methylhexanoic acid, 5-methylhexanoic acid and heptanoic acid) and C1-C7 aliphatic monoamines (methylamine, ethylamine, propylamine, isobutylamine, butylamine, isoamylamine, amylamine, 1,3-dimethylbutylamine, hexylamine, 2-heptylamine and heptylamine) was attempted with C8 aliphatic monocarboxylic acids (2-propylvaleric acid, 2-ethylhexanoic acid, 2-methylheptanoic acid and octanoic acid) and C8 aliphatic monoamines (1,5-dimethylhexylamine, 2-ethylhexylamine, 1-methylheptylamine and octylamine) as eluents, respectively. Using 1 mM 2-methylheptanoic acid at pH 4.0 as the eluent, excellent separation and relatively high sensitive detection for these C1-C7 carboxylic acids were achieved on a TSKgel G3000PWXL column (150 mm x 6 mm i.d.) in 60 min. Using 2 mM octylamine at pH 11.0 as the eluent, excellent separation and relatively high sensitive detection for these C1-C7 amines were also achieved on the TSKgel G3000PWXL column in 60 min.

  20. Ion-exclusion chromatographic behavior of aliphatic carboxylic acids and benzenecarboxylic acids on a sulfonated styrene--divinylbenzene co-polymer resin column with sulfuric acid containing various alcohols as eluent.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi

    2003-05-16

    The addition of C1-C7 alcohols (methanol, ethanol, propanol, butanol, heptanol, hexanol and heptanol) to dilute sulfuric acid as eluent in ion-exclusion chromatography using a highly sulfonated styrene-divinylbenzene co-polymer resin (TSKgel SCX) in the H+ form as the stationary phase was carried out for the simultaneous separations of both (a) C1-C7 aliphatic carboxylic acids (formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, 2-methylvaleric, isocaproic, caproic, 2,2-dimethyl-n-valeric, 2-methylhexanoic, 5-methylhexanoic and heptanoic acids) and (b) benzenecarboxylic acids (pyromellitic, hemimellitic, trimellitic, o-phthalic, m-phthalic, p-phthalic, benzoic and salicylic acids and phenol). Heptanol was the most effective modifier in ion-exclusion chromatography for the improvement of peak shapes and a reduction in retention volumes for higher aliphatic carboxylic acids and benzenecarboxylic acids. Excellent simultaneous separation and relatively highly sensitive conductimetric detection for these C1-C7 aliphatic carboxylic acids were achieved on the TSKgel SCX column (150 x 6 mm I.D.) in 30 min using 0.5 mM sulfuric acid containing 0.025% heptanol as eluent. Excellent simultaneous separation and highly sensitive UV detection at 200 nm for these benzenecarboxylic acids were also achieved on the TSKgel SCX column in 30 min using 5 mM sulfuric acid containing 0.075% heptanol as eluent.

  1. Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method.

    PubMed

    Kumihashi, Mitsuru; Ameno, Kiyoshi; Shibayama, Takayuki; Suga, Keisuke; Miyauchi, Hiroshi; Jamal, Mostofa; Wang, Weihuan; Uekita, Ikuo; Ijiri, Iwao

    2007-01-01

    We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.

  2. Separation of hexabromocyclododecane diastereomers: Application of C18 and phenyl-hexyl ultra-performance liquid chromatography columns.

    PubMed

    Baek, Song-Yee; Lee, Sunyoung; Kim, Byungjoo

    2017-03-10

    This study was performed to establish the proper liquid chromatographic conditions for the separation of hexabromocyclododecane (HBCD) diastereomers. Column selectivity towards HBCD diastereomers was evaluated for C18 and phenyl-hexyl stationary phases. First, the baseline separation of the primary HBCDs (α-, β-, and γ-HBCD) was obtained using the ultra-performance liquid chromatography (UPLC) column with C18 stationary phase chosen in most previous studies for HBCD analysis; however, co-elution of δ- and ε-HBCD with the primary HBCD diastereomers was observed. To prevent the interference from δ- and ε-HBCD, we adopted a phenyl-hexyl UPLC column to resolve the HBCD diastereomers. The phenyl-hexyl UPLC column showed significantly different selectivity for the HBCD diastereomers compared with the C18 column, which allowed the clear isolation of δ-HBCD and ε-HBCD from the primary HBCD diastereomers. In addition, by checking the retention times of all HBCD diastereomers using both C18 and phenyl-hexyl columns, we confirmed the presence of δ-, ε-, η-, and θ-HBCDs in two technical HBCD mixtures.

  3. Simultaneous determination of acidic pesticides in vegetables and fruits by liquid chromatography--tandem mass spectrometry.

    PubMed

    Shida, Shizuka S; Nemoto, Satoru; Matsuda, Rieko

    2015-01-01

    A sensitive and efficient method has been developed for the simultaneous determination of 73 multi-class acidic pesticides, such as phenoxy acid and sulfonylurea herbicides, in vegetables and fruits. The sample preparation procedure was carefully optimized for the efficient removal of co-extracted matrix components. The method involves extraction of acidic pesticides with acetonitrile containing hydrochloric acid, removal of water from crude extract by salting out, and sequential cleanup by octadecylsilyl silica gel and silica gel columns. For samples containing high amounts of pigments, such as spinach, additional cleanup using a graphitized carbon column was performed prior to liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Recovery tests were performed for five times for each sample of cabbage, spinach, potato, eggplant, orange, and apple fortified at 0.01 mg kg-1. Out of the 73 tested pesticides, 70 for cabbage, 67 for spinach, 69 for potato, 67 for eggplant, 64 for orange, and 70 for apple were within the range of 70-120%, with relative standard deviations below 25%. Nitenpyram and pyrasulfotole showed low recoveries for all the samples tested, probably due to low recoveries from silica gel column. The developed method effectively removed co-extracted matrix components and was highly selective, with no interfering peaks found in the chromatograms of blank samples. The overall results indicate that the developed method is suitable for the quantitative analysis of acidic pesticide residues in vegetables and fruits.

  4. Radial heterogeneity of some analytical columns used in high-performance liquid chromatography

    SciTech Connect

    Mriziq, Khaled S; Guiochon, Georges A

    2009-01-01

    An on-column electrochemical microdetector was used to determine accurately the radial distribution of the mobile phase velocity and of the column efficiency at the exit of three common analytical columns, namely a 100 mm x 4.6 mm C18 bonded silica-based monolithic column, a 150 mm x 4.6 mm column packed with 2.7 {micro}m porous shell particles of C18 bonded silica (HALO), and a 150 mm x 4.6 mm column packed with 3 {micro}m fully porous C18 bonded silica particles (LUNA). The results obtained demonstrate that all three columns are not radially homogeneous. In all three cases, the efficiency was found to be lower in the wall region of the column than in its core region (the central core with a radius of 1/3 the column inner radius). The decrease in local efficiency from the core to the wall regions was lower in the case of the monolith (ca. 25%) than in that of the two particle-packed columns (ca. 35-50%). The mobile phase velocity was found to be ca. 1.5% higher in the wall than in the core region of the monolithic column while, in contrast, it was ca. 2.5-4.0% lower in the wall region for the two particle-packed columns.

  5. Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

    PubMed

    Zacharis, Constantinos K; Kalaitzantonakis, Eftichios A; Podgornik, Ales; Theodoridis, Georgios

    2007-03-09

    In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

  6. Determination of chlorophylls in Taraxacum formosanum by high-performance liquid chromatography-diode array detection-mass spectrometry and preparation by column chromatography.

    PubMed

    Loh, Chin Hoe; Inbaraj, Baskaran Stephen; Liu, Man Hai; Chen, Bing Huei

    2012-06-20

    Taraxacum formosanum, a well-known Chinese herb shown to be protective against hepatic cancer as well as liver and lung damage, may be attributed to the presence of abundant carotenoids and chlorophylls. However, the variety and content of chlorophylls remain uncertain. The objectives of this study were to develop an high-performance liquid chromatography-diode array detection-mass spectrometry method for determination of chlorophylls in T. formosanum and preparation by column chromatography. An HyPURITY C18 column and a gradient mobile phase of water (A), methanol (B), acetonitrile (C), and acetone (D) could resolve 10 chlorophylls and an internal standard Fast Green FCF within 30 min with a flow rate at 1 mL/min and detection at 660 nm. Both chlorophylls a and a' were present in the largest amount (1389.6 μg/g), followed by chlorophylls b and b' (561.2 μg/g), pheophytins a and a' (31.7 μg/g), hydroxychlorophyll b (26.5 μg/g), hydroxychlorophylls a and a' (9.8 μg/g), and chlorophyllides a and a' (0.35 μg/g). A glass column containing 52 g of magnesium oxide-diatomaceous earth (1:3, w/w) could elute chlorophylls with 800 mL of acetone containing 50% ethanol at a flow rate of 10 mL/min. Some new chlorophyll derivatives including chlorophyllide b, pyropheophorbide b, hydroxypheophytin a, and hydroxypheophytin a' were generated during column chromatography but accompanied by a 63% loss in total chlorophylls. Thus, the possibility of chlorophyll fraction prepared from T. formosanum as a raw material for future production of functional food needs further investigation.

  7. Analysis of sugar phosphates in plants by ion chromatography on a titanium dioxide column with pulsed amperometric detection.

    PubMed

    Sekiguchi, Yoko; Mitsuhashi, Naoto; Inoue, Yoshinori; Yagisawa, Hitoshi; Mimura, Tetsuro

    2004-06-11

    This paper describes the development of a practical method for the analysis of sugar phosphates from the model higher plant Arabidopsis thaliana by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The extraction method of sugar phosphates from higher plants was first optimized for HPAEC-PAD analysis. In order to improve the resolution in HPAEC-PAD, a column packed with titanium dioxide resin was used. The titanium dioxide column was used as a trap-column for sugar phosphates and nucleotides, for the removal of sample matrices. Sample pretreatment was achieved in-line and automatically using a six-port valve placed after the injection valve.

  8. A simple procedure for the preparation of fritless columns by entrapping conventional high performance liquid chromatography sorbents.

    PubMed

    Chirica, G S; Remcho, V T

    2000-09-01

    A rapid and direct method for immobilizing conventional high performance liquid chromatography (HPLC) packing material inside fritless capillaries has been developed. Due to the simple composition of the entrapment matrix (tetraethoxysilane, alkyltriethoxysilane, ethanol and water), straightforward manufacturing procedure and modest equipment requirement, the method can readily be transferred to any laboratory and easily automated. The entrapment procedure has minimal influence on the structure and chromatographic properties of the original reverse-phase sorbent. Various immobilization solutions have been tested, and a comparison between columns entrapped with different immobilization mixtures and conventional packed capillaries is presented. High efficiency separations were obtained using tert-butyl-triethoxysilane entrapped columns in both capillary electrochromatography (reduced plate heights of 1.1-1.4 were measured) and microliquid chromatography (reduced plate heights of 2.2-2.6 were observed) formats. Elimination of frits, stabilization of the packed bed and on-the-fly customization of column length render mechanically robust columns that are remarkably stable over time, from which manufacturing imperfections can be removed easily.

  9. Fingerprinting of traditional Chinese medicines on the C18-Diol mixed-mode column in online or offline two-dimensional liquid chromatography on the single column modes.

    PubMed

    Wang, Qing; Tong, Ling; Yao, Lin; Zhang, Peng; Xu, Li

    2016-06-05

    In the present study, a mixed-mode stationary phase, C18-Diol, was applied for fingerprint analysis of traditional Chinese medicines. Hydrophobic, hydrogen bonding and electrostatic interactions were demonstrated to contribute the retention separately or jointly, which endowed the C18-Diol stationary phase with distinct selectivity compared to the bare C18 one. The separation of total alkaloids extracted from Fritillaria hupehensis was compared on the C18-Diol and conventional C18 column with the greater resolving power and better symmetry responses on the former one. Besides, a novel two-dimensional liquid chromatography on the single column (2D-LC-1C) was realized on C18-Diol with the offline mode for the alcohol extract of Fritillaria hupehensis and online mode for Ligusticum chuanxiong Hort. The early co-eluted extracted components with great polarity on the first dimension were reinjected on the same column and well separated on the second dimension. The results exhibited that the two complementary RPLC and HILIC modes on C18-Diol stationary phase enhanced the separation capacity and revealed more abundant chemical information of the sample, which was a powerful tool in analyzing complex herbal medicines.

  10. Muramic Acid Measurements for Bacterial Investigations in Marine Environments by High-Pressure Liquid Chromatography

    PubMed Central

    Mimura, Toru; Romano, Jean-Claude

    1985-01-01

    Muramic acid, a constituent of procaryotic cell walls, was assayed by high-pressure liquid chromatography in samples from several marine environments (water column, surface microlayer, and sediment) and a bacterial culture. It is used as a microbial biomass indicator. The method gave a good separation of muramic acid from interfering compounds with satisfactory reproducibility. A pseudomonad culture had a muramic acid content of 4.7 × 10−10 to 5.3 × 10−10 μg per cell during growth. In natural water samples, highly significant relationships were found between muramic acid concentrations and bacterial numbers for populations of 108 to 1011 cells per liter. The muramic acid content in natural marine water decreased from 5.3 × 10−10 to 1.6 × 10−10 μg per cell with increasing depth. In coastal sediments exposed to sewage pollution, concentrations of muramic acid, ATP, organic carbon, and total amino acids displayed a parallel decrease with increasing distance from the sewage outlet. Advantages of muramic acid measurement by high-pressure liquid chromatography are its high sensitivity and reduction of preparation steps, allowing a short time analysis. PMID:16346848

  11. Quantitative Evaluation of Models for Solvent-based, On-column Focusing in Liquid Chromatography

    PubMed Central

    Groskreutz, Stephen R.; Weber, Stephen G.

    2015-01-01

    On-column focusing or preconcentration is a well-known approach to increase concentration sensitivity by generating transient conditions during the injection that result in high solute retention. Preconcentration results from two phenomena: 1) solutes are retained as they enter the column. Their velocities are k′-dependent and lower than the mobile phase velocity and 2) zones are compressed due to the step-gradient resulting from the higher elution strength mobile phase passing through the solute zones. Several workers have derived the result that the ratio of the eluted zone width (in time) to the injected time width is the ratio k2/k1 where k1 is the retention factor of a solute in the sample solvent and k2 is the retention factor in the mobile phase (isocratic). Mills et al. proposed a different factor. To date, neither of the models has been adequately tested. The goal of this work was to evaluate quantitatively these two models. We used n-alkyl esters of p-hydroxybenzoic acid (parabens) as solutes. By making large injections to create obvious volume overload, we could measure accurately the ratio of widths (eluted/injected) over a range of values of k1 and k2. The Mills et al. model does not fit the data. The data are in general agreement with the factor k2/k1, but focusing is about 10% better than the prediction. We attribute the extra focusing to the fact that the second, compression, phenomenon provides a narrower zone than that expected for the passage of a step gradient through the zone. PMID:26210110

  12. New silica gel-based monolithic column for nano-liquid chromatography, used in the HILIC mode.

    PubMed

    Silva, Raquel G C; Bottoli, Carla B G; Collins, Carol H

    2012-09-01

    This paper describes the synthesis and chromatographic and morphologic characterization of two monolithic silica nano-columns (50 µm i.d.) prepared by sol-gel processes, using hydrophilic interaction (HILIC) mode separations to evaluate their performance. Two types of monoliths were prepared by varying the precursors (tetraethoxysilane or a tetraethoxysilane-methyltrimethoxysilane mixture) and by changing the type of catalyst (urea and acetic acid or ammonium hydroxide). The monoliths were characterized by scanning electron microscopy, thermogravimetric analysis, infrared spectroscopy and nitrogen adsorption-desorption isotherms. The columns were tested for the separation of several mixtures, with the organically modified silica (ormosil) column successfully separating two challenging mixtures using HILIC conditions.

  13. Polymethacrylate monolithic and hybrid particle-monolithic columns for reversed-phase and hydrophilic interaction capillary liquid chromatography.

    PubMed

    Jandera, Pavel; Urban, Jirí; Skeríková, Veronika; Langmaier, Pavel; Kubícková, Romana; Planeta, Josef

    2010-01-01

    We prepared hybrid particle-monolithic polymethacrylate columns for micro-HPLC by in situ polymerization in fused silica capillaries pre-packed with 3-5microm C(18) and aminopropyl silica bonded particles, using polymerization mixtures based on laurylmethacrylate-ethylene dimethacrylate (co)polymers for the reversed-phase (RP) mode and [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl) zwitterionic (co)polymers for the hydrophilic interaction (HILIC) mode. The hybrid particle-monolithic columns showed reduced porosity and hold-up volumes, approximately 2-2.5 times lower in comparison to the pure monolithic columns prepared in the whole volume of empty capillaries. The elution volumes of sample compounds are also generally lower in comparison to packed or pure monolithic columns. The efficiency and permeability of the hybrid columns are intermediate in between the properties of the reference pure monolithic and particle-packed columns. The chemistries of the embedded solid particles and of the interparticle monolithic moiety in the hybrid capillary columns contribute to the retention to various degrees, affecting the selectivity of separation. Some hybrid columns provided improved separations of proteins in comparison to the reference particle-packed columns in the reversed-phase mode. Zwitterionic hybrid particle-monolithic columns show dual mode retention HILIC/RP behaviour depending on the composition of the mobile phase and allow separations of polar compounds such as phenolic acids in the HILIC mode at lower concentrations of acetonitrile and, often in shorter analysis time in comparison to particle-packed and full-volume monolithic columns.

  14. Direct determination of resin and fatty acids in process waters of paper industries by liquid chromatography/mass spectrometry.

    PubMed

    Rigol, A; Latorre, A; Lacorte, S; Barceló, D

    2003-04-01

    Liquid chromatography/mass spectrometry (LC/MS)-based methods were developed for the analysis of 10 resin acids and five fatty acids in process waters of paper industries. No fragmentation of target compounds was observed using atmospheric pressure chemical ionization (APCI) with negative ionization. The [M - H](-) ion permitted the individual quantification of fatty and aromatic resin acids, whereas the non-aromatic resin acids presented a single and common ion at m/z 301. Separation with two columns of different polarity permitted peak confirmation. The method that used a C(8) column with 2-propanol in the mobile phase allowed a certain separation and identification of the non-aromatic resin acids, whereas the method using a C(18) column provided detection limits 10-fold lower for fatty acids. Limits of detection were 0.10 ng for all compounds. Direct sample introduction was compared with liquid-liquid extraction, with similar recoveries (70-101%). Whereas slightly lower detection limits were obtained with liquid-liquid extraction, better reproducibility was observed for direct sample introduction. Resin and fatty acids were determined in process waters of several paper industries. Palmitic, dehydroabietic and non-aromatic resin acids were encountered in most water samples, at levels between 22 and 403 micro g l(-1). LC/MS with direct sample introduction was found to be a good alternative to traditional liquid-liquid extraction and gas chromatography for the analysis of such compounds since no derivatization was required and sample manipulation was minimal.

  15. Exogenous factors contributing to column bed heterogeneity: Part 1: Consequences of 'air' injections in liquid chromatography.

    PubMed

    Samuelsson, Jörgen; Fornstedt, Torgny; Shalliker, Andrew

    2015-08-07

    It has been shown that not only the packing homogeneity, but also factors external to the column bed, such as, frits and distributors can have important effects on the column performance. This current communication is the first in a series focusing on the impact of exogenous factors on the column bed heterogeneity. This study is based on several observations by us and others that chromatographic runs often, for technical reasons, include more or less portions of air in the injections. It is therefore extremely important to find out the impact of air on the column performance, the reliability of the results derived from analyses where air was injected, and the effect on the column homogeneity. We used a photographic approach for visualising the air transport phenomena, and found that the air transport through the column is comprised of many different types of transport phenomena, such as laminal flow, viscous fingering like flows, channels and bulbs, and pulsations. More particularly, the air clouds within the column definitely interact in the adsorption, i.e. mobile phase adsorbed to the column surface is displaced. In addition, irrespective of the type of air transport phenomena, the air does not penetrate the column homogeneously. This process is strongly flow dependent. In this work we study air transport both in an analytical scale and a semi-prep column.

  16. [Determination of trace haloacetic acids in drinking water using ion chromatography coupled with solid phase extraction].

    PubMed

    Sun, Yingxue; Huang, Jianjun; Gu, Ping

    2006-05-01

    The combined solid phase extraction (SPE)-ion chromatography (IC) method was developed for the analysis of trace haloacetic acids (HAAs) in drinking water. The tested HAAs included monochloroacetic acid (MCAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), monobromoacetic acid (MBAA) and dibromoacetic acid (DBAA). For trace determination of HAAs in real drinking water samples, conditions of LiChrolut EN SPE cartridge were investigated for HAAs preconcentration and matrix elimination. Elution was carried out by 2 mL of sodium hydroxide (10 mmol/L) with the flow rate of 2 mL/min. The Dionex IonPac AS16 column (250 mm x 4 mm i. d.), a high capacity and hydroxide-selective anion-exchange column designed for the determination of polarizable anions, was chosen for chromatographic separation. HAAs were analyzed with a concentration gradient of NaOH with the flow rate of 0.8 mL/min and detected by suppressed conductivity. A 500 microL sample loop was used. The detection limits of this SPE-IC method for MCAA, DCAA, DBAA and TCAA were 0.38-1.69 microg/L and MBAA was 12.5 microg/L under 25-fold preconcentration. The results demonstrate that the method is suitable for the analysis of trace haloacetic acids in drinking water.

  17. Determination of ricin by nano liquid chromatography/mass spectrometry after extraction using lactose-immobilized monolithic silica spin column.

    PubMed

    Kanamori-Kataoka, Mieko; Kato, Haruhito; Uzawa, Hirotaka; Ohta, Shigenori; Takei, Yoshiyuki; Furuno, Masahiro; Seto, Yasuo

    2011-08-01

    Ricin is a glycosylated proteinous toxin that is registered as toxic substance by Chemical Weapons convention. Current detection methods can result in false negatives and/or positives, and their criteria are not based on the identification of the protein amino acid sequences. In this study, lactose-immobilized monolithic silica extraction followed by tryptic digestion and liquid chromatography/mass spectrometry (LC/MS) was developed as a method for rapid and accurate determination of ricin. Lactose, which was immobilized on monolithic silica, was used as a capture ligand for ricin extraction from the sample solution, and the silica was supported in a disk-packed spin column. Recovery of ricin was more than 40%. After extraction, the extract was digested with trypsin and analyzed by LC/MS. The accurate masses of molecular ions and MS/MS spectra of the separated peptide peaks were measured by Fourier transform-MS and linear iontrap-MS, respectively. Six peptides, which were derived from the ricin A-(m/z 537.8, 448.8 and 586.8) and B-chains (m/z 701.3, 647.8 and 616.8), were chosen as marker peptides for the identification of ricin. Among these marker peptides, two peptides were ricin-specific. This method was applied to the determination of ricin from crude samples. The monolithic silica extraction removed most contaminant peaks from the total ion chromatogram of the sample, and the six marker peptides were clearly detected by LC/MS. It takes about 5 h for detection and identification of more than 8 ng/ml of ricin through the whole handling, and this procedure will be able to deal with the terrorism using chemical weapon.

  18. Conventional and enantioselective gas chromatography with microfabricated planar columns for analysis of real-world samples of plant volatile fraction.

    PubMed

    Cagliero, C; Galli, S; Galli, M; Elmi, I; Belluce, M; Zampolli, S; Sgorbini, B; Rubiolo, P; Bicchi, C

    2016-01-15

    Within a project exploring the application of lab-on-chip GC to in-field analysis of the plant volatile fraction, this study evaluated the performance of a set of planar columns (also known as microchannels, MEMS columns, or microfabricated columns) of different dimensions installed in a conventional GC unit. Circular double-spiral-shaped-channel planar columns with different square/rectangular sections up to 2m long were applied to the analysis of both essential oils and headspace samples of a group of medicinal and aromatic plants (chamomile, peppermint, sage, rosemary, lavender and bergamot) and of standard mixtures of related compounds; the results were compared to those obtained with reference narrow-bore columns (l:5m, dc:0.1mm, df:0.1 μm). The above essential oils and headspaces were first analyzed quali-and quantitatively with planar columns statically coated with conventional stationary phases (5%-phenyl-polymethylsiloxane and auto-bondable nitroterephthalic-acid-modified polyethylene glycol), and then submitted to chiral recognition of their diagnostic markers, by enantioselective GC with a planar columns coated with a cyclodextrin derivative (30% 6(I-VII)-O-TBDMS-3(I-VII)-O-ethyl-2(I-VII)-O-ethyl-β-cyclodextrin in PS-086). Column characteristics and analysis conditions were first optimized to obtain suitable retention and efficiency for the samples investigated. The planar columns tested showed performances close to the reference conventional narrow-bore columns, with theoretical plate numbers per meter (N/m) ranging from 6100 to 7200 for those coated with the conventional stationary phases, and above 5600 for those with the chiral selector.

  19. Survey of organic acid eluents for anion chromatography

    SciTech Connect

    Book, D.E.

    1981-10-01

    Of all the potential eluents surveyed (including aromatic, sulfonic, phosphonic, among other acids), only the carboxylic acids and the nitrophenols are recommended as eluents for anion chromatography. The concentration of the eluent should be in the range 5 x 10/sup -5/ to 1 x 10/sup -3/ M. The eluent should have the same charge as inorganic anions, a higher charge than organic acid samples. Choice of eluents for separation of halides, chloride and sulfate, multivalent inorganic anions, small alkyl acids, and aromatic acids is discussed. (DLC)

  20. Robust design of adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography.

    PubMed

    Nestola, Piergiuseppe; Silva, Ricardo J S; Peixoto, Cristina; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2015-11-10

    A simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86 percent and DNA and HCP clearances of 90 and 89 percent, respectively, without any fine tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process the best set of operating parameters is selected only among candidate solutions that are robust feasible, that is, remain feasible for all parameter perturbations within their uncertainty intervals. This robust approach to optimal design replaces the nominal problem by a worst case problem. Computational tractability is ensured by formulating the robust design problem with only the vertices of the uncertainty region that have the worst effect on the product purity and recovery. The robust design is exemplified on the case where the column volume and interparticle porosity are subject to uncertainty. As expected, to increase the robustness of the 2CSMB-SEC process it is necessary to reduce its productivity and increase its solvent consumption. Nevertheless, the design solution given by our robust approach is the least detrimental of all feasible operating conditions for the 2CSMB-SEC process.

  1. Isolation and purification of diastereoisomeric flavonolignans from silymarin by binary-column recycling preparative high-performance liquid chromatography.

    PubMed

    Zhao, Weiquan; Yang, Guang; Zhong, Fanyi; Yang, Nan; Zhao, Xin; Qi, Yunpeng; Fan, Guorong

    2014-09-01

    Silymarin extracted from Silybum marianum (L.) Gaertn consists of a large number of flavonolignans, of which diastereoisomeric flavonolignans including silybin A and silybin B, and isosilybin A and isosilybin B are the main bioactive components, whose preparation from the crude extracts is still a difficult task. In this work, binary-column recycling preparative high-performance liquid chromatography systems without sample loop trapping, where two columns were switched alternately via one or two six-port switching valves, were established and successfully applied to the isolation and purification of the four diastereoisomeric flavonolignans from silymarin. The proposed system showed significant advantages over conventional preparative high-performance liquid chromatography with a single column in increasing efficiency and reducing the cost. To obtain the same amounts of products, the proposed system spends only one tenth of the time that the conventional system spends, and needs only one eleventh of the solvent that the conventional system consumes. Using the proposed system, the four diastereoisomers were successfully isolated from silymarin with purities over 98%.

  2. Analysis of oil-biodiesel samples by high performance liquid chromatography using the normal phase column of new generation and the evaporative light scattering detector.

    PubMed

    Fedosov, Sergey N; Fernandes, Natalia A; Firdaus, Mohd Y

    2014-01-24

    Conversion of vegetable oil to biodiesel is usually monitored by gas chromatography. This is not always convenient because of (i) an elaborate derivatization of the samples; (ii) inhibition of this process by methanol and water; (iii) low stability of the derivatives under storage. HPLC methods are apparently more convenient, but none of the described variants had won a wide recognition so far. This can be ascribed to the problems of reproducibility (in the case of normal phase chromatography) and limited separation of some analytes (in the case of reverse phase chromatography). Here we report an HPLC procedure suitable for separation of biodiesel, free fatty acids, glycerides, glycerol and lecithin. The normal phase column of new generation (Poroshell 120 HILIC) and the novel gradient were used. The method was tested on both the artificial mixtures and the crude reaction samples. Elution of the analytes was monitored by an evaporative light scattering detector. This method is usually confined to a very limited range of masses, where only a part of the complex calibration curve is used. We have analyzed the light scattering signal within a very broad range of masses, whereupon the calibration curves were produced. The data were approximated by the appropriate equations used afterward to recalculate the signal to the mass in a convenient way. An experimental conversion of rapeseed oil to biodiesel was performed by a liquid lipase formulation. This process was monitored by HPLC to illustrate advantages of the suggested registration method.

  3. Monolithic columns with organic sorbent based on poly-1-vinylimidazole for high performance liquid chromatography

    NASA Astrophysics Data System (ADS)

    Patrushev, Y. V.; Sidelnikov, V. N.; Yudina, Y. S.

    2017-03-01

    Monolithic chromatographic columns for HPLC with sorbent based on 1-vinylimidazole are prepared. It is shown that changing the 1-vinylimidazole content in the initial solution allows us to change the polarity of columns. An example of aromatic hydrocarbons separation is presented.

  4. Planar gas chromatography column on glass plate with nanodispersed silica as the stationary phase

    NASA Astrophysics Data System (ADS)

    Platonov, I. A.; Platonov, V. I.; Pavelyev, V. S.; Agafonov, A. N.

    2016-04-01

    The paper presents the GC column in the plane of the glass plate with the adsorption layer nanodispersed silica. Created gas chromatographic column allows to separate a mixture of five alkanes from pentane to nonane in isothermal (90 ° C) mode less than one minute.

  5. Effects of a malfunctional column on conventional and FeedCol-simulated moving bed chromatography performance.

    PubMed

    Song, Ji-Yeon; Oh, Donghoon; Lee, Chang-Ha

    2015-07-17

    The effects of a malfunctional column on the performance of a simulated moving bed (SMB) process were studied experimentally and theoretically. The experimental results of conventional four-zone SMB (2-2-2-2 configuration) and FeedCol operation (2-2-2-2 configuration with one feed column) with one malfunctional column were compared with simulation results of the corresponding SMB processes with a normal column configuration. The malfunctional column in SMB processes significantly deteriorated raffinate purity. However, the extract purity was equivalent or slightly improved compared with the corresponding normal SMB operation because the complete separation zone of the malfunctional column moved to a lower flow rate range in zones II and III. With the malfunctional column configuration, FeedCol operation gave better experimental performance (up to 7%) than conventional SMB operation because controlling product purity with FeedCol operation was more flexible through the use of two additional operating variables, injection time and injection length. Thus, compared with conventional SMB separation, extract with equivalent or slightly better purity could be produced from FeedCol operation even with a malfunctional column, while minimizing the decrease in raffinate purity (less than 2%).

  6. Quasi-adiabatic vacuum-based column housing for very high-pressure liquid chromatography.

    PubMed

    Gritti, Fabrice; Gilar, Martin; Jarrell, Joseph A

    2016-07-22

    A prototype vacuum-based (10(-6)Torr) column housing was built to thermally isolate the chromatographic column from the external air environment. The heat transfer mechanism is solely controlled by surface radiation, which was minimized by wrapping the column with low-emissivity aluminum tape. The adiabaticity of the column housing was quantitatively assessed from the measurement of the operational pressure and fluid temperature at the outlet of a 2.1mm×100mm column (sub-2 μm particles). The pressure drop along the column was raised up to 1kbar. The enthalpy balance of the eluent (water, acetonitrile, and one water/acetonitrile mixture, 70/30, v/v) showed that less than 1% of the viscous heat generated by friction of the fluid against the packed bed was lost to the external air environment. Such a vacuum-based column oven minimizes the amplitude of the radial temperature gradients across the column diameter and maximizes its resolving power.

  7. Separation of intact proteins on γ-ray-induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high-performance liquid chromatography with high-resolution mass spectrometry.

    PubMed

    Simone, Patrizia; Pierri, Giuseppe; Foglia, Patrizia; Gasparrini, Francesca; Mazzoccanti, Giulia; Capriotti, Anna Laura; Ursini, Ornella; Ciogli, Alessia; Laganà, Aldo

    2016-01-01

    Polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion-pair reversed-phase capillary liquid chromatography with high-resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow-split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high-resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ-ray-induced monolithic column lies in the batch-to-batch reproducibility and long-term high-temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene-divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.

  8. Determination of phenoxy acid herbicides in water by electron-capture and microcoulometric gas chromatography

    USGS Publications Warehouse

    Goerlitz, D.F.; Lamar, William L.

    1967-01-01

    A sensitive gas chromatographic method using microcoulometric titration and electron-capture detection for the analysis of 2,4-D, silvex, 2,4,5-T, and other phenoxy acid herbicides in water is described. The herbicides are extracted from unfiltered water samples (800-1,000 ml) by use of ethyl ether ; then the herbicides are concentrated and esterilied. To allow the analyst a choice, two esterilication procedures--using either boron trifluoride-methanol or diazomethane--are evaluated. Microcoulometric gas chromatography is specific for the detection of halogenated compounds such as the phenoxy acid herbicides whereas it does not respond to nonhalogenated components. Microcoulometric gas chromatography requires care and patience. It is not convenient for rapid screening of l-liter samples that contain less than 1 microgram of the herbicide. Although electroncapture gas chromatography is less selective and more critically affected by interfering substances, it is, nevertheless, convenient and more sensitive than microcoulometric gas chromatography. Two different liquid phases are used in the gas chromatographic columns--DC-200 silicone in one column and QF-1 silicone in the other. The performance of both columns is improved by the addition of Carbowax 20M. The Gas Chrom Q support is coated with the liquid phases by the 'frontal-analysis' technique. The practical lower limits for measurement of the phenoxy acid herbicides in water primarily depend upon the sample size, interferences present, anal instrumentation used. With l-liter samples of water, the practical lower limits of measurement are 10 ppt (parts per trillion) for 2,4-D and 2 ppt for silvex and 2,4,5-T when electron-capture detection is used, and approximately 20 ppt for each herbicide when analyzed by microcoulometric-titration gas chromatography. Recoveries of the herbicides immediately after addition to unfiltered water samples averaged 92 percent for 2,4-D, 90 percent for silvex, and 98 percent for 2

  9. Amino acid analysis in micrograms of meteorite sample by nanoliquid chromatography-high-resolution mass spectrometry.

    PubMed

    Callahan, Michael P; Martin, Mildred G; Burton, Aaron S; Glavin, Daniel P; Dworkin, Jason P

    2014-03-07

    Amino acids and their enantiomers in a 360 microgram sample of Murchison meteorite were unambiguously identified and quantified using chemical derivatization and nanoliquid chromatography coupled to nanoelectrospray ionization high resolution orbitrap mass spectrometry techniques. The distribution and abundance of amino acids were similar to past studies of Murchison meteorite but the samples used here were three orders of magnitude lower. The analytical method was also highly sensitive, and some amino acid reference standards were successfully detected at a level of ∼200 attomoles (on column). These results may open up the possibility for investigating other less studied, sample-limited extraterrestrial samples (e.g., micrometeorites, interplanetary dust particles, and cometary particles) for biologically-relevant organic molecules.

  10. Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography.

    PubMed

    Arakawa, Tsutomu; Tsumoto, Kouhei; Ejima, Daisuke; Kita, Yoshiko; Yonezawa, Yasushi; Tokunaga, Masao

    2007-04-10

    In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.

  11. Two-column sequential injection chromatography for fast isocratic separation of two analytes of greatly differing chemical properties.

    PubMed

    Šatínský, Dalibor; Chocholouš, Petr; Válová, Olga; Hanusová, Lucia; Solich, Petr

    2013-09-30

    This paper deals with a novel approach to separate two analytes with different chemical properties and different lipophilicity. The newly described methodology is based on the two column system that was used for isocratic separation of two analytes with very different lipophilicity-dexamethasone and cinchocaine. Simultaneous separation of model compounds cinchocaine and dexamethasone was carried under the following conditions in two-column sequential injection chromatography system (2-C SIC). A 25×4.6 mm C-18 monolithic column was used in the first dimension for retention and separation of dexamethasone with mobile phase acetonitrile:water 30:70 (v/v), flow rate 0.9 mL min(-1) and consumption of 1.7 mL. A 10×4.6 mm C-18 monolithic column with 5×4.6 mm C-18 precolumn was used in the second dimension for retention and separation of cinchocaine using mobile phase acetonitrile:water 60:40 (v/v), flow rate 0.9 mL min(-1) and consumption 1.5 mL. Whole analysis time including both mobile phase's aspirations and both column separations was performed in less than 4 min. The method was fully validated and used for determination of cinchocaine and dexamethasone in pharmaceutical otic drops. The developed 2-C SIC method was compared with HPLC method under the isocratic conditions of separation on monolithic column (25×4.6 mm C-18). Spectrophotometric detection of both compounds was performed at wavelength 240 nm. System repeatability and method precision were found in the range (0.39-3.12%) for both compounds. Linearity of determination was evaluated in the range 50-500 μg mL(-1) and coefficients of determination were found to be r(2)=0.99912 for dexamethasone and r(2)=0.99969 for cinchocaine.

  12. Separation of beta-human chorionic gonadotropin and immunoglobulin G by a miniaturized size exclusion chromatography column

    NASA Astrophysics Data System (ADS)

    Yang, Yongmo; Chae, Junseok

    2009-04-01

    This report describes a miniaturized size exclusion chromatography column that effectively preseparates raw samples for medical point-of-care testing (POCT) devices. The minicolumn is constructed of polydimethylsiloxane fabricated on a glass slide. The minicolumn separates 300 ng/ml of beta-human chorionic gonadotropin (β-hCG) from an immunoglobulin G (IgG)-rich solution (100 μg/ml) in 7.7 min, with 2.23 resolution and 0.018 mm plate height. The complete analyte discrimination shows potential for the sample preparation stage of POCT devices for cancer screening, prognosis, and monitoring.

  13. High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

    PubMed

    Li, D Q; Zhao, J; Li, S P

    2014-06-06

    Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures.

  14. Determination of trace levels of haloacetic acids and perchlorate in drinking water by ion chromatography with direct injection.

    PubMed

    Liu, Yongjian; Mou, Shifen

    2003-05-16

    Disinfection by products of haloacetic acids and perchlorate pose significant health risks, even at low microg/l levels in drinking water. A new method for the simultaneous determination of nine haloacetic acids (HAAs) and perchlorate as well as some common anions in one run with ion chromatography was developed. The HAAs tested included mono-, di-, trichloroacetic acids, mono, di-, tribromoacetic acids, bromochloroacetic acid, dibromochloroacetic acid, and bromodichloroacetic acid. Two high-capacity anion-exchange columns, a carbonate-selective column and a hydroxide-selective hydrophilic one, were used for the investigation. With the carbonate-selective column, the nine HAAs as well as fluoride, chloride, nitrite, nitrate, phosphate and sulfate could be well separated and determined in one run. With the very hydrophilic column and a gradient elution of sodium hydroxide, methanol and deionized water, the nine HAAs, fluoride, chloride, nitrite, nitrate as well as perchlorate could be simultaneously determined in one run within 34 min. The detection limits for HAAs were between 1.11 and 9.32 microg/l. For perchlorate, it was 0.60 microg/l.

  15. Micro-fabricated packed gas chromatography column based on laser etching technology.

    PubMed

    Sun, J H; Guan, F Y; Zhu, X F; Ning, Z W; Ma, T J; Liu, J H; Deng, T

    2016-01-15

    In this work, a micro packed gas chromatograph column integrated with a micro heater was fabricated by using laser etching technology (LET) for analyzing environmental gases. LET is a powerful tool to etch deep well-shaped channels on the glass wafer, and it is the most effective way to increase depth of channels. The fabricated packed GC column with a length of over 1.6m, to our best knowledge, which is the longest so far. In addition, the fabricated column with a rectangular cross section of 1.2mm (depth) × 0.6mm (width) has a large aspect ratio of 2:1. The results show that the fabricated packed column had a large sample capacity, achieved a separation efficiency of about 5800 plates/m and eluted highly symmetrical Gaussian peaks.

  16. Direct determination of benzamides in serum by column-switching high-performance liquid chromatography.

    PubMed

    Chiba, Ryoko; Ogasawara, Ayako; Kubo, Teppei; Yamazaki, Hiroyuki; Umino, Masuo; Ishizuka, Yoichi

    2003-05-01

    A column-switching high-performance liquid chromatographic method with fluorescence detection was developed for the simultaneous determination of four benzamide-type anti-psychotic drugs: sulpiride, tiapride, sultopride and metoclopramide in human serum. In this method, a TSKgel Super-ODS column was used as an analytical column, and a TSKgel G 2000SW was prepared as a pretreatment column. Under the optimized analytical conditions, four benzamide-type anti-psychotic drugs were eluted within 18 min. The detection limits (S/N = 3) for sulpiride, tiapride, sultopride and metoclopramide are 1 ng/ml, 4 ng/ml, 2 ng/ml and 0.5 ng/ml, respectively. Finally, the method was applied to the determination of sulpiride in human serum samples obtained after a single oral dose of sulpiride.

  17. Zero-derivative boundary condition for pulsed distributed systems. [column chromatography example

    NASA Technical Reports Server (NTRS)

    Lashmet, P. K.; Woodrow, P. T.

    1975-01-01

    To permit use of experimentally determined Peclet numbers in numerical simulations of pulsed distributed flow systems such as chromatograph columns, substitution of the zero-derivative boundary condition for the infinite boundary condition used in treating data is examined. Moment analysis shows that application of the zero-derivative condition external to the column will yield equivalent numerical results for the two boundary conditions. Criteria for locating this position are provided as a function of the Peclet number.

  18. Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

    SciTech Connect

    Evans, D.M.; Williams, K.P.; McGuinness, B.

    1996-04-01

    The authors have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A {open_quotes}target{close_quotes} column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against {beta}-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughput screening of chemical libraries or natural product extracts. 21 refs., 4 figs.

  19. Complete temperature profiles in ultra-high-pressure liquid chromatography columns.

    PubMed

    Gritti, Fabrice; Guiochon, Georges

    2008-07-01

    The temperature profiles were calculated along and across seven packed columns (lengths 30, 50, 100, and 150 mm, i.d., 1 and 2.1 mm, all packed with Acquity UPLC, BEH-C 18 particles, average d(p) approximately 1.7 microm) and their stainless steel tubes (o.d. 4.53 and 6.35 mm). These columns were kept horizontal and sheltered from forced air convection (i.e., under still air conditions), at room temperature. They were all percolated with pure acetonitrile, either under the maximum pressure drop (1034 bar) or at the maximum flow rate (2 mL/min) permitted by the chromatograph. The heat balance equation of chromatographic columns was discretized and solved numerically with minimum approximation. Both the compressibility and the thermal expansion of the eluent were taken into account. The boundary conditions were determined from the experimental measurements of the column inlet pressure and of the temperature profile along the column wall, which were made with a precision better than +/-0.1 K. These calculation results provide the 3-D temperature profiles along and across the columns. The axial and radial temperature gradients are discussed in relationship with the experimental conditions used. The temperature map obtained permits a prediction of the chromatographic data obtained under a very high pressure gradient.

  20. The Binding of Biotin to Sepharose-Avidin Column: Demonstration of the Affinity Chromatography Technique

    ERIC Educational Resources Information Center

    Landman, A. D.; Landman, N. N.

    1976-01-01

    Describes a biochemistry experiment that illustrates the methodology of affinity chromatography by attaching avidin, a glycoprotein in egg white, to a Sepharose matrix in order to bind biotin-containing proteins. (MLH)

  1. Analysis of Free Fatty Acids on the Fingertips by High Performance Liquid Chromatography.

    DTIC Science & Technology

    1978-12-20

    This investigation studied the efficiency of high performance liquid chromatography in the determination of free fatty acids present on the...utilized to eliminate the microbial contamination. The high performance liquid chromatography provided excellent separation of skin fatty acids for

  2. [Simultaneous determination of 16 organic acids in feed additives by on-line enrichment and ion chromatography-mass spectrometry].

    PubMed

    Xiong, Zhiyu; Dong, Ying; Zhou, Hongbin; Yu, Yang; Li, Jing; Sun, Li

    2014-02-01

    A novel analytical method for simultaneous determination of sixteen organic acids by on-line enrichment and ion chromatography-mass spectrometry (IC-MS) was developed. Online enrichment and separation of the organic acids were performed by ion chromatography on a homemade enrichment column and a homemade separation column. The qualitative and quantitative analyses of the organic acids were performed by mass spectrometry in selected ion monitoring (SIM) mode on the basis of atmospheric pressure chemical ionization (APCI) source in negative mode. The sample of 200 microL was injected for the analysis, and the on-line enrichment time was 3 min. The sodium hydroxide solution was used as a gradient elution system. The two columns made it possible to have a low limit of detection due to the good enrichment and separation capability. The sixteen organic acids were separated completely within 30 min. All curves showed good linearity within the test concentration ranges. The limits of detection (LODs) were between 0.01 and 0.22 mg/L, and the average recoveries were between 70.6% and 110.8%. The relative standard deviations (RSDs) were less than 6.3%. The results indicate that this method is simple, rapid, sensitive and accurate for the determination of the organic acids in feed additives.

  3. Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography.

    PubMed

    Cao, Xue-Li; Xu, Ya-Tao; Zhang, Guang-Ming; Xie, Sheng-Meng; Dong, Ying-Mao; Ito, Yoichiro

    2006-09-15

    High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.

  4. Free amino acids analysis by liquid chromatography with tandem mass spectrometry in several botanicals with antioxidant character.

    PubMed

    Moldoveanu, Serban C; Zhu, Jeff; Qian, Nancy

    2015-07-01

    A novel method based on liquid chromatography with tandem mass spectrometry for the analysis of 19 amino acids in plant materials is described. For the analysis, the plant material is extracted with 0.1 N hydrochloric acid with internal standards present in the extraction solution. The filtered extracts are injected using no clean-up into the liquid chromatographic system coupled with a triple-quadrupole tandem mass spectrometer with an electrospray ionization source. The analytes are separated using ion pair chromatography on a reversed-phase column. The detection is performed in multiple-reaction monitoring positive-ion mode. Quantitation is obtained using calibrations. The validated procedure has been applied for the analysis of amino acids in 18 samples of plant material including botanicals with antioxidant character. The analysis requires 16 min separation time, has excellent precision and accuracy allowing amino acid analysis in a wide range of concentrations.

  5. Characterization of a multiple endogenously expressed adenosine triphosphate-binding cassette transporters using nuclear and cellular membrane affinity chromatography columns.

    PubMed

    Habicht, K-L; Singh, N S; Khadeer, M A; Shimmo, R; Wainer, I W; Moaddel, R

    2014-04-25

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP-Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN-229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN-229)) and (CMAC(LN-229)), respectively. Pgp, MRP1 and BCRP transporters co-immobilized on both columns were characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs. 3.7μM), verapamil (0.6 vs. 0.7μM) and prazosin (0.099 vs. 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of eight compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN-229) column and decreased it (-5%) on the NMAC(LN-229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences.

  6. Comparison of chromatographic band profiles obtained under microwave irradiated and non-irradiated reversed-phase liquid chromatography column

    SciTech Connect

    Galinada, Wilmer; Guiochon, Georges A

    2005-08-01

    The possible influence of the application of microwave energy to a reversed-phase liquid chromatography column on the mass transfer kinetics and the thermodynamics of equilibrium between mobile and stationary phases was examined. Chromatograms of propylbenzene and phenol were recorded under the same experimental conditions, on the same column, successively irradiated and not. The effect of microwave irradiation on the mass transfer kinetics was determined by measuring the second moment of small pulses of propylbenzene in a 70:30 (v/v) solution of methanol in water and microwave outputs of 15 and 30 W. The effect of microwave irradiation on the equilibrium thermodynamics was determined by measuring the elution time of breakthrough curves of phenol at high concentrations in a 20:80 (v/v) solution of methanol and water and microwave outputs of 15, 50, and 150 W. A qualitative comparison of the profiles of the propylbenzene peaks obtained with and without irradiation suggests that this irradiation affects significantly the peak shapes. However, a qualitative comparison of the profiles of the breakthrough curves of phenol obtained with and without irradiation suggests that this irradiation has no significant effect on their shapes. The peak sharpening observed may be due to an increase in the diffusivity, resulting from the dielectric polarization under microwave irradiation. This effect is directly related to an increase of the rate of mass transfers in the column. In contrast, the similarity of the overloaded band profiles at high concentrations suggests that the equilibrium thermodynamics is unaffected by microwave irradiation. This may be explained by the transparence of the stationary phase to microwaves at 2.45 GHz. The column temperature was measured at the column outlet under irradiation powers of 15, 30, 50, and 150 W. It increases with increasing power, the corresponding effluent temperatures being 25 {+-} 1, 30 {+-} 1, 35 {+-} 1, and 45 {+-} 1 C, respectively.

  7. Determination of ambroxol hydrochloride, methylparaben and benzoic acid in pharmaceutical preparations based on sequential injection technique coupled with monolithic column.

    PubMed

    Satínský, Dalibor; Huclová, Jitka; Ferreira, Raquel L C; Montenegro, Maria Conceição B S M; Solich, Petr

    2006-02-13

    The porous monolithic columns show high performance at relatively low pressure. The coupling of short monoliths with sequential injection technique (SIA) results in a new approach to implementation of separation step to non-separation low-pressure method. In this contribution, a new separation method for simultaneous determination of ambroxol, methylparaben and benzoic acid was developed based on a novel reversed-phase sequential injection chromatography (SIC) technique with UV detection. A Chromolith SpeedROD RP-18e, 50-4.6 mm column with 10 mm precolumn and a FIAlab 3000 system with a six-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in our study. The mobile phase used was acetonitrile-tetrahydrofuran-0.05M acetic acid (10:10:90, v/v/v), pH 3.75 adjusted with triethylamine, flow rate 0.48 mlmin(-1), UV-detection was at 245 nm. The analysis time was <11 min. A new SIC method was validated and compared with HPLC. The method was found to be useful for the routine analysis of the active compounds ambroxol and preservatives (methylparaben or benzoic acid) in various pharmaceutical syrups and drops.

  8. Problems in the size exclusion chromatography of poly( N-isopropylacrylamide) on styragel columns

    NASA Astrophysics Data System (ADS)

    Estrin, Ya. I.; Perepelitsina, E. O.; Grishchuk, A. A.

    2016-07-01

    The molecular weights of poly( N-isopropylacrylamide) (PNIPA), calculated according to polystyrene calibration standards upon the elution of THF on styragel columns, appear to be much lower than their actual values determined using independent approaches. This is likely due to interactions between the nitrogen-containing units of PNIPA polymer chains and the sorbent, so the polymer is eluted in the mode intermediate between exclusion and critical. An effective exclusion mode during the elution of PNIPA on a styragel column can be achieved by using an eluent more polar than tetrahydrofuran (particularly, 1-methylpyrrolidone).

  9. Preparation and evaluation of 3 m open tubular capillary columns with a zwitterionic polymeric porous layer for liquid chromatography.

    PubMed

    Peng, Li; Zhu, Manman; Zhang, Lingyi; Liu, Haiyan; Zhang, Weibing

    2016-10-01

    A 3 m zwitterionic polymeric porous layer open tubular column (3 m × 25 μm id × 375 μm od) with a polymeric porous layer thickness of 4 μm was fabricated by the copolymerization of [2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide and N,N'-methylenebis(acrylamide). The effects of the diameter of the capillary, reaction temperature, and polymerization time on the preparation of the open tubular column were investigated. Characterized by scanning electron microscopy, the zwitterionic layer was observed to be rough and throughout the fused-silica capillary homogenously, which increased the phase ratio. The separation of neutral, basic, and acidic compounds demonstrates the strong hydrophilicity of the poly[2-(methacryloyloxy)ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide coating. In addition, the poly[2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide porous layer open tubular column was applied for the analysis of flavonoids from the rootstalk of licorice, revealing the potential in separating complex samples. The relative standard deviation of retention time for run-to-run (n = 5), day-to-day (n = 3), and column-to-column (n = 3) of toluene, N,N-dimethylformamide, formamide, and thiourea were below 1.2%, exhibiting good repeatability.

  10. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    PubMed Central

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  11. Separation and purification of epigallocatechin-3-gallate (EGCG) from green tea using combined macroporous resin and polyamide column chromatography.

    PubMed

    Jin, Xin; Liu, Mingyan; Chen, Zaixing; Mao, Ruikun; Xiao, Qinghuan; Gao, Hua; Wei, Minjie

    2015-10-01

    Epigallocatechin-3-gallate (EGCG) is a major bioactive ingredient of green tea that produces beneficial neuroprotective effects. In this paper, to optimize the EGCG enrichment, thirteen macroporous resins with different chemical and physical properties were systemically evaluated. Among the thirteen tested resins, the H-bond resin HPD826 exhibited best adsorption/desorption capabilities and desorption ratio, as well as weakest affinity for caffeine. The absorption of EGCG on the HPD826 resin followed the pseudo-second-order kinetics and Langmuir isotherm model. The separation parameters of EGCG were optimized by dynamic adsorption/desorption experiments with the HPD826 resin column. Under the optimal condition, the content of EGCG in the 30% ethanol eluent increased by 5.8-fold from 7.7% to 44.6%, with the recovery yield of 72.1%. After further purification on a polyamide column, EGCG with 74.8% purity was obtained in the 40-50% ethanol fraction with a recovery rate of 88.4%. In addition, EGCG with 95.1% purity could be easily obtained after one-step crystallization in distilled water. Our study suggests that the combined macroporous resin and polyamide column chromatography is a simple method for large-scale separation and purification of EGCG from natural plants for food and pharmaceutical applications.

  12. Rapid analysis of benzodiazepines in whole blood by high-performance liquid chromatography: use of a monolithic column.

    PubMed

    Bugey, Aurélie; Staub, Christian

    2004-05-28

    In a previous work [J. Pharm. Biomed. Anal. 23 (2000) 447] a rapid high-performance liquid chromatography (HPLC) method, using a monolithic column in HPLC coupled with a diode-array detector, was developed for the quantitative determination of benzodiazepines in whole blood. The present method has been applied to the assay of eight benzodiazepines amongst the most frequently encountered in forensic toxicology: clonazepam, desalkylflurazepam, diazepam, flunitrazepam, lorazepam, midazolam, nordiazepam and oxazepam. The sample pre-treatment involved a liquid-liquid extraction of blood samples by n-butyl chloride. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e 100 x 4.6 mm) column. The mobile phase was composed of a phosphate buffer (35 mM, pH 2.1) and acetonitrile (70:30, v/v) and the flow-rate was 2 ml/min. The duration of the analysis was less than 4 min and the results of validation, including linearity, precision, recovery, limit of quantification, were satisfactory. The therapeutic and toxic concentrations usually encountered for these substances could be measured. The compounds were separated by a monolithic column which, on account of its particular structure, could bear higher flow-rates than usually found for this kind of analysis. The present method has been applied to two real cases and was tested with about 30 compounds.

  13. PERIODS OF VERTEBRAL COLUMN SENSITIVITY TO BORIC ACID TREATMENT IN CD-1 MICE IN UTERO

    EPA Science Inventory

    Periods of vertebral column sensitivity to boric acid treatment in CD-1 mice in utero.

    Cherrington JW, Chernoff N.

    Department of Toxicology, North Carolina State University, Raleigh, NC 27695, USA. jana_cherrington@hotmail.com

    Boric acid (BA) has many uses as...

  14. Vacancy ion-exclusion/adsorption chromatography of aliphatic amines on a polymethacrylate-based weakly basic anion-exchange column.

    PubMed

    Mori, Masanobu; Helaleh, Murad I H; Xu, Qun; Hu, Wenzhi; Ikedo, Mikaru; Ding, Ming-Yu; Taoda, Hiroshi; Tanaka, Kazuhiko

    2004-06-11

    Vacancy ion-exclusion/adsorption chromatography has been applied to investigate the separation behavior of five aliphatic amines (ethylamine, propylamine, butylamine, pentylamine and hexylamine) on a polymethacrylate-based weakly basic anion-exchange column (Tosoh TSKgel DEAE-5PW). This system is consisted of analytes as a mobile phase and water as an injected sample. In the vacancy ion-exclusion/adsorption chromatography, the elution order was as follows: ethylamine < propylamine < butylamine < pentylamine < hexylamine, depending on their hydrophobicity. The retention times of the amines were decreased with decreasing their concentrations in the mobile phase. The retention times and resolutions of the amines were increased by adding a basic compound (e.g., lithium hydroxide or heptylamine) and by increasing the pH of mobile phase (pH > 11). This was because the dissociations of amine samples in the mobile phase were suppressed and thus the hydrophobic adsorption effects were enhanced. The linearity of calibration graphs could be obtained from the peak areas of the amine samples injected to the 0.05, 0.5 and 5 mM of amine mobile phase at pH 11 by heptylamine. The detection limits of aliphatic amines as injected samples were around 1 microM for five aliphatic amines at three different amine mobile phases. From these results, the retention behaviors of aliphatic amines on vacancy ion-exclusion/adsorption chromatography were concluded to be governed by the hydrophobic adsorption effect.

  15. Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid.

    PubMed

    Berthod, Alain; Mekaoui, Nazim

    2011-09-09

    There is some confusion in chromatography between terms such as solute distribution ratio, distribution constant and partition coefficient. These terms are very precisely defined in the field of liquid-liquid systems and liquid-liquid extraction as well as in the field of chromatography with sometimes conflicting definitions. Countercurrent chromatography (CCC) is a chromatographic technique in which the stationary phase is a support-free liquid. Since the mobile phase is also liquid, biphasic liquid systems are used. This work focuses on the exact meaning of the terms since there are consequences on experimental results. The retention volumes of solutes in CCC are linearly related to their distribution ratios. The partition coefficient that should be termed (IUPAC recommendation) distribution constant is linked to a single definite species. Using benzoic acid that can dimerize in heptane and ionize in aqueous phase and an 18 mL hydrodynamic CCC column, the role and relationships between parameters and the consequences on experimental peak position and shape are discussed. If the heptane/water distribution constant (marginally accepted to be called partition coefficient) of benzoic acid is 0.2 at 20 °C and can be tabulated in books, its CCC measured distribution ratio or distribution coefficient can change between zero (basic aqueous mobile phase) and more than 25 (acidic aqueous mobile phase and elevated concentration). Benzoic acid distribution ratio and partition coefficient coincide only when both dimerization and ionization are quenched, i.e. at very low concentration and pH 2. It is possible to quench dimerization adding butanol in the heptane/water system. However, butanol additions also affect the partition coefficient of benzoic acid greatly by increasing it.

  16. Ethyl-bridged hybrid column as an efficient alternative for HPLC analysis of plasma amino acids by pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

    PubMed

    Castellanos, Mar; Van Eendenburg, Cecile Van; Gubern, Carme; Sanchez, Juan M

    2016-09-01

    Conventional C18 silica columns have proven to be useful for the analysis of amino acids (AA) from protein hydrolysates but undesirable peak overlapping is usually found when analyzing body fluids given that a large number of AAs are present in the samples. As an alternative to silica packings, an ethyl-bridged packing for reversed-phase liquid chromatography of derivatized AAs with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) has been evaluated. The new packing material improves the separation efficiency allowing better separations when analyzing biological fluids. Moreover, this packing has advantages for routine AA analysis, such as a decrease in the total running time and an increase in the life-time of the columns. The pH of the mobile phase has a significant effect on the elution behavior of the AQC hydrolysis product (AMQ) and on the AA derivatives. It is not possible to elute AMQ before detecting the first AA derivative, which requires an accurate adjustment of the pH in the range of 5.30-5.35 to obtain good separation and resolution for the most polar compounds. Under the conditions proposed, it is possible to separate all AAs except the Gly-Gln pair, which is not a problem when hydrolyzed samples are analyzed. The AMQ-Ser pair requires either the use of a different mobile phase pH for its baseline separation or the use of fluorescence detection. Two different procedures for protein removal from plasma samples have been evaluated, solvent precipitation and ultrafiltration (UF) and it has been found that UF gives better results as no significant losses of AAs were observed. The validation of the proposed method with UV detection gives method detection limits in the range of 8-12μM, with repeatability values<8% (n=6) and inter-day precision in plasma samples ranging from 4 to 13% (n=4).

  17. Three column intermittent simulated moving bed chromatography: 1. Process description and comparative assessment.

    PubMed

    Jermann, Simon; Mazzotti, Marco

    2014-09-26

    The three column intermittent simulated moving bed (3C-ISMB) process is a new type of multi-column chromatographic process for binary separations and can be regarded as a modification of the I-SMB process commercialized by Nippon Rensui Corporation. In contrast to conventional I-SMB, this enables the use of only three instead of four columns without compromising product purity and throughput. The novel mode of operation is characterized by intermittent feeding and product withdrawal as well as by partial recycling of the weakly retained component from section III to section I. Due to the smaller number of columns with respect to conventional I-SMB, higher internal flow rates can be applied without violating pressure drop constraints. Therefore, the application of 3C-ISMB allows for a higher throughput whilst using a smaller number of columns. As a result, we expect that the productivity given in terms of throughput per unit time and unit volume of stationary phase can be significantly increased. In this contribution, we describe the new process concept in detail and analyze its cyclic steady state behavior through an extensive simulation study. The latter shows that 3C-ISMB can be easily designed by Triangle Theory even under highly non-linear conditions. The simple process design is an important advantage to other advanced SMB-like processes. Moreover, the simulation study demonstrates the superior performance of 3C-ISMB, namely productivity increases by roughly 60% with respect to conventional I-SMB without significantly sacrificing solvent consumption.

  18. Sugar Determination in Foods with a Radially Compressed High Performance Liquid Chromatography Column.

    ERIC Educational Resources Information Center

    Ondrus, Martin G.; And Others

    1983-01-01

    Advocates use of Waters Associates Radial Compression Separation System for high performance liquid chromatography. Discusses instrumentation and reagents, outlining procedure for analyzing various foods and discussing typical student data. Points out potential problems due to impurities and pump seal life. Suggests use of ribose as internal…

  19. A two-column flash chromatography approach to pyoverdin production from Pseudomonas putida GB1.

    PubMed

    Duckworth, Owen W; Markarian, Dawn S; Parker, Dorothy L; Harrington, James M

    2017-04-01

    Our knowledge of the biological and environmental reactivity of siderophores is limited by the difficulty and cost of obtaining reasonable quantities by purification or synthesis. In this note, we describe a modified procedure for the low-cost, mg-scale purification of pyoverdin-type siderophores using a dual-flash chromatography (reverse-phase absorption and size exclusion) approach.

  20. Determination of ortho-phenylphenol residues in lemon rind by high-performance liquid chromatography with electrochemical detection using a microbore column.

    PubMed

    Yang, Lan; Kotani, Akira; Hakamata, Hideki; Kusu, Fumiyo

    2004-01-01

    A simple and highly sensitive method has been developed for determining ortho-phenylphenol (OPP) in lemon rind by high-performance liquid chromatography with electrochemical detection using a microbore column (microHPLC-ECD). Based on the voltammetric behavior of OPP, microHPLC-ECD was established using a CAPCELL PAK C-18 UG 120 microbore ODS column, 17 mM acetic acid-sodium acetate buffer (pH 4.0)/acetonitrile (60/40, v/v) as a mobile phase and an applied potential at +0.9 V vs. Ag/AgCl. The current peak height was found to be linearly related to the amount of OPP injected from 3.4 pg to 1.7 ng (r > 0.999). The detection limit (S/N = 3) was 3.4 pg (20 fmol), which was 100 times greater in terms of sensitivity when compared to conventional HPLC with UV detection. Standard OPP at 0.425 ng was detected with a relative standard deviation (RSD) of 1.9% (n = 10). The OPP contents in several lemon samples were determined by the present method. The recoveries of OPP from lemon rind exceeded 98% with an RSD (n = 5) of less than 3.01%.

  1. Fractionation of equine antivenom using caprylic acid precipitation in combination with cationic ion-exchange chromatography.

    PubMed

    Raweerith, Rutai; Ratanabanangkoon, Kavi

    2003-11-01

    A combined process of caprylic acid (CA) precipitation and ion-exchange chromatography on SP-Sepharose was studied as a means to fractionate pepsin-digested horse antivenom F(ab')(2) antibody. In the CA precipitation, the optimal concentration for fractionation of F(ab')(2) from pepsin-digested horse plasma was 2%, in which 89.61% of F(ab')(2) antibody activity was recovered in the supernatant with 1.5-fold purification. A significant amount of pepsin was not precipitated and remained active under these conditions. An analytical cation exchanger Protein-Pak SP 8HR HPLC column was tested to establish optimal conditions for the effective separation of IgG, albumin, pepsin and CA from the F(ab')(2) product. From these results, the supernatant from CA precipitation of pepsin-digested plasma was subjected to a SP-Sepharose column chromatography using a linear salt gradient. With stepwise elution, a peak containing F(ab')(2) antibody could be obtained by elution with 0.25 M NaCl. The total recovery of antibody was 65.56% with 2.91-fold purification, which was higher than that achieved by ammonium sulfate precipitation. This process simultaneously and effectively removed residual pepsin, high molecular weight aggregates and CA in the final F(ab')(2) product, and should be suitable for large-scale fractionation of therapeutic equine antivenoms.

  2. Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to anion exchange chromatography and integrated pulsed electrochemical detection using post-column switching.

    PubMed

    Torto, N; Gorton, L; Marko-Varga, G; Emnéus, J; Akerberg, C; Zacchi, G; Laurell, T

    1997-12-05

    A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable alpha-amylase (endo-1,4-alpha-d-glucan, glucanohydrolase; EC 3.2.1.78), is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefication step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90 degrees C. Characteristic fingerprint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 546-554, 1997.

  3. Monitoring of enzymatic hydrolysis of starch by microdialysis sampling coupled on-line to anion exchange chromatography and integrated pulsed electrochemical detection using post-column switching

    SciTech Connect

    Torto, N.; Gorton, L.; Emneus, J.; Laurell, T.; Marko-Varga, G.; Akerberg, C.; Zacchi, G. |

    1997-12-05

    A quantitative evaluation of the hydrolysis of wheat starch using Termamyl, a thermostable {alpha}-amylase, is reported. Data from the monitoring of the hydrolysis of wheat starch indicated that, after 1 h, glucose and maltooligosaccharides up to DP 7 were the main hydrolysis products and thus enabled optimization of a liquefaction step during the production of L-lactic acid. The monitoring system used, both in the on- and off-line mode, was based on continuous flow microdialysis sampling (CFMS) coupled to anion exchange chromatography and integrated pulsed electrochemical detection (IPED). A microdialysis probe equipped with a 5-mm polysulfone (SPS 4005) membrane, with a molecular-weight cut-off of 5 kDa, was used to sample the hydrolysis products of native wheat starch at 90 C. Characteristic fingerpoint separations were achieved by anion exchange chromatography after enzymatic hydrolysis. Post-column switching improved the detection and, consequently, also quantification of the hydrolysates as fouling of the electrode could be reduced. Maltooligosaccharide standards were used for quantification and to verify the elution of the hydrolysates by spiking the off-line samples.

  4. Sensitivity improvement in hydrophilic interaction chromatography negative mode electrospray ionization mass spectrometry using 2-(2-methoxyethoxy)ethanol as a post-column modifier for non-targeted metabolomics.

    PubMed

    Koch, Wendelin; Forcisi, Sara; Lehmann, Rainer; Schmitt-Kopplin, Philippe

    2014-09-26

    The application of ammonia acetate buffered liquid chromatography (LC) eluents is known to concomitantly lead to ion suppression when electrospray ionization mass spectrometry (ESI-MS) detection is used. In negative ESI mode, post column infusion of 2-(2-methoxyethoxy)ethanol (2-MEE) was shown in the literature to help to compensate this adverse effect occurring in reversed phase liquid chromatography mass spectrometry (RP-LC-MS) analyses. Here a setup of direct infusion and hydrophilic interaction chromatography (HILIC) post-column infusion experiments was established in order to investigate systematically the beneficial effects of 2-MEE. We demonstrate that, 2-MEE can help to improve ESI-MS sensitivity in HILIC too and reveal analyte structure specific behaviors. Our study indicates that 2-MEE especially improves ESI response for small and polar molecules. The ESI response of stable isotope labeled amino acids spiked into biological matrices increases up to 50-fold (i.e. D5-l-glutamic acid) when post column infusion of 2-MEE is applied. A non-targeted analysis of a pooled urine sample via HILIC-ESI-QTOF-MS supports this hypothesis. In direct infusion, the combined application of an ammonia acetate buffered solution together with 2-MEE results in an improved ESI response compared to a non-buffered solution. We observed up to 60-fold increased ESI response of l-lysine. We propose this effect is putatively caused by the formation of smaller ESI droplets and stripping of positive charge from ESI droplets due to evaporation of acetic acid anions. In summary, post-column infusion of 2-MEE especially enhances ESI response of small and polar molecules. Therefore it can be regarded as a valuable add-on in targeted or non-targeted metabolomic HILIC-MS studies since this method sets a focus on this molecule category.

  5. Fabrication and characterization of microelectromechanical systems-based gas chromatography column with embedded micro-posts for separation of environmental carcinogens.

    PubMed

    Sun, Jianhai; Cui, Dafu; Chen, Xing; Zhang, Lulu; Cai, Haoyuan; Li, Hui

    2013-05-24

    In this paper, a micro gas chromatography (μGC) column with embedded micro-posts was developed for increasing overall surface area of the columns which is able to support more of the stationary phase and reducing the effective width of the column, leading to higher separation efficiency. The proposed columns have a higher sample capacity as the overall surface area is about 3 times larger than that of open columns with the same dimensions. In order to achieve an even flow velocity in the channels, the location of the micro-posts in the linear channels and the configuration of curved channels were optimized by numerical simulation. The results have indicated that the proposed column separated 5 environmental carcinogens in less than 50s, achieved a separation efficiency of about 9500plates/m and eluted highly symmetrical Gaussian peaks.

  6. Self-interaction chromatography in pre-packed columns: a critical evaluation of self-interaction chromatography methodology to determine the second virial coefficient.

    PubMed

    Rakel, Natalie; Schleining, Kristina; Dismer, Florian; Hubbuch, Juergen

    2013-06-07

    The characterization of protein-protein interactions is commonly conducted via self-interaction chromatography to describe magnitude and direction of the interactions with the resulting osmotic second virial coefficient (B22). However, the method is invasive and protein immobilization on the adsorber surface can influence the results obtained. In order to replace batch immobilization procedures followed by a column packing, direct on-column preparation was optimized in terms of protein immobilization under a continuous flow. Surface load was measured applying a novel method based on partial least squares analysis of spectral scans to reduce analytical error when determining the amount of immobilized protein. Subsequently influencing parameters such as the effects of absolute surface load, injected protein concentration and distribution of protein orientation were analyzed and system performance evaluated. The results disprove the consistency of the SIC method regarding the non-random orientation of proteins on adsorber particles. Thus the determined B22-values differ quantitatively from those determined with static light scattering. Furthermore, variations in immobilization conditions influence the results obtained. These results make clear that SIC does not fulfill the theoretical framework of B22-analysis. It is rather a qualitative measure of protein-protein interactions in the respective system used for experimentation.

  7. Behavior of short silica monolithic columns in high pressure gas chromatography.

    PubMed

    Maniquet, Adrien; Bruyer, Nicolas; Raffin, Guy; Baco-Antoniali, Franck; Demesmay, Claire; Dugas, Vincent; Randon, Jérôme

    2016-08-19

    In order to analyze light hydrocarbons mixtures with silica monolithic columns, a conventional gas chromatograph was modified to work with carrier gas pressure as high as 60bar. To understand hydrodynamic flow and retention with short columns (less than 30cm), special attention was required due to the temperature difference between the oven area and the FID detector which contain a significant length of the column. Efficiency and selectivity using various carrier gases (helium, nitrogen and carbon dioxide) at different inlet pressure for different oven temperature were studied. Carrier gas nature was a very significant parameter: on one side, linked to adsorption mechanism for gases like nitrogen and carbon dioxide onto the stationary phase modifying retention and selectivity, on the other side in relation to the minimum theoretical plate height which was as low as 15μm (66 000 platem(-1)) using carbon dioxide as carrier gas. The chromatographic system was then used to separate methane, ethane, ethylene, acetylene, propane, cyclopropane, and butane in less than 30s.

  8. Evolution in miniaturized column liquid chromatography instrumentation and applications: An overview.

    PubMed

    Nazario, Carlos E D; Silva, Meire R; Franco, Maraíssa S; Lanças, Fernando M

    2015-11-20

    The purpose of this article is to underline the miniaturized LC instrumental system and describe the evolution of commercially available systems by discussing their advantages and drawbacks. Nowadays, there are already many miniaturized LC systems available with a great variety of pump design, interface and detectors as well as efficient columns technologies and reduced connections devices. The solvent delivery systems are able to drive the mobile phase without flow splitters and promote gradient elution using either dual piston reciprocating or syringe-type pumps. The mass spectrometry as detection system is the most widely used detection system; among many alternative ionization sources direct-EI LC-MS is a promising alternative to APCI. In addition, capillary columns are now available showing many possibilities of stationary phases, inner diameters and hardware materials. This review provides a discussion about miniaturized LC demonstrating fundamentals and instrumentals' aspects of the commercially available miniaturized LC instrumental system mainly nano and micro LC formats. This review also covers the recent developments and trends in instrumentation, capillary and nano columns, and several applications of this very important and promising field.

  9. Critical evaluation and comparison of fluid distribution systems for industrial scale expanded bed adsorption chromatography columns.

    PubMed

    Arpanaei, A; Heebøll-Nielsen, A; Hubbuch, J J; Thomas, O R T; Hobley, T J

    2008-07-11

    The hydrodynamic properties of an expanded bed contactor with 30 cm or 150 cm internal diameter, which employs a rotating or oscillating fluid distributor, were compared to prototype columns of 60 cm or 150 cm diameter employing local stirring (fixed wall nozzles plus central bottom mounted stirrer) for fluid distribution. Fluid introduction through a rotating fluid distributor was found to give superior hydrodynamic characteristics in the 30 cm and 150 cm diameter column compared to using the local stirrer in both the 60 cm and 150 cm diameter columns. The shortcomings of the local stirring distributor at large scale were apparent: dead zones were present which could not be removed by increasing rotation rates or flow rates, and such changes led to a deterioration in hydrodynamic properties. In contrast, during fluid introduction through a rotating distributor no dead zones were observed, and residence time distribution tests showed that plate numbers remained constant or increased slightly as flow rate was raised from 200 cm h(-1) to 470 cm h(-1). Under the conditions studied, oscillation of the rotating fluid distributor led to increased mixing and poorer performance than rotary movement. The results imply that further improvement in distributor design is needed and careful attention should be given to the trade off between turbulence and adequate fluid distribution.

  10. Enantioselective separation of dansyl-DL-amino acids and some racemates on "click" functionalized native α-cyclodextrin based sub-2 μm columns.

    PubMed

    Ai, Feng; Wang, Yong; Chen, Hui; Yang, Yanhui; Tan, Timothy Thatt Yang; Ng, Siu-Choon

    2013-04-21

    The current work demonstrates that native α-cyclodextrin, anchored onto sub-2 μm silica particles via "click" reactions and packed into a 5 cm column, was found to be effective for the resolution of 11 pairs of dansyl-DL-amino acids (DAAs) using ultra-high performance liquid chromatography (UHPLC). All DAAs were completely or partially separated on the column and the resolution achieved for 7 pairs of DAAs was significantly greater than 1.5. It was found that the buffer type exerted a profound impact on the separation. The effects of analyte substituents adjacent to the chiral center of analytes as well as operation conditions with respect to the separation efficiency were discussed. Five racemic compounds with single or double rings also got resolved on this short α-CD column to some extent.

  11. Separation of Long and Short Chain Fatty Acids as Naphthacyl and Substituted Phenacyl Esters by High Performance Liquid Chromatography.

    DTIC Science & Technology

    High performance liquid chromatography of various C2 - C24 fatty acids was run on their p-bromophenacyl, p-nitrophenacyl, p-chlorophenacyl, and 2--naphthacyl esters. All the separations were accomplished using reversed phase columns with the eluent consisting of an acetonitrile:water gradient. For all derivatives tested the separations were well defined and analogous although certain esters eluted together as one peak. Quantitative results indicate that the limit of detection in the present study was two picograms of n-caproic acid and 10 picograms of

  12. Preparation of quaternary amine monolithic column for strong anion-exchange chromatography and its application to the separation of Enterovirus 71.

    PubMed

    Gu, Huimin; Yin, Dezhong; Ren, Jie; Zhang, Baoliang; Zhang, Qiuyu

    2016-10-15

    Large size virion is unable to diffuse into pores of conventional porous chromatography particles. Therefore, separation of virion by conventional column-packing materials is not quite efficient. To solve this problem, a monolithic column with large convective pores and quaternary amine groups was prepared and was applied to separate Enterovirus 71 (EV71, ≈5700-6000kDa). Cross-section, pore structure, hydrodynamic performance, adsorption property and dynamic binding capacity of prepared monolithic column were determined. Double-pore structures, macropore at 2472nm and mesopore at 5-60nm, were formed. The porosity was up to 63.3%, which enable higher permeability and lower back pressure of the monolithic column than commercial UNO™ Q1 column. Based on the breakthrough curves, the loading capacity of bovine serum albumin was calculated to be 42.0mg per column. In addition, prepared quaternary amine monolithic column was proved to be suitable for the separation of protein mixture by strong anion-exchange chromatography. As a practical application, prepared monolith column presents excellent performance to the separation of EV71 from virus-proteins mixture.

  13. Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography.

    PubMed

    Peram, Thanmaya; McDonald, Paul; Carter-Franklin, Jayme; Fahrner, Robert

    2010-01-01

    The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.

  14. A Continuous Procedure Based on Column Chromatography to Purify Anthocyanins from Schisandra chinensis by a Macroporous Resin plus Gel Filtration Chromatography.

    PubMed

    Yue, Daran; Yang, Lei; Liu, Shouxin; Li, Jian; Li, Wei; Ma, Chunhui

    2016-02-06

    In our previous study, as natural food colorants and antioxidants, the color and content stabilities of Schisandra chinensis (S. chinensis) anthocyanins were investigated. In this work, the purification process parameters of S. chinensis anthocyanins using a macroporous resin and gel filtration chromatography were evaluated. The optimized parameters of static adsorption and desorption were as follows. The selected resin is HPD-300 (nonpolar copolymer styrene type resin), and the anthocyanins adsorption saturation capacity of HPD-300 resin was 0.475 mg/g dry resin. Adsorption time was 4 h, and 0.517 mg/mL of S. chinensis anthocyanins was adsorbed on the resin column with a flow rate of 39 mL/h (3 BV/h). After adsorption, the anthocyanins were completely desorpted with 2.5 BV of 90% (v/v) ethanol solution, and the desorption flow rate was 13 mL/h (1 BV/h). After purification by dynamic adsorption and desorption, the anthocyanins content in the effluent increased from 47.6 mg/g to 128.4 mg/g, the purity of anthocyanins increased six-fold from 5.08% to 30.43%, and the anthocyanins recovery was 96.5%. The major constituent of S. chinensis anthocyanins was isolated with Bio-Gel P2 gel filtration chromatography, and it was detected by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS) as cyanidin-3-O-xylosylrutinoside. Moreover, the antioxidant activities of S. chinensis anthocyanins were investigated. After purification using the HPD-300 resin, the antioxidant activities of anthocyanins were increased 1.2-fold (FRAP) and 1.7-fold (ABTS).

  15. Chromatography.

    ERIC Educational Resources Information Center

    Brantley, L. Reed, Sr.; Demanche, Edna L.; Klemm, E. Barbara; Kyselka, Will; Phillips, Edwin A.; Pottenger, Francis M.; Yamamoto, Karen N.; Young, Donald B.

    This booklet presents some activities on chromatography. Directions for preparing leaf pigment extracts using alcohol are given, and paper chromatography and thin-layer chromatography are described as modifications of the basic principles of chromatography. (KHR)

  16. Investigation of sorbic acid volatile degradation products in pharmaceutical formulations using static headspace gas chromatography.

    PubMed

    Yarramraju, Sitaramaraju; Akurathi, Vamsidhar; Wolfs, Kris; Van Schepdael, Ann; Hoogmartens, Jos; Adams, Erwin

    2007-06-28

    An analytical method that allows simultaneous analysis of sorbic acid and its degradation products was developed using static headspace gas chromatography (HS-GC). AT-Aquawax-DA, the capillary column used, showed good selectivity and separation towards sorbic acid and its degradation products. Sorbic acid degradation was investigated in both acidic and aqueous media at room and elevated temperatures. In total 12 sorbic acid degradation products were found, 8 of which could be characterized. The method was investigated for its accuracy towards estimation of degradation products. Using the HS-GC method different batches of pharmaceutical preparations such as cold cream, cetomacrogol cream and vaseline were investigated for sorbic acid degradation products which were estimated by applying the standard addition method. Acetaldehyde was found to be the major degradation product. The other identified degradation products were: acetone; 2-methylfuran; crotonaldehyde; alfa-angelicalactone; 2-acetyl, 5-methylfuran; toluene and 2,5-dimethylfuran. Both mass spectrometeric (MS) and flame ionization detection (FID) were used. The qualitative investigation was done on HS-GC-MS and the quantitative work on HS-GC-FID.

  17. Evaluation of octanol-water partition coefficients using capillary gas chromatography with cold on-column injection

    SciTech Connect

    Haky, J.E.; Leja, B.

    1986-01-01

    A general procedure is described for the determination of the octanol-water partition coefficients (log P) of very lipophilic compounds by the shake flask method, employing the analytical technique of capillary gas chromatography with cold on-column injection. The solute and an internal reference with a known log P value are partitioned between the layers of an octanol-water mixture and their relative concentrations are determined by analysis of each phase, using highly standardized chromatographic conditions. The overall method is demonstrated to be convenient, reproducible, and useful for the determination of the partition coefficients of a wide variety of compounds with log P values as high as 5.7. 14 references, 1 figure, 1 table.

  18. Selenium speciation by high-performance anion-exchange chromatography-post-column UV irradiation coupled with atomic fluorescence spectrometry.

    PubMed

    Liang, Lina; Mo, Shumin; Zhang, Ping; Cai, Yaqi; Mou, Shifen; Jiang, Guibin; Wen, Meijuan

    2006-06-16

    A technique for the speciation of selenomethylcysteine (SeMeCys), selenocystine (SeCys), selenite [Se(IV)] and selenomethionine (SeMet) was established in this paper using high-performance anion-exchange chromatography coupled with atomic fluorescence spectrometry (HPAEC-AFS). Analytes were separated on an AminoPac PA10 column and then digested by on-line ultraviolet (UV) irradiation, which destroyed organic compound structure. Hydride generation was used as an available sample introduction technique for atomic fluorescence detection. The detection limits of four compounds were 1-5 microg/L (250 microL injection, 10 times of the baseline noise). The relative standard deviations (RSDs), calculated from seven consecutive injections of 100 microg/L standard mixtures, were from 2 to 4%. Selenious yeast tablet, which had been proposed as selenium supplement, and human urine collected from a volunteer were analyzed. Good spiked recoveries from 86 to 103% were obtained.

  19. Separation of the fatty acids in menhaden oil as methyl esters with a highly polar ionic liquid gas chromatographic column and identification by time of flight mass spectrometry.

    PubMed

    Fardin-Kia, Ali Reza; Delmonte, Pierluigi; Kramer, John K G; Jahreis, Gerhard; Kuhnt, Katrin; Santercole, Viviana; Rader, Jeanne I

    2013-12-01

    The fatty acids contained in marine oils or products are traditionally analyzed by gas chromatography using capillary columns coated with polyethylene glycol phases. Recent reports indicate that 100 % cyanopropyl siloxane phases should also be used when the analyzed samples contain trans fatty acids. We investigated the separation of the fatty acid methyl esters prepared from menhaden oil using the more polar SLB-IL111 (200 m × 0.25 mm) ionic liquid capillary column and the chromatographic conditions previously optimized for the separation of the complex mixture of fatty acid methyl esters prepared from milk fat. Identifications of fatty acids were achieved by applying Ag(+)-HPLC fractionation and GC-TOF/MS analysis in CI(+) mode with isobutane as the ionization reagent. Calculation of equivalent chain lengths confirmed the assignment of double bond positions. This methodology allowed the identification of 125 fatty acids in menhaden oil, including isoprenoid and furanoid fatty acids, and the novel 7-methyl-6-hexadecenoic and 7-methyl-6-octadecenoic fatty acids. The chromatographic conditions applied in this study showed the potential of separating in a single 90-min analysis, among others, the short chain and trans fatty acids contained in dairy products, and the polyunsaturated fatty acids contained in marine products.

  20. Analysis of perfluorinated carboxylic acids in soils II: optimization of chromatography and extraction.

    PubMed

    Washington, John W; Henderson, W Matthew; Ellington, J Jackson; Jenkins, Thomas M; Evans, John J

    2008-02-15

    With the objective of detecting and quantitating low concentrations of perfluorinated carboxylic acids (PFCAs), including perfluorooctanoic acid (PFOA), in soils, we compared the analytical suitability of liquid chromatography columns containing three different stationary phases, two different liquid chromatography-tandem mass spectrometry (LC/MS/MS) systems, and eight combinations of sample-extract pretreatments, extractions and cleanups on three test soils. For the columns and systems we tested, we achieved the greatest analytical sensitivity for PFCAs using a column with a C(18) stationary phase in a Waters LC/MS/MS. In this system we achieved an instrument detection limit for PFOA of 270 ag/microL, equating to about 14 fg of PFOA on-column. While an elementary acetonitrile/water extraction of soils recovers PFCAs effectively, natural soil organic matter also dissolved in the extracts commonly imparts significant noise that appears as broad, multi-nodal, asymmetric peaks that coelute with several PFCAs. The intensity and elution profile of this noise is highly variable among soils and it challenges detection of low concentrations of PFCAs by decreasing the signal-to-noise contrast. In an effort to decrease this background noise, we investigated several methods of pretreatment, extraction and cleanup, in a variety of combinations, that used alkaline and unbuffered water, acetonitrile, tetrabutylammonium hydrogen sulfate, methyl-tert-butyl ether, dispersed activated carbon and solid-phase extraction. For the combined objectives of complete recovery and minimization of background noise, we have chosen: (1) alkaline pretreatment; (2) extraction with acetonitrile/water; (3) evaporation to dryness; (4) reconstitution with tetrabutylammonium-hydrogen-sulfate ion-pairing solution; (5) ion-pair extraction to methyl-tert-butyl ether; (6) evaporation to dryness; (7) reconstitution with 60/40 acetonitrile/water (v/v); and (8) analysis by LC/MS/MS. Using this method, we

  1. The use of a temperature-responsive column for the direct analysis of drugs in serum by two-dimensional heart-cutting liquid chromatography.

    PubMed

    Mikuma, Toshiyasu; Uchida, Ryo; Kajiya, Mizuki; Hiruta, Yuki; Kanazawa, Hideko

    2017-02-01

    A novel pretreatment method, which was performed using a two-dimensional high-performance liquid chromatography (2D-HPLC) system, was proposed for the direct analysis of drugs in human serum. A temperature-responsive column was used as a pretreatment column. The stationary phase of the temperature-responsive column exhibits temperature-regulated hydrophilic/hydrophobic characteristics. Controlling the ionic strength of the eluent enables human serum albumin (HSA) to pass through the column without retention. When serum samples containing barbiturates or benzodiazepines were injected into the temperature-responsive column using 10 mM of ammonium acetate (pH 6.5) as the mobile phase and in the temperature range of 10-40 °C, HSA was eluted from the column near the dead time, followed by the individual drugs. When the column temperature was changed, the retention times of the drugs were altered owing to surface property changes within the pretreatment column. These closely eluted compounds were subsequently introduced into the analytical column using a column-switching valve, with a minimal gap time to avoid foreign substance contamination. This new 2D-HPLC method afforded high-quality chromatograms of multiple drugs without unwanted peaks from foreign substances. The present technique could be an attractive choice in selecting the analytical method for drug analysis.

  2. Formation of Iron Complexes from Trifluoroacetic Acid Based Liquid Chromatography Mobile Phases as Interference Ions in LC-ESI-MS Analysis

    PubMed Central

    Shukla, Anil; Zhang, Rui; Orton, Daniel; Zhao, Rui; Clauss, Therese; Moore, Ronald; Smith, Richard

    2011-01-01

    Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization-mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid that severely interfered with sample analysis. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are composed of three components; clusters of trifluoroacetic acid, clusters of mass 159 and iron. Formation of these ions is inhibited by removing trifluoroacetic acid from the mobile phases and using formic acid in its place, replacing the stainless steel union with a titanium union or by adding a small blank fused silica capillary column between the chromatography column and the electrospray tip via a stainless steel union without any adverse effects to chromatographic separation, peak broadening or peptide identifications. PMID:21504012

  3. Kinetic performance of a 50mm long 1.8μm chiral column in supercritical fluid chromatography.

    PubMed

    Berger, Terry A

    2016-08-12

    Reduced plate heights (hr) of <2 were observed for the first time during the chiral separation of enantiomers, on sub-2μm particles with supercritical fluid chromatography (SFC). The enantiomers of trans-stilbene oxide, were separated on a 4.6×50mm, 1.8μm R,R-Whelk-O1 column, with hr as low as 1.93. The plumbing of a commercial SFC instrument was modified to create a low dispersion version. Without the modification performance was considerably worse. vanDeemter like plots of reduced plate height vs. flow rate, for trans-stilbene oxide, indicate that the optimum flow varied with% modifier. On a 4.6×250mm, 5μm R,R- Whelk-O1 column, the optimum flow was >4mL/min for 5% methanol in CO2, decreasing to <2mL/min for 40% methanol (more than a factor of 2). For a 4.6×50mm column packed with 1.8μm particles the optimum appeared to be near, or >5mL/min with 2.5%, 5%, and 10% methanol, decreasing to between 3 and 3.5mL/min at 40% methanol. This is the first time such shifts have been characterized. Since the solutes were the same in all cases, the differences are likely due to changes in solute diffusion coefficients caused by changes in modifier concentration, and pressure. Pump pressure requirements sometimes exceeded 500bar. It is shown that a 5mL/min flow rate is inadequate for use with 1.8μm particles in a 4.6mm ID column format. Instead, it is suggested to decrease the ID of the column to 3mm, where the optimum flow rates are on the order of 2mL/min with decreased tubing variance. Nevertheless, a number of sub-1min chromatograms are presented.

  4. Combined thin layer chromatography and gas chromatography with mass spectrometric analysis of lipid classes and fatty acids in malnourished polar bears (Ursus maritimus) which swam to Iceland.

    PubMed

    Eibler, Dorothee; Krüger, Sabine; Skírnisson, Karl; Vetter, Walter

    2017-03-01

    Between 2008 and 2011, four polar bears (Ursus maritimus) from the Greenland population swam and/or drifted on ice to Iceland where they arrived in very poor body condition. Body fat resources in these animals were only between 0% and 10% of the body weight (usually 25%). Here we studied the lipid composition in different tissues (adipose tissue if available, liver, kidney and muscle). Lipid classes were determined by thin layer chromatography (TLC) and on-column gas chromatography with mass spectrometry (GC/MS). The fatty acid pattern of total lipids and free fatty acids was analyzed by GC/MS in selected ion monitoring (SIM) mode. Additionally, cholesteryl esters and native fatty acid methyl esters, initially detected as zones in thin layer chromatograms, were enriched by solid phase extraction and quantified by GC/MS. The ratio of free fatty acids to native fatty acid methyl esters could be correlated with the remained body lipids in the polar bears and thus may also serve as a marker for other starving animals or even for humans.

  5. Aflatoxin evaluation in ready-to-eat brazil nuts using reversed-phase liquid chromatography and post-column derivatisation.

    PubMed

    Iamanaka, Beatriz Thie; Nakano, Felipe; Lemes, Daniel Ponciano; Ferranti, Larissa Souza; Taniwaki, Marta Hiromi

    2014-01-01

    A high-performance liquid chromatography-fluorescence (HPLC-FD) method for aflatoxin quantification in brazil nuts was developed. Samples of brazil nuts collected in Brazilian markets were extracted with methanol:water and cleaned using an immunoaffinity column. Aflatoxins were eluted with methanol and a post-column derivatisation was performed with bromine, using a Kobra Cell system. The optimised method for total aflatoxins was sensitive, with detection and quantification limits of 0.05 and 0.25 µg kg⁻¹, respectively. The method was accurate, with recovery values of 87.6%; 85.3% and 85.0% for 0.5, 5.0 and 14.6 µg kg⁻¹ spiked levels, respectively. It was shown that the method was applicable to brazil nuts. From a total of 95 brazil nut samples analysed from 21 São Paulo supermarket samples and 51 Manaus and 23 Belém street markets samples, 37.9% showed detectable levels of aflatoxins and three exceeded the recommended Codex Alimentarius limit of 10 µg kg⁻¹ for ready-to-eat brazil nuts.

  6. Packing of large-scale chromatography columns with irregularly shaped glass based resins using a stop-flow method

    PubMed Central

    Siu, Sun Chau; Chia, Celeste; Mok, Yanglin; Pattnaik, Priyabrata

    2014-01-01

    Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure-flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow-pack method when applied to irregularly shaped particles does not yield well-consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop-flow packing method was developed as an improvement over the flow-pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large-scale columns packed using the stop-flow method over multiple cycles has shown a two- to three-fold reduction of change in bed integrity values as compared to a flow-packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (As). PMID:25080096

  7. Preparative separation of gallocatechin gallate from Camellia ptilophylla using macroporous resins followed by sephadex LH-20 column chromatography.

    PubMed

    Li, Kaikai; Zhou, Xuelin; Liu, Cheuk-Lun; Yang, Xiaorong; Han, Xiaoqiang; Shi, Xianggang; Song, Xiaohong; Ye, Chuangxing; Ko, Chun-hay

    2016-02-01

    Gallocatechin gallate (GCG) possesses multiple potential biological activities. However, the content of GCG in traditional green tea is too low which limits its in-depth pharmacological research and application. In the present study, a simple, efficient and environment-friendly chromatographic separation method was developed for preparative enrichment and separation of GCG from cocoa tea (Camellia ptilophylla) which contains high content of GCG. In the first step, the adsorption properties of selected resins were evaluated, and XAD-7HP resin was chosen by its adsorption and desorption properties for GCG. In order to maximize column efficiency for GCG collection, the operating parameters (e.g., flow rate, ethanol concentration, and bed height) were optimized. We found that the best combination was the feed concentration at 20mg/mL, flow rate at 0.75 BV/h and the ratio of diameter to bed heights as 1:12. Under these conditions, the purity of GCG was 45% with a recovery of 89%. In order to obtain pure target, a second step was established using column chromatography with sephadex LH-20 gel and 55% ethanol-water solution as eluent. After this step, the purity of the GCG was 91% with a recovery of 68% finally.

  8. Spiral column configuration for protein separation by high-speed countercurrent chromatography

    PubMed Central

    Ito, Yoichiro

    2009-01-01

    Retention of the stationary phase of aqueous-aqueous polymer phase systems is improved by a spiral column configuration which utilizes the radially acting centrifugal force along the spiral pitch to retain the heavier phase in the outer portion and the lighter phase in the inner portion of the spiral channel. For the separation of proteins which has low mass transfer rates, the system needs further modification of the separation channel to interrupt the laminar flow and enhance mixing of the two phases. Two spiral column assemblies were developed, one using a disk with spiral grooves and the other, the spiral tube support which accommodates the multiple spiral layers made from a single piece of fluorinated plastic tubing. In the spiral disk assembly, the best protein separation is achieved by the mixer-settler system which vigorously mixes two phases by vibrating glass beads placed in every other section of barricaded spiral channel, while in the spiral tube assembly the partition efficiency of proteins is enhanced by compressing the tubing to interrupt the laminar flow of the mobile phase. In both systems protein samples were well resolved by choosing the suitable elution modes. PMID:20823942

  9. Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets.

    PubMed

    Zhou, Xingding; Kini, R Manjunatha; Sivaraman, J

    2011-02-01

    This protocol describes a method for identifying unknown target proteins from a mixture of biomolecules for a given drug or a lead compound. This method is based on a combination of chromatography and isothermal titration calorimetry (ITC) where ITC is used as a tracking tool. The first step involves the use of ITC to confirm the binding of ligand to a component in the biomolecular mixture. Subsequently, the biomolecular mixture is fractionated by chromatography, and the binding of the ligand with individual fractions (or subfractions) is verified by ITC. The iteration of chromatographic purification on the fractions combined with ITC results in identifying the target protein. This method is useful when the target protein or ligand is unknown and/or not amenable to labeling, chemical modification or immobilization. This protocol has been successfully used by our team and by others to identify both low-abundance and highly abundant target proteins present in biomolecular mixtures. With this protocol, it takes approximately 3-5 d to identify the target protein from a mixture.

  10. Residual on column host cell protein analysis during lifetime studies of protein A chromatography.

    PubMed

    Lintern, Katherine; Pathak, Mili; Smales, C Mark; Howland, Kevin; Rathore, Anurag; Bracewell, Daniel G

    2016-08-26

    Capacity reduction in protein A affinity chromatography with extended cycling during therapeutic antibody manufacture is well documented. Identification of which residual proteins remain from previous cycles during the lifetime of these adsorbent materials is required to understand their role in this ageing process, but represents a significant metrological challenge. Scanning electron microscopy (SEM) and liquid chromatography mass spectrometry (LC-MS/MS) are combined to detect and map this phenomenon of protein carry-over. We show that there is a morphological change at the surface of the agarose resin, revealing deposits on the polymer fibres increasing with cycle number. The amount of residual host cell proteins (HCPs) by LC-MS/MS present on the resin is shown to increase 10-fold between 50 and 100 cycles. During this same period the functional class of the predominant HCPs associated with the resin increased in diversity, with number of proteins identified increasing 5-fold. This ageing is observed in the context of the product quality of the eluate HCP and protein A leachate concentration remaining constant with cycle number.

  11. [Simultaneous determination of erdosteine and its active metabolite in human plasma by liquid chromatography-tandem mass spectrometry with pre-column derivatization].

    PubMed

    Jin, Jing; Chen, Xiao-Yan; Zhang, Yi-Fan; Ma, Zhi-Yu; Zhong, Da-Fang

    2013-03-01

    A sensitive, rapid and accurate liquid chromatography-tandem mass spectrometric (LC-MS/MS) method with pre-column derivatization was developed for the simultaneous determination of erdosteine and its thiol-containing active metabolite in human plasma. Paracetamol and captopril were chosen as the internal standard of erdosteine and its active metabolite, respectively. Aliquots of 100 microL plasma sample were derivatized by 2-bromine-3'-methoxy acetophenone, then separated on an Agilent XDB-C18 (50 mm x 4.6 mm ID, 1.8 microm) column using 0.1% formic acid methanol--0.1% formic acid 5 mmol x L(-1) ammonium acetate as mobile phase, in a gradient mode. Detection of erdosteine and its active metabolite were achieved by ESI MS/MS in the positive ion mode. The linear calibration curves for erdosteine and its active metabolite were obtained in the concentration ranges of 5-3 000 ng x mL(-1) and 5-10 000 ng x mL(-1), respectively. The lower limit of quantification of erdosteine and its active metabolite were both 5.00 ng x mL(-1). The pharmacokinetic results of erdosteine and its thiol-containing active metabolite showed that the area under curve (AUC) of the thiol-containing active metabolite was 6.2 times of that of erdosteine after a single oral dose of 600 mg erdosteine tables in 32 healthy volunteers, The mean residence time (MRT) of the thiol-containing active metabolite was (7.51 +/- 0.788) h, which provided a pharmacokinetic basis for the rational dosage regimen.

  12. Rapid bioanalysis of vancomycin in serum and urine by high-performance liquid chromatography tandem mass spectrometry using on-line sample extraction and parallel analytical columns.

    PubMed

    Cass, R T; Villa, J S; Karr, D E; Schmidt, D E

    2001-01-01

    A novel high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS) method is described for the determination of vancomycin in serum and urine. After the addition of internal standard (teicoplanin), serum and urine samples were directly injected onto an HPLC system consisting of an extraction column and dual analytical columns. The columns are plumbed through two switching valves. A six-port valve directs extraction column effluent either to waste or to an analytical column. A ten-port valve simultaneously permits equilibration of one analytical column while the other is used for sample analysis. Thus, off-line analytical column equilibration time does not require mass spectrometer time, freeing the detector for increased sample throughput. The on-line sample extraction step takes 15 seconds followed by gradient chromatography taking another 90 seconds. Having minimal sample pretreatment the method is both simple and fast. This system has been used to successfully develop a validated positive-ion electrospray bioanalytical method for the quantitation of vancomycin. Detection of vancomycin was accurate and precise, with a limit of detection of 1 ng/mL in serum and urine. The calibration curves for vancomycin in rat, dog and primate were linear in a concentration range of 0.001-10 microg/mL for serum and urine. This method has been successfully applied to determine the concentration of vancomycin in rat, dog and primate serum and urine samples from pharmacokinetic and urinary excretion studies.

  13. Application and comparison of high-speed countercurrent chromatography and high performance liquid chromatography in preparative enantioseparation of α-substitution mandelic acids.

    PubMed

    Tong, Shengqiang; Zhang, Hu; Shen, Mangmang; Ito, Yoichiro; Yan, Jizhong

    2015-04-01

    Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by high-speed countercurrent chromatography (HSCCC) and high performance liquid chromatography (HPLC) were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C18 reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L(-1) phosphate buffer at pH 2.68 containing 20 mmol L(-1) HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L(-1) SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n-hexane-methyl tert.-butyl ether-0.1 molL(-1) phosphate buffer solution at pH 2.67 containing 0.1 mol L(-1) HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L(-1) SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, total 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 105-110 mg of enantiomers with 95-98% purity and 85-90% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper preparative enantioseparation by HSCCC and HPLC was compared from various aspects.

  14. Application and comparison of high-speed countercurrent chromatography and high performance liquid chromatography in preparative enantioseparation of α-substitution mandelic acids

    PubMed Central

    Tong, Shengqiang; Zhang, Hu; Shen, Mangmang; Ito, Yoichiro; Yan, Jizhong

    2014-01-01

    Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by high-speed countercurrent chromatography (HSCCC) and high performance liquid chromatography (HPLC) were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C18 reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L−1 phosphate buffer at pH 2.68 containing 20 mmol L−1 HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L−1 SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n-hexane-methyl tert.-butyl ether-0.1 molL−1 phosphate buffer solution at pH 2.67 containing 0.1 mol L−1 HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L−1 SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, total 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 105-110 mg of enantiomers with 95-98% purity and 85-90% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper preparative enantioseparation by HSCCC and HPLC was compared from various aspects. PMID:25983356

  15. Automatic analyzer for highly polar carboxylic acids based on fluorescence derivatization-liquid chromatography.

    PubMed

    Todoroki, Kenichiro; Nakano, Tatsuki; Ishii, Yasuhiro; Goto, Kanoko; Tomita, Ryoko; Fujioka, Toshihiro; Min, Jun Zhe; Inoue, Koichi; Toyo'oka, Toshimasa

    2015-03-01

    A sensitive, versatile, and reproducible automatic analyzer for highly polar carboxylic acids based on a fluorescence derivatization-liquid chromatography (LC) method was developed. In this method, carboxylic acids were automatically and fluorescently derivatized with 4-(N,N-dimethylaminosulfonyl)-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ) in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride by adopting a pretreatment program installed in an LC autosampler. All of the DBD-PZ-carboxylic acid derivatives were separated on the ODS column within 30 min by gradient elution. The peak of DBD-PZ did not interfere with the separation and the quantification of all the acids with the exception of lactic acid. From the LC-MS/MS analysis, we confirmed that lactic acid was converted to an oxytriazinyl derivative, which was further modified with a dimethoxy triazine group of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM). We detected this oxytriazinyl derivative to quantify lactic acid. The detection limits (signal-to-noise ratio = 3) for the examined acids ranged from 0.19 to 1.1 µm, which correspond to 95-550 fmol per injection. The intra- and inter-day precisions of typical, highly polar carboxylic acids were all <9.0%. The developed method was successfully applied to the comprehensive analysis of carboxylic acids in various samples, which included fruit juices, red wine and media from cultured tumor cells.

  16. Profiling of soil fatty acids using comprehensive two-dimensional gas chromatography with mass spectrometry detection.

    PubMed

    Zeng, Annie Xu; Chin, Sung-Tong; Patti, Antonio; Marriott, Philip J

    2013-11-22

    Profiling of phospholipid fatty acids (PLFA) represents a challenging goal for distinguishing the diversity of microbial communities and biomass in the complex and heterogeneous soil ecosystem. Comprehensive two-dimensional gas chromatography (GC×GC) coupled with simultaneous flame ionisation and mass spectrometry detection was applied as a culture-independent method for PLFA profiling of microbial classification in forest soil. A number of column sets were evaluated for the GC×GC separation of fatty acid methyl esters (FAME). Due to better isomeric separation and compound patterns on the 2D contour plot, an apolar-polar column combination was selected for soil microbial PLFA characterisation. A comprehensive view of PLFA composition with carbon chain length varying from 12 to 20 was observed in forest soil samples, with the commonly reported bacterial FAME of iso-/anteiso-, methyl-branched-, cyclopropyl-, and hydroxyl-substituted FA identified by their mass spectral and retention time according to authentic standards. Notably, some uncommon oxygenated FAME were found in high abundance and were further characterised by GC×GC coupled with high resolution mass spectrometry. This tentatively revealed geometric pairs of methyl 9,10-epoxyoctadecanoate isomers.

  17. Fast simultaneous determination of prominent polyphenols in vegetables and fruits by reversed phase liquid chromatography using a fused-core column.

    PubMed

    Martí, Raúl; Valcárcel, Mercedes; Herrero-Martínez, José Manuel; Cebolla-Cornejo, Jaime; Roselló, Salvador

    2015-02-15

    A reversed-phase high-performance liquid chromatography method with photodiode array detection has been developed enabling the joint determination of 17 prominent flavonoids and phenolic acids in vegetables and fruits. A multi-segmented gradient program using a fused-core column for the separation of several phenolic classes (phenolic acids and flavonoids) has been optimised. The influence of extraction conditions (sample freeze-drying, ultrasound extraction, solvent composition and extraction time) has been also optimised using response surface methodology with tomato samples as a model. Complete recoveries (76-108%) were obtained for the phenolic compounds present in tomato. The developed method provided satisfactory repeatability in terms of peak area (RSD<2.9%) and retention time (RSD<0.2%) both for standards and real samples. Detection limits ranged between 3 and 44μgkg(-1) for the detected polyphenols. This method is recommended for routine analysis of large number of samples typical of production quality systems or plant breeding programs.

  18. Micellar and sub-micellar ultra-high performance liquid chromatography of hydroxybenzoic acid and phthalic acid positional isomers.

    PubMed

    Fasciano, Jennifer M; Danielson, Neil D

    2016-03-18

    Micellar liquid chromatography (MLC) has been used primarily for the separation of neutral analytes of varying polarities, most commonly phenols and polyaromatic hydrocarbons, but does not seem to have been used to study aromatic hydroxy acids in detail. We have studied the separation of hydroxybenzoic acid mixtures, including monohydroxybenzoic and dihydroxybenzoic acid positional isomers by MLC. Sodium dodecylsulfate (SDS) is investigated as the modifying surfactant on a C18 ultra-high performance liquid chromatography (UHPLC) column (100 × 2.1mm, 1.8 μm). The addition of only SDS (no organic solvent) to the mobile phase reduced the influence of hydrophobic interactions while improving the retention times, resolution, and peak shapes, even at concentrations below the critical micellization concentration (CMC). The UHPLC separation of 7 hydroxybenzoic acids, including 6 dihydroxybenzoic acid positional isomers and one trihydroxybenzoic acid, is achieved with high efficiency using 0.1% SDS in 1.84 mM sulfuric acid (pH 2.43) mobile phase, in less than 6 min with a flow rate of 0.3 mL min(-1), and in less than four min with a flow rate of 0.7 mL min(-1). Six monohydroxybenzoic acid isomers are also effectively separated by MLC, using a 0.5% SDS mobile phase modifier, in less than 20 min with a flow rate of 0.3 mL min(-1), and in less than 14 min with a flow rate of 0.7 mL min(-1). The 3 phthalic acid isomers could be separated using a similar mobile phase and flow rates in less than 6 and 4 min. Solute-micelle equilibrium constants and partition coefficients are calculated for 6 monohydroxybenzoic acids based on a plot of MLC retention factor vs. mobile phase micelle concentration. All aromatic acid isomers studied can be classified as binding solutes in the MLC retention mechanism. Less effective separations are observed with shorter chain surfactants, leading to higher retention times and poor peak shapes. It is concluded that increasing chain length led to more

  19. [Determination of glycyrrhizic acid in glycyrrhiza preparations with capillary electrophoresis and high performance liquid chromatography].

    PubMed

    Peng, J; Wang, F; Zhu, M

    1999-01-01

    High performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography(MECC) have been used to determine glycyrrhizic acid in glycyrrhiza preparations. By HPLC, mobile phase was V(methanol):V(water):V(acetic acid) = 75:23.5:1.5. By CZE, experiment was performed with 15 kV power, 0.075 mm i.d. x 800 mm fused-silica capillary column and UV detector. Samples were injected into the capillary by electromigration injection for 20 s. Absorbance detection was at 254 nm. The running buffer was made up of 0.02 mol/L dipotassium hydrogenphosphate and borax (pH 9.0). By MECC, the running buffer was made up of 0.025 mol/L sodium dodecyl sulfate (SDS), 0.02 mol/L dipotassium hydrogenphosphate and borax (pH 9.0). Each new capillary was washed with 0.1 mol/L NaOH, deionized water and buffer, each for 3 min, before use. Comparison of the results of analysis with HPLC, CZE and MECC has been made. It was found that result of MECC was very close to that of HPLC. MECC has been satisfactorily applied to plant drug analysis.

  20. Individual volatile fatty acids determination by chromogenic derivatization coupled to multi-syringe chromatography.

    PubMed

    Robert-Peillard, Fabien; Boudenne, Jean-Luc; Coulomb, Bruno

    2013-10-15

    In this paper, a new multisyringe chromatography (MSC) system is proposed for a simple and accurate measurement of individual volatile fatty acids (VFA) in anaerobic treatment processes. The determination method is based on the derivatization of VFA with N-(1-naphthyl) ethylenediamine (EDAN) followed by the separation of VFA derivatives on an Onyx C18 monolithic column (25 mm × 4.6mm i.d.). Chromatographic separation conditions have been investigated and were found to be optimal with a mixture of acetonitrile and formic acid 0.1% (ratio 35/65), providing good separation of C2-C5 VFA in 8 min. Optimization of the derivatization reaction was also carried out with special attention paid to the buffering capacity of the reaction medium, so as to be able to deal with samples of various characteristics in terms of alkalinity or of VFA concentration range. Individual VFA could be quantified between 0.05-2.5 g L(-1) with LOD of 0.01-0.02 g L(-1). Overall procedure time was about 18 min for one analytical cycle, which fulfils the requirement of real-time monitoring of an anaerobic digester. Validation of the system developed has been assessed by application of the procedure to sludge samples from various origins, and comparative results with gas chromatography analyses showed satisfactory correlation (R²>0.98).

  1. High sensitivity measurement of amino acid isotope enrichment using liquid chromatography-mass spectrometry.

    PubMed

    van Eijk, Hans M H; Wijnands, Karolina A P; Bessems, Babs A F M; Olde Damink, Steven W; Dejong, Cornelis H C; Poeze, Martijn

    2012-09-15

    Measurement of the incorporation or conversion of infused stable isotope enriched metabolites in vivo such as amino acids plays a key role in metabolic research. Specific routes are frequently probed in knockout mouse models limiting the available amount of sample. Although less precise as compared to combustion-isotope ratio mass spectrometry (C-IRMS), gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-mass spectrometry (LC-MS) techniques are therefore often the method of choice to measure isotopic enrichment of target metabolites. However, under conditions of metabolic depletion, the precision of these systems becomes limiting. In this paper, studies were performed to enhance the sensitivity and precision of isotope enrichment measurements using LC-MS. Ion-statistics and resolution were identified as critical factors for this application when using a linear trap mass spectrometer. The combination with an automated pre-column derivatization and a carefully selected solvent mix allowed us to measure isotopic enrichments down to 0.005% at plasma concentrations as low as 5 μmol/l, an improvement by a factor of 100 compared to alternative methods. The resulting method now allowed measurement of the in vivo conversion of the amino acid arginine into citrulline as a marker for the production of nitric oxide in an in vivo murine endotoxemia model with depleted plasma levels of arginine and citrulline.

  2. Effect of the detergent Tween-20 on the DNA affinity chromatography of Gal4, C/EBPalpha, and lac repressor with observations on column regeneration.

    PubMed

    Robinson, F Darlene; Moxley, Robert A; Jarrett, Harry W

    2004-01-23

    C/EBPalpha, Gal4, and lac repressor, representing three different transcription factor homology families, were expressed as fusion proteins and used to characterize the effects of column aging, Mg2+, the nonionic detergent Tween-20, column loading, and bovine serum albumin on DNA-affinity chromatography. When lac-repressor-beta-galactosidase fusion protein is loaded onto a new DNA-Sepharose column, less elutes from a new column than one that has been used two or more times. Higher amounts of lac repressor, the Green Fluorescent Protein fusions with CAAT enhancer binding protein (C/EBPalpha) and Gal4, elute from the columns when 0.1% Tween-20 is added to the mobile phase. The amount of improvement found depends upon the transcription factor studied and the amount of the protein loaded on the column; lac repressor and Gal4 are eluted in higher amounts over a large range of protein loads while C/EBP shows the greatest effect at low protein loads. This detergent effect is seen when either Sepharose or silica is used for the stationary phase. Including bovine serum albumin in the mobile phase gives a similar though lesser improvement to that observed with Tween-20. Mg2+ or EDTA in the mobile phase gave similar chromatography for C/EBP; since EDTA protects columns from DNases, its inclusion in the mobile phase is preferred. After extended use, the DNA affinity columns no longer bind transcription factors and this is not due to losses of DNA from the columns. Two simple methods (sodium dodecylsulfate and KSCN) were developed to regenerate such worn out columns.

  3. A column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry method to determine anandamide and 2-arachidonoylglycerol in plasma samples.

    PubMed

    Marchioni, Camila; de Souza, Israel Donizeti; Grecco, Caroline Fernandes; Crippa, José Alexandre; Tumas, Vitor; Queiroz, Maria Eugênia Costa

    2017-03-23

    This study reports a fast, sensitive, and selective column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG) in plasma samples. This bidimensional system used a restricted access media column (RP-8 ADS, 25 mm × 4 mm × 25 μM) in the first dimension and a core-shell Kinetex C18 (100 mm × 2, 1.7 mm × 1 μM) column in the second dimension, followed by detection in a mass spectrometer triple quadrupole (multiple reactions monitoring mode) operating in the positive mode. RP-8 ADS was used for trace enrichment of eCBs (reverse phase partitioning) and macromolecular matrix size exclusion; the core-shell column was used for the chromatographic separation. The column switching UHPLC-MS/MS method presented a linear range spanning from 0.1 ng mL(-1) (LOQ) to 6 ng mL(-1) for AEA and from 0.04 ng mL(-1) (LOQ) to 10 ng mL(-1) for 2-AG. Excluding the LLOQ values, the precision assays provided coefficients of variation lower than 8% and accuracy with relative standard error values lower than 14%. Neither carryover nor matrix effects were detected. This high-throughput column switching method compared to conventional methods is time saving as it involves fewer steps, consumes less solvent, and presents lower LLOQ. The column switching UHPLC-MS/MS method was successfully applied to determine AEA and 2-AG in plasma samples obtained from Alzheimer's disease patients. Graphical abstract A column switching ultra high-performance liquid chromatography-tandem mass spectrometry method using RP-8 ADS column and core shell column to determine endocannabinoids in plasma samples.

  4. Temperature-based on-column solute focusing in capillary liquid chromatography reduces peak broadening from pre-column dispersion and volume overload when used alone or with solvent-based focusing.

    PubMed

    Groskreutz, Stephen R; Horner, Anthony R; Weber, Stephen G

    2015-07-31

    On-column focusing is essential for satisfactory performance using capillary scale columns. On-column focusing results from generating transient conditions at the head of the column that lead to high solute retention. Solvent-based on-column focusing is a well-known approach to achieve this. Temperature-assisted on-column focusing (TASF) can also be effective. TASF improves focusing by cooling a short segment of the column inlet to a temperature that is lower than the column temperature during the injection and then rapidly heating the focusing segment to the match the column temperature. A troublesome feature of an earlier implementation of TASF was the need to leave the capillary column unpacked in that portion of the column inside the fitting connecting it to the injection valve. We have overcome that problem in this work by packing the head of the column with solid silica spheres. In addition, technical improvements to the TASF instrumentation include: selection of a more powerful thermo-electric cooler to create faster temperature changes and electronic control for easy incorporation into conventional capillary instruments. Used in conjunction with solvent-based focusing and with isocratic elution, volumes of paraben samples (esters of p-hydroxybenzoic acid) up to 4.5-times the column liquid volume can be injected without significant bandspreading due to volume overload. Interestingly, the shapes of the peaks from the lowest volume injections that we can make, 30nL, are improved when using TASF. TASF is very effective at reducing the detrimental effects of pre-column dispersion using isocratic elution. Finally, we show that TASF can be used to focus the neuropeptide galanin in a sample solvent with elution strength stronger than the mobile phase. Here, the stronger solvent is necessitated by the need to prevent peptide adsorption prior to and during analysis.

  5. The Trace Analysis of DEET in Water using an On-line Preconcentration Column and Liquid Chromatography with UV Photodiode Array Detection

    EPA Science Inventory

    A method for the detection of trace levels of N,N-diethyl-m-toluamide (DEET) in water is discussed. The method utilizes an on-line preconcentration column in series with high performance liquid chromatography (HPLC) and UV photodiode array detection. DEET, a common insect repel...

  6. Determination of citrus limonoid glucosides by high performance liquid chromatography coupled to post-column reaction with Ehrlich’s Reagent

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method for the identification and quantification of citrus limonoid glucosides in juices based upon high performance liquid chromatography (HPLC) separation coupled to post-column reaction with Ehrlichs’s reagent has been developed. This method utilizes a phenyl stationary phase and an isocratic ...

  7. Separation of the Components of a Commercial Analgesic Tablet: A Two-Week Sequence Comparing Purification by Two-Base Extraction and Column Chromatography

    ERIC Educational Resources Information Center

    Revell, Kevin D.

    2011-01-01

    A new laboratory experiment is described in which students compare two benchtop separation methods to isolate the three active components of the commercial analgesic Excedrin. In the two-week sequence, aspirin, acetaminophen, and caffeine are separated using either a two-base liquid-liquid extraction or silica column chromatography. Students then…

  8. Research on the interaction of hydrogen-bond acidic polymer sensitive sensor materials with chemical warfare agents simulants by inverse gas chromatography.

    PubMed

    Yang, Liu; Han, Qiang; Cao, Shuya; Huang, Feng; Qin, Molin; Guo, Chenghai; Ding, Mingyu

    2015-06-02

    Hydrogen-bond acidic polymers are important high affinity materials sensitive to organophosphates in the chemical warfare agent sensor detection process. Interactions between the sensor sensitive materials and chemical warfare agent simulants were studied by inverse gas chromatography. Hydrogen bonded acidic polymers, i.e., BSP3, were prepared for micro-packed columns to examine the interaction. DMMP (a nerve gas simulant) and 2-CEES (a blister agent simulant) were used as probes. Chemical and physical parameters such as heats of absorption and Henry constants of the polymers to DMMP and 2-CEES were determined by inverse gas chromatography. Details concerning absorption performance are also discussed in this paper.

  9. Determination of ascorbic acid and carotenoids in food commodities by liquid chromatography with mass spectrometry detection.

    PubMed

    Frenich, A Garrido; Torres, M E Hernández; Vega, A Belmonte; Vidal, J L Martínez; Bolaños, P Plaza

    2005-09-21

    Two methods, one to determine ascorbic acid and one to determine lycopene and beta-carotene, in vegetables and fruits by liquid chromatography coupled with mass spectrometry (LC-MS) have been established. The chromatographic separation of the studied compounds and their MS parameters were optimized to improve selectivity and sensitivity. In both methods, separation was carried out with two coupled columns, first a C(18) and then a dC(18), using as mobile phase 70% methanol (0.005% acetic acid) and 30% acetic acid 0.05% for ascorbic acid determination and a mixture of methanol, tetrahydrofuran, and acetonitrile (60:30:10 v/v/v) for carotenoid analysis in isocratic mode. The molecular ion was selected for the quantification in selective ion monitoring (SIM) mode. Ascorbic acid was detected with electrospray ionization probe (ESI) in negative mode, while chemical ionization atmospheric pressure (APCI) in positive mode was used for the target carotenoids. The methodology for ascorbic acid analysis is based on an extraction with polytron using methanol and a mixture of methaphosphoric acid and acetic acid. Extraction of the carotenoids was carried out with tetrahydrofuran/methanol (1:1) (v/v). The proposed methods were applied, after their corresponding validations, to the analysis of four varieties of tomatoes, tomato in tin enriched and dried tomato, and to the analysis of mango and kiwi fruits, to compare the content in these compounds. Moreover, the influence of the process of freezing and the effect that the manipulation/preservation has in the content of ascorbic acid in tomato have also been studied.

  10. Rapid and reliable quantitation of amino acids and myo-inositol in mouse brain by high performance liquid chromatography and tandem mass spectrometry.

    PubMed

    Bathena, Sai P; Huang, Jiangeng; Epstein, Adrian A; Gendelman, Howard E; Boska, Michael D; Alnouti, Yazen

    2012-04-15

    Amino acids and myo-inositol have long been proposed as putative biomarkers for neurodegenerative diseases. Accurate measures and stability have precluded their selective use. To this end, a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on multiple reaction monitoring was developed to simultaneously quantify glutamine, glutamate, γ-aminobutyric acid (GABA), aspartic acid, N-acetyl aspartic acid, taurine, choline, creatine, phosphocholine and myo-inositol in mouse brain by methanol extractions. Chromatography was performed using a hydrophilic interaction chromatography silica column within in a total run time of 15 min. The validated method is selective, sensitive, accurate, and precise. The method has a limit of quantification ranging from 2.5 to 20 ng/ml for a range of analytes and a dynamic range from 2.5-20 to 500-4000 ng/ml. This LC-MS/MS method was validated for biomarker discovery in models of human neurological disorders.

  11. Analytical approach to determining human biogenic amines and their metabolites using eVol microextraction in packed syringe coupled to liquid chromatography mass spectrometry method with hydrophilic interaction chromatography column.

    PubMed

    Konieczna, Lucyna; Roszkowska, Anna; Synakiewicz, Anna; Stachowicz-Stencel, Teresa; Adamkiewicz-Drożyńska, Elżbieta; Bączek, Tomasz

    2016-04-01

    Analysis of biogenic amines (BAs) in different human samples provides insight into the mechanisms of various biological processes, including pathological conditions, and thus may be very important in diagnosing and monitoring several neurological disorders and cancerous tumors. In this work, we developed a simple and fast procedure using a digitally controlled microextraction in packed syringe (MEPS) coupled to liquid chromatography mass spectrometry (LC-MS) method for simultaneous determination of biogenic amines, their precursors and metabolites in human plasma and urine samples. The separation of 12 low molecular weight and hydrophilic molecules with a wide range of polarities was achieved with hydrophilic interaction chromatography (HILIC) column without derivatization step in 12 min. MEPS was implemented using the APS sorbent in semi-automated analytical syringe (eVol(®)) and small volume of urine and plasma samples, 5 0µL and 100 μL, respectively. We evaluated important parameters influencing MEPS efficiency, including stationary phase selection, sample pH and volume, number of extraction cycles, and washing and elution volumes. In optimized MEPS conditions, the analytes were eluted by 3 × 50 μL of methanol with 0.1% formic acid. The chromatographic separation of analytes was performed on XBridge Amide™ BEH analytical column (3.0mm × 100 mm, 3.5 µm) using gradient elution with mobile phase consisting of phase A: 10mM ammonium formate buffer in water pH 3.0 and phase B: 10mM ammonium formate buffer in acetonitrile pH 3.0. The LC-HILIC-MS method was validated and, in optimum conditions, presented good linearity in concentration range within 10-2000 ng/mL for all the analytes with a determination coefficient (r(2)) higher than 0.999 for plasma and urine samples. Method recovery ranged within 87.6-104.3% for plasma samples and 84.2-98.6% for urine samples. The developed method utilizing polar APS sorbent along with polar HILIC column was applied for

  12. Trace anion determination in concentrated hydrofluoric acid solutions by two-dimensional ion chromatography I. Matrix elimination by ion-exclusion chromatography.

    PubMed

    Vermeiren, Koen

    2005-08-26

    Since years, ion exclusion chromatography (ICE) has been the standard method to separate strong acid analyte anions from concentrated weak acid matrices such as hydrofluoric acid (HF). In this work, the commercially available IonPac ICE-AS 1 column was used to separate trace levels of chloride, nitrate, sulfate and phosphate from HF solutions at 20% (w/w). The efficiency of the separation was studied in more detail using techniques such as ion chromatography (IC), inductively coupled plasma optical emission spectrometry (ICP-OES) and ICP-mass spectrometry (ICP-MS). For 20% (w/w) HF solutions and at a water carrier flow-rate of 0.50 ml/min, the cut window was set from 8.5 to 14.5 min. Under these conditions, analyte recoveries of better than 90% were obtained for chloride, nitrate and sulfate, but only about 75% for phosphate. The HF rejection efficiency was better than 99.9%. It was found that the ICP techniques, measuring total element levels and not species, yielded significantly higher recoveries for phosphorus and sulfur compared to IC. Evidence will be given that part of the added phosphorus (approximately 15% for an addition of 10 mg PO4/kg) is present as mono-fluorophosphoric acid (H2FPO3). In the case of sulfate, the difference between IC and ICP-MS could be attributed to an important matrix effect from the residual HF concentration.

  13. Reversed-phase high-performance liquid chromatography of the stable electrophoretic fractions of soil humic acids

    NASA Astrophysics Data System (ADS)

    Trubetskoi, O. A.; Trubetskaya, O. E.

    2015-02-01

    Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used for the hydrophobicity analysis of soil humic acids and their stable electrophoretic fractions A, B, and C + D preliminarily prepared by the combination of gel permeation chromatography on Sephadex with polyacrylamide gel electrophoresis. In two humic acid preparations of different genesis, the electrophoretic fraction A of the larger molecular size was the most hydrophobic (60-73% of the fraction was irreversibly adsorbed on a hydrophobic reversed-phase (RF) column C18), and the fraction C + D of the smallest molecular size was the most hydrophilic. The fraction B of medium size occupied an intermediate position (33-47% of the fraction was irreversibly adsorbed on the column). The use of RP-HPLC allowed for the first time detecting the hydrophobic electrophoretic fraction A of the largest molecular size mainly composed of aliphatic long-chained hydrocarbon, protein, and carbohydrate fragments in soil humic acids. Data on the degree of hydrophobicity and the earlier obtained physicochemical characteristics of stable electrophoretic fractions are discussed in terms of the supramolecular and macromolecular structure of soil humic acids.

  14. Formation of iron complexs from trifluoroacetic acid based liquid chromatography mobile phases as interference ions in liquid chromatography/electrospray ionization mass spectrometric analysis

    SciTech Connect

    Shukla, Anil K.; Zhang, Rui; Orton, Daniel J.; Zhao, Rui; Clauss, Therese RW; Moore, Ronald J.; Smith, Richard D.

    2011-05-30

    Two unexpected singly charged ions at m/z 1103 and 944 have been observed in mass spectra obtained from electrospray ionization-mass spectrometric analysis of liquid chromatography effluents with mobile phases containing trifluoroacetic acid. Accurate mass measurement and tandem mass spectrometry studies revealed that these two ions are not due to any contamination from solvents and chemicals used for mobile and stationary phases or from the laboratory atmospheric environment. Instead these ions are clusters of trifluoroacetic acid formed in association with acetonitrile, water and iron from the stainless steel union used to connect the column with the electrospray tip and to apply high voltage; the molecular formulae are Fe+((OH)(H2O)2)9(CF3COOH)5 and Fe+((OH)(H2O)2)6 (CF3COOH)5.

  15. Characterization of warfarin unusual peak profiles on oligoproline chiral high performance liquid chromatography columns.

    PubMed

    Lao, Wenjian; Gan, Jay

    2010-10-15

    Unusual peak profiles of warfarin were characterized on two oligoproline chiral stationary phases (CSPs). The pattern of 1st peak (S(-)) broadening and the 2nd peak (R(+)) compression was observed under mobile phase of hexane (0.1% TFA)/2-propanol (IPA) on a triproline CSP 1, and with other alcohol modifier such as ethanol, 1-propanol, 1-butanol, 2-butanol, and tert-butanol as well. Through analyzing system peak of additives, the unusual peak profile was interpreted by perturbation of TFA additive system peak. The unusual peak profile was also found in enantioseparation of coumachlor and on a covalently bonded doubly tethered diproline CSP 2. The pattern of 1st peak (S(-)) broadening and the 2nd peak (R(+)) compression can change to pattern of 1st peak compression and the 2nd peak broadening from 15 to 50°C. Chiral separation of warfarin created nonlinear van't Hoff plots on CSP. No peak broadening/compression were observed with methyl tertiary butyl ether or ethyl acetate as the modifier. The peak shapes of the two warfarin enantiomers can thus be tuned by varying alcohol concentration and column temperature. High separation factor and resolution may be carried out to tune the peak profiles into Langmuir/anti-Langmuir band-shape composition. Using none hydrogen donor modifier may avoid interference of the TFA system peak.

  16. Packed column supercritical fluid chromatography of hydrophilic analytes via water-rich modifiers.

    PubMed

    Taylor, Larry T

    2012-08-10

    The use of additives to dramatically extend the range of solute polarity amenable to CO(2) based supercritical fluid chromatography (pcSFC) was predicted over 20 years ago. At that time additives were predicted to have multiple functions such as enhancement of mobile phase solvating power, ion suppression, and ion pairing. The adsorption of mobile phase components on the stationary phase causing a modification of its surface was predicted, but the implications for separations were not defined. Reports published in the late 1980s showed that while water could not function as a primary modifier due to it poor solubility in carbon dioxide, its use as an additive was more promising. The past decade has seen very little published work concerning water and pcSFC. Now reports are beginning to appear that demonstrate enhanced selectivity with water, and application of the technology to polypeptide salts, drug molecules, and nucleobases. This review attempts to bridge the past with the present. As evidenced by the studies described in this review, water may offer much potential as an additive in that it could (a) enhance the solvating power of the mobile phase, (b) introduce HILIC-like analyte partitioning, (c) simplify preparative purifications, and (d) offer a more mass spectrometrically compatible interface.

  17. Performance limits and kinetic optimization of parallel and serially connected multi-column systems spanning a wide range of efficiencies for liquid chromatography.

    PubMed

    Cabooter, Deirdre; Desmet, Gert

    2012-01-06

    Using a set of experimentally determined liquid chromatography column performance data, it has been investigated how a range of efficiencies can best be covered when using a multi-column system. Two main variants are considered: a serially-connected variant (realizing different column lengths by connecting a different number of column segments in series) and a parallel-connected variant (realizing different column lengths by simply switching between columns with a different length arranged in parallel). Both variants are compared for their ability to keep the average analysis time along a given range of efficiencies as close as possible to the intrinsic Knox & Saleem-speed limit. It was found that the serial connection mode offers a better compromise between average speed and amount of required silica (total required column length) than the parallel connection mode for all efficiency ranges running from 5000-10,000 plates up to 75,000-150,000 plates. Considering an ultra-high performance liquid chromatography (UHPLC) operation at 1200 bar, the best possible serial connection system can get within about within 15-25% of the Knox & Saleem-speed limit, whereas a three-column parallel system can only get to within 40-50% of the speed limit, while needing 50-100% more total column length. In absolute terms, the serially-connected system with individually optimized segment lengths should be able to cover a range of 5000-75,000 theoretical plates (dynamic range of 25) in an average analysis time of 14.3 min when using a 1200 bar instrument. At 400 bar, this would be 37.9 min, showing that the construction of wide-efficiency range systems would be one of the application areas where the advantages of UHPLC-conditions would be most fully realized.

  18. Column leaching test to evaluate the use of alkaline industrial wastes to neutralize acid mine tailings

    SciTech Connect

    Doye, I.; Duchesne, J.

    2005-08-01

    Acid mine drainage is a serious environmental problem caused by the oxidation of sulfide minerals that releases highly acidic, sulfate, and metals-rich drainage. In this study, alkaline industrial wastes were mixed with acid mine tailings in order to obtain neutral conditions. A series of column leaching tests were performed to evaluate the behavior of reactive mine tailings amended with alkaline-additions under dynamic conditions. Column tests were conducted of oxidized mine tailings combined with cement kiln dust, red mud bauxite, and mixtures of cement kiln dust with red mud bauxite. The pH results show the addition of 10% of alkaline materials permits the maintenance of near neutral conditions. In the presence of 10% alkaline material, the concentration of toxic metals such as Al, Cu, Fe, Zn are significantly reduced as well as the number of viable cells (Thiobacillus ferrooxidans) compared to control samples.

  19. Scale-up protein separation on stainless steel wide bore toroidal columns in the type-J counter-current chromatography.

    PubMed

    Guan, Yue Hugh; Hewitson, Peter; van den Heuvel, Remco N A M; Zhao, Yan; Siebers, Rick P G; Zhuang, Ying-Ping; Sutherland, Ian

    2015-12-11

    Manufacturing high-value added biotech biopharmaceutical products (e.g. therapeutic proteins) requires quick-to-develop, GMP-compliant, easy-to-scale and cost effective preparatory chromatography technologies. In this work, we describe the construction and testing of a set of 5-mm inner diameter stainless steel toroidal columns for use on commercially available preparatory scale synchronous J-type counter-current chromatography (CCC) machinery. We used a 20.2m long column with an aqueous two-phase system containing 14% (w/w) PEG1000 and 14% (w/w) potassium phosphate at pH 7, and tested a sample loading of 5% column volume and a mobile phase flow rate of 20ml/min. We then satisfactorily demonstrated the potential for a weekly protein separation and preparation throughput of ca. 11g based on a normal weekly routine for separating a pair of model proteins by making five stacked injections on a single portion of stationary phase with no stripping. Compared to our previous 1.6mm bore PTFE toroidal column, the present columns enlarged the nominal column processing throughput by nearly 10. For an ideal model protein injection modality, we observed a scaling up factor of at least 21. The 2 scales of protein separation and purification steps were realized on the same commercial CCC device.

  20. Quantitation of triacylglycerols in edible oils by off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column.

    PubMed

    Wei, Fang; Hu, Na; Lv, Xin; Dong, Xu-Yan; Chen, Hong

    2015-07-24

    In this investigation, off-line comprehensive two-dimensional liquid chromatography-atmospheric pressure chemical ionization mass spectrometry using a single column has been applied for the identification and quantification of triacylglycerols in edible oils. A novel mixed-mode phenyl-hexyl chromatographic column was employed in this off-line two-dimensional separation system. The phenyl-hexyl column combined the features of traditional C18 and silver-ion columns, which could provide hydrophobic interactions with triacylglycerols under acetonitrile conditions and can offer π-π interactions with triacylglycerols under methanol conditions. When compared with traditional off-line comprehensive two-dimensional liquid chromatography employing two different chromatographic columns (C18 and silver-ion column) and using elution solvents comprised of two phases (reversed-phase/normal-phase) for triacylglycerols separation, the novel off-line comprehensive two-dimensional liquid chromatography using a single column can be achieved by simply altering the mobile phase between acetonitrile and methanol, which exhibited a much higher selectivity for the separation of triacylglycerols with great efficiency and rapid speed. In addition, an approach based on the use of response factor with atmospheric pressure chemical ionization mass spectrometry has been developed for triacylglycerols quantification. Due to the differences between saturated and unsaturated acyl chains, the use of response factors significantly improves the quantitation of triacylglycerols. This two-dimensional liquid chromatography-mass spectrometry system was successfully applied for the profiling of triacylglycerols in soybean oils, peanut oils and lord oils. A total of 68 triacylglycerols including 40 triacylglycerols in soybean oils, 50 triacylglycerols in peanut oils and 44 triacylglycerols in lord oils have been identified and quantified. The liquid chromatography-mass spectrometry data were analyzed

  1. [Determination of free formaldehyde in cosmetics by pre-column derivatization, extraction inhibition and high performance liquid chromatography].

    PubMed

    Lü, Chunhua; Huang, Chaoqun; Chen, Mei; Xie, Wen; Chen, Xiaomei

    2012-12-01

    Pre-column derivatization and inhibition by solvent extraction were applied to determine free formaldehyde in cosmetics by high performance liquid chromatography (HPLC). Due to the rapid decomposition of formaldehyde donors in the derivatization, it is hard to detect the amount of the free formaldehyde in cosmetics. The formaldehyde directly reacted with 2,4-dinitrophenylhydrazine in acetonitrile-phosphate buffer (pH 2) (1:1, v/v) solution for 2 min, then dichloromethane extraction was used to induce the decomposition of formaldehyde donors. The extract was diluted with acetonitrile and then determined by HPLC. The formaldehyde derivative was separated on an Agilent C18 column (250 mm x 4.6 mm, 5 microm) at 30 degrees C with acetonitrile-water (60:40, v/v) as mobile phase at a flow rate of 1.0 mL/min, and detected at the wavelength of 355 nm. The recoveries were from 81% to 106% at the spiked levels of 50, 100, 500, 1 000 microg/g of formaldehyde in shampoo, milk, cream, hand cleaner, toothpaste, nail polish, powder separately, and the relative standard deviations (n = 6) were less than 5.0%. The limit of quantification of the formaldehyde in cosmetics was 50 microg/g. The method has been applied to the determination of free formaldehyde in real samples and the results showed that the release by formaldehyde donors was inhibited. The method has the advantages of simple operation, good accuracy and meets the requirement of determination of free formaldehyde in cosmetics.

  2. Effect of high pH column regeneration on the separation performances in reversed phase chromatography of peptides.

    PubMed

    Gétaz, David; Gencoglu, Mumun; Forrer, Nicola; Morbidelli, Massimo

    2010-05-21

    Caustic regeneration procedures are often used in chromatographic purification processes of peptides and proteins to remove irreversibly bound impurities from the stationary phase. Silica-based materials are the most commonly used materials in reversed phase chromatography of peptides. Their limited chemical stability at high pH can be, however, problematic when high pH column regeneration (i.e. cleaning in place) is required. The effect of cleaning in place on the surface chemistry of the stationary phase has been investigated using the Tanaka test. It has been shown that the high pH treatment does not significantly affect the hydrophobicity of the material, but it strongly increases its silanol activity. A representative peptide purification process has been used to investigate the impact of cleaning in place on the separation performance. It has been shown that the caustic regeneration increases the peptide retention at high pH (pH 6.5), due to the interactions between the peptide and the negatively charged silanol groups. These unwanted interactions reduce the separation performances by decreasing the selectivity between the late eluting impurities and the main peptide. However, it has been shown that the effect of the silanol groups on the peptide adsorption and on the separation performance can be minimized by carrying out the purification process at low pH (pH approximately 2). In this case, the silanol groups are protonated and their electrostatic interactions with the positively charged analyte (i.e. peptides) are suppressed. In these conditions, the peptide adsorption and the impurity selectivity is not changing upon high pH column regeneration and the separation performance is not affected.

  3. Simultaneous identification and quantification of tetrodotoxin in fresh pufferfish and pufferfish-based products using immunoaffinity columns and liquid chromatography/quadrupole-linear ion trap mass spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Mengmeng; Wu, Haiyan; Jiang, Tao; Tan, Zhijun; Zhao, Chunxia; Zheng, Guanchao; Li, Zhaoxin; Zhai, Yuxiu

    2016-08-01

    In this study, we established a comprehensive method for simultaneous identification and quantification of tetrodotoxin (TTX) in fresh pufferfish tissues and pufferfish-based products using liquid chromatography/quadrupole-linear ion trap mass spectrometry (LC-QqLIT-MS). TTX was extracted by 1% acetic acid-methanol, and most of the lipids were then removed by freezing lipid precipitation, followed by purification and concentration using immunoaffinity columns (IACs). Matrix effects were substantially reduced due to the high specificity of the IACs, and thus, background interference was avoided. Quantitation analysis was therefore performed using an external calibration curve with standards prepared in mobile phase. The method was evaluated by fortifying samples at 1, 10, and 100 ng/g, respectively, and the recoveries ranged from 75.8%-107%, with a relative standard deviation of less than 15%. The TTX calibration curves were linear over the range of 1-1 000 μg/L, with a detection limit of 0.3 ng/g and a quantification limit of 1 ng/g. Using this method, samples can be further analyzed using an information-dependent acquisition (IDA) experiment, in the positive mode, from a single liquid chromatography-tandem mass spectrometry injection, which can provide an extra level of confirmation by matching the full product ion spectra acquired for a standard sample with those from an enhanced product ion (EPI) library. The scheduled multiple reaction monitoring method enabled TTX to be screened for, and TTX was positively identified using the IDA and EPI spectra. This method was successfully applied to analyze a total of 206 samples of fresh pufferfish tissues and pufferfish-based products. The results from this study show that the proposed method can be used to quantify and identify TTX in a single run with excellent sensitivity and reproducibility, and is suitable for the analysis of complex matrix pufferfish samples.

  4. Effect of pressure pulses at the interface valve on the stability of second dimension columns in online comprehensive two-dimensional liquid chromatography.

    PubMed

    Talus, Eric S; Witt, Klaus E; Stoll, Dwight R

    2015-01-23

    Users of online comprehensive two-dimensional liquid chromatography (LCxLC) frequently acknowledge that the mechanical instability of HPLC columns installed in these systems, particularly in the second dimension, is a significant impediment to its use. Such instability is not surprising given the strenuous operating environment to which these columns are subjected, including the large number (thousands per day) of fast and large pressure pulses resulting from interface valve switches (on the timescale of tens of milliseconds) associated with very fast second dimension separations. There appear to be no published reports of systematic studies of the relationship between second dimension column lifetime and any of these variables. In this study we focused on the relationship between the lifetimes of commercially available columns and the pressure pulses observed at the inlet of the second dimension column that occur during the switching of the valve that interfaces the two dimensions of a LCxLC system. We find that the magnitude of the pressure drop at the inlet of the second dimension column during the valve switch, which may vary between 10 and 95% of the column inlet pressure, is dependent on valve switching speed and design, and has a dramatic impact on column lifetime. In the worst case, columns fail within the first few hours of use in an LCxLC system. In the best case, using a valve that exhibits much smaller pressure pulses, the same columns exhibit much improved lifetimes and have been used continuously under LCxLC conditions for several days with no degradation in performance. This result represents a first step in understanding the factors that affect second dimension column lifetime, and will significantly improve the usability of the LCxLC technique in general.

  5. Profiling of triacylglycerols in plant oils by high-performance liquid chromatography-atmosphere pressure chemical ionization mass spectrometry using a novel mixed-mode column.

    PubMed

    Hu, Na; Wei, Fang; Lv, Xin; Wu, Lin; Dong, Xu-Yan; Chen, Hong

    2014-12-01

    In this investigation, a rapid and high-throughput method for profiling of TAGs in plant oils by liquid chromatography using a single column coupled with atmospheric pressure chemical ionization (APCI) mass spectrometry was reported. A novel mixed-mode phenyl-hexyl chromatographic column was employed in this separation system. The phenyl-hexyl column could provide hydrophobic interactions as well as π-π interactions. Compared with two traditionally columns used in TAG separation - the C18 column and silver-ion column, this column exhibited much higher selectivity for the separation of TAGs with great efficiency and rapid speed. By comparison with a novel mix-mode column (Ag-HiSep OTS column), which can also provide both hydrophobic interactions as well as π-π interactions for the separation of TAGs, phenyl-hexyl column exhibited excellent stability. LC method using phenyl-hexyl column coupled with APCI-MS was successfully applied for the profiling of TAGs in soybean oils, peanut oils, corn oils, and sesame oils. 29 TAGs in peanut oils, 22 TAGs in soybean oils, 19 TAGs in corn oils, and 19 TAGs in sesame oils were determined and quantified. The LC-MS data was analyzed by barcodes and principal component analysis (PCA). The resulting barcodes constitute a simple tool to display differences between different plant oils. Results of PCA also enabled a clear identification of different plant oils. This method provided an efficient and convenient chromatographic technology for the fast characterization and quantification of complex TAGs in plant oils at high selectivity. It has great potential as a routine analytical method for analysis of edible oil quality and authenticity control.

  6. Copper(II) complexes of lipophilic aminoglycoside derivatives for the amino acid enantiomeric separation by ligand-exchange liquid chromatography.

    PubMed

    Zaher, Mustapha; Baussanne, Isabelle; Ravelet, Corinne; Halder, Somnath; Haroun, Mohamed; Fize, Jennifer; Décout, Jean-Luc; Peyrin, Eric

    2008-03-28

    In this paper, a new class of ligand-exchange chiral stationary phase (LE-CSP) based on the copper complexes of lipophilic aminoglycoside derivatives was reported. Different stationary phases were developed by coating reversed-phase liquid chromatography supports with three neamine derivatives carrying a lipophilic octadecyl chain at the 4', 5 and 6 positions, respectively. The enantioselective ability of these LE neamine-based CSPs was evaluated and the 4'-derivative coated column was found to be the most interesting one for the amino acid resolution. The effects of the variation of several chromatographic parameters on the enantioseparation were evaluated in order to identify the analysis optimal conditions.

  7. Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine.

    PubMed

    Zaher, Mustapha; Ravelet, Corinne; Baussanne, Isabelle; Ravel, Anne; Grosset, Catherine; Décout, Jean-Luc; Peyrin, Eric

    2009-01-01

    In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 micromol/m(2)) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids.

  8. Simultaneous determination of amino acids and carbohydrates in culture media of Clostridium thermocellum by valve-switching ion chromatography.

    PubMed

    Fa, Yun; Yang, Haiyan; Ji, Chengshuai; Cui, He; Zhu, Xinshu; Du, Juan; Gao, Jun

    2013-10-10

    An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5-4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.

  9. Application of polymethacrylate resin as stationary phase in liquid chromatography with UV detection for C1-C7 aliphatic monocarboxylic acids and C1-C7 aliphatic monoamines.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi; Takeuchi, Toyohide

    2004-06-11

    The application of unfunctionized polymethacrylate resin (TSKgel G3000PWXL) as a stationary phase in liquid chromatography with UV detection for C1-C7 aliphatic monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid, 3,3-dimethylbutyric acid, 4-methylvaleric acid, hexanoic acid, 2-methylhexanoic acid, 5-methylhexanoic acid and heptanoic acid) and C1-C7 aliphatic monoamines (methylamine, ethylamine, propylamine, isobutylamine, butylamine, isoamylamine, amylamine, 1,3-dimethylbutylamine, hexylamine, 2-heptylamine and heptylamine) was carried out. Using dilute sulfuric acid as the eluent, the TSKgel G3000PWXL, resin acted as an advanced stationary phase for these C1-C7 carboxylic acids. Excellent simultaneous separation and symmetrical peaks for these C1-C7 carboxylic acids were achieved on a TSKgel G3000PWXL column (150 mm x 6 mm i.d.) in 60 min with 0.25 mM sulfuric acid containing 1 mM 2-methylheptanoic acid at pH 3.3 as the eluent. Using dilute sodium hydroxide as the eluent, the TSKgel G3000PWXL resin also behaved as an advanced stationary phase for these C1-C7 amines. Excellent simultaneous separation and good peaks for these C1-C7 amines were achieved on the TSKgel G3000PWXL column in 60 min with 10 mM sodium hydroxide containing 0.5 mM 1-methylheptylamine at pH 11.9 as the eluent.

  10. Liquid chromatography coupled to on-line post column derivatization for the determination of organic compounds: a review on instrumentation and chemistries.

    PubMed

    Zacharis, Constantinos K; Tzanavaras, Paraskevas D

    2013-10-10

    Analytical derivatization either in pre or post column modes is one of the most widely used sample pretreatment techniques coupled to liquid chromatography. In the present review article we selected to discuss the post column derivatization mode for the analysis of organic compounds. The first part of the review focuses to the instrumentation of post-column setups including not only fundamental components such as pumps and reactors but also less common parts such as static mixers and back-pressure regulators; the second part of the article discusses the most popular "chemistries" that are involved in post column applications, including reagent-less approaches and new sensing platforms such as the popular gold nanoparticles. Some representative recent applications are also presented as tables.

  11. A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization.

    PubMed

    Oguri, Shigeyuki; Nomura, Michiko; Fujita, Youko

    2005-06-17

    A new strategy for the selective determination of D-amino acids (DAAs) employing a pre-column derivatization was designed with concepts based on both enzymatic and chemical modifications. Selective determination of DAAs was accomplished by following: DAA was enzymatically modified with D-amino acid oxidase (DAAO: EC 1.4.3.3) to form an alpha-keto acid. Subsequently, resulting alpha-keto acid was detected by high-performance liquid chromatography (HPLC) after chemical modification with o-phenylenediamine (PDA) in the presence of 2-mercaptoethanol (2ME) to give the corresponding quinoxalinol derivative (PDA-alpha-keto acid derivative). After optimizing the pre-column derivatization and HPLC separation, five peaks corresponding to DAAs (D-alanine, D-leucine, D-methionine, D-phenylalanine, D-valine (as the standard mixture of DAAs in this paper) were separately eluted and monitored by means of a conventional HPLC system with a gradient elution on octadecyl silica gel (ODS) column and a fluorescence detector (Ex.: 341 nm, Em.: 413 nm), respectively. It was confirmed that the present method was incapable of detecting L-amino acids (LAA) when a sample solution consisting of both LAAs and DAAs was examined. The linearity of the peak-area responses to their concentration range of DAAs from 10 to 500 microM is 0.994-1.000, and their detection limits were 0.2-1 microM (signal/noise = 3). When this method was applied to a methanolic extract of short-necked clams, Ruditapes philippinarum (in Japanese, Asari), a big peak, corresponding to D-alanine was detected, corresponding to 2.9 mg/g D-alanine. In this paper, we present an example of pre-column derivatization method that was newly configured to take into account both the biological and chemical properties of the substances in question.

  12. Determination of histamine in wines with an on-line pre-column flow derivatization system coupled to high performance liquid chromatography.

    PubMed

    García-Villar, Natividad; Saurina, Javier; Hernández-Cassou, Santiago

    2005-09-01

    A new rapid and sensitive high performance liquid chromatography (HPLC) method for determining histamine in red wine samples, based on continuous flow derivatization with 1,2-naphthoquinone-4-sulfonate (NQS), is proposed. In this system, samples are derivatized on-line in a three-channel flow manifold for reagent, buffer and sample. The reaction takes place in a PTFE coil heated at 80 degrees C and with a residence time of 2.9 min. The reaction mixture is injected directly into the chromatographic system, where the histamine derivative is separated from other aminated compounds present in the wine matrix in less than ten minutes. The HPLC procedure involves a C18 column, a binary gradient of 2% acetic acid-methanol as a mobile phase, and UV detection at 305 nm. Analytical parameters of the method are evaluated using red wine samples. The linear range is up to 66.7 mg L(-1) (r = 0.9999), the precision (RSD) is 3%, the detection limit is 0.22 mg L(-1), and the average histamine recovery is 101.5% +/- 6.7%. Commercial red wines from different Spanish regions are analyzed with the proposed method.

  13. Speciation analysis of inorganic tin by on-column complexation ion chromatography with inductively coupled plasma mass spectrometry and electrospray mass spectrometry.

    PubMed

    Huang, Li; Yang, Die; Guo, Xiangquan; Chen, Zuliang

    2014-11-14

    Inductively coupled plasma-mass spectrometry (ICP-MS) and electrospray mass spectrometry (ESI-MS) were used as complementary methods to identify Sn-pentaacetic acid (DTPA) complex formation. ESI-MS was used to initially confirm the formation of [Sn(DTPA)](3-) and [Sn(DTPA)](1-) and their MS spectra suggest these tin complexes were stable in solution. On-column complexation of tin with DTPA and the separation of [Sn(DTPA)](3-) and [Sn(DTPA)](1-) was performed on anion-exchange chromatography with an mobile phase containing 20mM NH4NO3 and 3mM DTPA at pH 6.0, and the subsequent detection of [Sn(DTPA)](3-) and [Sn(DTPA)](1-) was achieved by ICP-MS. Linear plots were obtained in a concentration range of 1.0-1000 μg L(-1) with detection limits ranging from 0.1 to 0.3 μg L(-1). The developed procedure allows the simultaneous determination of [Sn(DTPA)](3-) and [Sn(DTPA)](1-) in less than 5 min with a RSD between 2.1 and 2.7%. The recoveries of [Sn(DTPA)](3-) and [Sn(DTPA)](1-) were found to be 96.8 and 99.4%, respectively, when the sample was spiked with 20 μg L(-1) standard. Finally, the proposed procedure was used for the determination of tin species in contaminated water.

  14. Tracing novel hemostatic compounds from heating products of total flavonoids in Flos Sophorae by spectrum-effect relationships and column chromatography.

    PubMed

    Chen, Yeqing; Yu, Hongli; Wu, Hao; Pan, Yaozong; Wang, Kuilong; Liu, Liping; Jin, Yangping; Zhang, Chenchao

    2015-05-01

    Flos Sophorae and its processed product have been clinically used to treat hemorrhage. In this study, the total ion chromatographic fingerprints of the heating products of total flavonoids in Flos Sophorae were established by high-performance liquid chromatography with tandem mass spectrometry and the hemostatic activities were studied by hemostatic screening tests in vivo. The spectrum-effect relationships between fingerprints and hemostatic activities were investigated using canonical correlation analysis to trace the peaks responsible for the hemostatic effects. The predicted active peaks in fingerprints were isolated by column chromatography and their structures were identified by NMR spectroscopy and mass spectrometry. The hemostatic activities of them were verified by platelet aggregation and procoagulation assays in vitro. Canonical correlation analysis results showed that peak 8 and peak 11 were correlated most closely, thus probably being the main hemostatic compounds. Through column chromatography separation, peak 8 (compound I) and peak 11 (compound II) were obtained with purities of 95.61 and 93.38%, respectively, and were discovered new hemostatic compounds named as huaicarbon A (I) and huaicarbon B (II), respectively. This study provides a universal model to trace the active compounds of other herbs which have bioactivity enhancement after processing by spectrum-effect relationships and column chromatography.

  15. A soil-column gas chromatography (SCGC) approach to explore the thermal desorption behavior of hydrocarbons from soils.

    PubMed

    Yu, Ying; Liu, Liang; Shao, Ziying; Ju, Tianyu; Sun, Bing; Benadda, Belkacem

    2016-01-01

    A soil-column gas chromatography approach was developed to simulate the mass transfer process of hydrocarbons between gas and soil during thermally enhanced soil vapor extraction (T-SVE). Four kinds of hydrocarbons-methylbenzene, n-hexane, n-decane, and n-tetradecane-were flowed by nitrogen gas. The retention factor k' and the tailing factor T f were calculated to reflect the desorption velocities of fast and slow desorption fractions, respectively. The results clearly indicated two different mechanisms on the thermal desorption behaviors of fast and slow desorption fractions. The desorption velocity of fast desorption fraction was an exponential function of the reciprocal of soil absolute temperature and inversely correlated with hydrocarbon's boiling point, whereas the desorption velocity of slow desorption fraction was an inverse proportional function of soil absolute temperature, and inversely proportional to the log K OW value of the hydrocarbons. The higher activation energy of adsorption was found on loamy soil with higher organic content. The increase of carrier gas flow rate led to a reduction in the apparent activation energy of adsorption of slow desorption fraction, and thus desorption efficiency was significantly enhanced. The obtained results are of practical interest for the design of high-efficiency T-SVE system and may be used to predict the remediation time.

  16. Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.

    PubMed

    Zhuang, Ran; Zhang, Yuan; Zhang, Rui; Song, Chaojun; Yang, Kun; Yang, Angang; Jin, Boquan

    2008-05-01

    GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.

  17. Potential of chiral anion-exchangers operated in various subcritical fluid chromatography modes for resolution of chiral acids.

    PubMed

    Pell, Reinhard; Lindner, Wolfgang

    2012-07-06

    Anion-exchange-type chiral stationary phases (CSPs) derived from quinine or quinidine were applied in subcritical fluid chromatography (SFC) for the direct separation of chiral acidic compounds. Employing subcritical (sc) mobile phase modes (CO₂ + methanol as co-solvent and acids and bases as additives) first the influence of type and amount of acidic and basic additives on separation performance was investigated. Secondly, water was tested as a neutral additive and the influence of temperature variation on enantioselectivity was studied. Thirdly, we could chromatographically confirm that the often verbalized "inherent acidity" of sc CO₂ + methanol is manifested by the in situ formation of methylcarbonic acids in the sc mobile phase and thus functioning as acidic additive. Accordingly the dissociated methylcarbonic acid, acting as a counterion, enables an anion exchange mechanism between the cationic CSP and the corresponding acidic analyte. In the absence of a dissociable acid in the mobile phase such an ion exchange mode would not work following a stoichiometric displacement model. This finding is further corroborated by the use of ammonia in methanol as co-solvent thus generating in situ the ammonium salt of methylcarbonic acid. In summary, we report on ion-exchange mediated chromatographic separations in SFC modes by merely using (i) sc CO₂ and MeOH, (ii) sc CO₂ and ammonia in MeOH, and (iii) sc CO₂ and MeOH plus acids and bases as additives. Comparisons to HPLC mode have been undertaken to evaluate merits and limitations. This mode exhibits high potential for preparative chromatography of chiral acids combining pronounced enantioselectivity with high column loadability and avoiding possibly troublesome mobile phase additives, as the in situ formed methylcarbonic acid disintegrates to CO₂ and methanol upon pressure release.

  18. Determination of mono- and dichloroacetic acids in betaine media by liquid chromatography.

    PubMed

    Ghassempour, Alireza; Chalavi, Soheila; Abdollahpour, Assem; Mirkhani, Seyed Amir

    2006-02-15

    A simple and sensitive method has been developed for the analysis of residue amounts of chloroacetic acids in betaine samples based on derivatization by 1-naphthylamine (NA). The derivatized compounds are analyzed by reverse phase high performance liquid chromatography using methanol and water as mobile phase in the ratio of 32/68 (v/v) and phenyl column and PDA detection at 222nm. The detection limits (LOD) of monochloroacetic acid (MCA) and dichloroacetic acid (DCA) are 0.1 and 0.15mugmL(-1), respectively. The limits of quantification (LOQ) and the linear dynamic ranges (LDR) of MCA are found to be 1 and 1-400mugmL(-1), respectively, and for DCA are found to be 3 and 3-400mugmL(-1), respectively. The precision at the 5ppm level for MCA and DCA are about 3% and 2%, (n=5), respectively. The average recovery for MCA and DCA spiked to betaine samples are 98% and 97%, respectively.

  19. Quantification of malachite green in fish feed utilising liquid chromatography-tandem mass spectrometry with a monolithic column.

    PubMed

    Abro, Kamran; Mahesar, Sarfaraz Ahmed; Iqbal, Seema; Perveen, Shahnaz

    2014-01-01

    The purpose of this study was to develop a rapid and sensitive method for the quantification of malachite green (MG) in fish feed using LC-ESI-MS/MS with a monolithic column as stationary phase. Fish feed was cleaned using ultrasonic assisted liquid-liquid extraction. The separation was achieved on a Chromolith® Performance RP-18e column (100 × 4.6 mm) using gradient mobile phase composition of methanol and 0.1% formic acid at the flow rate of 1.0 ml min⁻¹. The analyte was ionised using electrospray ionisation in positive mode. Mass spectral transitions were recorded in selected reaction monitoring (SRM) mode at m/z 329.78 → m/z 314.75 with a collision energy (CE) of 52% for MG. The system suitability responses were calculated for reproducibility tests of the retention time, number of theoretical plates and capacity factor. System validation was evaluated for precision, specificity and linearity of MG. The linearity and calibration graph was plotted in the range of 15.0-250 ng ml⁻¹ with the regression coefficient of >0.997. The lower limits of detection and quantification for MG were 0.55 and 1.44 ng ml⁻¹, respectively, allowing easy determination in fish feed with accuracy evaluated as a percentage recovery of 92.1% and precision determined as % CV of < 5. The method was also extended to the determination of MG in an actual fish feed. The sensitivity and selectivity of LC-ESI-MS/MS using monolithic column offers a valuable alternative to the methodologies currently employed for the quantification of MG in fish feeds.

  20. Versatile, sensitive liquid chromatography mass spectrometry – Implementation of 10 μm OT columns suitable for small molecules, peptides and proteins

    PubMed Central

    Vehus, T.; Roberg-Larsen, H.; Waaler, J.; Aslaksen, S.; Krauss, S.; Wilson, S. R.; Lundanes, E.

    2016-01-01

    We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 μm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today’s capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 μg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-μg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is “fit-for-all”, well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics. PMID:27897190

  1. [Fast analysis of common fatty acids in edible vegetable oils by ultra-performance convergence chromatography-mass spectrometry].

    PubMed

    Lin, Chunhua; Xie, Xianqing; Fan, Naili; Tu, Yuanhong; Chen, Yan; Liao, Weilin

    2015-04-01

    A fast analytical method for five common fatty acids in six edible vegetable oils was developed by ultra-performance convergence chromatography-mass spectrometry (UPC2-MS). The five fatty acids are palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid. Their contents in the corn oil, sunflower oil, soybean oil, tea oil, rapeseed oil and peanut oil were compared. The chromatographic separation was performed on an ACQUITY UPC2 BEH 2-EP column (100 mm x 2.1 mm, 1.7 µm) using the mobile phases of carbon dioxide and methanol/acetonitrile (1:1, v/v) with gradient elution. The separated compounds were detected by negative electrospray ionization ESF-MS. The results showed that the reasonable linearities were achieved for all the analytes over the range of 0.5-100 mg/L with the correlation coefficients (R2) of 0.9985-0.9998. The limits of quantification (S/N ≥ 10) of the five fatty acids were 0.15-0.50 mg/L. The recoveries of the five fatty acids at three spiked levels were in the range of 89.61%-108.50% with relative standard deviations of 0.69%-3.01%. The developed method showed high performance, good resolution and fast analysis for the underivatized fatty acids. It has been successfully used to detect the five fatty acids from corn oil, sunflower oil, soybean oil, tea oil rapeseed oil and peanut oil.

  2. [Determination of antidangdruff agent salicylic acid, zinc pyrithione, octopirox, climbazole and ketoconazole in shampoo by high performance liquid chromatography].

    PubMed

    Yang, Yan-Wei; Zhu, Ying; Su, Xiao-Qing

    2005-09-01

    A high performance liquid chromatography method was established for determination of antidangdruff agent salicylic acid,zinc pyrithione, octopirox, climbazole and ketoconazole in shampoo on a C18 column using acetonitrile-metholaqueous solution (10 mmol/L KH2 PO4 and 5 mmol/L EDTANa2, pH is adjusted to 4.0 with H3 PO4) (50:10:40) as mobile phase at a flow rate of 1.0 ml/min, with the column temperature 25 degrees C and detection wave 230nm. The precision was less than 3.8% and recovery varied from 92.7% to 104.9%. The experimental results showed that the method was simple, precise and accurate.

  3. Hydrophilic interaction liquid chromatography-mass spectrometry of (lyso)phosphatidic acids, (lyso)phosphatidylserines and other lipid classes.

    PubMed

    Cífková, Eva; Hájek, Roman; Lísa, Miroslav; Holčapek, Michal

    2016-03-25

    The goal of this work is a systematic optimization of hydrophilic interaction liquid chromatography (HILIC) separation of acidic lipid classes (namely phosphatidic acids-PA, lysophosphatidic acids-LPA, phosphatidylserines-PS and lysophosphatidylserines-LPS) and other lipid classes under mass spectrometry (MS) compatible conditions. The main parameters included in this optimization are the type of stationary phases used in HILIC, pH of the mobile phase, the type and concentration of mobile phase additives. Nine HILIC columns with different chemistries (unmodified silica, modified silica using diol, 2-picolylamine, diethylamine and 1-aminoanthracene and hydride silica) are compared with the emphasis on peak shapes of acidic lipid classes. The optimization of pH is correlated with the theoretical calculation of acidobasic equilibria of studied lipid classes. The final method using the hydride column, pH 4 adjusted by formic acid and the gradient of acetonitrile and 40 mmol/L of aqueous ammonium formate provides good peak shapes for all analyzed lipid classes including acidic lipids. This method is applied for the identification of lipids in real samples of porcine brain and kidney extracts.

  4. Trace level haloacetic acids in drinking water by direct injection ion chromatography and single quadrupole mass spectrometry.

    PubMed

    Mathew, Johnson; McMillin, Rick; Gandhi, Jay; Mohsin, Sheher; Czyborra, Stefanie

    2009-08-01

    Chlorine has been widely used to kill disease-causing microbes in drinking water. During the disinfection process, organic and inorganic material in source waters can combine with chlorine and certain other chemical disinfectants to form disinfection by-products. The kind of disinfectant used can produce different types and levels of disinfectant byproducts in the drinking water, such as trihalomethanes and haloacetic acids (5HAAs). Currently, USEPA Method 552 utilizes a methyl tert-butyl ether extraction and diazomethane derivatization of HAAs and phenolic disinfectant by-products, and a gas chromatograph equipped with a capillary column to perform the separation of methyl-haloacetates and anisoles. To detect, gas chromatography and electron capture detector are used. This article demonstrates a simple method using direct injection ion chromatography hyphenated with mass spectrometry for the analysis of 5HAAs.

  5. Pervious concrete reactive barrier for removal of heavy metals from acid mine drainage - column study.

    PubMed

    Shabalala, Ayanda N; Ekolu, Stephen O; Diop, Souleymane; Solomon, Fitsum

    2017-02-05

    This paper presents a column study conducted to investigate the potential use of pervious concrete as a reactive barrier for treatment of water impacted by mine waste. The study was done using acid mine drainage (AMD) collected from a gold mine (WZ) and a coalfield (TDB). Pervious concrete mixtures consisting of Portland cement CEM I 52.5R with or without 30% fly ash (FA) were prepared at a water-cementitious ratio of 0.27 then used to make cubes which were employed in the reactor columns. It was found that the removal efficiency levels of Al, Fe, Mn, Co and Ni were 75%, 98%, 99%, 94% and 95% for WZ; 87%, 96%, 99%, 98% and 90% for TDB, respectively. The high rate of acid reduction and metal removal by pervious concrete is attributed to dissolution of portlandite which is a typical constituent of concrete. The dominant reaction product in all four columns was gypsum, which also contributed to some removal of sulphate from AMD. Formation of gypsum, goethite, and Glauber's salt were identified. Precipitation of metal hydroxides seems to be the dominant metal removal mechanism. Use of pervious concrete offers a promising alternative treatment method for polluted or acidic mine water.

  6. Pressure, temperature and density drops along supercritical fluid chromatography columns. I. Experimental results for neat carbon dioxide and columns packed with 3- and 5-micron particles.

    PubMed

    Poe, Donald P; Veit, Devon; Ranger, Megan; Kaczmarski, Krzysztof; Tarafder, Abhijit; Guiochon, Georges

    2012-08-10

    The pressure drop and temperature drop on columns packed with 3- and 5-micron particles were measured using neat CO(2) at a flow rate of 5 mL/min, at temperatures from 20°C to 100°C, and outlet pressures from 80 to 300 bar. The density drop was calculated based on the temperature and pressure at the column inlet and outlet. The columns were suspended in a circulating air bath either bare or covered with foam insulation. The results show that the pressure drop depends on the outlet pressure, the operating temperature, and the thermal environment. A temperature drop was observed for all conditions studied. The temperature drop was relatively small (less than 3°C) for combinations of low temperature and high pressure. Larger temperature drops and density drops occurred at higher temperatures and low to moderate pressures. Covering the column with thermal insulation resulted in larger temperature drops and corresponding smaller density drops. At 20°C the temperature drop was never more than a few degrees. The largest temperature drops occurred for both columns when insulated at 80°C and 80 bar, reaching a maximum value of 21°C for the 5-micron column, and 26°C for the 3-micron column. For an adiabatic column, the temperature drop depends on the pressure drop, the thermal expansion coefficient, and the density and the heat capacity of the mobile phase fluid, and can be described by a simple mathematical relationship. For a fixed operating temperature and outlet pressure, the temperature drop increases monotonically with the pressure drop.

  7. Optimization of post-column reactor radius in capillary high performance liquid chromatography Effect of chromatographic column diameter and particle diameter.

    PubMed

    Xu, Hongjuan; Weber, Stephen G

    2006-04-28

    A post-column reactor consisting of a simple open tube (Capillary Taylor Reactor) affects the performance of a capillary LC in two ways: stealing pressure from the column and adding band spreading. The former is a problem for very small radius reactors, while the latter shows itself for large reactor diameters. We derived an equation that defines the observed number of theoretical plates (N(obs)) taking into account the two effects stated above. Making some assumptions and asserting certain conditions led to a final equation with a limited number of variables, namely chromatographic column radius, reactor radius and chromatographic particle diameter. The assumptions and conditions are that the van Deemter equation applies, the mass transfer limitation is for intraparticle diffusion in spherical particles, the velocity is at the optimum, the analyte's retention factor, k', is zero, the post-column reactor is only long enough to allow complete mixing of reagents and analytes and the maximum operating pressure of the pumping system is used. Optimal ranges of the reactor radius (a(r)) are obtained by comparing the number of observed theoretical plates (and theoretical plates per time) with and without a reactor. Results show that the acceptable reactor radii depend on column diameter, particle diameter, and maximum available pressure. Optimal ranges of a(r) become narrower as column diameter increases, particle diameter decreases or the maximum pressure is decreased. When the available pressure is 4000 psi, a Capillary Taylor Reactor with 12 microm radius is suitable for all columns smaller than 150 microm (radius) packed with 2-5 microm particles. For 1 microm packing particles, only columns smaller than 42.5 microm (radius) can be used and the reactor radius needs to be 5 microm.

  8. [Determination of six main components in compound theophylline tablet by convolution curve method after prior separation by column partition chromatography

    NASA Technical Reports Server (NTRS)

    Zhang, S. Y.; Wang, G. F.; Wu, Y. T.; Baldwin, K. M. (Principal Investigator)

    1993-01-01

    On a partition chromatographic column in which the support is Kieselguhr and the stationary phase is sulfuric acid solution (2 mol/L), three components of compound theophylline tablet were simultaneously eluted by chloroform and three other components were simultaneously eluted by ammonia-saturated chloroform. The two mixtures were determined by computer-aided convolution curve method separately. The corresponding average recovery and relative standard deviation of the six components were as follows: 101.6, 1.46% for caffeine; 99.7, 0.10% for phenacetin; 100.9, 1.31% for phenobarbitone; 100.2, 0.81% for theophylline; 99.9, 0.81% for theobromine and 100.8, 0.48% for aminopyrine.

  9. Gas Chromatography.

    ERIC Educational Resources Information Center

    Karasek, Francis W.; And Others

    1984-01-01

    This review covers fundamental developments in gas chromatography during 1982 and 1983. Literature is considered under these headings: columns; liguid phases; solid supports; sorption processes and solvents; open tubular column gas chromatography; instrumentation; high-resolution columns and applications; other techniques; qualitative and…

  10. Luminescent determination of quinolones in milk samples by liquid chromatography/post-column derivatization with terbium oxide nanoparticles.

    PubMed

    Yánez-Jácome, G S; Aguilar-Caballos, M P; Gómez-Hens, A

    2015-07-31

    The usefulness of terbium oxide nanoparticles (Tb4O7NPs) as post-column derivatizing reagent for the liquid chromatographic determination of residues of quinolone antibiotics in milk samples has been studied. Seven quinolones of veterinary use have been chosen as model analytes to develop this method. The derivatization step is based on the formation of luminescent chelates of quinolones with Tb4O7NPs, which are monitored at λex=340nm and λem=545nm. Another relevant feature of the method is that the use of a 10-cm column and a ternary mixture of methanol, acetonitrile and acetic acid as mobile phase in gradient elution mode allow the chromatographic separation of the quinolones in about 13min, whereas previously described chromatographic methods require about 20min. The dynamic ranges of the calibration graphs and limits of detection are, respectively: 65-900ngmL(-1) and 35ngmL(-1) for marbofloxacin, 7.2-900ngmL(-1) and 2.5ngmL(-1) for ciprofloxacin, 6-900ngmL(-1) and 2ngmL(-1) for danofloxacin, 50-900ngmL(-1) and 20ngmL(-1) for enrofloxacin, 35-900ngmL(-1) and 12ngmL(-1) for sarafloxacin, 5-900ngmL(-1) and 2ngmL(-1) for oxolinic acid, and 7-900ngmL(-1) and 2.5ngmL(-1) for flumequine. The precision, established at two concentration levels of each analyte and expressed as the percentage of the relative standard deviation is in the range of 1.9-8.1% using standards, and of 3.4-10.7% in the presence of milk samples. The method has been satisfactorily applied to the analysis of skimmed, semi-skimmed and whole milk samples, with recoveries ranging from 89.0 to 106.5%.

  11. A microfluidic device for preparing next generation DNA sequencing libraries and for automating other laboratory protocols that require one or more column chromatography steps.

    PubMed

    Tan, Swee Jin; Phan, Huan; Gerry, Benjamin Michael; Kuhn, Alexandre; Hong, Lewis Zuocheng; Min Ong, Yao; Poon, Polly Suk Yean; Unger, Marc Alexander; Jones, Robert C; Quake, Stephen R; Burkholder, William F

    2013-01-01

    Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

  12. Chromatography

    MedlinePlus

    Chromatography is a way of separating two or more chemical compounds. Chemical compounds are chemicals that are ... of chemical compound. There are different kinds of chromatography. These include gas, high pressure liquid, or ion ...

  13. Hair analysis of histamine after fluorescence labeling by column-switching reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair.

    PubMed

    Toyo'oka, Toshimasa; Suzuki, Ayako; Fukushima, Takeshi; Kato, Masaru

    2004-10-15

    Sensitive determination of histamine (HA) in hair was carried out by column-switching reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). HA was labeled with excess amounts of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 60 degrees C for 30 min in a mixture of 0.1 M borax (pH 9.3) and acetonitrile (CH(3)CN). The resulting DBD-HA derivative was roughly separated by a Mightysil RP-18 GP (100 x 2mm i.d., 3 microm) with an acidic mobile phase containing 0.1% trifluoroacetic acid. DBD-HA in the fraction flowing due to a position change in the six-port column-switching valve was then completely separated by a Wakopak Navi C30 (150 x 2mm i.d., 5 microm) with 20 mM AcONH(4)-CH(3)CN (8:2). The mass spectrometer was operated in the selected reaction monitoring (SRM) mode for the product ion (m/z 292) obtained from MS-MS measurement using the protonated molecular ion [M+H](+) (m/z 337) as the precursor ion. Good linearity was achieved from the calibration curve obtained by plotting peak area ratios of the internal standard (HA-d(4)) against the injected amounts of HA (1.66-16.6 pmol, r(2)=0.999). The coefficients of variation, at 1.66- and 16.6-pmol injections, were 5.6 and 3.7%, respectively (n=6). Furthermore, the detection limit was 0.167 pmol. The efficiency of the recommended procedure was identified from the determination in the rat hair root after intraperitoneal administration of HA. The proposed method was applied to HA determination in the hair shaft of Dark Agouti rats and healthy volunteers. The variations in the concentrations in 1mg of hair shaft were 0.80-1.84 pmol (mean+/-SD=1.33+/-0.33, n=12) in rats and 0.94-72.3 pmol (17.2+/-21.5, n=16) in humans. The determination of HA in the plasma of rats and humans was also performed successfully by this method. Because the proposed method provides good precision and trace detection of HA in hair, the analytical technique

  14. The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae Prasiola crispa and Porphyra umbilicalis

    NASA Astrophysics Data System (ADS)

    Karsten, Ulf; Escoubeyrou, Karine; Charles, François

    2009-09-01

    Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.

  15. Development and characterization of a chitosan-supported immunoaffinity chromatography column for the selective extraction of methandrostenolone from food and feed samples.

    PubMed

    Wang, Yun; Xu, Yan; Zhang, Xun; Wang, Enlan; Dong, Ying

    2011-10-01

    The development of a chitosan-supported immunoaffinity chromatography (IAC) column and its application to the selective extraction of methandrostenolone (MA) from food and feed samples were described in this paper. Using hybridoma technique, a monoclonal antibody (mAb) against MA was produced. The IAC column was prepared by coupling the produced antibody with crosslinked chitosan. Scanning electron microscopy and IR spectroscopy was used to characterize the chitosan crosslinking and antibody coupling. 2% and 90% methanol were respectively selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was 1790ng/mL gel. The extraction recoveries of the column for MA at three different spiked concentrations ranged from 83.7 to 98.5%. After 2 cycles of usage, the column capacity and extraction recovery still remained 84.6% and 80.5%. To further verify the effect of matrix on the IAC cleanup, MA-fortified food and feed samples were extracted using the prepared IAC column, and MA recovery rates were found to be 86.2% and 70.4%, respectively.

  16. Kinetic studies of clavulanic acid recovery by ion exchange chromatography.

    PubMed

    Barboza, M; Almeida, R M; Hokka, C O

    2001-01-01

    Clavulanic acid (CA) is a beta-lactamase inhibitor produced by strains of Streptomyces clavuligerus. Nowadays, the combination of CA with amoxycillin is the most successful example of the use of a beta-lactam antibiotic sensitive to beta-lactamases together with an inhibitor of these enzymes. Clavulanic acid is purified from fermentation broth by a series of steps consisting mainly of two-phase separation processes such as liquid-liquid extraction, adsorption or ion-exchange chromatography, among others. Amberlite IRA 400, a strong anion-exchange resin, has a very high adsorption capacity for CA (Mayer et al. 1997). This resin can be pre-treated with NaCl (chloride cycle), to remove selectively only those anions, which are able to displace chloride from the resin or with NaOH (hydroxyl cycle), to remove all species of anions. In order to decide the best operating conditions for CA recovery by ion-exchange resins and then to construct a model of this separation process, batch experiments were conducted using Amberlite IRA 400 in the chloride cycle. These runs were carried out in a 200 ml stirred tank, at two different initial solution pH, 6.2 and 4.0; the temperature was maintained at 10 degrees C and 20 degrees C during adsorption and 30 degrees C during the desorption step. It was possible, on the basis of these batch results, to model the separation process, including the adsorption kinetics, equilibrium data and mass transfer limitations.

  17. Purification of quercetin-3-O-sophoroside and isoquercitrin from Poacynum hendersonii leaves using macroporous resins followed by Sephadex LH-20 column chromatography.

    PubMed

    Li, Hongjuan; Liu, Ying; Yi, Yuetao; Miao, Qin; Liu, Sujing; Zhao, Feng; Cong, Wei; Wang, Chunhua; Xia, Chuanhai

    2017-03-24

    In China, Poacynum hendersonii is frequently used as a substitute for Apoacynum venetum L (Luobuma), which is a famous traditional Chinese medicine. Quercetin-3-O-sophoroside and isoquercitrin are two major flavonoids in Poacynum hendersonii leaves. In this work, a suitable method was established for the large-scale preparation of quercetin-3-O-sophoroside (QOS) and isoquercitrin (ISO) from Poacynum hendersonii leaves using macroporous resin combined with Sephadex LH-20 column chromatography. The adsorption/desorption capacities and desorption ratios of six macroporous resins were evaluated using static experiments. The HPD-300 resin had the best adsorption performance because it had the largest surface area, and was selected for further study. Compared with pseudo-first-order and intraparticle diffusion kinetics models, the pseudo-second-order model could better fit the adsorption kinetics of both QOS and ISO on the HPD-300 resin. In addition, the adsorption isotherms of the two compounds on the HPD-300 resin were fitted well to the Langmuir model. Under optimal conditions, the purities of QOS and ISO in the product were increased from 2.16% and 1.26% to 21.34% and 10.70% with recovery yields of 82.1% and 77.3%, respectively. Subsequently, Sephadex LH-20 column chromatography was employed for improving the purities of the two compounds. After separation by Sephadex LH-20 column chromatography, the purities of QOS and ISO achieved 93.5% and 95.6%, respectively.

  18. Analysis of naphthenic acid mixtures as pentafluorobenzyl derivatives by gas chromatography-electron impact mass spectrometry.

    PubMed

    Gutierrez-Villagomez, Juan Manuel; Vázquez-Martínez, Juan; Ramírez-Chávez, Enrique; Molina-Torres, Jorge; Trudeau, Vance L

    2017-01-01

    In this study, we report for the first time the efficiency of pentafluorobenzyl bromide (PFBBr) for naphthenic acid (NA) mixtures derivatization, and the comparison in the optimal conditions to the most common NAs derivatization reagents, BF3/MeOH and N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA). Naphthenic acids are carboxylic acid mixtures of petrochemical origin. These compounds are important for the oil industry because of their corrosive properties, which can damage oil distillation infrastructure. Moreover, NAs are commercially used in a wide range of products such as paint and ink driers, wood and fabric preservatives, fuel additives, emulsifiers, and surfactants. Naphthenic acids have also been found in sediments after major oils spills in the United States and South Korea. Furthermore, the toxicity of the oil sands process-affected water (OSPW), product of the oil sands extraction activities in Canada's oil sands, has largely been attributed to NAs. One of the main challenges for the chromatographic analysis of these mixtures is the resolution of the components. The derivatization optimization was achieved using surface response analysis with molar ratio and time as factors for derivatization signal yield. After gas chromatography-electron impact mass spectrometry (GC/EIMS) analysis of a mixture of NA standards, it was found that the signal produced by PFB-derivatives was 2.3 and 1.4 times higher than the signal produced by methylated and MTBS-derivatives, respectively. The pentafluorobenzyl derivatives have a characteristic fragment ion at 181m/z that is diagnostic for the differentiation of carboxylic and non-carboxylic acid components within mixtures. In the analysis of a Sigma and a Merichem derivatized oil extract NA mixtures, it was found that some peaks lack the characteristic fragment ion; therefore they are not carboxylic acids. Open column chromatography was used to obtain a hexane and a methanol fraction of the Sigma and

  19. The use of phospholipid modified column for the determination of lipophilic properties in high performance liquid chromatography.

    PubMed

    Godard, Tal; Grushka, Eli

    2011-03-04

    A new chromatographic stationary phase obtained by coating a reversed phase amide column with phosphatidylcholine based liposomes solution to yield a phospholipid modified column (PLM). The modification is achieved by the dynamic coating method which recycles the coating solution through the column in a closed loop for a period of 24 h. The chromatographic properties of the new column have changed significantly as compared to the original amide column due to the phospholipid coating. A good correlation was observed between n-octanol/water logP values and the logarithm of the retention factor obtained on the PLM column for a large number of solutes. In addition the PLM column was characterized using the linear solvation energy relationship (LSER). The values of the LSER system constants for the PLM column were calculated and were found to be very close to those of the n-octanol/water extraction system thus suggesting that the PLM column can be used for the estimation of n-octanol/water partition coefficient and serve as a possible alternative to the shake-flask method for lipophilicity determination. In addition, the results suggest that the PLM column can provide an alternative to other phospholipid-based column such as the IAM and the DPC columns.

  20. Development of an immunoaffinity chromatography column for selective extraction of a new agonist phenylethylamine A from feed, meat and liver samples.

    PubMed

    Mei, Liyun; Cao, Biyun; Yang, Hong; Xie, Yun; Xu, Shouming; Deng, Anping

    2014-01-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist that has been illegally used as an animal feed additive for growth promotion in China. In this study, an immunoaffinity chromatography (IAC) column for selective extraction of PA from swine feed, meat and liver samples was developed. The IAC column was constructed by covalently coupling specific polyclonal antibody (Ab) against PA to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, selectivity, extraction recovery and stability. The maximum capacity of the ICA for PA extraction was found to be 239.4ng. For selectivity testing, 100ng of other three β-adrenergic agonists (clenbuterol, ractopamine and salbutamol) was separately loaded onto the column, and it was observed that the tested compounds could not be captured on the column, e.g. the column could only selectively recognize PA. The recovery of the IAC for PA extraction was found within 96.47-101.98% when 10, 50 and 100ng PA were separately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in range of 1.0-20ngg(-1). The spiked and unspiked samples were extracted by IAC column and measured by high performance liquid chromatography (HPLC). It was found that there was no detectable PA in the blank samples, and the extraction recoveries of the IAC for PA from the spiked samples were within 89.48-104.89%. The stability of the column was also tested. It was showed that after 35 times repeated usage, 60% of the maximum capacity was still remained. The proposed IAC was proven to be a feasible extraction method for PA from different matrices with the properties of high maximum capacity, selectivity, extraction efficiency and

  1. Simultaneous determination of trace oxyhalides and haloacetic acids using suppressed ion chromatography-electrospray mass spectrometry.

    PubMed

    Barron, Leon; Paull, Brett

    2006-05-15

    A new analytical procedure for the simultaneous determination of trace oxyhalides and haloacetic acids (HAs) in drinking water and aqueous soil extracts is described. The method uses micro-bore ion chromatography (IC) coupled with suppressed conductivity (SC) and electrospray ionization mass spectrometric detection (ESI-MS). The IC-SC-ESI-MS system included a secondary flow of 100% MeOH, which was added to the column eluate (post-suppressor) and resulted in a significant increase in sensitivity for all analytes. All ESI-MS parameters were optimized for HA analysis and sensitivity quantitatively compared to suppressed conductivity. Full analytical performance characteristics for the developed method are presented for monochloro-, monobromo-, dichloro-, dibromo-, trichloro-, bromochloro, chlorodifluoro-, trifluoro-, dichlorobromo- and dibromochloroacetic acid, as well as the oxyhalides iodate, bromate, chlorate and perchlorate. In the case of the HAs, an optimised 25-fold SPE preconcentration method meant all analytes could be readily detected well below the USEPA 60mug/L regulatory limit using conductivity and/or ESI-MS. The IC-ESI-MS method was applied to the determination of oxyhalides and HAs in both soil extracts and drinking water samples. Soil samples were extracted using ultra pure water with subsequent determination of perchlorate at 1.68mug/g of soil. A drinking water sample containing HAs was preconcentrated using LiChrolut EN solid phase extraction cartridges with subsequent sulphate and chloride removal. Total HAs were determined at 13mug/L.

  2. High-performance liquid chromatography with conductimetric detection of perfluorocarboxylic acids and perfluorosulfonates.

    PubMed

    Hori, Hisao; Hayakawa, Etsuko; Yamashita, Nobuyoshi; Taniyasu, Sachi; Nakata, Fumiya; Kobayashi, Yoshimi

    2004-10-01

    A rapid and simple method for separating and determining various environmentally harmful perfluorocarboxylic acids and perfluorosulfonates was successfully developed using high- performance liquid chromatography with conductimetric detection, for product and waste management of these compounds at manufacturing and processing sites. Compounds having C(3)-C(8) perfluoroalkyl groups were separated using a Tosoh TSKgel Super-ODS column and a mobile phase consisting of a mixture of methanol and aqueous NaH(2)PO(4) at several mixing ratios. The best detection limits for the compounds ranged from 0.12 to 0.66 mg l(-1) (ppm), and linear calibration graphs were obtained up to 87-109 mg l(-1). The combination of this method with concentration of the sample by solid-phase extraction with cartridges based on styrene-divinylbenzene-copolymer enabled the determination of approximately 50 microg l(-1) (ppb) for compounds with C(4)-C(8) perfluoroalkyl groups. This method was successfully used to monitor the artificial decomposition of the perfluorocarboxylic acid n-C(4)F(9)COOH induced by a photocatalyst.

  3. The synthesis of surface-glycosylated porous monolithic column via aqueous two-phase graft copolymerization and its application in capillary-liquid chromatography.

    PubMed

    Xiong, Xiyue; He, Haiqin; Shu, Yan; Li, Yuxin; Yang, Zihui; Chen, Yingzhuang; Ma, Ming; Chen, Bo

    2016-12-01

    A facile, flexible process was developed for the preparation of surface-glycosylated porous monolithic columns via aqueous two-phase graft copolymerization of polyethylene glycol diacrylate (PEGDA) and water-soluble dextran (dextran sulfate). The formation of poly(PEGDA) porous skeletons and surface glycosylation were achieved via a one-step process without pre-modification of the dextran. The synthesis conditions were thoroughly optimized. The optimal monolithic column exhibited a large dry state surface area (greater than 400m(2)/g), and it was evaluated as a hydrophilic liquid chromatography (HILIC) stationary phase. A typical HILIC mechanism was observed at high organic solvent content (≥65% acetonitrile). In addition, the resulting monolithic column demonstrated the potential use in analysis of complex biological sample and enviroment water.

  4. A novel ion chromatography cycling-column-switching system for the determination of low-level chlorate and nitrite in high salt matrices.

    PubMed

    Wang, Nani; Wang, Rui Qi; Zhu, Yan

    2012-10-15

    A novel ion chromatography cycling-column-switching system was developed for the determination of chlorate and nitrite in high salt matrices. The simple system included a pump, two valves, a single eluent, and a conductivity detector. Both online pre-concentration and matrix elimination were achieved by this method. The target anions were eluted from the concentrator column to the analytical columns circularly. Chloride matrix was then eliminated completely. The method was applied to the determination of low-level chlorate and nitrite in the chloride matrix. Our experimental results demonstrated that this system is of advantages such as high sensitivity, facile automation and simple sample pretreatment, which might be a promising approach for environmental researches and food control.

  5. Extraction of amphetamines and methylenedioxyamphetamines from urine using a monolithic silica disk-packed spin column and high-performance liquid chromatography-diode array detection.

    PubMed

    Namera, Akira; Nakamoto, Akihiro; Nishida, Manami; Saito, Takeshi; Kishiyama, Izumi; Miyazaki, Shota; Yahata, Midori; Yashiki, Mikio; Nagao, Masataka

    2008-10-24

    To overcome the limitations of solid-phase extraction, we developed a device comprising a spin column packed with octadecyl silane-bonded monolithic silica for extracting amphetamines and methylenedioxyamphetamines from urine. Urine (0.5mL), buffer (0.4mL), and methoxyphenamine (internal standard) were directly put into the preactivated column. The column was centrifuged (3000rpm, 5min) for sample loading and washed. The adsorbed analytes were eluted and analyzed by high-performance liquid chromatography, without evaporation. The results were as follows: linear curves (drug concentrations of 0.2-20microg/mL); correlation coefficients >0.99; detection limit, 0.1microg/mL. The proposed method is not only useful for drugs from biological materials but also highly reproducible for the analysis of these drugs in urine.

  6. Analysis of underivatized amino acids in geological samples using ion-pairing liquid chromatography and electrospray tandem mass spectrometry.

    PubMed

    Liu, De-Ling; Beegle, Luther W; Kanik, Isik

    2008-04-01

    The capability of detecting biomarkers, such as amino acids, in chemically complex field samples is essential to establishing the knowledge required to search for chemical signatures of life in future planetary explorations. However, due to the complexities of in situ investigations, it is important to establish a new analytical scheme that utilizes a minimal amount of sample preparation. This paper reports the feasibility of a novel and sensitive technique, which has been established to quantitate amino acids in terrestrial crust samples directly without derivatization using volatile ion-pairing liquid chromatography and tandem mass spectrometry equipped with an electrospray ionization source. Adequate separation of 20 underivatized amino acids was achieved on a C(18) capillary column within 26 min with nonafluoropentanoic acid (NFPA) as ion-pairing reagent. Each amino acid was identified from its retention time as well as from its characteristic parent-to-daughter ion transition. Using tandem mass spectrometry as a detection technique allows co-elution of some amino acids, as it is more specific than traditional spectrophotometric methods. In the present study, terrestrial samples collected from 3 different locations were analyzed for their water-extractable free amino acid contents, following the removal of metal and organic interferences via ion exchange procedures. This is the first time that amino acids in geological samples were directly determined quantitatively without complicated derivatization steps. Depending on the amino acid, the detection limits varied from 0.02 to 5.7 pmol with the use of a 1 microl sample injection loop.

  7. Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of chlorinated pesticides in aquatic tissue by capillary-column gas chromatography with electron-capture detection

    USGS Publications Warehouse

    Leiker, Thomas J.; Madsen, J.E.; Deacon, J.R.; Foreman, W.T.

    1995-01-01

    A method for the determination of chlorinated organic compounds in aquatic tissue by dual capillary-column gas chromatography with electron-capture detection is described. Whole-body-fish or corbicula tissue is homogenized, Soxhlet extracted, lipid removed by gel permeation chromatography, and fractionated using alumina/silica adsorption chromatography. The extracts are analyzed by dissimilar capillary-column gas chromatography with electron-capture detection. The method reporting limits are 5 micrograms per kilogram (μg/kg) for chlorinated compounds, 50 μg/kg for polychlorinated biphenyls, and 200 μg/kg for toxaphene.

  8. Rapid simultaneous determination of amines and organic acids in citrus using high-performance liquid chromatography.

    PubMed

    Uckoo, Ram M; Jayaprakasha, Guddadarangavvanahally K; Nelson, Shad D; Patil, Bhimanagouda S

    2011-01-15

    Rapid analytical method for the simultaneous separation and determination of amines and organic acids is a vital interest for quality control of citrus and their products. In the present study, a simultaneous high performance liquid chromatography (HPLC) method for the rapid separation of three amines and two organic acids was developed. Chromatographic separation of compounds was achieved using Xbridge C(18) column at ambient temperature, with an isocratic mobile phase of 3mM phosphoric acid at a flow rate of 1.0 mL min(-1). A photodiode array (PDA) detector was used to monitor the eluent at 223 nm and 254 nm with a total analysis time of 10 min. Extraction of amines and organic acids from citrus juice was optimized. The method was validated by tests of linearity, recovery, precision and ruggedness. The limit of detection (LOD) and limit of quantification (LOQ) for amines and ascorbic acid were determined to be 5 ng and 9.8 ng, respectively. All calibration curves showed good linearity (R(2) ≥ 0.9999) within the test ranges. The recoveries of the amines and organic acids ranged between 84% and 117%. The identity of each peak was confirmed by mass spectral (MS) analysis. The developed method was successfully applied to analyze the content of amines and organic acids in six different species and two varieties of citrus. Results indicate that mandarin and Marrs sweet orange contain high level of amines, while pummelo and Rio Red grapefruit had high content of ascorbic acid (137-251 μg mL(-1)) and citric acid (5-22 mg mL(-1)). Synephrine was the major amine present in Clementine (114 μg mL(-1)) and Marrs sweet orange (85 μg mL(-1)). To the best of our knowledge, this is the first report on simultaneous separation and quantification of amines and organic acids in Marrs sweet orange, Meyer lemon, Nova tangerine, Clementine, Ugli tangelo and Wekiwa tangelo.

  9. Effects of increasing acidity on metal(loid) bioprecipitation in groundwater: column studies

    SciTech Connect

    Alexander C. Davis; Bradley M. Patterson; Michelle E. Grassi; Blair S. Robertson; Henning Prommer; Allan J. McKinley

    2007-10-15

    Large-scale column experiments were carried out over a period of 545 days to assess the effect of increasing acidity on bacterial denitrification, sulfate reduction, and metal(loid) bioprecipitation in groundwater affected by acid mine drainage. At a groundwater pH of 5.5, denitrification and Cu{sup 2+} removal, probably via malachite (Cu{sub 2}(OH){sub 2}CO{sub 3}) precipitation, were observed in the ethanol-amended column. Sulfate reduction, sulfide production, and Zn{sup 2+} removal were also observed, with Zn{sup 2+} removal observed in the zone of sulfate reduction, indicating likely precipitation as sphalerite (ZnS). Se{sup 6+} removal was also observed in the sulfate reducing zone, probably as direct bioreduction to elemental selenium via ethanol/acetate oxidation or sulfide oxidation precipitating elemental sulfur. A step decrease in groundwater pH from 5.5 to 4.25 resulted in increased denitrification and sulfate reduction half-lives, migration of both these redox zones along the ethanol-amended column, and the formation of an elevated Cu{sup 2+} plume. Additionally, an elevated Zn{sup 2+} plume formed in the previous sulfate reducing zone of the ethanol-amended column, suggesting dissolution of precipitated sphalerite as a result of the reduction in groundwater pH. As Cu{sup 2+} passed through the zone of sphalerite dissolution, SEM imaging and EDS detection suggested that Cu{sup 2+} removal had occurred via chalcocite (Cu{sub 2}S) or covellite (CuS) precipitation. 23 refs., 8 figs.

  10. Treatment of Selenium and Nitrate in Acid Mine Drainage: A Column Study

    NASA Astrophysics Data System (ADS)

    An, H.; Jeen, S. W.

    2015-12-01

    Treatment efficiency of selenium and nitrate in acid mine drainage (AMD) by two types of reactive mixtures, i.e., organic carbon-limestone (OC-LS) and organic carbon-zero valent iron (OC-ZVI), was evaluated through column experiments. The influent AMD, collected at an abandoned metal mine site in Korea, had pH of 2.9 and contained 1600 mg/ L of SO42- and elevated concentrations of metals (e.g., Al, Cd, Co, Cu, Fe, Zn). Selenium (40 mg/L) and nitrate (100 mg/L as NO3-N initially and 10 mg/L as NO3-N after 55 days) were spiked into the AMD. The columns were operated for a total of 90 days. The results showed the increase of pH from 2.9 to 7.0 and the decreases in concentrations of most of major ions including selenium and nitrate in both the OC-LS and OC-ZVI columns. The OC-ZVI column had higher removal rates of selenium and nitrate and created a more reduced environment than the OC-LS column due to the abiotic reactions of ZVI. However, a notable amount of ammonia was produced as a reaction product in the OC-ZVI column, while the OC-LS produced a minimum amount of ammonia, suggesting formation of N2 by denitrification. In both columns, removal rates of selenium were substantially increased when the influent NO3-N concentration was changed from 100 mg/L to 10 mg/L. Sulfate was reduced as much as 390 mg/L, as indicated by detection of hydrogen sulfide. The reduction of most metals is considered to be due to precipitation of metal-containing secondary minerals (e.g., sulfides, hydroxides, carbonates). This study shows that treatment of selenium and nitrate in AMD can be achievable using organic carbon-based reactive mixtures through reduction of selenium and nitrate. However, the use of ZVI is not recommended when selenium and nitrate coexist in AMD because of production of ammonia by abiotic reaction between ZVI and nitrate. This study also shows that concentration of nitrate in AMD is an important factor to determine the rate of selenium removal.

  11. Simultaneous determination of iodide and iodate in povidone iodine solution by ion chromatography with homemade and exchange capacity controllable columns and column-switching technique.

    PubMed

    Huang, Zhongping; Zhu, Zuoyi; Subhani, Qamar; Yan, Wenwu; Guo, Weiqiang; Zhu, Yan

    2012-08-17

    A simple ion chromatographic method for simultaneous detection of iodide and iodate in a single running was proposed, with columns packed with homemade functionalized polystyrene-divinylbenzene (PS-DVB) resins and column-switching technique. Homemade resins were functionalized with controllable amounts of quaternary ammonium groups. The low-capacity anion-exchange column and high-capacity anion-exchange column were prepared, due to the resins having different exchange capacities. With this method, iodide and iodate in povidone iodine solution were detected simultaneously in a short time with iodide being eluted off first. A series of standard solutions consisting of target anions of various concentrations from 0.01 mg/L to 100 mg/L were analyzed. Each anion exhibited satisfactory linearity, with correlation coefficient r ≥ 0.9990. The detection limits (LODs) for iodide and iodate obtained by injecting 100 μL of sample were 5.66 and 14.83 μg/L (S/N=3), respectively. A spiking study was performed with satisfactory recoveries between 101.2% and 100.6% for iodide and iodate.

  12. Monolithic stationary phases with incorporated fumed silica nanoparticles. Part II. Polymethacrylate-based monolithic column with "covalently" incorporated modified octadecyl fumed silica nanoparticles for reversed-phase chromatography.

    PubMed

    Aydoğan, Cemil; El Rassi, Ziad

    2016-05-06

    This study is concerned with the incorporation of surface modified fumed silica nanoparticles (FSNPs) into polymethacrylate based monolithic columns for use in reversed phase chromatography (RPC) of small solutes and proteins. First, FSNPs were modified with 3-(trimethoxysilyl)propylmethacrylate (TMSPM) to yield the "hybrid" methacryloyl fumed silica nanoparticle (MFSNP) monomer. The resulting MFSNP was then mixed with glyceryl monomethacrylate (GMM) and ethylene dimethacrylate (EDMA) in a binary porogenic solvent composed of cyclohexanol and dodecanol, and the in situ copolymerization of MFSNP, GMM and EDMA was performed in a stainless steel column of 4.6 mm i.d. The silanol groups of the hybrid monolith thus obtained were grafted with octadecyl ligands by perfusing the hybrid monolithic column with a solution of 4% w/v of dimethyloctadecylchlorosilane (DODCS) in toluene while the column was maintained at 110°C for 6h (in a heated HPLC oven). One of the originalities of this study was to demonstrate MFSNP as a novel derivatized "hybrid monomer" in making RPC monolithic columns with surface bound octadecyl ligands. In this respect, the RPC behavior of the monolithic column with "covalently" incorporated FNSPs having surface grafted octadecyl ligands was evaluated with alkylbenzenes, aniline derivatives and phenolic compounds. The results showed that the hybrid poly(GMA-EDMA-MFSNP) having surface bound octadecyl ligands exhibited hydrophobic interactions under reversed phase elution conditions. Furthermore, six standard proteins were baseline separated on the column using a 10min linear gradient elution at increasing ACN concentration in the mobile phase at a flow rate of 1.0mL/min using a 10 cm×4.6mm i.d. column. The relative standard deviations (RSDs) for the retention times of the tested solutes were lower than 2.1% and 2.4% under isocratic elution and gradient elution conditions, respectively.

  13. Relating pressure tuned coupled column ensembles with the solvation parameter model for tunable selectivity in gas chromatography.

    PubMed

    Sharif, Khan M; Kulsing, Chadin; Chin, Sung-Tong; Marriott, Philip J

    2016-07-15

    The differential pressure drop of carrier gas by tuning the junction point pressure of a coupled column gas chromatographic system leads to a unique selectivity of the overall separation, which can be tested using a mixture of compounds with a wide range of polarity. This study demonstrates a pressure tuning (PT) GC system employing a microfluidic Deans switch located at the mid-point of the two capillary columns. This PT system allowed variations of inlet-outlet pressure differences of the two columns in a range of 52-17psi for the upstream column and 31-11psi for the downstream column. Peak shifting (differential migration) of compounds due to PT difference are related to a first order regression equation in a Plackett-Burman factorial study. Increased first (upstream) column pressure drop makes the second column characteristics more significant in the coupled column retention behavior, and conversely increased second (downstream) column pressure drop makes the first column characteristics more apparent; such variation can result in component swapping between polar and non-polar compounds. The coupled column system selectivity was evaluated in terms of linear solvation energy relationship (LSER) parameters, and their relation with different pressure drop effects has been constructed by applying multivariate principle component analysis (PCA). It has been found that the coupled column PT system descriptors provide a result that shows a clear clustering of different pressure settings, somewhat intermediate between those of the two commercial columns. This is equivalent to that obtained from a conventional single-column GC analysis where the interaction energy contributed from the stationary phases can be significantly adjusted by choice of midpoint PT. This result provides a foundation for pressure differentiation for selectivity enhancement.

  14. A new N-hydroxyethyliminodiacetic acid modified core-shell silica phase for chelation ion chromatography of alkaline earth, transition and rare earth elements.

    PubMed

    McGillicuddy, Nicola; Nesterenko, Ekaterina P; Nesterenko, Pavel N; Stack, Elaine M; Omamogho, Jesse O; Glennon, Jeremy D; Paull, Brett

    2013-12-20

    Bare core-shell silica (1.7μm) has been modified with iminodiacetic acid functional groups via standard silane chemistry, forming a new N-hydroxyethyliminodiacetic acid (HEIDA) functionalised core-shell stationary phase. The column was applied in high-performance chelation ion chromatography and evaluated for the retention of alkaline earth, transition and heavy metal cations. The influence of nitric acid eluent concentration, addition of complexing agent dipicolinic acid, eluent pH and column temperature on the column performance was investigated. The efficiencies obtained for transition and heavy metal cations (and resultant separations) were comparable or better than those previously obtained for alternative fully porous silica based chelation stationary phases, and a similarly modified monolithic silica column, ranging from ∼15 to 56μm HETP. Increasing the ionic strength of the eluent with the addition of KNO3 (0.75M) and increasing the column temperature (70°C) facilitated the isocratic separation of a mixture of 14 lanthanides and yttrium in under 12min, with HETP averaging 18μm (7μm for Ce(III)).

  15. Ion-exclusion chromatographic separations of C1-C6 aliphatic carboxylic acids on a sulfonated styrene-divinylbenzene co-polymer resin column with 5-methylhexanoic acid as eluent.

    PubMed

    Ohta, Kazutoku; Towata, Atsuya; Ohashi, Masayoshi

    2003-05-16

    The application of C7 aliphatic carboxylic acids (heptanoic, 2-methylhexanoic, 5-methylhexanoic and 2,2-dimethyl-n-valeric acids) as eluents in ion-exclusion chromatography with conductimetric detection for C1-C6 aliphatic carboxylic acids (formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic and caproic acids) was carried out using a highly sulfonated styrene-divinylbenzene co-polymer resin (TSKgel SCX) in the H+ form as a stationary phase. When using 0.05 mM sulfuric acid at pH 4.0 as the eluent, peak shapes of hydrophobic carboxylic acids (isovaleric, valeric, isocaproic and caproic acids) were tailed strongly. In contrast, when using 1 mM these C7 carboxylic acids at pH ca. 4 as the eluents, although system peaks (vacant peaks) corresponding to these C7 carboxylic acids appeared, peak shapes of these hydrophobic acids were improved drastically. Excellent simultaneous separation and relatively high sensitive conductimetric detection for these C1-C6 aliphatic carboxylic acids were achieved in 25 min on the TSKgel SCX column (150 x 6 mm I.D.) using 1 mM 5-methylhexanoic acid at pH 4.0 as the eluent.

  16. Comparison of chiral separations of aminophosphonic acids and their aminocarboxylic acid analogs using a crown ether column.

    PubMed

    Barnhart, Wesley W; Xia, Xiaoyang; Jensen, Randy; Gahm, Kyung H

    2013-07-01

    Crown ethers are capable of complexing with primary amines and have been utilized in chromatography to separate amino acid racemates. This application has been extended to resolve (1-amino-1-phenylmethyl)phosphonic acid and (1-aminoethyl)phosphonic acid racemates, along with their aminocarboxylic acid analogs (2-phenylglycine and alanine, respectively), via a ChiroSil RCA crown ether based chiral stationary phase. Effects of the organic modifier, temperature, and acid type and concentration on retention and selectivity were also investigated. Trends in retention and selectivity varied between aminophosponic acids and their aminocarboxylic analogs. Computer modeling and (1)H NMR analyses were performed to potentially gain a better understanding of interactions of the aforementioned molecules with the ChiroSil RCA chiral stationary phase. Theoretical predictions of the most stable conformations for (R)- and (S)-enantiomers were compared to elution order; it was found that the elution order agreed with molecular modeling such that the longest retention correlated with the predicted most stable complex between the enantiomer and crown ether. (1)H NMR demonstrated interactions of aminophosphonic and aminocarboxylic racemates with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in solution and was utilized to determine enantiomeric excess of (1-amino-1-phenylmethyl)phosphonic acid after its enantioenrichment via crystallization through diastereomeric salt formation with the crown ether followed by filtration.

  17. Characterization and comparison of methacrylic acid with 2-acrylamido-2-methyl-1-propanesulfonic acid in the preparation of monolithic column for capillary electrochromatography.

    PubMed

    Horiguchi, Daisuke; Ohyama, Kaname; Masunaga, Tomoko; Fujita, Yoshiko; Ali, Marwa Fathy Bakr; Kishikawa, Naoya; Kuroda, Naotaka

    2013-01-01

    Butyl methacrylate (BMA)-ethylene dimethacrylate (EDMA)-methacrylic acid (MAA) and BMA-EDMA-2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monolithic columns were prepared by varying the percentage of ionic monomers for capillary electrochromatography. Monolithic columns with a higher content of ionic monomers provided better column efficiency, and the performance of BMA-EDMA-MAA monoliths was better than BMA-EDMA-AMPS. To characterize and optimize BMA-EDMA-MAA monoliths, the effects of the content of cross-linker and the total monomer in the polymerization mixture on column performance were also studied. Plate heights of 8.2 µm for the unretained solute (thiourea) and 12.6 µm for the retained solute (naphthalene) were achieved with a monolithic column using 2.5% MAA (Column I).

  18. Use of Gas-Liquid Chromatography to Determine the End Products of Growth of Lactic Acid Bacteria

    PubMed Central

    Thornhill, Patrick J.; Cogan, Timothy M.

    1984-01-01

    A simple gas-liquid chromatographic procedure for analyzing ethanol, acetic acid, acetoin, and racemic and meso-2,3-butylene glycol in broth media is described. Overnight broth cultures were filtered or centrifuged, and the filtrate or supernatant was treated with formic acid to aid separation of volatile fatty acids. Samples were then directly analyzed by gas-liquid chromatography on a 20% Tween 80-Chromosorb W-AW column and propionic acid as an internal standard. A complete analysis took ca. 8 min. The method can be used to distinguish homofermentative from heterofermentative lactic acid bacteria based on the level of ethanol produced and citrate-utilizing from non-citrate-utilizing lactic acid bacteria based on the levels of acetic acid produced. The method also has potential in distinguishing other bacterial fermentations. Of the 13 species of lactic acid bacteria tested, Streptococcus lactis subsp. diacetylactis was the major producer of 2,3-butylene glycol (total range, 0.3 to 3.5 mM), and, except for strain DRC1, both the racemic and meso isomers were produced in approximately equal amounts. PMID:16346562

  19. A practical strategy for using miniature chromatography columns in a standardized high-throughput workflow for purification development of monoclonal antibodies.

    PubMed

    Welsh, John P; Petroff, Matthew G; Rowicki, Patricia; Bao, Haiying; Linden, Thomas; Roush, David J; Pollard, Jennifer M

    2014-01-01

    The emergence of monoclonal antibody (mAb) therapies has created a need for faster and more efficient bioprocess development strategies in order to meet timeline and material demands. In this work, a high-throughput process development (HTPD) strategy implementing several high-throughput chromatography purification techniques is described. Namely, batch incubations are used to scout feasible operating conditions, miniature columns are then used to determine separation of impurities, and, finally, a limited number of lab scale columns are tested to confirm the conditions identified using high-throughput techniques and to provide a path toward large scale processing. This multistep approach builds upon previous HTPD work by combining, in a unique sequential fashion, the flexibility and throughput of batch incubations with the increased separation characteristics for the packed bed format of miniature columns. Additionally, in order to assess the applicability of using miniature columns in this workflow, transport considerations were compared with traditional lab scale columns, and performances were mapped for the two techniques. The high-throughput strategy was utilized to determine optimal operating conditions with two different types of resins for a difficult separation of a mAb monomer from aggregates. Other more detailed prediction models are cited, but the intent of this work was to use high-throughput strategies as a general guide for scaling and assessing operating space rather than as a precise model to exactly predict performance.

  20. Proteomic analysis with integrated multiple dimensional liquid chromatography/mass spectrometry based on elution of ion exchange column using pH steps.

    PubMed

    Dai, Jie; Shieh, Chia Hui; Sheng, Quan-Hu; Zhou, Hu; Zeng, Rong

    2005-09-15

    A novel integrated multidimensional liquid chromatography (IMDL) method is demonstrated for the separation of peptide mixtures by two-dimensional HPLC coupled with ion trap mass spectrometry. The method uses an integrated column, containing both strong cation exchange and reversed-phase sections for two-dimensional liquid chromatography. The peptide mixture was fractionated by a pH step using a series of pH buffers, followed by reversed-phase chromatography. Since no salt was used during separation, the integrated multidimensional liquid chromatography can be directly connected to mass spectrometry for peptide analysis. The pH buffers were injected from an autosampler, and the entire process can be carried out on a one-dimensional liquid chromatography system. In a single analysis, the IMDL system, coupled with linear ion trap mass spectrometry, identified more than 2000 proteins in mouse liver. The peptides were eluted according to their pI distribution. The resolution of the pH fractionation is approximately 0.5 pH unit. The method has low overlapping across pH fractions, good resolution of peptide mixture, and good correlation of peptide pIs with pH steps. This method provides a technique for large-scale protein identification using existing one-dimensional HPLC systems.

  1. Chromatography on DEAE ion-exchange and Protein G affinity columns in tandem for the separation and purification of proteins.

    PubMed

    Qi, Y; Yan, Z; Huang, J

    2001-10-30

    A high-performance liquid-chromatographic method based on coupled DEAE anion-exchange and Protein G affinity columns has been developed for the simultaneous separation and purification of immunoglobulin G and albumin from mouse serum. The diluted mouse serum was injected directly into this system, and the proteins were eluted separately from the DEAE and Protein G columns, coupled in series, by the column-switching technique. The advantages of this method are that IgG and albumin can be separated and purified simultaneously, the expensive affinity column is protected from contamination by the impurities in the mouse serum, and it is fast, selective, robust, and reproducible.

  2. Accelerated quantification of amphetamine enantiomers in human urine using chiral liquid chromatography and on-line column-switching coupled with tandem mass spectrometry.

    PubMed

    Hädener, Marianne; Bruni, Pia S; Weinmann, Wolfgang; Frübis, Matthias; König, Stefan

    2017-02-01

    Amphetamine (AM) is a powerful psychostimulant existing in two enantiomeric forms. Stereoselective analysis of AM in biosamples can assist clinicians and forensic experts in differentiating between abuse of illicitly synthesized racemic AM and ingestion of pharmaceutical AM formulations containing either S-AM or different proportions of the S- and R-enantiomers. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying AM enantiomers in urine was newly developed. The method comprised dilution with water, followed by injection of the diluted sample onto an achiral C18 trapping column for purification and subsequent backflush elution to a chiral Lux 3 μm AMP LC column by means of a switching valve. An isocratic mobile phase of 25 % acetonitrile in 0.1 M aqueous ammonia was used for enantiomeric separation. Injection, cleanup, and backflush of the next sample were performed before the previous sample had eluted from the analytical column, thus enabling simultaneous enantioseparation of up to three samples within the analytical column. This novel chromatographic concept allowed for increased sample throughput by accelerating both the sample preparation and the LC analysis. Analyte detection was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was successfully validated through assessment of its linearity, lower limit of quantification, accuracy and precision, selectivity, matrix effect, carry-over, dilution integrity, and re-injection reproducibility. Linearity ranged from 0.05 to 25 mg/L for both enantiomers. Proof of the method included analysis of urine samples obtained from drug abusers and patients receiving an S-AM prodrug. Graphical Abstract Enantioselective determination of amphetamine in human urine using liquid chromatography with achiral-chiral column-switching and tandem mass spectrometry.

  3. Advantages of monolithic over particulate columns for multiresidue analysis of organic pollutants by in-tube solid-phase microextraction coupled to capillary liquid chromatography.

    PubMed

    Moliner-Martínez, Y; Molins-Legua, C; Verdú-Andrés, J; Herráez-Hernández, R; Campíns-Falcó, P

    2011-09-16

    The performance of a monolithic C(18) column (150 mm×0.2 mm i.d.) for multiresidue organic pollutants analysis by in-tube solid-phase microextraction (IT-SPME)-capillary liquid chromatography has been studied, and the results have been compared with those obtained using a particulate C(18) column (150 mm×0.5 mm i.d., 5 μm). Chromatographic separation has been carried out under isocratic elution conditions, and for detection and identification of the analytes a UV-diode array detector has been employed. Several compounds of different chemical structure and hydrophobicity have been used as model compounds: simazine, atrazine and terbutylazine (triazines), chlorfenvinphos and chlorpyrifos (organophosphorous), diuron and isoproturon (phenylureas), trifluralin (dinitroaniline) and di(2-ethylhexyl)phthalate. The results obtained revealed that the monolithic column was clearly advantageous in the context of multiresidue organic pollutants analysis for a number of reasons: (i) the selectivity was considerably improved, which is of particular interest for the most polar compounds triazines and phenyl ureas that could not be resolved in the particulate column, (ii) the sensitivity was enhanced, and (iii) the time required for the chromatographic separation was substantially shortened. In this study it is also proved that the mobile-phase flow rates used for separation in the capillary monolithic column are compatible with the in-valve IT-SPME methodology using extractive capillaries of dimensions similar to those used in conventional scale liquid chromatography (LC). On the basis of these results a new method is presented for the assessment of pollutants in waters, which permits the characterization of whole samples (4 mL) in less than 30 min, with limits of detection in the range of 5-50 ng/L.

  4. Separation of transition metals on a poly-iminodiacetic acid grafted polymeric resin column with post-column reaction detection utilising a paired emitter-detector diode system.

    PubMed

    Barron, Leon; O'Toole, Martina; Diamond, Dermot; Nesterenko, Pavel N; Paull, Brett

    2008-12-05

    The selectivity, retention and separation of transition metals on a short (2 mm x 50 mm) column packed with a poly-iminodiacetic acid functionalised polymer 10 microm resin (Dionex ProPac IMAC-10) are presented. This stationary phase, typically used for the separation of proteins, is composed of long chain poly-iminodiacetic acid groups grafted to a hydrophilic layer surrounding a 10 microm polymeric bead. Through the use of a combination of a multi-step pH and picolinic acid gradient, the separation of magnesium, iron, cobalt, cadmium, zinc, lead and copper was possible, followed by post-column reaction with 4-(2-pyridylazo) resorcinol (PAR) and absorbance detection at 510 nm using a novel and inexpensive optical detector, comprised of two light emitting diodes with one acting as a light source and the other as a detector. Column efficiency for selective transition metals was in excess of N=10,000, with the baseline separation of seven metal cations in <3 min possible under optimised conditions. Detection limits of between 5 and 81 microg/L were possible based upon a 50 microL injection volume.

  5. Quantification of endogenous brassinosteroids in plant by on-line two-dimensional microscale solid phase extraction-on column derivatization coupled with high performance liquid chromatography-tandem mass spectrometry.

    PubMed

    Wu, Qian; Wu, Dapeng; Shen, Zheng; Duan, Chunfeng; Guan, Yafeng

    2013-07-05

    An on-line two-dimensional microscale solid phase extraction (2DμSPE)-on column derivatization (OCD)-high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed for quantification of brassinosteroids (BRs) in plant tissues. Five BRs with widest distribution in plant species and high bioactivity (24-epibrassinolide, 24-epicastasterone, 6-deoxo-24-epicastasterone, teasterone and typhastero) were selected as target analytes. 2DμSPE column packed sequentially with phenyl boronic acid silica sorbent (the first dimension) and C18 silica sorbent (the second dimension) was used to selectively extract and enrich BRs by 110-146 times. OCD was carried out on the second dimension of 2DμSPE column with m-aminophenylboronic acid (m-APBA) as a derivatization reagent, enhancing the sensitivity of MS/MS to BRs by 13-8437 times. It was also found that pre-trap of derivatization reagent on the C18 section of 2DμSPE column could increase reaction efficiency by 3-10 times. The whole process time of the on-line system was less than 30min. The detection limits of the method for five BRs were between 1.4 and 6.6pg with RSDs less than 10%. Endogeneous BRs in tomato leaves were analyzed by using this method. Owing to the high selectivity of this on-line 2DμSPE system, BRs in plant extracts could be quantified using matrix-free standard calibration method with relative recoveries in the range of 80-124%.

  6. Separation of oligo/polymers of 5-N-acetylneuraminic acid, 5-N-glycolylneuraminic acid, and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid by high-performance anion-exchange chromatography with pulsed amperometric detector.

    PubMed

    Zhang, Y; Inoue, Y; Inoue, S; Lee, Y C

    1997-08-01

    A sensitive and efficient method to analyze oligo/ poly-sialic acids containing alpha2-8-linked 5-N-acetylneuraminic acid (Neu5Ac), 5-N-glycolylneuraminic acid (Neu5Gc), and deaminated neuraminic acid (KDN) using high-performance anion-exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD-2) has been developed. Using a CarboPac PA-100 column and sodium nitrate as the pushing agent, polymers in colominic acid with degree of polymerization (DP) up to 80 were separated in 68 min. A similar DP-based resolution was also obtained on a CarboPac PA-1 column. The elution ladders of the Neu5Ac, Neu5Gc, and KDN series were sufficiently different to be used as diagnostic indices. This technique was applied to identification of the sialic acid components in a polysialoglycoprotein (PSGP) sample as well as monitoring the oligo/poly-KDN-containing fractions during the purification of KDN-containing glycoprotein (KDN-gp). The maximum DPs of oligo-Neu5Gc and oligo-KDN that can be detected in PSGP and KDN-gp hydrolysates were 11 and 8, respectively. The high sensitivity of this method was demonstrated by the quantification of Neu5Ac oligomers. Distributions of the monomer and oligo/polymers in the acid and enzymatic hydrolysates of colominic acid and PSGP under different conditions were also studied.

  7. Low-level bromate analysis in drinking water by ion chromatography with optimized suppressed conductivity cell current followed by a post-column reaction and UV/Vis detection.

    PubMed

    Fotsing, Marcellin; Barbeau, Benoit; Prevost, Michele

    2011-01-01

    In the present work, a high capacity anion exchange column was used to efficiently and simultaneously separate traces of oxyhalide disinfection byproducts (DBP) anions and bromide by an ion chromatography system followed by a post-column reaction (PCR). The PCR generates in situ hydroiodic (HI) acid from the excess of potassium iodate that combines with bromate from the column effluent to form the triiodide anion detectable by UV/Vis absorbance at 352 nm. The suppressed conductivity cell current was optimized at 70 mA, with a flow rate of 1.0 mL/min and a 9 mM carbonate eluent. Its performance was investigated on a trace-level determination of bromate in ozonated municipal and bottled drinking water. Based on ozonated municipal drinking water matrix, the method detection limit of 0.27 μg BrO(-)(3)/L was evaluated with the Method Quantification Limit (MQL) of 0.89 μg BrO(-)(3)/L. However, in ultrapure water, a MDL of 0.015 μg BrO(-)(3)/L and a MRL of 0.052 μg BrO(-)(3)/L were achieved. The recovery for spiked municipal samples was in the range of 90%-115%.

  8. Phenylboronic acid-salicylhydroxamic acid bioconjugates. 2. Polyvalent immobilization of protein ligands for affinity chromatography.

    PubMed

    Wiley, J P; Hughes, K A; Kaiser, R J; Kesicki, E A; Lund, K P; Stolowitz, M L

    2001-01-01

    Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

  9. Development of molecular imprinted column-on line-two dimensional liquid chromatography for selective determination of clenbuterol residues in biological samples.

    PubMed

    Guo, Pengqi; Luo, Zhimin; Xu, Xinya; Zhou, Yulan; Zhang, Bilin; Chang, Ruimiao; Du, Wei; Chang, Chun; Fu, Qiang

    2017-02-15

    A novel method coupling molecular imprinted monolithic column with two-dimensional liquid chromatography was developed and validated for the analysis of clenbuterol in pork liver and swine urine samples. The polymers were characterized by using Fourier transform infrared spectroscopy, nitrogen adsorption desorption analyses, frontal analysis and the adsorption of selectivity. The results indicated that the imprinted columns were well prepared and possessed high selectivity adsorption capacity. Subsequently, the MIMC-2D-LC (molecular imprinted monolithic column-two dimensional liquid chromatography) method was developed for the selective analysis of clenbuterol in practical samples. The accuracy ranged from 94.3% to 99.7% and from 93.7% to 99.6% for liver and urine, respectively. The relative standard deviation (RSD) of repeatability was lower than 8.6% for both analyses. The limit of detections was 16ng·mL(-1) for liver and 25ng·mL(-1) for urine, respectively. Compared with the reported methods, the disturbance of endogenous impurity could be avoided by the 2D-LC method.

  10. On-line coupling of macroporous resin column chromatography with direct analysis in real time mass spectrometry utilizing a surface flowing mode sample holder.

    PubMed

    Zeng, Shanshan; Wang, Lu; Chen, Teng; Qu, Haibin

    2014-02-06

    A surface flowing mode sample holder was designed as an alternative sampling strategy for direct analysis in real time mass spectrometry (DART-MS). With the sample holder, the on-line coupling of macroporous resin column chromatography with DART-MS was explored and the new system was employed to monitor the column chromatography elution process of Panax notoginseng. The effluent from macroporous resin column was first diluted and mixed with a derivatization reagent on-line, and the mixture was then directly transferred into the ionization region of DART-MS by the sample holder. Notoginsenosides were methylated and ionized in a metastable helium gas stream, and was introduced into MS for detection. The on-line system showed reasonable repeatability with a relative standard deviation of 12.3% for the peak area. Three notoginsenosides, i.e. notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rg1, were simultaneously determined during the eluting process. The alteration of the chemical composition in the effluent was accurately identified in 9 min, agreeing well with the off-line analysis. The presented technique is more convenient compared to the traditional UPLC method. These results suggest that the surface flowing mode DART-MS has a good potential for the on-line process monitoring in the pharmaceutical industry.

  11. An Eco-Friendly Direct Injection HPLC Method for Methyldopa Determination in Serum by Mixed-Mode Chromatography Using a Single Protein-Coated Column.

    PubMed

    Emara, Samy; Masujima, Tsutomu; Zarad, Walaa; Kamal, Maha; Fouad, Marwa; El-Bagary, Ramzia

    2015-09-01

    A simple, rapid and environment-friendly direct injection HPLC method for the determination of methyldopa (MTD) in human serum has been developed and validated. The method was based on cleanup and separation of MTD from serum by mixed-mode liquid chromatography using a single protein-coated TSK gel ODS-80 TM analytical column (50 × 4.0 mm i.d., 5 µm). The protein-coated column exhibited excellent resolution, selectivity and functioned in two chromatographic modes: size-exclusion chromatography [i.e., solid-phase extraction (SPE) for serum proteins] and reversed-phase chromatography for the final separation of MTD. SPE and HPLC separation were carried out simultaneously with a green mobile phase consisting of acetate buffer (0.1 M, pH 2.4) at a flow rate of 1 mL/min and at room temperature (23 ± 1°C). The eluent was monitored at emission and excitation wavelengths of 320 and 270 nm, respectively. A calibration curve was linear over the range of 0.1-30 µg/mL with a detection limit of 0.027 µg/mL. This online SPE method was successfully applied to real samples obtained from patients receiving MTD therapy.

  12. [Determination of urinary trans, trans-muconic acid by high performance liquid chromatography].

    PubMed

    Liu, Liwen; Song, Shizhen; Hu, Xiamin; Ye, Fangli

    2006-05-01

    A method for the determination of urinary trans, trans-muconic acid (tt-MA) (benzene metabolite) by high performance liquid chromatography (HPLC) was developed. The separation was carried out on a C18 column (Spherisorb C18, 150 mm x 4.6 mm i. d. , 5 microm) at 25 degrees C with glacial acetic acid-tetrahydrofuran-methanol-water (1 : 2 : 10: 87, v/v) as mobile phase. Urinary sample was acidified by 2 mol/L hydrochloric acid and pretreated by liquid-liquid extraction using diethyl ether. After removal of diethyl ether with a stream of nitrogen, the residue was re-dissolved in 1 mL of mobile phase for HPLC injection. Good linearity was observed within the range from 0.10 mg/L to 10.00 mg/L (r =0.9999) and the detection limit was 0.10 mg/L. The average recoveries for tt-MA were 95.1%-100.5%. Relative standard deviations (RSD) for intra-day and inter-day determinations were 4.0%-9.0% and 6.2%-8.8%, respectively. The method was applied to 56 benzene-exposed workers and 24 controll workers. Urinary tt-MA in benzen-exposed workers were significantly higher than that in the control group. They can be correlated with benzene exposure concentrations (P <0.01). This analytical method for tt-MA is sensitive, rapid, and convenient. It is suitable for monitoring of occupational exposure to benzene and toxic kinetics studies.

  13. [Determination of glyphosate and aminomethylphosphonic acid in rice using liquid chromatography-tandem mass spectrometry].

    PubMed

    Cao, Zhaoyun; Mou, Renxiang; Chen, Mingxue

    2010-08-01

    A method was developed for the determination of glyphosate (GLY) and aminomethylphosphonic acid (AMPA) in rice using liquid chromatography tandem mass spectrometry (LC-MS/MS). The sample was extracted with water followed by a simple cleanup with a C18 solid phase extraction (SPE) cartridge, and then GLY and AMPA were derivatized using 9-fluorenylmethoxycarbonyl (FMOC-Cl) in borate buffer. The derivatives of GLY and AMPA were separated on a C18 column with gradient elution with the mobile phase of acetonitrile and 5 mmol/L ammonium acetate (pH 9), and finally detected with negative ion electrospray ionization-mass spectrometry (ESI-MS) in multiple reaction monitoring (MRM) mode. The results showed that the linearities of GLY and AMPA were in the concentration range of 0.000 50 to 1.0 mg/L with the correlation coefficients of 0.999 7 and 0.999 9, respectively. The mean spiked recoveries of GLY and AMPA at 3 spiked levels ranged from 72.5% to 113.6% with the relative standard deviations (RSD, n = 5) of 3.8% - 16.2%. The limits of detection were 2.0 and 3.0 microg/kg for GLY and AMPA, respectively. This method is rapid, sensitive, and suitable for simultaneous determination of GLY and AMPA in rice.

  14. Determination of short chain carboxylic acids in vegetable oils and fats using ion exclusion chromatography electrospray ionization mass spectrometry.

    PubMed

    Viidanoja, Jyrki

    2015-02-27

    A new method for quantification of short chain C1-C6 carboxylic acids in vegetable oils and fats by employing Liquid Chromatography Mass Spectrometry (LC-MS) has been developed. The method requires minor sample preparation and applies non-conventional Electrospray Ionization (ESI) liquid phase chemistry. Samples are first dissolved in chloroform and then extracted using water that has been spiked with stable isotope labeled internal standards that are used for signal normalization and absolute quantification of selected acids. The analytes are separated using Ion Exclusion Chromatography (IEC) and detected with Electrospray Ionization Mass Spectrometry (ESI-MS) as deprotonated molecules. Prior to ionization the eluent that contains hydrochloric acid is modified post-column to ensure good ionization efficiency of the analytes. The averaged within run precision and between run precision were generally lower than 8%. The accuracy was between 85 and 115% for most of the analytes. The Lower Limit of Quantification (LLOQ) ranged from 0.006 to 7mg/kg. It is shown that this method offers good selectivity in cases where UV detection fails to produce reliable results.

  15. Passive treatment of acid mine drainage using coal combustion by-products and spent mushroom substrate: Results of column study

    SciTech Connect

    Crisp, T.E.; Nairn, R.W.; Strevett, K.A.; Everett, J.

    1998-12-31

    A column study was conducted to evaluate the feasibility of using of coal combustion by-products (CCB) as alkaline materials in a field scale downflow constructed wetlands for acid mine drainage treatment. Five columns (15.24 cm in diameter and 91.44 cm high) were constructed and filled with a combination of spent mushroom substrate (SMS) and one of three alkaline materials (limestone, hydrated fly ash, or fluidized bed ash). The five mixtures utilized were 10% fluidized bed ash/40% limestone (FBA/LS), 10% fluidized bed ash (FBA), 50% limestone (LS), 50% hydrated fly ash (HFA),m and 50% sieved (>1.5 cm) hydrated fly ash (S. HFA) with the remainder as SMS on a w/w basis. Column received synthetic acid mine drainage containing: 400 mg/L iron, 59 mg/L aluminum, 11 mg/L manganese, 50% mg/L magnesium, 40 mg/L calcium, and 1200 mg/L sulfate for 5 months. Anoxic conditions in the influent reservoirs were maintained by a positive nitrogen pressure head. Flow rates of 2.0 mL/minute to each column were maintained by a multichannel peristaltic pump. For all columns, effluent acidity concentrations were less than influent acidity concentration (877{sup {minus}}30, n = 75f). Mean effluent acidity concentrations were 241 mg/L (FBA/LS), 186 mg/L (FBA), 419 mg/L (LS), {minus}28.5 mg/L (HFA), and 351 mg/L (S. HFA), respectively. While all column produced measurable alkalinity, only the HFA column produced a net alkaline discharge. The results of these column studies are applicable to the design and sizing of innovative field scale systems using alkaline-rich CCB`s.

  16. Using active flow technology columns for high through-put and efficient analyses: The drive towards ultra-high through-put high-performance liquid chromatography with mass spectral detection.

    PubMed

    Kocic, Danijela; Shalliker, R Andrew

    2015-11-20

    The performance of active flow technology chromatography columns in parallel segmented flow mode packed with 5 μm Hypersil GOLD particles was compared to conventional UHPLC columns packed with 1.9 μm Hypersil GOLD particles. While the conventional UHPLC columns produced more theoretical plates at the optimum flow rate, when separations were performed at maximum through-put the larger particle size AFT column out-performed the UHPLC column. When both the AFT column and the UHPLC column were operated such that they yielded the same number of theoretical plates per separation, the separation on the AFT column was twice as fast as that on the UHPLC column, with the same level of sensitivity and at just 70% of the back pressure. Furthermore, as the flow velocity further increased the performance gain on the AFT column compared to the UHPLC column improved. An additional advantage of the AFT column was that the flow stream at the exit of the column was split in the radial cross section of the peak profile. This enables the AFT column to be coupled to a flow limiting detector, such as a mass spectrometer. When operated under high through-put conditions separations as fast as six seconds, using mobile phase flow rates in the order of 5-6 mL/min have been recorded.

  17. Determination of the polar drug dimiracetam in human plasma and serum by column-switching high-performance liquid chromatography.

    PubMed

    Torchio, L; Lombardi, F; Visconti, M; Doyle, E

    1995-04-07

    A sensitive and fully automated column-switching HPLC method was developed for the determination of a novel cognition enhancer, dimiracetam, in human plasma and serum. A sample of plasma was ultrafiltered and then directly injected onto a first mu Bondapak C18 column. The sample was eluted with water. The portion of the eluate (heart-cut) from this column containing the drug was selected and loaded on a second mu Bondapak C18 column and eluted with water. During the elution of the second column, the first column was automatically rinsed with water-acetonitrile (50:50, v/v) and then equilibrated for a new injection. The total analysis time for each sample was 35 min. This corresponded to a 30% decrease in analysis time compared with the time that would have been needed with a simple run with two mu Bondapak C18 columns assembled in series. Ultraviolet detection at 200 nm was used to monitor the eluate. The method was fully validated over the quantitation range 27 ng/ml-15 micrograms/ml. The applicability of the method was demonstrated by analysis of serum samples from a study performed in human healthy volunteers.

  18. Direct detection of free fatty acids in edible oils using supercritical fluid chromatography coupled with mass spectrometry.

    PubMed

    Qu, Shuping; Du, Zhenxia; Zhang, Yun

    2015-03-01

    Determination of free fatty acids (FFAs) in food products is of enormous interest mainly because they are related to the quality and authenticity of the oils. In this study, supercritical fluid chromatography (SFC), followed by an electrospray ionisation triple-quadrupole mass spectrometry (ESI-MS), is shown to provide a novel method for the separation and detection of FFAs in edible oils without any pretreatment. Eight FFAs were separated on a HSS C18 SB column with gradient elution within 3 min. Effects of different columns, modifiers and column temperature changes were evaluated. The results indicated the feasibility of this method for the high-throughput determination of individual FFAs with satisfactory correlation coefficients (R(2)>0.994) and good reproducibility of RSD<13.5% (intraday) and <15.0% (interday). By combined with principal component analysis (PCA), different types of edible oil were successfully distinguished into several categories, showing a potential application for the determination of oil quality or authenticity.

  19. Determination of volatile fatty acids in landfill leachates by ion-exclusion chromatography.

    PubMed

    Yamamoto, Atsushi; Yasuhara, Akio; Kodama, Shuji; Matsunaga, Akinobu; Suzuki, Shigeru; Mohri, Shino; Yamada, Masato

    2004-03-01

    An ion-exclusion chromatographic method with on-line desalinization for the determination of volatile fatty acids in landfill leachates is described. Highly sensitive conductivity detection of the organic acids was achieved by using dilute p-hydroxybenzoic acid solution as an eluent. Interference with mineral acids was reduced by treatment with barium chloride solution prior to desalinization. A silver-loaded cation-exchange guard column for the desalinization was installed in series with the analytical column to avoid the contamination of organic acids. This method features detection limits of 0.01 mg L(-1) formic acid, 0.02 mg L(-1) acetic acid, 0.05 mg L(-1) propionic acid, and 0.1 mg L(-1) butyric acid, respectively, with an injection of 20 microL sample. Application of the on-line desalinization LC method is illustrated for leachate samples from a Japanese sanitary landfill.

  20. Identification of l-carnitine and its impurities in food supplement formulations by online column-switching liquid chromatography coupled with linear ion trap mass spectrometry.

    PubMed

    Wang, Hang; Xie, Sijun

    2017-01-01

    The identification of impurities in l-carnitine by mass spectrometry is difficult because derivative reagents or ion pair reagents are usually used to separate and increase the retention of l-carnitine on the reversed-phase column. In this study, four impurities including 3-chloro-2-hydroxy-N,N,N-trimethylpropan-1-aminium, 3-cyano-2-hydroxy-N,N,N-trimethylpropan-1-aminium, 3-carboxy-N,N,N-trimethylprop-2-en-1-aminium, and 4-chloro-2,3,4-trihydroxy-N,N,N-trimethylbutan-1-aminium were identified in l-carnitine and its tablets by using two-dimensional column-switching high-performance liquid chromatography coupled with linear ion trap mass spectrometry. The first column was a C8 column at a flow rate of 0.15 mL/min; the detection wavelength was 220 nm. The second column was an Acclaim Q1 column using a gradient elution program with aqueous 30 mM ammonium acetate (pH 5.0) and acetonitrile as the mobile phase at a flow rate of 0.5 mL/min. The mass fragmentation patterns and structural assignments of impurities were studied, and the quantitative validation of three impurities was further investigated. The linearity (r(2) ) was found to be >0.99, with ranges from 0.2 to 50 ng/mL and 0.1 to 10 ng/mL. The method was used successfully for determination of impurities in five samples of l-carnitine and tablets.

  1. 1.1 μm superficially porous particles for liquid chromatography: part II: column packing and chromatographic performance.

    PubMed

    Blue, Laura E; Jorgenson, James W

    2015-02-06

    The predicted advantages of superficially porous particles over totally porous particles are decreased eddy dispersion, longitudinal diffusion, and resistance to mass transfer contributions to the theoretical plate height. While sub-2 micron superficially porous particles are commercially available, further improvements in performance are predicted by decreasing the particle diameter and decreasing the porous layer thickness. 1.1 μm superficially porous particles with 187Å pores have been synthesized using a layer-by-layer method tuned for production of smaller diameter particles. Following synthesis, these particles were packed into 30 μm i.d. capillary columns and their chromatographic performance evaluated using electrochemical detection. Based on the initial studies, the column efficiency did not meet theory, but was similar to the commercially available products tested. It is believed that the column packing process plays a critical role in the sub-par column performance. To determine if column efficiency could be predicted by solvent-particle interactions, in-solution optical microscopy and sedimentation velocity of particles in various slurry solvents were investigated and compared to column performance. Aggregating slurry solvents, such as methanol were found to produce columns with increased efficiency. The hmin for a column packed with an acetone slurry and a methanol slurry at 3mg/mL were found to be 6.3 and 3.5, respectively. Increasing the slurry concentration to 25mg/mL further improved the efficiency, producing a column with an hmin of 2.6. These efficiency results were accurately predicted by in-solution optical microscopy.

  2. Isolation and identification of arctiin and arctigenin in leaves of burdock (Arctium lappa L.) by polyamide column chromatography in combination with HPLC-ESI/MS.

    PubMed

    Liu, Shiming; Chen, Kaoshan; Schliemann, Willibald; Strack, Dieter

    2005-01-01

    A simple method involving polyamide column chromatography in combination with HPLC-PAD and HPLC-ESI/MS for isolating and identifying two kinds of lignans, arctiin and arctigenin, in the leaves of burdock (Arctium lappa L.) has been established. After extraction of burdock leaves with 80% methanol, the aqueous phase of crude extracts was partitioned between water and chloroform and the aqueous phase was fractionated on a polyamide glass column. The fraction, eluting with 100% methanol, was concentrated and gave a white precipitate at 4 degrees C from which two main compounds were purified by semi-preparative HPLC. In comparison with the UV and ESI-MS spectra and the HPLC retention time of authentic standards, the compounds were determined to be arctiin and arctigenin. The extraction/separation technique was validated using an internal standard method.

  3. On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column.

    PubMed

    Kulsing, Chadin; Komaromy, Andras Z; Boysen, Reinhard I; Hearn, Milton T W

    2016-10-21

    This study documents the use of an integrated approach, involving on-line hydrophobic interaction chromatography interfaced with Small Angle X-ray Scattering (HIC-SAXS) measurements, to monitor the conformational status of proteins immediately upon elution from a chromatographic column operated at different temperatures. Moreover, this approach provides an additional avenue to interrogate the changes in protein shape that may occur across the eluted chromatographic peak. To this end, radii of gyration were extrapolated from the Guinier approximation with the HIC-SAXS data, whilst pair distribution functions and bead model simulations were generated by using the indirect transform program GNOM and ab initio reconstruction with GASBOR to provide further insight into protein conformational changes that occur during hydrophobic interaction chromatography.

  4. Chemometrics-assisted high performance liquid chromatography-diode array detection strategy to solve varying interfering patterns from different chromatographic columns and sample matrices for beverage analysis.

    PubMed

    Yin, Xiao-Li; Wu, Hai-Long; Gu, Hui-Wen; Hu, Yong; Wang, Li; Xia, Hui; Xiang, Shou-Xia; Yu, Ru-Qin

    2016-02-26

    This work reports a chemometrics-assisted high performance liquid chromatography-diode array detection (HPLC-DAD) strategy to solve varying interfering patterns from different chromatographic columns and sample matrices for the rapid simultaneous determination of six synthetic colorants in five kinds of beverages with little sample pretreatment. The investigation was performed using two types of LC columns under the same elution conditions. Although analytes using different columns have different co-elution patterns that appear more seriously in complex backgrounds, all colorants were properly resolved by alternating trilinear decomposition (ATLD) method and accurate chromatographic elution profiles, spectral profiles as well as relative concentrations were obtained. The results were confirmed by those obtained from traditional HPLC-UV method at a particular wavelength and the results of both methods were consistent with each other. All results demonstrated that the proposed chemometrics-assisted HPLC-DAD method is accurate, economical and universal, and can be promisingly applied to solve varying interfering patterns from different chromatographic columns and sample matrices for the analysis of complex food samples.

  5. Determination of benzalkonium chloride in eye care products by high-performance liquid chromatography and solid-phase extraction or on-line column switching.

    PubMed

    Elrod, L; Golich, T G; Morley, J A

    1992-11-20

    Benzalkonium chloride (BAK) is a mixture of alkylbenzyldimethylammonium chlorides, which is commonly used as a bacteriostat. In this work, the three major homologues of BAK are quantitated in the over-the-counter eye care products Murine and Murine Plus using high-performance liquid chromatography (HPLC). The analytes are separated from various product excipients and concentrated by either solid-phase extraction onto Sep-Pak C18 cartridges or by an on-line column-switching technique using 1-cm reversed-phase precolumns. Absolute recoveries of BAK homologues by the solid-phase extraction technique ranged from 97.2 to 98.7% for standards and from 98.0 to 98.4% for samples. Absolute recovery of the BAK homologues by the column-switching technique was 101.3% for standards and ranged from 99.9 to 103.7% for samples. Relative recoveries were quantitative by both techniques. Assay precision (R.S.D. values) were +/- 2.2% to +/- 2.6% and +/- 0.4% to +/- 0.8% by solid-phase extraction and column-switching techniques, respectively. The method provides advantages of high sample throughput, excellent column life and automation.

  6. [Intersection point rule for the retention value with mobile phase composition and boiling point of the homologues and chlorobenzenes in soil leaching column chromatography].

    PubMed

    Xu, F; Liang, X; Lin, B; Su, F

    1999-03-01

    Based on the linear retention equation of the logarithm of the capacity factor (logk') vs. the methanol volume fraction (psi) of aqueous binary mobile phase in soil leaching column chromatography, the intersection point rule for the logk' of homologues and weak polar chlorobenzenes, with psi, as well as with boiling point, has been derived due to existence of the similar interactions among solutes of the same series, stationary phase (soil) and eluent (methanol-water). These rules were testified by experimental data of homologues (n-alkylbenzenes, methylbenzenes) and weak polar chlorobenzenes.

  7. Development Of Ion Chromatography Methods To Support Testing Of The Glycolic Acid Reductant Flowsheet In The Defense Waste Processing Facility

    SciTech Connect

    Wiedenman, B. J.; White, T. L.; Mahannah, R. N.; Best, D. R.; Stone, M. E.; Click, D. R.; Lambert, D. P.; Coleman, C. J.

    2013-10-01

    Ion Chromatography (IC) is the principal analytical method used to support studies of Sludge Reciept and Adjustment Tank (SRAT) chemistry at DWPF. A series of prior analytical ''Round Robin'' (RR) studies included both supernate and sludge samples from SRAT simulant, previously reported as memos, are tabulated in this report.2,3 From these studies it was determined to standardize IC column size to 4 mm diameter, eliminating the capillary column from use. As a follow on test, the DWPF laboratory, the PSAL laboratory, and the AD laboratory participated in the current analytical RR to determine a suite of anions in SRAT simulant by IC, results also are tabulated in this report. The particular goal was to confirm the laboratories ability to measure and quantitate glycolate ion. The target was + or - 20% inter-lab agreement of the analyte averages for the RR. Each of the three laboratories analyzed a batch of 12 samples. For each laboratory, the percent relative standard deviation (%RSD) of the averages on nitrate, glycolate, and oxalate, was 10% or less. The three laboratories all met the goal of 20% relative agreement for nitrate and glycolate. For oxalate, the PSAL laboratory reported an average value that was 20% higher than the average values reported by the DWPF laboratory and the AD laboratory. Because of this wider window of agreement, it was concluded to continue the practice of an additional acid digestion for total oxalate measurement. It should also be noted that large amounts of glycolate in the SRAT samples will have an impact on detection limits of near eluting peaks, namely Fluoride and Formate. A suite of scoping experiments are presented in the report to identify and isolate other potential interlaboratory disceprancies. Specific ion chromatography inter-laboratory method conditions and differences are tabulated. Most differences were minor but there are some temperature control equipment differences that are significant leading to a recommendation of

  8. Isolation of fluorescent constituents from soil humic and fulvic acids by hydrophilic interaction chromatography

    NASA Astrophysics Data System (ADS)

    Aoyama, Masakazu

    2014-05-01

    Humic acids (HAs) and fulvic acids (FAs) are the most abundant components of soil organic matter and exhibit fluorescence. Our previous studies using high performance size-exclusion chromatography (HPSEC) and polyacrylamide gel electrophoresis demonstrated that the fluorescence of soil HAs was mainly due to the minor constituents with relatively small molecular sizes. In order to clarify the nature of the fluorescence of soil organic matter, it is necessary to isolate the fluorescent constituents from HAs and FAs. I succeeded in isolating the fluorescent constituents from soil HAs and FAs by using hydrophilic interaction chromatography (HILIC). When HILIC of soil HAs and FAs was carried out under isocratic conditions using a SeQuant ZIC-HILIC column and acetonitrile-water as a mobile phase, the complete separation of fluorescent and non-fluorescent peaks was achieved at the acetonitrile concentration of 90%. Another fluorescent peak was eluted with decreasing concentration of acetonitrile from 90% to 50%. The use of a TSKgel Amide-80 column gave the same results. The best resolution was obtained when HILIC was performed under gradient conditions from 90% to 50% acetonitrile using the ZIC-HILIC and Amide-80 columns linked in series. For both HAs and FAs, a sharp non-fluorescent peak (peak A) followed by a sharp fluorescent peak (peak B) and a broad fluorescent peak (peak C) were eluted under the above optimum operating conditions. The intensity of peak A relative to that of peak B was significantly less in the FAs than in the HAs. The fluorescent peaks (peaks B and C) of the FAs showed considerable UV absorption, whereas those of the HAs did little UV absorption. When the fluorescence emission spectra (excitation at 280 nm) were measured for the fluorescent peaks, two emission peaks were located at 460 and 520 nm for the HAs, while for the FAs, a broad emission peak at 400-450 nm with a small shoulder at around 500 nm was observed. The peaks were collected

  9. Tidally driven water column hydro-geochemistry in a remediating acidic wetland

    NASA Astrophysics Data System (ADS)

    Johnston, Scott G.; Keene, Annabelle F.; Bush, Richard T.; Sullivan, Leigh A.; Wong, Vanessa N. L.

    2011-10-01

    SummaryManaged tidal inundation is a newly evolved technique for remediating coastal acid sulphate soil (CASS) wetlands. However, there remains considerable uncertainty regarding the hydro-geochemical pathways and spatiotemporal dynamics of residual H + and metal(loid) mobilisation into the tidal fringe surface waters of these uniquely iron-rich landscapes. Here, we examine the hydrology and water column chemistry across the intertidal slope of a remediating CASS wetland during several tide cycles. There was extreme spatial and temporal dynamism in water column chemistry, with pH fluctuating by ˜3 units (˜3.5-6.5) during a single tide cycle. Acute acidity was spatially confined to the upper intertidal slope, reflecting surface sediment properties, and tidal overtopping is an important pathway for mobilisation of residual H + and Al 3+ to the water column. Marine derived HCO3- was depleted from surface waters migrating across the intertidal slope and a strong gradient in HCO3- was observed from the tidal fringe to the adjacent tributary channel and nearby estuary. Tidal forcing generated oscillating hydraulic gradients in the shallow fringing aquifer, favouring ebb-tide seepage and driving rapid, heterogeneous advection of groundwater on the lower intertidal slope via surface connected macropores. A combination of diffusive and advective flux across the sediment-water interface led to persistent, elevated surface water Fe 2+ (˜10-1000 μM). The geochemical processes associated with Fe 2+ mobilisation displayed distinct spatial zonation, with low pH, proton-promoted desorption occurring on the upper intertidal slope, whilst circum-neutral pH, Fe(III)-reducing processes dominated the lower intertidal slope. Arsenic was also mobilised into surface waters on the lower intertidal slope under moderate pH (˜6.0) conditions and was strongly positively correlated with Fe 2+. Saturation index values for aragonite were substantially depressed (-1 to -5) and significantly

  10. Enhancing detection sensitivity in gradient liquid chromatography via post-column refocusing and strong-solvent remobilization.

    PubMed

    De Vos, Jelle; Desmet, Gert; Eeltink, Sebastiaan

    2016-07-15

    We developed earlier the post-column refocusing strategy for isocratic separations, which employs trapping target analytes after an analytical separation and additionally focusing them using a strong remobilization solvent prior to detection, and have now extended it to high-speed gradient LC. A gradient separation of antibiotics and its metabolites, applying a linear aqueous acetonitrile gradient from 2 to 65% (v/v) ACN containing 0.1% FA in 10min, performed on an analytical column was selected as an application. Eluted heart-cut fractions were directed from the analytical silica C18 column to a trap column packed with Hypercarb particles. The remobilization of the target analytes was performed in back-flush mode using solvent mixtures tuned to maximize the solvent strength by mixing isopropanol into the remobilization solvent containing acetonitrile. Additionally, a viscosity-calibration experiment showed that the viscosity difference between trapping and remobilization solvents should be smaller than 0.15mPa·s to prevent viscous fingering. To keep the viscosity difference below this limit, during the gradient separation performed on the analytical column, the composition of the remobilization solvent was changed in time. An empirical equation is provided that allows for the selection of the optimal remobilization-solvent composition. To maximize the signal enhancement, the loading time of target analytes on the trap column should be optimized. Peak dispersion was further minimized by applying a flow rate that corresponded to the optimal van-Deemter flow rate of the trap column (20μL/min). Finally, decreasing the diameter of the trap column from 1mm to 0.3mm led to a significant enhancement of the detection sensitivity with more than one order of magnitude. Using an optimized trap configuration and elution/remobilization conditions, a signal enhancement of a factor of 14 was achieved for sulfaguanidine (early-eluting compound in the gradient separation) and 7

  11. Serial lectin affinity chromatography with concavalin A and wheat germ agglutinin demonstrates altered asparagine-linked sugar-chain structures o