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.

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.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,

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 gelsmore » 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.« less

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.

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…

Determination of 3-hydroxypropylmercapturic acid in urine by three column-switching high-performance liquid chromatography with electrochemical detection using a diamond electrode.

PubMed

Higashi, Kyohei; Shibasaki, Mana; Kuni, Kyoshiro; Uemura, Takeshi; Waragai, Masaaki; Uemura, Kenichi; Igarashi, Kazuei; Toida, Toshihiko

2017-09-29

A three column-switching high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was established to determine 3-hydroxypropylmercapturic acid (3-HPMA) in urine. An extracted urine sample was consecutively fractionated using a strong anion-exchange column (first column) and a C8 column (second column) via a switching valve before application on an Octa Decyl Silyl (ODS) column (third column), followed by ECD analysis. The% recovery of 3-HPMA standard throughout the three-column process and limit of detection (LOD) were 94±1% and 0.1pmol, respectively. A solid phase extraction step is required for the sensitive analysis of 3-HPMA in urine by column-switching HPLC-ECD despite a decreased% recovery (55%) of urine sample spiked with 100pmol of 3-HPMA. To test the utility of our column-switching HPLC-ECD method, 3-HPMA levels of 27 urine samples were determined, and the correlation between HPLC-ECD and LC-Electrospray ionization (ESI)-MS/MS method was examined. As a result, the median values of μmol 3-HPMA/g Creatinine (Cre) in urine obtained by column-switching HPLC-ECD and LC-MS/MS were 2.19±2.12μmol/g Cre and 2.13±3.38μmol/g Cre, respectively, and the calibration curve (y=1.5171x-1.007) exhibited good linearity within a defined range (r 2 =0.907). These results indicate that the combination of column-switching HPLC and ECD is a powerful tool for the specific, reliable detection of 3-HPMA in urine. Copyright © 2017 Elsevier B.V. All rights reserved.

"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.

Fiber-based monolithic columns for liquid chromatography.

PubMed

Ladisch, Michael; Zhang, Leyu

2016-10-01

Fiber-based monoliths for use in liquid chromatographic separations are defined by columns packed with aligned fibers, woven matrices, or contiguous fiber structures capable of achieving rapid separations of proteins, macromolecules, and low molecular weight components. A common denominator and motivating driver for this approach, first initiated 25 years ago, was reducing the cost of bioseparations in a manner that also reduced residence time of retained components while achieving a high ratio of mass to momentum transfer. This type of medium, when packed into a liquid chromatography column, minimized the fraction of stagnant liquid and resulted in a constant plate height for non-adsorbing species. The uncoupling of dispersion from eluent flow rate enabled the surface chemistry of the stationary phase to be considered separately from fluid transport phenomena and pointed to new ways to apply chemistry for the engineering of rapid bioseparations. This paper addresses developments and current research on fiber-based monoliths and explains how the various forms of this type of chromatographic stationary phase have potential to provide new tools for analytical and preparative scale separations. The different stationary phases are discussed, and a model that captures the observed constant plate height as a function of mobile phase velocity is reviewed. Methods that enable hydrodynamically stable fiber columns to be packed and operated over a range of mobile phase flow rates, together with the development of new fiber chemistries, are shown to provide columns that extend the versatility of liquid chromatography using monoliths, particularly at the preparative scale. Graphical Abstract Schematic representation of a sample mixture being separated by a rolled-stationary phase column, resulting separated peaks shown in the chromatogram.

Immunoglobulin G elution in protein A chromatography employing the method of chromatofocusing for reducing the co-elution of impurities.

PubMed

Pinto, Nuno D S; Uplekar, Shaunak D; Moreira, Antonio R; Rao, Govind; Frey, Douglas D

2017-01-01

Purification processes for monoclonal Immunoglobulin G (IgG) typically employ protein A chromatography as a capture step to remove most of the impurities. One major concern of the post-protein A chromatography processes is the co-elution of some of the host cell proteins (HCPs) with IgG in the capture step. In this work, a novel method for IgG elution in protein A chromatography that reduces the co-elution of HCPs is presented where a two-step pH gradient is self-formed inside a protein A chromatography column. The complexities involved in using an internally produced pH gradient in a protein A chromatography column employing adsorbed buffering species are discussed though equation-based modeling. Under the conditions employed, ELISA assays show a 60% reduction in the HCPs co-eluting with the IgG fraction when using the method as compared to conventional protein A elution without affecting the IgG yield. Evidence is also obtained which indicates that the amount of leached protein A present in free solution in the purified product is reduced by the new method. Biotechnol. Bioeng. 2017;114: 154-162. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

A reversed phase high performance liquid chromatography method for the determination of fumonisins B1 and B2 in food and feed using monolithic column and positive confirmation by liquid chromatography/tandem mass spectrometry.

PubMed

Khayoon, Wejdan Shakir; Saad, Bahruddin; Salleh, Baharuddin; Ismail, Nor Azliza; Abdul Manaf, Normaliza Hj; Abdul Latiff, Aishah

2010-10-29

The development of a reversed phase high performance liquid chromatography fluorescence method for the determination of the mycotoxins fumonisin B(1) and fumonisin B(2) by using silica-based monolithic column is described. The samples were first extracted using acetonitrile:water (50:50, v/v) and purified by using a C(18) solid phase extraction-based clean-up column. Then, pre-column derivatization for the analyte using ortho-phthaldialdehyde in the presence of 2-mercaptoethanol was carried out. The developed method involved optimization of mobile phase composition using methanol and phosphate buffer, injection volume, temperature and flow rate. The liquid chromatographic separation was performed using a reversed phase Chromolith(®) RP-18e column (100 mm × 4.6 mm) at 30 °C and eluted with a mobile phase of a mixture of methanol and phosphate buffer pH 3.35 (78:22, v/v) at a flow rate of 1.0 mL min(-1). The fumonisins separation was achieved in about 4 min, compared to approximately 20 min by using a C(18) particle-packed column. The fluorescence excitation and emission were at 335 nm and 440 nm, respectively. The limits of detections were 0.01-0.04 μg g(-1) fumonisin B(1) and fumonisin B(2), respectively. Good recoveries were found for spiked samples (0.1, 0.5, 1.5 μg g(-1) fumonisins B(1) and B(2)), ranging from 84.0 to 106.0% for fumonisin B(1) and from 81.0 to 103.0% for fumonisin B(2). Fifty-three samples were analyzed including 39 food and feeds and 14 inoculated corn and rice. Results show that 12.8% of the food and feed samples were contaminated with fumonisin B(1) (range, 0.01-0.51 μg g(-1)) and fumonisin B(2) (0.05 μg g(-1)). The total fumonisins in these samples however, do not exceed the legal limits established by the European Union of 0.8 μg g(-1). Of the 14 inoculated samples, 57.1% contained fumonisin B(1) (0.16-41.0 μg g(-1)) and fumonisin B(2) (range, 0.22-50.0 μg g(-1)). Positive confirmation of selected samples was carried out using

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

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…

Hydrodynamic chromatography of macromolecules using polymer monolithic columns.

PubMed

Edam, Rob; Eeltink, Sebastiaan; Vanhoutte, Dominique J D; Kok, Wim Th; Schoenmakers, Peter J

2011-12-02

The selectivity window of size-based separations of macromolecules was tailored by tuning the macropore size of polymer monolithic columns. Monolithic materials with pore sizes ranging between 75 nm and 1.2 μm were prepared in situ in large I.D. columns. The dominant separation mechanism was hydrodynamic chromatography in the flow-through pores. The calibration curves for synthetic polymers matched with the elution behavior by HDC separations in packed columns with 'analyte-to-pore' aspect ratios (λ) up to 0.2. For large-macropore monoliths, a deviation in retention behavior was observed for small polystyrene polymers (M(r)<20 kDa), which may be explained by a combined HDC-SEC mechanism for λ<0.02. The availability of monoliths with very narrow pore sizes allowed investigation of separations at high λ values. For high-molecular weight polymers (M(r)>300,000 Da) confined in narrow channels, the separation strongly depended on flow rate. Flow-rate dependent elution behavior was evaluated by calculation of Deborah numbers and confirmed to be outside the scope of classic shear deformation or slalom chromatography. Shear-induced forces acting on the periphery of coiled polymers in solution may be responsible for flow-rate dependent elution. Copyright © 2011 Elsevier B.V. All rights reserved.

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography.

PubMed

Ruprecht, Benjamin; Koch, Heiner; Domasinska, Petra; Frejno, Martin; Kuster, Bernhard; Lemeer, Simone

2017-01-01

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO 2 or Ti-IMAC beads, Fe-IMAC HPLC columns do not suffer from issues regarding incomplete phosphopeptide binding or elution and enrichment efficiency scales linearly with the amount of starting material. Here, we provide a step-by-step protocol for the entire phosphopeptide enrichment procedure including sample preparation (lysis, digestion, desalting), Fe-IMAC column chromatography (column setup, operation, charging), measurement by LC-MS/MS (nHPLC gradient, MS parameters) and data analysis (MaxQuant). To increase throughput, we have optimized several key steps such as the gradient time of the Fe-IMAC separation (15 min per enrichment), the number of consecutive enrichments possible between two chargings (>20) and the column recharging itself (<1 h). We show that the application of this protocol enables the selective (>90 %) identification of more than 10,000 unique phosphopeptides from 1 mg of HeLa digest within 2 h of measurement time (Q Exactive Plus).

Flexible and Accessible Automated Operation of Miniature Chromatography Columns on a Liquid Handling Station.

PubMed

Konstantinidis, Spyridon; Goh, Hai-Yuan; Martin Bufájer, José M; de Galbert, Paul; Parau, Maria; Velayudhan, Ajoy

2018-03-01

The High Throughput (HT) investigation of chromatographic separations is an important element of downstream bioprocess development due to the importance of chromatography as a technique for achieving stringent regulatory requirements on product purity. Various HT formats for chromatography exist, but the miniature column approach has characteristics resembling large scale packed bed column chromatography the most. The operation of such columns on robotic stations can be automated, but this is not always a straightforward procedure; the robotic manipulations are highly dependent on the settings of each experiment and the standard commands of the supporting software may not provide readily the required flexibility and accessibility for "plug and play" functionality. These can limit the potential of this technique in laboratories engaging on HT activities. In this work, we present an application which aims to overcome this challenge by providing end-users with a flexible operation of the miniature column technique on an automated liquid handler. The application includes a script which is written on Freedom EVOware, and is supplemented by custom compiled executables. Here, the manipulations carried out by the application are described in detail and its functionality is demonstrated through typical experiments based on bind and elute miniature column chromatography. The application is shown to allow for the unsupervised "on-the-fly" programming of the robotic station and to ultimately make the technique accessible to non-automation experts. This application is therefore well suited to simplifying development activities based on the robotic deployment of the miniature column chromatography technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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…

Determination of soluble immunoglobulin G in bovine colostrum products by Protein G affinity chromatography-turbidity correction and method validation.

PubMed

Holland, Patrick T; Cargill, Anne; Selwood, Andrew I; Arnold, Kate; Krammer, Jacqueline L; Pearce, Kevin N

2011-05-25

Immunoglobulin-containing food products and nutraceuticals such as bovine colostrum are of interest to consumers as they may provide health benefits. Commercial scale colostrum products are valued for their immunoglobulin G (IgG) content and therefore require accurate analysis. One of the most commonly used methods for determining total soluble IgG in colostrum products is based on affinity chromatography using a Protein G column and UV detection. This paper documents improvements to the accuracy of the Protein G analysis of IgG in colostrum products, especially those containing aggregated forms of IgG. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) analysis confirmed that aggregated IgG measured by Protein G does not contain significant amounts of casein or other milk proteins. Size exclusion chromatography identified the content of soluble IgG as mainly monomeric IgG and aggregated material MW > 450 kDa with small amounts of dimer and trimer. The turbidity of the eluting IgG, mainly associated with aggregated IgG, had a significant effect on the quantitative results. Practical techniques were developed to correct affinity LC data for turbidity on an accurate, consistent, and efficient basis. The method was validated in two laboratories using a variety of colostrum powders. Precision for IgG was 2-3% (RSD(r)) and 3-12% (RSD(R)). Recovery was 100.2 ± 2.4% (mean ± RSD, n = 10). Greater amounts of aggregated IgG were solubilized by a higher solution:sample ratio and extended times of mixing or sonication, especially for freeze-dried material. It is concluded that the method without acid precipitation and with turbidity correction provides accurate, precise, and robust data for total soluble IgG and is suitable for product specification and quality control of colostrum products.

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.

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…

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. Copyright © 2017 Elsevier B.V. All rights reserved.

Integration of On-Column Chemical Reactions in Protein Characterization by Liquid Chromatography/Mass Spectrometry: Cross-Path Reactive Chromatography.

PubMed

Pawlowski, Jake W; Carrick, Ian; Kaltashov, Igor A

2018-01-16

Profiling of complex proteins by means of mass spectrometry (MS) frequently requires that certain chemical modifications of their covalent structure (e.g., reduction of disulfide bonds), be carried out prior to the MS or MS/MS analysis. Traditionally, these chemical reactions take place in the off-line mode to allow the excess reagents (the majority of which interfere with the MS measurements and degrade the analytical signal) to be removed from the protein solution prior to MS measurements. In addition to a significant increase in the analysis time, chemical reactions may result in a partial or full loss of the protein if the modifications adversely affect its stability, e.g,, making it prone to aggregation. In this work we present a new approach to solving this problem by carrying out the chemical reactions online using the reactive chromatography scheme on a size exclusion chromatography (SEC) platform with MS detection. This is achieved by using a cross-path reaction scheme, i.e., by delaying the protein injection onto the SEC column (with respect to the injection of the reagent plug containing a disulfide-reducing agent), which allows the chemical reactions to be carried out inside the column for a limited (and precisely controlled) period of time, while the two plugs overlap inside the column. The reduced protein elutes separately from the unconsumed reagents, allowing the signal suppression in ESI to be avoided and enabling sensitive MS detection. The new method is used to measure fucosylation levels of a plasma protein haptoglobin at the whole protein level following online reduction of disulfide-linked tetrameric species to monomeric units. The feasibility of top-down fragmentation of disulfide-containing proteins is also demonstrated using β 2 -microglobulin and a monoclonal antibody (mAb). The new online technique is both robust and versatile, as the cross-path scheme can be readily expanded to include multiple reactions in a single experiment (as

Fast preparation of hybrid monolithic columns via photo-initiated thiol-yne polymerization for capillary liquid chromatography.

PubMed

Ma, Shujuan; Zhang, Haiyang; Li, Ya; Li, Yanan; Zhang, Na; Ou, Junjie; Ye, Mingliang; Wei, Yinmao

2018-02-23

Although several approaches have been developed to fabricate hybrid monoliths, it would still take a few hours to finish the formation of monoliths. Herein, photo-initiated thiol-yne polymerization was first adopted to in situ fabricate hybrid monoliths within the confines of UV-transparent fused-silica capillary. A silicon-containing diyne (1,3-diethynyltetramethyl-disiloxane, DYDS) was copolymerized with three multithiols, 1,6-hexanedithiol, trimethylolpropane tris(3-mercaptopropionate) and pentaerythriol tetrakis(3-mercaptopropionate), by using a binary porogenic system of diethylene glycol diethyl ether (DEGDE)/poly(ethylene glycol) (PEG200) within 10 min. Several characterizations of three hybrid monoliths (assigned as I, II and III, respectively) were performed. The results showed that these hybrid monoliths possessed bicontinuous porous structure, which was remarkably different from that via typical free-radical polymerization. The highest column efficiency of 76,000 plates per meter for butylbenzene was obtained on the column I in reversed-phase liquid chromatography (RPLC). It was observed that the efficiencies for strong-retained butylbenzene were almost close to those of weak-retained benzene, indicating a retention-independent efficient performance of small molecules on hybrid column I. The surface area of this hybrid monolith was very small in the dry state (less than 10.0 m 2 /g), and the chromatographic behavior of hybrid monolithic columns would be possibly explained by radical-mediated step-growth process of thiol-yne polymerization. Finally, the column I was applied for separation of BSA tryptic digest by cLC-MS/MS, indicating satisfactory separation ability for complicated samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Cellufine sulfate column chromatography as a simple, rapid, and effective method to purify dengue virus.

PubMed

Kanlaya, Rattiyaporn; Thongboonkerd, Visith

2016-08-01

Conventional method to purify/concentrate dengue virus (DENV) is time-consuming with low virus recovery yield. Herein, we applied cellufine sulfate column chromatography to purify/concentrate DENV based on the mimicry between heparan sulfate and DENV envelope protein. Comparative analysis demonstrated that this new method offered higher purity (as determined by less contamination of bovine serum albumin) and recovery yield (as determined by greater infectivity). Moreover, overall duration used for cellufine sulfate column chromatography to purify/concentrate DENV was approximately 1/20 of that of conventional method. Therefore, cellufine sulfate column chromatography serves as a simple, rapid, and effective alternative method for DENV purification/concentration. Copyright © 2016 Elsevier B.V. All rights reserved.

Column chromatography isolation of nicotine from tobacco leaf extract (Nicotiana tabaccum L.)

NASA Astrophysics Data System (ADS)

Fathi, Raden Muhammad; Fauzantoro, Ahmad; Rahman, Siti Fauziyah; Gozan, Misri

2018-02-01

Restrictions on the use of dried tobacco leaf for cigarette production must be accompanied by the development of non-cigarette alternative products that are made from tobacco leaves. One of the alternative that can be done is to use the nicotine compound in tobacco leaf extract as medical product, such as Parkinson's medication or to be used as active substance in biopesticide. Nicotine was isolated using column chromatography method with the variation of mobile phase mixture ratio (petroleum ether and ethanol), started from 8:2, 6:4, 4:6, 2:8, to 0:10. All of the chromatographic fraction from each mobile phase's ratio was then tested qualitatively using thin layer chromatography (TLC) and also quantitatively using HPLC instrument. The column chromatography process could isolate 4.006% of nicotine compound from 4.19% tobacco leaf extract's nicotine. It is also known that ethanol is a good solution to be used as chromatography's mobile phase for nicotine isolation from tobacco leaf extract.

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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Effect of the thermal environment on the efficiency of packed columns in supercritical fluid chromatography.

PubMed

Zauner, Jordan; Lusk, Ryan; Koski, Steven; Poe, Donald P

2012-11-30

When a packed column is operated at temperatures and pressures near the critical point in supercritical fluid chromatography, the thermal environment in which it is placed has a significant impact on retention and efficiency. We measured the retention factors, plate heights, and related parameters for elution of a test mixture of alkylbenzenes with 5% methanol/95% carbon dioxide mobile phase on a 250 mm × 4.6 mm i.d. column packed with 5-micron Luna-C18 particles. Separations were performed at outlet pressures from 100 to 150 bar and a column oven temperature of 323K. For a bare column thermostated with convective air, significant efficiency losses were observed for outlet pressures equal to or less than 120 bar. These large efficiency losses are attributed to radial temperature gradients. Addition of foam insulation resulted in significant improvements in efficiency. Operating the column in still air using a commercially available column heater provided the best overall performance, with no measurable efficiency loss over the entire range of pressures studied. A reduced plate height of 1.88 was obtained at an optimum flow rate of 3.0 mL/min at 100 bar outlet pressure and with the temperature of the incoming mobile phase set approximately 2.3K above the temperature of the column oven. Retention time repeatability for all three thermal conditions was equal to or less than 0.5% RSD. These results demonstrate that it is possible to perform fast, efficient separations with excellent repeatability using SFC under near-critical conditions if the thermal environment is optimized to minimize the generation of radial temperature gradients. Copyright © 2012 Elsevier B.V. All rights reserved.

Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

PubMed Central

1977-01-01

A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

Behavior of macroporous vinyl silica and silica monolithic columns in high pressure gas chromatography.

PubMed

Maniquet, Adrien; Bruyer, Nicolas; Raffin, Guy; Baco-Antionali, Franck; Demesmay, Claire; Dugas, Vincent; Randon, Jérôme

2017-06-30

80% vinyltrimethoxysilane-based hybrid silica monoliths (80-VTMS), which have been initially developed for separation in reversed-phase liquid chromatography, have been investigated in high pressure gas chromatography separations (carrier gas pressure up to 60bar) and compared to silica monolithic columns. The behavior of both silica and 80-VTMS monolithic columns was investigated using helium, nitrogen and carbon dioxide as carrier gas. The efficiency of 80-VTMS monolithic columns was shown to vary differently than silica monolithic columns according to the temperature and the carrier gas used. Carrier gas nature was a significant parameter on the retention for both silica and vinyl columns in relation to its adsorption onto the stationary phase in such high pressure conditions. The comparison of retention and selectivity between 80-VTMS monoliths and silica was performed under helium using the logarithm of the retention factor according to the number of carbon atoms combined to Kovats indexes. The very good performances of these columns were demonstrated, allowing the separation of 8 compounds in less than 1min. Copyright © 2017 Elsevier B.V. All rights reserved.

Gradient stationary phase optimized selectivity liquid chromatography with conventional columns.

PubMed

Chen, Kai; Lynen, Frédéric; Szucs, Roman; Hanna-Brown, Melissa; Sandra, Pat

2013-05-21

Stationary phase optimized selectivity liquid chromatography (SOSLC) is a promising technique to optimize the selectivity of a given separation. By combination of different stationary phases, SOSLC offers excellent possibilities for method development under both isocratic and gradient conditions. The so far available commercial SOSLC protocol utilizes dedicated column cartridges and corresponding cartridge holders to build up the combined column of different stationary phases. The present work is aimed at developing and extending the gradient SOSLC approach towards coupling conventional columns. Generic tubing was used to connect short commercially available LC columns. Fast and base-line separation of a mixture of 12 compounds containing phenones, benzoic acids and hydroxybenzoates under both isocratic and linear gradient conditions was selected to demonstrate the potential of SOSLC. The influence of the connecting tubing on the deviation of predictions is also discussed.

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. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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. Copyright © 2015 Elsevier Ltd. All rights reserved.

High-capacity cation-exchange column for enhanced resolution of adjacent peaks of cations in ion chromatography.

PubMed

Rey, M A

2001-06-22

One of the advantages of ion chromatography [Anal Chem. 47 (1975) 1801] as compared to other analytical techniques is that several ions may be analyzed simultaneously. One of the most important contributions of cation-exchange chromatography is its sensitivity to ammonium ion, which is difficult to analyze by other techniques [J. Weiss, in: E.L. Johnson (Ed.), Handbook of Ion Chromatography, Dionex, Sunnyvale, CA, USA]. The determination of low concentrations of ammonium ion in the presence of high concentrations of sodium poses a challenge in cation-exchange chromatography [J. Weiss, Ion Chromatography, VCH, 2nd Edition, Weinheim, 1995], as both cations have similar selectivities for the common stationary phases containing either sulfonate or carboxylate functional groups. The task was to develop a new cation-exchange stationary phase (for diverse concentration ratios of adjacent peaks) to overcome limitations experienced in previous trails. Various cation-exchange capacities and column body formats were investigated to optimize this application and others. The advantages and disadvantages of two carboxylic acid columns of different cation-exchange capacities and different column formats will be discussed.

A flow-through column electrolytic cell for supercritical fluid chromatography.

PubMed

Yamamoto, Kazuhiro; Ueki, Tatsuya; Higuchi, Naoyuki; Takahashi, Kouji; Kotani, Akira; Hakamata, Hideki

2017-10-01

A novel flow-through column electrolytic cell was proposed as a detector to obtain current signals for supercritical fluid chromatography. The electrochemical cell consisted of two electrodes and its holder, and a working and a counter electrode were fabricated from 192 carbon strings, which were composed of 400 carbon fibers of 10 μm in diameter filled into a heat-shrinkable tube. These electrodes were placed in the center of a holder made from polyether ether ketone blocks and they were separated by polytetrafluoroethylene membrane filters. To evaluate the sensitivity of this cell, a standard solution of ferrocene was injected into the supercritical fluid chromatography system connected to the electrolytic cell. The ferrocene was eluted through a silica gel column using a mixture of a mobile phase of supercritical CO 2 and a modifier of methanol containing ammonium acetate. The current peak area of ferrocene correlated to the ferrocene concentration in the range of 10-400 μmol/L (r = 0.999). Moreover, the limit of detection on the column estimated from a signal-to-noise ratio of 3 was 9.8  × 10 -13  mol. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography.

PubMed

Horie, Kanta; Ikegami, Tohru; Hosoya, Ken; Saad, Nabil; Fiehn, Oliver; Tanaka, Nobuo

2007-09-14

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

PubMed

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Preparation of polymer monolithic column functionalized by arsonic acid groups for mixed-mode capillary liquid chromatography.

PubMed

Qin, Zhang-Na; Yu, Qiong-Wei; Wang, Ren-Qi; Feng, Yu-Qi

2018-04-27

A mixed-mode polymer monolithic column functionalized by arsonic acid groups was prepared by single-step in situ copolymerization of monomers p-methacryloylaminophenylarsonic acid (p-MAPHA) and pentaerythritol triacrylate (PETA). The prepared poly(p-MAPHA-co-PETA) monolithic column has a homogeneous monolithic structure with good permeability and mechanical stability. Zeta potential measurements reveal that the monolithic stationary phase holds a negative surface charge when the mobile phase resides in the pH range of 3.0-8.0. The retention mechanisms of prepared monolithic column are explored by the separation of selected polycyclic aromatic hydrocarbons (PAHs), nucleosides, and three basic compounds. The results indicate that the column functions in three different separation modes associated with reversed-phase chromatography based on hydrophobic interaction, hydrophilic interaction chromatography, and cation-exchange chromatography. The column efficiency of prepared monolithic column is estimated to be 70,000 and 76,000 theoretical plates/m for thiourea and naphthalene, respectively, at a linear flow velocity of 0.85 mm/s using acetonitrile/H 2 O (85/15, v/v) as the mobile phase. Furthermore, an analysis of the retention factors obtained for the PAHs indicates that the prepared monolithic column exhibits good reproducibility with relative standard deviations of 2.9%, 4.0%, and 4.7% based on run-to-run injections, column-to-column preparation, and batch-to-batch preparation, respectively. Finally, we investigate the separation performance of the proposed monolithic column for select phenols, sulfonamides, nucleobases and nucleosides. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Linear solvation energy relationships regarding sorption and retention properties of hydrophobic organic compounds in soil leaching column chromatography.

PubMed

Xu, Feng; Liang, Xinmiao; Lin, Bingcheng; Su, Fan; Schramm, Karl-Werner; Kettrup, Antonius

2002-08-01

The capacity factors of a series of hydrophobic organic compounds (HOCs) were measured in soil leaching column chromatography (SLCC) on a soil column, and in reversed-phase liquid chromatography on a C18 column with different volumetric fractions (phi) of methanol in methanol-water mixtures. A general equation of linear solvation energy relationships, log(XYZ) XYZ0 + mV(I)/100 + spi + bbetam + aalpham, was applied to analyze capacity factors (k'), soil organic partition coefficients (Koc) and octanol-water partition coefficients (P). The analyses exhibited high accuracy. The chief solute factors that control logKoc, log P, and logk' (on soil and on C18) are the solute size (V(I)/100) and hydrogen-bond basicity (betam). Less important solute factors are the dipolarity/polarizability (pi*) and hydrogen-bond acidity (alpham). Log k' on soil and log Koc have similar signs in four fitting coefficients (m, s, b and a) and similar ratios (m:s:b:a), while log k' on C18 and logP have similar signs in coefficients (m, s, b and a) and similar ratios (m:s:b:a). Consequently, logk' values on C18 have good correlations with logP (r > 0.97), while logk' values on soil have good correlations with logKoc (r > 0.98). Two Koc estimation methods were developed, one through solute solvatochromic parameters, and the other through correlations with k' on soil. For HOCs, a linear relationship between logarithmic capacity factor and methanol composition in methanol-water mixtures could also be derived in SLCC.

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. © 2014 American Institute of Chemical Engineers.

Practical issues relating to soil column chromatography for sorption parameter determination.

PubMed

Bi, Erping; Schmidt, Torsten C; Haderlein, Stefan B

2010-08-01

Determination of sorption distribution coefficients (K(d)) of organic compounds by a dynamic soil column chromatography (SCC) method was developed and validated. Eurosoil 4, quartz, and alumina were chosen as exemplary packing materials. Heterocyclic aromatic compounds were selected in the validation of SCC. The prerequisites of SCC with regard to column dimension, packing procedure, and sample injection volume are discussed. Reproducible soil column packing was achieved by addition of a pre-column and an HPLC pump for subsequent compression of the packed material. Various methods to determine retention times from breakthrough curves are discussed and the use of the half mass method is recommended. To dilute soil with inert material can prevent column-clogging and help to complete experiments in a reasonable period of time. For the chosen probe compounds, quartz rather than alumina proved a suitable dilution material. Non-equilibrium issue can be overcome by conducting the experiments under different flowrates and/or performing numerical simulation. Copyright 2010 Elsevier Ltd. All rights reserved.

Schinus terebinthifolius countercurrent chromatography (Part III): Method transfer from small countercurrent chromatography column to preparative centrifugal partition chromatography ones as a part of method development.

PubMed

das Neves Costa, Fernanda; Hubert, Jane; Borie, Nicolas; Kotland, Alexis; Hewitson, Peter; Ignatova, Svetlana; Renault, Jean-Hugues

2017-03-03

Countercurrent chromatography (CCC) and centrifugal partition chromatography (CPC) are support free liquid-liquid chromatography techniques sharing the same basic principles and features. Method transfer has previously been demonstrated for both techniques but never from one to another. This study aimed to show such a feasibility using fractionation of Schinus terebinthifolius berries dichloromethane extract as a case study. Heptane - ethyl acetate - methanol -water (6:1:6:1, v/v/v/v) was used as solvent system with masticadienonic and 3β-masticadienolic acids as target compounds. The optimized separation methodology previously described in Part I and II, was scaled up from an analytical hydrodynamic CCC column (17.4mL) to preparative hydrostatic CPC instruments (250mL and 303mL) as a part of method development. Flow-rate and sample loading were further optimized on CPC. Mobile phase linear velocity is suggested as a transfer invariant parameter if the CPC column contains sufficient number of partition cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Controlled crosslinking of trimethylolpropane trimethacrylate for preparation of organic monolithic columns for capillary liquid chromatography.

PubMed

Gama, Mariana R; Aggarwal, Pankaj; Lee, Milton L; Bottoli, Carla B G

2017-11-01

Organic monolithic columns based on single crosslinking of trimethylolpropane trimethacrylate (TRIM) monomer were prepared in a single step by living/controlled free-radical polymerization. Full optimization of the preparation, such as using different percentages of TRIM and different amounts of radical promoter as well as various porogen solvents were explored. The resulting monolithic columns were characterized by scanning electronic microscopy and nitrogen sorption for structure morphology studies and surface area measurements, respectively. Using capillary liquid chromatography, 150 μm i.d. columns were applied to separate a mixture of small hydrophobic molecules. The results indicated that column performance is highly sensitive to the type and the amount of porogen solvents used in the polymerization mixture composition. Good resolution factors and methylene selectivity were obtained, indicating the promising potential of this material for capillary liquid chromatography separations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two new oxygen-containing biomarkers isolated from the Chinese Maoming oil shale by silica gel column chromatography and preparative gas chromatography.

PubMed

Zhang, Xiangyun; Lu, Hong; Liao, Jing; Tang, Caiming; Sheng, Guoying; Peng, Ping'an

2017-02-01

Two biomarkers, 5,9-dimethyl-6-isopropyl-2-decanone (1) and 4,9,11-trimethyl-6-isopropyl-2-dodecanone (2), were isolated from Chinese Maoming oil shale by silica gel column chromatography and preparative gas chromatography. Their structures were elucidated by using spectroscopic techniques. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Q Sepharose micro-column chromatography: A simple screening method for identifying beta thalassemia traits and hemoglobin E carriers.

PubMed

Wong, Peerapon; Sritippayawan, Suchila; Suwannakhon, Narutchala; Tapprom, Akamon; Deoisares, Rawisut; Sanguansermsri, Torpong

2016-11-01

For beta thalassemia control program in pregnancy, mass screening of the carrier state by determination of the hemoglobin (Hb) A 2 and Hb E proportions and mutation analysis is a preferred method for making prenatal diagnoses. Q Sepharose micro-column chromatography, developed for the determination of Hb A 2 and Hb E for screening purposes, was compared with high performance liquid chromatography (HPLC) to ascertain its relative accuracy and reliability. Results using Q Sepharose micro-column chromatography in 350 blood specimens, including 50 samples genetically proven to be beta thalassemia heterozygotes, were compared to HPLC for validation. An additional study was conducted to test a clinical application on a large-scale survey for beta thalassemia in 1581 pregnant women and their spouses. The mean (±SD) Hb A 2 proportions in the normal and genetically proven beta thalassemia heterozygotes were 2.70±0.40% and 6.30±1.23%, respectively, as determined by Q-Sepharose micro-column chromatography, and 2.65±0.31% and 5.37±0.96%, respectively, as determined by HPLC. The mean Hb E proportions in the Hb E heterozygotes were 23.25±4.13% and 24.72±3.5% as determined by Q Sepharose micro-column chromatography and HPLC, respectively. In the large-scale survey for beta thalassemia, 23 at risk couples were detected. Seven affected fetuses were identified by prenatal diagnosis. Q Sepharose micro-column chromatography was found to be reliable, reproducible and well-suited for large-scale surveys. Additionally, by being reusable and convenient, this simple and economical chromatography method may be an alternative means to screen for beta thalassemia and Hb E carriers in the mass population. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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).

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

Advantages of core-shell particle columns in Sequential Injection Chromatography for determination of phenolic acids.

PubMed

Chocholouš, Petr; Vacková, Jana; Srámková, Ivana; Satínský, Dalibor; Solich, Petr

2013-01-15

Currently, for Sequential Injection Chromatography (SIC), only reversed phase C18 columns have been used for chromatographic separations. This article presents the first use of three different stationary phases: three core-shell particle-packed reversed phase columns in flow systems. The aim of this work was to extend the chromatographic capabilities of the SIC system. Despite the particle-packed columns reaching system pressures of ≤ 610 PSI, their conditions matched those of a commercially produced and optimised SIC system (SIChrom™ (FIAlab(®), USA)) with a 8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with a 4 mL reservoir and maximum system pressure of ≤ 1000 PSI. The selectivity of each of the tested columns, Ascentis(®) Express RP-Amide, Ascentis(®) Express Phenyl-Hexyl and Ascentis(®) Express C18 (30 mm × 4.6mm, core-shell particle size 2.7 μm), was compared by their ability to separate seven phenolic acids that are secondary metabolite substances widely distributed in plants. The separations of all of the components were performed by isocratic elution using binary mobile phases composed of acetonitrile and 0.065% phosphoric acid at pH 2.4 (a specific ratio was used for each column) at a flow-rate of 0.60 mL/min. The volume of the mobile phase was 3.8 mL for each separation. The injection volume of the sample was 10 μL for each separation. The UV detection wavelengths were set to 250, 280 and 325 nm. The RP-Amide column provided the highest chromatographic resolution and allowed for complete baseline separation of protocatechuic, syringic, vanillic, ferulic, sinapinic, p-coumaric and o-coumaric acids. The Phenyl-Hexyl and C18 columns were unable to completely separate the tested mixture, syringic and vanillic acid and ferulic and sinapinic acids could not be separated from one another. The analytical parameters were a LOD of 0.3 mg L(-1), a LOQ of 1.0 mg L(-1), a calibration range of 1.0-50.0 (100.0) mg L(-1

Design of two-column batch-to-batch recirculation to enhance performance in ion-exchange chromatography.

PubMed

Persson, Oliver; Andersson, Niklas; Nilsson, Bernt

2018-01-05

Preparative liquid chromatography is a separation technique widely used in the manufacturing of fine chemicals and pharmaceuticals. A major drawback of traditional single-column batch chromatography step is the trade-off between product purity and process performance. Recirculation of impure product can be utilized to make the trade-off more favorable. The aim of the present study was to investigate the usage of a two-column batch-to-batch recirculation process step to increase the performance compared to single-column batch chromatography at a high purity requirement. The separation of a ternary protein mixture on ion-exchange chromatography columns was used to evaluate the proposed process. The investigation used modelling and simulation of the process step, experimental validation and optimization of the simulated process. In the presented case the yield increases from 45.4% to 93.6% and the productivity increases 3.4 times compared to the performance of a batch run for a nominal case. A rapid concentration build-up product can be seen during the first cycles, before the process reaches a cyclic steady-state with reoccurring concentration profiles. The optimization of the simulation model predicts that the recirculated salt can be used as a flying start of the elution, which would enhance the process performance. The proposed process is more complex than a batch process, but may improve the separation performance, especially while operating at cyclic steady-state. The recirculation of impure fractions reduces the product losses and ensures separation of product to a high degree of purity. Copyright © 2017 Elsevier B.V. All rights reserved.

Characterisation of capillary ionic liquid columns for gas chromatography-mass spectrometry analysis of fatty acid methyl esters.

PubMed

Zeng, Annie Xu; Chin, Sung-Tong; Nolvachai, Yada; Kulsing, Chadin; Sidisky, Leonard M; Marriott, Philip J

2013-11-25

Due to their distinct chemical properties, the application of ionic liquid (IL) compounds as gas chromatography (GC) stationary phases offer unique GC separation especially in the analysis of geometric and positional fatty acid methyl ester (FAME) isomers. Elution behaviour of FAME on several commercialised IL capillary columns including phosphonium based SLB-IL59, SLB-IL60, SLB-IL61 and SLB-IL76 and imidazolium based SLB-IL82, SLB-IL100, and SLB-IL111 as well as a general purpose column SLB-5ms, were evaluated in gas chromatography-mass spectrometry (GC-MS) analysis. The phases were further characterised by using a linear solvation energy relationship (LSER) approach according to the equivalent chain length (ECL) index of FAME. Among all tested IL columns, elution temperatures of saturated FAME increased as their McReynolds' polarity value decreased, except for IL60. ECL values increased markedly as the stationary phase polarity increased, particularly for the polyunsaturated FAME. The LSER study indicated a lowest l/e value at 0.864 for IL111, displaying phase selectivity towards unsaturated FAME, with higher peak capacity within a carbon number isomer group. s and e descriptors calculated from LSER were validated by excellent correlation with dipole moments and lowest unoccupied molecular orbital (LUMO) energies, with R(2) values of 0.99 and 0.92 respectively, calculated using GAUSSIAN. Copyright © 2013 Elsevier B.V. All rights reserved.

Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

PubMed

Ren, Jiangtao; Beckner, Matthew A; Lynch, Kyle B; Chen, Huang; Zhu, Zaifang; Yang, Yu; Chen, Apeng; Qiao, Zhenzhen; Liu, Shaorong; Lu, Joann J

2018-05-15

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances. Copyright © 2018 Elsevier B.V. All rights reserved.

Column chromatography as a useful step in purification of diatom pigments.

PubMed

Tokarek, Wiktor; Listwan, Stanisław; Pagacz, Joanna; Leśniak, Piotr; Latowski, Dariusz

2016-01-01

Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies.

Determination of nitrite, nitrate, bromide, and iodide in seawater by ion chromatography with UV detection using dilauryldimethylammonium-coated monolithic ODS columns and sodium chloride as an eluent.

PubMed

Ito, Kazuaki; Nomura, Ryosuke; Fujii, Takuya; Tanaka, Masahito; Tsumura, Tomoaki; Shibata, Hiroyuki; Hirokawa, Takeshi

2012-11-01

A method was developed for determination of inorganic anions, including nitrite (NO(2)(-)), nitrate (NO(3)(-)), bromide (Br(-)), and iodide (I(-)), in seawater by ion chromatography (IC). The IC system used two dilauryldimethylammonium bromide (DDAB)-coated monolithic ODS columns (50 × 4.6 mm i.d. and 100 × 4.6 mm i.d.) connected in series for separation of the ions. Aqueous NaCl (0.5 mol/L; flow rate, 3 mL/min) containing 5 mmol/L phosphate buffer (pH 5) was used as the eluent, and detection was with a UV detector at 225 nm. The monolithic ODS columns were coated and equilibrated with a 1-mmol/L DDAB solution (in H(2)O/methanol, 90:10 v/v). The hydrophilic ions (NO(2)(-), NO(3)(-), and Br(-)) were separated within 3 min and the retention time of I(-) was 16 min. No interferences from matrix ions, such as chloride and sulfate ions, were observed in 35 ‰ artificial seawater. The detection limits were 0.6 μg/L for NO(2)(-), 1.1 μg/L for NO(3)(-), 70 μg/L for Br(-), and 1.6 μg/L for I(-) with a 200-μL sample injection. The performance of the coated columns was maintained without addition of DDAB in the eluent. The IC system was successfully applied to real seawater samples with recovery rates of 94-108 % for all ions.

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. Copyright © 2014 John Wiley & Sons, Ltd.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

On-column reduction of catecholamine quinones in stainless steel columns during liquid chromatography.

PubMed

Xu, R; Huang, X; Kramer, K J; Hawley, M D

1995-10-10

The chromatographic behavior of quinones derived from the oxidation of dopamine and N-acetyldopamine has been studied using liquid chromatography (LC) with both a diode array detector and an electrochemical detector that has parallel dual working electrodes. When stainless steel columns are used, an anodic peak for the oxidation of the catecholamine is observed at the same retention time as a cathodic peak for the reduction of the catecholamine quinone. In addition, the anodic peak exhibits a tail that extends to a second anodic peak for the catecholamine. The latter peak occurs at the normal retention time of the catecholamine. The origin of this phenomenon has been studied and metallic iron in the stainless steel components of the LC system has been found to reduce the quinones to their corresponding catecholamines. The simultaneous appearance of a cathodic peak for the reduction of catecholamine quinone and an anodic peak for the oxidation of the corresponding catecholamine occurs when metallic iron in the exit frit reduces some of the quinones as the latter exits the column. This phenomenon is designated as the "concurrent anodic-cathodic response." It is also observed for quinones of of 3,4-dihydroxybenzoic acid and probably occurs with o- or p-quinones of other dihydroxyphenyl compounds. The use of nonferrous components in LC systems is recommended to eliminate possible on-column reduction of quinones.

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. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

Ideal versus real automated twin column recycling chromatography process.

PubMed

Gritti, Fabrice; Leal, Mike; McDonald, Thomas; Gilar, Martin

2017-07-28

The full baseline separation of two compounds (selectivity factors α<1.03) is either impractical (too long analysis times) or even impossible when using a single column of any length given the pressure limitations of current LC instruments. The maximum efficiency is that of an infinitely long column operated at infinitely small flow rates. It is determined by the maximum allowable system pressure, the column permeability (particle size), the viscosity of the eluent, and the intensity of the effective diffusivity of the analytes along the column. Alternatively, the twin-column recycling separation process (TCRSP) can overcome the efficiency limit of the single-column approach. In the TCRSP, the sample mixture may be transferred from one to a second (twin) column until its band has spread over one column length. Basic theory of chromatography is used to confirm that the speed-resolution performance of the TCRSP is intrinsically superior to that of the single-column process. This advantage is illustrated in this work by developing an automated TCRSP for the challenging separation of two polycyclic aromatic hydrocarbon (PAH) isomers (benzo[a]anthracene and chrysene) in the reversed-phase retention mode at pressure smaller than 5000psi. The columns used are the 3.0mm×150mm column packed with 3.5μm XBridge BEH-C 18 material (α=1.010) and the 3.0mm or 4.6mm×150mm columns packed with the same 3.5μm XSelect HSST 3 material (α=1.025). The isocratic mobile phase is an acetonitrile-water mixture (80/20, v/v). Remarkably, significant differences are observed between the predicted retention times and efficiencies of the ideal TCRSP (given by the number of cycles multiplied by the retention time and efficiency of one column) and those of the real TCRSP. The fundamental explanation lies in the pressure-dependent retention of these PAHs or in the change of their partial molar volume as they are transferred from the mobile to the stationary phase. A revisited retention and

Recent advances in monolithic columns for protein and peptide separation by capillary liquid chromatography.

PubMed

Liang, Yu; Zhang, Lihua; Zhang, Yukui

2013-03-01

Capillary liquid chromatography (cLC) has great potential for protein and peptide separation, with advantages of high efficiency, high resolution, low sample consumption, and high sensitivity when coupled with mass spectrometry. In recent years, monoliths have been widely used as the stationary phases for capillary columns, owing to easy preparation, high permeability, fast mass transfer, and low backpressure. This review summarizes recent advances (2007-2012) in monolithic columns for protein and peptide separation by cLC. After a brief introduction on the preparation of monolithic capillary columns, the emphasis of this review is focused on the recent application of such columns for protein and peptide separation by cLC. Furthermore, the challenges and potential hot points of monolithic capillary columns in the future are discussed.

Demonstration of motionless Knudsen pump based micro-gas chromatography featuring micro-fabricated columns and on-column detectors.

PubMed

Liu, Jing; Gupta, Naveen K; Wise, Kensall D; Gianchandani, Yogesh B; Fan, Xudong

2011-10-21

This paper reports the investigation of a micro-gas chromatography (μGC) system that utilizes an array of miniaturized motionless Knudsen pumps (KPs) as well as microfabricated separation columns and optical detectors. A prototype system was built to achieve a flow rate of 1 mL min(-1) and 0.26 mL min(-1) for helium and dry air, respectively, when they were used as carrier gas. This system was then employed to evaluate GC performance compromises and demonstrate the ability to separate and detect gas mixtures containing analytes of different volatilities and polarities. Furthermore, the use of pressure programming of the KP array was demonstrated to significantly shorten the analysis time while maintaining a high detection resolution. Using this method, we obtained a high resolution detection of 5 alkanes of different volatilities within 5 min. Finally, we successfully detected gas mixtures of various polarities using a tandem-column μGC configuration by installing two on-column optical detectors to obtain complementary chromatograms.

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.

Hydrodynamic flow in capillary-channel fiber columns for liquid chromatography.

PubMed

Stanelle, Rayman D; Sander, Lane C; Marcus, R Kenneth

2005-12-23

The flow characteristics of capillary-channel polymer (C-CP) fiber liquid chromatographic (LC) columns have been investigated. The C-CP fibers are manufactured with eight longitudinal grooves (capillary channels) extending the length of the fibers. Three C-CP fiber examples were studied, with fiber dimensions ranging from approximately 35 microm to 65 microm, and capillary-channel dimensions ranging from approximately 6 microm to 35 microm. The influence of fiber packing density and column inner diameter on peak asymmetry, peak width, and run-to-run reproducibility have been studied for stainless steel LC columns packed with polyester (PET) and polypropylene (PP) C-CP fibers. The van Deemter A-term was evaluated as a function of fiber packing density (approximately 0.3 g/cm(3)-0.75 g/cm(3)) for columns of 4.6 mm inner diameter (i.d.) and at constant packing densities for 1.5 mm, 3.2 mm, 4.6 mm, and 7.7 mm i.d. columns. Although column diameter had little influence on the eluting peak widths, peak asymmetry increased with increasing column diameter. The A-terms for the C-CP fiber packed columns are somewhat larger than current commercial, microparticulate-packed columns, and means for improvement are discussed. Applications in the area of protein (macromolecule) separations appear the most promising at this stage of the system development.

Longitudinal On-Column Thermal Modulation for Comprehensive Two-Dimensional Liquid Chromatography.

PubMed

Creese, Mari E; Creese, Mathew J; Foley, Joe P; Cortes, Hernan J; Hilder, Emily F; Shellie, Robert A; Breadmore, Michael C

2017-01-17

Longitudinal on-column thermal modulation for comprehensive two-dimensional liquid chromatography is introduced. Modulation optimization involved a systematic investigation of heat transfer, analyte retention, and migration velocity at a range of temperatures. Longitudinal on-column thermal modulation was realized using a set of alkylphenones and compared to a conventional valve-modulator employing sample loops. The thermal modulator showed a reduced modulation-induced pressure impact than valve modulation, resulting in reduced baseline perturbation by a factor of 6; yielding a 6-14-fold improvement in signal-to-noise. A red wine sample was analyzed to demonstrate the potential of the longitudinal on-column thermal modulator for separation of a complex sample. Discrete peaks in the second dimension using the thermal modulator were 30-55% narrower than with the valve modulator. The results shown herein demonstrate the benefits of an active focusing modulator, such as reduced detection limits and increased total peak capacity.

Characterization of retentivity of reversed phase liquid chromatography columns.

PubMed

Ying, P T; Dorsey, J G

1991-03-01

There are dozens of commercially available reversed phase columns, most marketed as C-8 or C-18 materials, but with no useful way of classifying their retentivity. A useful way of ranking these columns in terms of column "strength" or retentivity is presented. The method utilizes a value for ln k'(w), the estimated retention of a solute from a mobile phase of 100% water, and the slope of the plot of ln k' vsE(T)(30), the solvent polarity. The method is validated with 26 solutes varying in ln k'(w) from about 2 to over 20, on 14 different reversed phase columns. In agreement with previous work, it is found that the phase volume ratio of the column is the most important parameter in determining retentivity. It is strongly suggested that manufacturers adopt a uniform method of calculating this value and that it be made available in advertising, rather than the uninterpretable "% carbon".

Chromatographic properties PLOT multicapillary columns.

PubMed

Nikolaeva, O A; Patrushev, Y V; Sidelnikov, V N

2017-03-10

Multicapillary columns (MCCs) for gas chromatography make it possible to perform high-speed analysis of the mixtures of gaseous and volatile substances at a relatively large amount of the loaded sample. The study was performed using PLOT MCCs for gas-solid chromatography (GSC) with different stationary phases (SP) based on alumina, silica and poly-(1-trimethylsilyl-1-propyne) (PTMSP) polymer as well as porous polymers divinylbenzene-styrene (DVB-St), divinylbenzene-vinylimidazole (DVB-VIm) and divinylbenzene-ethylene glycol dimethacrylate (DVB-EGD). These MCCs have the efficiency of 4000-10000 theoretical plates per meter (TP/m) and at a column length of 25-30cm can separate within 10-20s multicomponent mixtures of substances belonging to different classes of chemical compounds. The sample amount not overloading the column is 0.03-1μg and depends on the features of a porous layer. Examples of separations on some of the studied columns are considered. Copyright © 2017 Elsevier B.V. All rights reserved.

Measurement of bromate in bread by liquid chromatography with post-column flow reactor detection.

PubMed

Himata, K; Noda, M; Ando, S; Yamada, Y

2000-01-01

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30,000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10-52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.

[Monitoring method for macroporous resin column chromatography process of salvianolic acids based on near infrared spectroscopy].

PubMed

Hou, Xiang-Mei; Zhang, Lei; Yue, Hong-Shui; Ju, Ai-Chun; Ye, Zheng-Liang

2016-07-01

To study and establish a monitoring method for macroporous resin column chromatography process of salvianolic acids by using near infrared spectroscopy (NIR) as a process analytical technology (PAT).The multivariate statistical process control (MSPC) model was developed based on 7 normal operation batches, and 2 test batches (including one normal operation batch and one abnormal operation batch) were used to verify the monitoring performance of this model. The results showed that MSPC model had a good monitoring ability for the column chromatography process. Meanwhile, NIR quantitative calibration model was established for three key quality indexes (rosmarinic acid, lithospermic acid and salvianolic acid B) by using partial least squares (PLS) algorithm. The verification results demonstrated that this model had satisfactory prediction performance. The combined application of the above two models could effectively achieve real-time monitoring for macroporous resin column chromatography process of salvianolic acids, and can be used to conduct on-line analysis of key quality indexes. This established process monitoring method could provide reference for the development of process analytical technology for traditional Chinese medicines manufacturing. Copyright© by the Chinese Pharmaceutical Association.

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.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen, Yufeng; Tolic, Nikola; Piehowski, Paul D.

We report development of an approach providing high-resolution RPLC of proteins and its utility for mass spectrometry-based top-down proteomics. A chromatographic peak capacity of ~450 was achieved for proteins and large polypeptides having MWs up to 43 kDa in the context of proteomics applications. RPLC column lengths from 20 to 200 cm, particle sizes from 1.5 to 5 m, bonding alkyl chains from C1 to C2, C4, C8, and C18, and particle surface structures that spanned porous, superficially porous (porous shell, core-shell), and nonporous were investigated at pressures up to14K psi. Column length was found as the most important factormore » for >20 kDa proteins in gradient RPLC, and shortening column length degraded RPLC resolution and sensitivity regardless of the size and surface structure of the packing particles used. The alkyl chains bonded to the silica particle surface significantly affected the RPLC recovery and efficiency, and short alkyl C1-C4 phases provided higher sensitivity and resolution than C8 and C18 phases. Long gradient separations (e.g., >10 hours) with long columns (e.g., 100 cm) were particularly effective in conjunction with use of high accuracy mass spectrometers (e.g., the Orbitrap Elite) for top-down proteomics with improved proteoform coverage by allowing multiple HCD, CID, and ETD dissociation modes. It was also found that HCD produced small fragments useful for proteoform identification, while low energy CID and ETD often complemented HCD by providing large fragments.« less

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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…

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.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Phenolic composition of pomegranate peel extracts using an liquid chromatography-mass spectrometry approach with silica hydride columns.

PubMed

Young, Joshua E; Pan, Zhongli; Teh, Hui Ean; Menon, Veena; Modereger, Brent; Pesek, Joseph J; Matyska, Maria T; Dao, Lan; Takeoka, Gary

2017-04-01

The peels of different pomegranate cultivars (Molla Nepes, Parfianka, Purple Heart, Wonderful and Vkunsyi) were compared in terms of phenolic composition and total phenolics. Analyses were performed on two silica hydride based stationary phases: phenyl and undecanoic acid columns. Quantitation was accomplished by developing a liquid chromatography with mass spectrometry approach for separating different phenolic analytes, initially in the form of reference standards and then with pomegranate extracts. The high-performance liquid chromatography columns used in the separations had the ability to retain a wide polarity range of phenolic analytes, as well as offering beneficial secondary selectivity mechanisms for resolving the isobaric compounds, catechin and epicatechin. The Vkunsyi peel extract had the highest concentration of phenolics (as determined by liquid chromatography with mass spectrometry) and was the only cultivar to contain the important compound punicalagin. The liquid chromatography with mass spectrometry data were compared to the standard total phenolics content as determined by using the Folin-Ciocalteu assay. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of pesticides in water by C-18 solid-phase extraction and capillary-column gas chromatography/mass spectrometry with selected-ion monitoring

USGS Publications Warehouse

Zaugg, Steven D.; Sandstrom, Mark W.; Smith, Steven G.; Fehlberg, Kevin M.

1995-01-01

A method for the isolation of 41 pesticides and pesticide metabolites in natural-water samples using C-18 solid-phase extraction and determination by capillary-column gas chromatography/mass spectrometry with selected-ion monitoring is described. Water samples are filtered to remove suspended particulate matter and then are pumped through disposable solid-phase extraction columns containing octadecyl-bonded porous silica to extract the pesticides. The columns are dried using carbon dioxide or nitrogen gas, and adsorbed pesticides are removed from the columns by elution with 3.0 milliliters of hexane-isopropanol (3:1). Extracted pesticides are determined by capillary- column gas chromatography/mass spectrometry with selected-ion monitoring of three characteristic ions. The upper concentration limit is 4 micrograms per liter (g/L) for most pesticides, with the exception of widely used corn herbicides--atrazine, alachlor, cyanazine, and metolachlor--which have upper concentration limits of 20 g/L. Single- operator method detection limits in reagent-water samples range from 0.001 to 0.018 g/L. Average short-term single-operator precision in reagent- water samples is 7 percent at the 0.1- and 1.0-g/L levels and 8 percent at the 0.01-g/L level. Mean recoveries in reagent-water samples are 73 percent at the 0.1- and 1.0-g/L levels and 83 percent at the 0.01-g/L level. The estimated holding time for pesticides after extraction on the solid-phase extraction columns was 7 days. An optional on-site extraction procedure allows for samples to be collected and processed at remote sites where it is difficult to ship samples to the laboratory within the recommended pre-extraction holding time.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

On-line hyphenation of solid-phase extraction to chromatographic separation of sulfonamides with fused-core columns in sequential injection chromatography.

PubMed

Batista, Alex D; Chocholouš, Petr; Satínský, Dalibor; Solich, Petr; Rocha, Fábio R P

2015-02-01

On-line sample pretreatment (clean-up and analyte preconcentration) is for the first time coupled to sequential injection chromatography. The approach combines anion-exchange solid-phase extraction and the highly effective pentafluorophenylpropyl (F5) fused-core particle column for separation of eight sulfonamide antibiotics with similar structures (sulfathiazole, sulfanilamide, sulfacetamide, sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxazole and sulfadimethoxine). The stationary phase was selected after a critical comparison of the performance achieved by three fused-core reversed phase columns (Ascentis(®) Express RP-Amide, Phenyl-Hexyl, and F5) and two monolithic columns (Chromolith(®) High Resolution RP-18 and CN). Acetonitrile and acetate buffer pH 5.0 at 0.60 mL min(-1) were used as mobile phase to perform the separations before spectrophotometric detection. The first mobile phase was successfully used as eluent from SPE column ensuring transfer of a narrow zone to the chromatographic column. Enrichment factors up to 39.2 were achieved with a 500 µL sample volume. The developed procedure showed analysis time <10.5 min, resolutions >1.83 with peak symmetry ≤1.52, LODs between 4.9 and 27 µg L(-1), linear response ranges from 30.0 to 1000.0 µg L(-1) (r(2)>0.996) and RSDs of peak heights <2.9% (n=6) at a 100 µg L(-1) level and enabled the screening control of freshwater samples contaminated at the 100 µg L(-1) level. The proposed approach expanded the analytical potentiality of SIC and avoided the time-consuming batch sample pretreatment step, thus minimizing risks of sample contamination and analyte losses. Copyright © 2014 Elsevier B.V. All rights reserved.

Low-density solvent based ultrasound-assisted emulsification microextraction and on-column derivatization combined with gas chromatography-mass spectrometry for the determination of carbamate pesticides in environmental water samples.

PubMed

Guo, Liang; Lee, Hian Kee

2012-04-27

A fast and efficient method for the determination of trace level of carbamate pesticides using a lower-density-than-water solvent for ultrasound-assisted emulsification microextraction coupled to on-column derivatization and analysis by GC-MS has been developed and studied. In this approach, a soft plastic Pasteur pipette was employed as a convenient extraction device. Fifty microliters of extraction solvent, of lower density than water, was injected into the sample solution held in the pipette. The latter was immediately immersed in an ultrasound water bath to form an emulsion. After 2 min extraction, the emulsion was fractionated into two layers by centrifugation. The upper layer (organic extract) could be collected conveniently by squeezing the bulb of the pipette, now held upside down, to move it into the narrow stem of the device, facilitating its retrieval for analysis. The extract was then combined with trimethylphenylammonium hydroxide and directly injected into a gas chromatography-mass spectrometry (GC-MS) system for on-column derivatization and analysis. The on-column derivatization provided an added convenience (since a separate step was not necessary). Parameters affecting the derivatization and extraction were investigated. Under the most favorable conditions, the method demonstrated high extraction efficiency with low limits of detection of between 0.01 and 0.1 μg/L, good linearity in the range of 0.05-50 μg/L, to 0.5-100 μg/L, and good repeatability (RSD below 9.2%, n=5). The proposed method was evaluated by determining carbamate pesticides in river water samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Online hyphenation of extraction, Sephadex LH-20 column chromatography, and high-speed countercurrent chromatography: A highly efficient strategy for the preparative separation of andrographolide from Andrographis paniculata in a single step.

PubMed

Zhang, Ying-Qi; Wang, Shan-Shan; Han, Chao; Xu, Jin-Fang; Luo, Jian-Guang; Kong, Ling-Yi

2017-12-01

A novel isolation strategy, online hyphenation of ultrasonic extraction, Sephadex LH-20 column chromatography combined with high-speed countercurrent chromatography, was developed for pure compounds extraction and purification. Andrographolide from Andrographis paniculata was achieved only in a single step purification protocol via the present strategy. The crude powder was ultrasonic extracted and extraction was pumped into Sephadex LH-20 column directly to cut the nontarget fractions followed by the second-dimensional high-speed countercurrent chromatography, hyphenated by a six-port valve equipped at the post-end of Sephadex LH-20 column, for the final purification. The results yielded andrographolide with the amount of 1.02 mg and a purity of 98.5% in a single step, indicating that the present method is effective to harvest target compound from medicinal plant. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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…

A unified classification of stationary phases for packed column supercritical fluid chromatography.

PubMed

West, C; Lesellier, E

2008-05-16

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94-111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.

Retention behavior of hydrophobic organic chemicals as a function of temperature in soil leaching column chromatography.

PubMed

Liang, Xinmiao; Xu, Feng; Lin, Bingcheng; Su, Fan; Schramm, Karl-Werner; Kettrup, Antonius

2002-11-01

To study the transport mechanism of hydrophobic organic chemicals (HOCs) and the energy change in soil/solvent system, a soil leaching column chromatographic (SLCC) experiment at an environmental temperature range of 20-40 degrees C was carried out, which utilized a reference soil (SP 14696) packed column and a methanol-water (1:4 by volume ratio) eluent. The transport process quickens with the increase of column temperature. The ratio of retention factors at 30 and 40 degrees C (k'30/k'40) ranged from 1.08 to 1.36. The lower enthalpy change of the solute transfer in SLCC (from eluent to soil) than in conventional reversed-phase liquid chromatography (e.g., from eluent to C18) is consistent with the hypothesis that HOCs were dominantly and physically partitioned between solvent and soil. The results were also verified by the linear solvation energy relationships analysis. The chief factor controlling the retention was found to be the solute solvophobic partition, and the second important factor was the solute hydrogen-bond basicity, while the least important factors were the solute polarizability-dipolarity and hydrogen-bond acidity. With the increase of temperature, the contributions of the solute solvophobic partition and hydrogen-bond basicity gradually decrease, and the latter decreases faster than the former.

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…

Rapid, direct determination of polyphenols in tea by reversed-phase column liquid chromatography.

PubMed

Ding, M; Yang, H; Xiao, S

1999-07-23

Column liquid chromatography on a C18-bonded silica column with water-methanol-acetic acid as eluent was used to determine polyphenols and caffeine in tea. Without any pretreatment, catechin, epicatechin gallate, epigallocatechin gallate, epigallocatechin, epicatechin and caffeine were separated successfully within 15 min. The detection limits (S/N = 3) of polyphenols studied were 1.8-24 mg/l at a detection wavelength 270 nm. The linear range of the peak area calibration curves for the analytes were over two orders of magnitude with a correlation coefficient of 0.996-0.999. Using this method, some Chinese tea samples were analyzed with a good reproducibility (RSD are below 5%).

An automated HPLC method for the fractionation of polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins, and polychlorinated dibenzofurans in fish tissue on a porous graphitic carbon column

USGS Publications Warehouse

Echols, Kathy R.; Gale, Robert W.; Tillitt, Donald E.; Schwartz, Ted R.; O'Laughlin, Jerome

1997-01-01

The Ah (aryl-hydrocarbon) hydroxylase-receptor active polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were fractionated by an automated high-performance liquid chromatography (HPLC) system using the Hypercarb™ porous graphitic carbon (PGC) column. This commercially available column was used to fractionate the di-, mono-, and non-ortho PCBs into three fractions for gas chromatography (GC)/electron capture detection analysis, and a fourth fraction containing the PCDDs/PCDFs for GC/mass spectrometry analysis. The recoveries of the PCBs ranged from 68 to 96%, and recoveries of the PCDDs/PCDFs ranged from 74 to 123%. The PGC column has the advantage of faster separations (110 min versus 446 min) and less solvent use (275 ml versus 1,100 ml) compared with automated fractionation of these compounds on activated carbon (PX-21), while still affording good separation of the classes. The PGC column may have an advantage over the pyrenyl-based HPLC method because it has a greater loading capacity (400 μg total PCBs versus 250 μg). Overall, the PGC is a standard column that provides reproducible fractionation of PCDD/PCDFs and PCBs for analytical measurement in environmental samples.

Hydroxylapatite chromatography.

PubMed

Broadhurst, A V

2001-05-01

Hydroxylapatite (also called hydroxyapatite), a form of calcium phosphate, can be used as a matrix for the chromatography of both proteins and nucleic acids. Protocols are provided for both standard low-pressure chromatography of a protein mixture using a hydroxylapatite column prepared in the laboratory, and an HPLC method, applicable to proteins and nucleic acids, that uses a commercially available column. Alternate protocols describe column chromatography using a step gradient or batch binding and step-gradient elution.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Quality by design: a systematic and rapid liquid chromatography and mass spectrometry method for eprosartan mesylate and its related impurities using a superficially porous particle column.

PubMed

Kalariya, Pradipbhai D; Kumar Talluri, Murali V N; Gaitonde, Vinay D; Devrukhakar, Prashant S; Srinivas, Ragampeta

2014-08-01

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous determination of eprosartan mesylate and its six impurities using quality-by-design principles. The method was developed in two phases, screening and optimization. During the screening phase, the most suitable stationary phase, organic modifier, and pH were identified. The optimization was performed for secondary influential parameters--column temperature, gradient time, and flow rate using eight experiments--to examine multifactorial effects of parameters on the critical resolution and generated design space representing the robust region. A verification experiment was performed within the working design space and the model was found to be accurate. This study also describes other operating features of the column packed with superficially porous particles that allow very fast separations at pressures available in most liquid chromatography instruments. Successful chromatographic separation was achieved in less than 7 min using a fused-core C18 (100 mm × 2.1 mm, 2.6 μm) column with linear gradient elution of 10 mM ammonium formate (pH 3.0) and acetonitrile as the mobile phase. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance with the International Conference on Harmonization Q2 (R1) guidelines. The impurities were identified by liquid chromatography with mass spectrometry. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enantioselective ultra-high and high performance liquid chromatography: a comparative study of columns based on the Whelk-O1 selector.

PubMed

Kotoni, Dorina; Ciogli, Alessia; D'Acquarica, Ilaria; Kocergin, Jelena; Szczerba, Ted; Ritchie, Harald; Villani, Claudio; Gasparrini, Francesco

2012-12-21

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150 mm, 100mm, 75 mm and 50mm columns, either 4.6 or 3.0mm ID. A 100 mm × 4.6 mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250 mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated H(min) values of 3.53 μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μ(inter)) of 5.07 mm/s, and H(min) of 4.26 and 4.17 μm for the two enantiomers of acenaphthenol, at μ(inter) of 4.85 mm/s and 4.24 mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150 mm, 100mm, 75 mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250 mm ×4.6 mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50 mm×3.0 mm ID column was used as a compromise between reduced mobile phase consumption (less than 1 mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600 bar for a wide range of flow rates, the

Isolation of biofunctional bovine immunoglobulin G from milk- and colostral whey with mixed-mode chromatography at lab and pilot scale.

PubMed

Heidebrecht, Hans-Jürgen; Kainz, Bernadette; Schopf, Roland; Godl, Klaus; Karcier, Züleyha; Kulozik, Ulrich; Förster, Beatrix

2018-05-23

The aim of the present work was to develop a new scalable and cost-efficient process to isolate bovine immunoglobulin G from colostral whey with high purity and minimal loss of activity. The mixed mode material Mercapto-Ethyl-Pyridine-Hypercel™ was identified appropriate for direct capture of immunoglobulin G. The binding mechanism is primarily based on hydrophobic interactions at physiological conditions. As compared to immunoglobulin G, all other low molecular whey proteins such as α-Lactalbumin or β-Lactoglobulin, except lactoperoxidase, are more hydrophilic and were therefore found in the flow-through fraction. In order to remove lactoperoxidase as an impurity the column was combined in series with a second mixed mode material (Capto™- with N-benzoyl-homocysteine as ligand) using the same binding conditions. At pH 7.5 the carboxyl group of this ligand is negatively charged and can hence bind the positively charged lactoperoxidase, whose isoelectric point is at pH 9.6. After sample application, the columns were eluted separately. By combining the two columns it was possible to obtain immunoglobulin G with a purity of >96.1% and yield of 65-80%. The process development was carried out using 1 mL columns and upscaling was performed in three steps up to a column volume of 8800 mL for the Hypercel™ column and 3000 mL for the Capto™- column. At this scale it is possible to obtain 130-150 g pure immunoglobulin G from 3 L colostrum within five hours, including the regeneration of both columns. Additionally, the impact of freeze-drying on the isolated immunoglobulin G was studied. The nativity of the freeze dried immunoglobulin was above 95%, which was proven by reversed phase liquid chromatography and validated by differential scanning calorimetry. The activity of immunoglobulin G was preserved over the isolation process and during drying as measured by enzyme-linked immunosorbent assay. In conclusion, by applying the proposed isolation process, it

Rapid trace level determination of sulfonamide residues in honey with online extraction using short C-18 column by high-performance liquid chromatography with fluorescence detection.

PubMed

Sajid, Muhammad; Na, Na; Safdar, Muhammad; Lu, Xin; Ma, Lin; He, Lan; Ouyang, Jin

2013-11-01

A sensitive and inexpensive quantification method with online extraction using a short C-18 column for sulfonamide residues in honey by high performance liquid chromatography with fluorescence detector was developed and validated. In sample preparation, acid hydrolysis was used to break the N-glycoside bond between the honey sugar and sulfonamide drugs and derivatization of sulfonamide residues with fluorescamine was conducted at pH 3.5 using a citrate buffer (0.5M) in the honey matrix. The chromatography was carried out on Zorbax Extended C-18 (250mm×4.6mm; 5μm) column, using a mixture of acetonitrile and an acetate buffer (pH 4.50, 20mM) as a mobile phase. A Zorbax Extended C-18 (12mm×4.6mm; 5μm) column was used for online extraction of fifteen sulfonamide residues from honey sample with the help of a two position valve. The limit of quantification of sulfonamide residues in honey was less than 3ngg(-1), and the percentage recovery of study compounds in spiked honey sample was from 80% for sulfacetamide to 100% of sulfachloropyridazine. The developed method has excellent linearity for all studied sulfonamides with a correlation coefficient 0.993. Copyright © 2013 Elsevier B.V. All rights reserved.

Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction-reversed-phase liquid chromatography mechanism, a review.

PubMed

Jandera, Pavel; Hájek, Tomáš

2018-01-01

Hydrophilic interaction liquid chromatography on polar columns in aqueous-organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed-phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one- and two-dimensional liquid chromatography separations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column.

PubMed

Narita, H; Takeda, Y; Takagaki, K; Nakamura, T; Harata, S; Endo, M

1995-11-20

Glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid) were labeled with a fluorescent reagent, 2-aminopyridine. The fluoro-labeled glycosaminoglycans were subjected to high-performance liquid chromatography on a hydroxyapatite column. The binding property of each glycosaminoglycan to hydroxyapatite was different. The structural properties of glycosaminoglycans bound to hydroxyapatite were then investigated using chemical desulfated or enzymic depolymerized glycosaminoglycans. This revealed that the sulfate content and molecular weight of the glycosaminoglycans correlated with their binding properties to hydroxyapatite. Desulfated dermatan sulfate but not desulfated chondroitin 6-sulfate bound to the hydroxyapatite. These data indicate that iduronic acid residues of glycosaminoglycans are important for the binding property. The method described which uses hydroxyapatite columns facilitates rapid separation and microanalysis of the glycosaminoglycans, especially dermatan sulfate and chondroitin sulfate.

Simultaneous multi-mycotoxin determination in nutmeg by ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection.

PubMed

Kong, Wei-Jun; Liu, Shu-Yu; Qiu, Feng; Xiao, Xiao-He; Yang, Mei-Hua

2013-05-07

A simple and sensitive analytical method based on ultrasound-assisted solid-liquid extraction and immunoaffinity column clean-up coupled with high performance liquid chromatography and on-line post-column photochemical derivatization-fluorescence detection (USLE-IAC-HPLC-PCD-FLD) has been developed for simultaneous multi-mycotoxin determination of aflatoxins B1, B2, G1, G2 (AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) in 13 edible and medicinal nutmeg samples marketed in China. AFs and OTA were extracted from nutmeg samples by ultrasonication using a methanol : water (80 : 20, v/v) solution, followed by an IAC clean-up step. Different USL extraction conditions, pre-processing ways for nutmeg sample and clean-up columns for mycotoxins, as well as HPLC-PCD-FLD parameters (mobile phase, column temperature, elution procedure, excitation and emission wavelengths) were optimized. This method, which was appraised for analyzing nutmeg samples, showed satisfactory results with reference to limits of detection (LODs) (from 0.02 to 0.25 μg kg(-1)), limits of quantification (LOQs) (from 0.06 to 0.8 μg kg(-1)), linear ranges (up to 30 ng mL(-1) for AFB1, AFG1 and OTA and 9 ng mL(-1) for AFB2 and AFG2), intra- and inter-day variability (all <2%) and average recoveries (from 79.6 to 90.8% for AFs and from 93.6 to 97.3% for OTA, respectively). The results of the application of developed method in nutmeg samples have elucidated that four samples were detected with contamination of AFs and one with OTA. AFB1 was the most frequently found mycotoxin in 30.8% of nutmeg samples at contamination levels of 0.73-16.31 μg kg(-1). At least two different mycotoxins were co-occurred in three samples, and three AFs were simultaneously detected in one sample.

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

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.

Significant viscosity dependent deviations from classical van Deemter theory in liquid chromatography with porous silica monolithic columns.

PubMed

Nesterenko, Pavel N; Rybalko, Marina A; Paull, Brett

2005-06-01

Significant deviations from classical van Deemter behaviour, indicative of turbulent flow liquid chromatography, has been recorded for mobile phases of varying viscosity on porous silica monolithic columns at elevated mobile phase flow rates.

Gas chromatography on wall-coated open-tubular columns with ionic liquid stationary phases.

PubMed

Poole, Colin F; Lenca, Nicole

2014-08-29

Ionic liquids have moved from novel to practical stationary phases for gas chromatography with an increasing portfolio of applications. Ionic liquids complement conventional stationary phases because of a combination of thermophysical and solvation properties that only exist for ionic solvents. Their high thermal stability and low vapor pressure makes them suitable as polar stationary phases for separations requiring high temperatures. Ionic liquids are good solvents and can be used to expand the chemical space for separations. They are the only stationary phases with significant hydrogen-bond acidity in common use; they extend the hydrogen-bond basicity of conventional stationary phases; they are as dipolar/polarizable as the most polar conventional stationary phases; and some ionic liquids are significantly less cohesive than conventional polar stationary phases. Problems in column coating techniques and related low column performance, column activity, and stationary phase reactivity require further exploration as the reasons for these features are poorly understood at present. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of underivatised low volatility compounds by comprehensive two-dimensional gas chromatography with a short primary column.

PubMed

Novaes, Fábio Junior Moreira; Kulsing, Chadin; Bizzo, Humberto Ribeiro; de Aquino Neto, Francisco Radler; Rezende, Claudia Moraes; Marriott, Philip John

2018-02-09

Comprehensive two-dimensional gas chromatography (GC×GC) approaches with cryogenic modulation were developed for the qualitative analysis of selected low volatility compounds in raw coffee bean extracts, without derivatisation. The approaches employed short first ( 1 D) and second ( 2 D) dimension columns, specifically a 1 D 65% phenyl methyl siloxane column (11m) and a 2 D 5% phenyl methyl siloxane column (1m), which allowed elution of high molar mass compounds (e.g.>600Da). Solutes included hydrocarbons, fatty acids, diterpenes, tocopherols, sterols, diterpene esters, and di- and triacylglycerides. An oven temperature program up to 370°C was employed. The effects of experimental conditions were investigated, revealing that the GC×GC results strongly depended on the cryogenic trap T, and oven T program. An appropriate condition was selected and further applied for group type analysis of low volatility compounds in green Arabica coffee beans. Retention indices were compiled for 1D GC analysis and were similar for the composite column data in GC×GC. The elution of some compounds was confirmed by use of authentic standards. The approach allowed direct analysis of coffee extract in ethyl acetate solution, with improved analyte peak capacity (approximately 200 compounds were detected) without prior fractionation or pre-treatment of the sample. This avoided potential hydrolysis of high molar mass conjugate esters as well as degradation of thermally labile compounds such as the derivatives of the diterpenes cafestol and kahweol. Copyright © 2017 Elsevier B.V. All rights reserved.

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%. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg6, D-Trp7,9, mePhe8]-substance P (6-11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate.

PubMed

Cummings, J; MacLellan, A J; Mark, M; Jodrell, D I

1999-09-24

[Arg6, D-Trp7,9 mePhe8]-substance P (6-11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met11 (M2) and G[Met11(O)] (M3). Gradient elution was employed using 40 mM ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (microBondapak C18, 30 cm X 2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40 degrees C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G-P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1-3 and G-P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1-3. Sample clean-up by solid-phase extraction using C2-bonded 40 microm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml-100 microg/ml) normally varied by < 10%, although at the highest concentrations of M1 and M2 studied (50 microg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.

Chiral separation of G-type chemical warfare nerve agents via analytical supercritical fluid chromatography.

PubMed

Kasten, Shane A; Zulli, Steven; Jones, Jonathan L; Dephillipo, Thomas; Cerasoli, Douglas M

2014-12-01

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G-type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical-scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(-) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(-) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc.

Chiral Separation of G-type Chemical Warfare Nerve Agents via Analytical Supercritical Fluid Chromatography

PubMed Central

Kasten, Shane A; Zulli, Steven; Jones, Jonathan L; Dephillipo, Thomas; Cerasoli, Douglas M

2014-01-01

Chemical warfare nerve agents (CWNAs) are extremely toxic organophosphorus compounds that contain a chiral phosphorus center. Undirected synthesis of G-type CWNAs produces stereoisomers of tabun, sarin, soman, and cyclosarin (GA, GB, GD, and GF, respectively). Analytical-scale methods were developed using a supercritical fluid chromatography (SFC) system in tandem with a mass spectrometer for the separation, quantitation, and isolation of individual stereoisomers of GA, GB, GD, and GF. Screening various chiral stationary phases (CSPs) for the capacity to provide full baseline separation of the CWNAs revealed that a Regis WhelkO1 (SS) column was capable of separating the enantiomers of GA, GB, and GF, with elution of the P(+) enantiomer preceding elution of the corresponding P(–) enantiomer; two WhelkO1 (SS) columns had to be connected in series to achieve complete baseline resolution. The four diastereomers of GD were also resolved using two tandem WhelkO1 (SS) columns, with complete baseline separation of the two P(+) epimers. A single WhelkO1 (RR) column with inverse stereochemistry resulted in baseline separation of the GD P(–) epimers. The analytical methods described can be scaled to allow isolation of individual stereoisomers to assist in screening and development of countermeasures to organophosphorus nerve agents. Chirality 26:817–824, 2014. © 2014 The Authors. Chirality published by John Wiley Periodicals, Inc. PMID:25298066

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

PubMed

Abia, Jude A; Mriziq, Khaled S; Guiochon, Georges A

2009-04-10

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 4.6mm C18 bonded silica-based monolithic column, a 150 mm x 4.6mm column packed with 2.7 microm porous shell particles of C18 bonded silica (HALO), and a 150 mm x 4.6mm column packed with 3 microm 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.

Chromatography resin support

DOEpatents

Dobos, James G.

2002-01-01

An apparatus and method of using an improved chromatography resin support is disclosed. The chromatography support platform is provided by a stainless steel hollow cylinder adapted for being inserted into a chromatography column. An exterior wall of the stainless steel cylinder defines a groove for carrying therein an "O"-ring. The upper surface of the stainless steel column is covered by a fine stainless steel mesh welded to the edges of the stainless steel cylinder. When placed upon a receiving ledge defined within a chromatography column, the "O"-ring provides a fluid tight seal with the inner edge wall of the chromatography cylinder. The stainless steel mesh supports the chromatography matrix and provides a back flushable support which is economical and simple to construct.

Multivariate data analysis to characterize gas chromatography columns for dioxin analysis.

PubMed

Do, Lan; Geladi, Paul; Haglund, Peter

2014-06-20

dipolar moment. Finally, the PCA and PLS analyses were complemented with linear regression analysis to identify the most orthogonal column combinations, which could be used in comprehensive two-dimensional gas chromatography (GC×GC) to enhance PCDD/F separation and congener profiling. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Separation and purification of astaxanthin from Phaffia rhodozyma by preparative high-speed counter-current chromatography.

PubMed

Du, Xiping; Dong, Congcong; Wang, Kai; Jiang, Zedong; Chen, Yanhong; Yang, Yuanfan; Chen, Feng; Ni, Hui

2016-09-01

An effective high-speed counter-current chromatography (HSCCC) method was established for the preparative isolation and purification of astaxanthin from Phaffia rhodozyma. With a two-phase solvent system composed of n-hexane-acetone-ethanol-water (1:1:1:1, v/v/v/v), 100mg crude extract of P. rhodozyma was separated to yield 20.6mg of astaxanthin at 92.0% purity. By further one step silica gel column chromatography, the purity reached 99.0%. The chemical structure of astaxanthin was confirmed by thin layer chromatography (TLC), UV spectroscopy scanning, high performance liquid chromatography with a ZORBAX SB-C18 column and a Waters Nova-pak C18 column, and ESI/MS/MS. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study.

PubMed

Stroka, J; Anklam, E; Jörissen, U; Gilbert, J

2000-01-01

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and

Extending the limits of operating pressure of narrow-bore column liquid chromatography instrumentation.

PubMed

Pauw, Ruben De; Degreef, Bart; Ritchie, Harald; Eeltink, Sebastiaan; Desmet, Gert; Broeckhoven, Ken

2014-06-20

The increase of the operating pressure in Liquid Chromatography, has been one of the crucial steps toward faster and more efficient separations. In the present contribution, it was investigated if the pressure limits for narrow-bore columns (2.1mm ID) could be increased beyond those of commercially available (1300bar) instrumentation without performance loss. Whereas previous studies applying pressures higher than 2000bar were limited to the use of columns with a diameter smaller or equal to 1mm, it is a difficult feat to expand this to 2.1mm ID given that viscous-heating effects increase according to the fifth power of the column radius. A prototype LC set-up was realized, allowing to operate at pressures up to 2600bar (260MPa) for large separation volumes (>5mL). The performance of an in-house-built injector was compared at 800bar to commercially available injectors, yielding equal performance but twice the maximum pressure rating. The performance of (coupled) custom columns packed with fully porous and superficially porous particles were assessed at ultra-high-pressure conditions. Increasing the inlet pressure from 800 to 2400bar and scaling the column length proportionally (from 150mm to 450mm), resulted in the theoretically expected linear increase in plate count from 20,000 to 59,000. A maximum plate number of 81,000 was realized using a 600mm long (coupled) column at 2600bar. Viscous-heating effects were diminished by insulating coupled columns and applying an intermediate-cooling strategy in a forced-air oven. Copyright © 2014 Elsevier B.V. All rights reserved.

Intensified Separation of Steviol Glycosides from a Crude Aqueous Extract of Stevia rebaudiana Leaves Using Centrifugal Partition Chromatography.

PubMed

Hubert, Jane; Borie, Nicolas; Chollet, Sébastien; Perret, Joël; Barbet-Massin, Claire; Berger, Monique; Daydé, Jean; Renault, Jean-Hugues

2015-11-01

Aqueous extracts of Stevia rebaudiana leaves have been approved since 2008 by the Joint Expert Committee for Food Additives as sugar substitutes in many food and beverages in Western and Far East Asian countries. The compounds responsible for the natural sweetness of Stevia leaves include a diversity of diterpenoid glycosides derived from a steviol skeleton. These steviol glycosides also exhibit a low calorific value as well as promising therapeutic applications, particularly for the treatment of sugar metabolism disturbances. In this work, centrifugal partition chromatography is proposed as an efficient technical alternative to purify steviol glycosides from crude aqueous extracts of Stevia leaves on a multigram scale. Two different commercial instruments, including an ASCPC250® and a FCPE300® made of columns containing 1890 and 231 twin-cells, respectively, were evaluated and compared. All experiments were performed with a polar biphasic solvent system composed of ethyl acetate, n-butanol, and water in a gradient elution mode. When using the 1890 partition cell centrifugal partition chromatography column of 250 mL, 42 mg of stevioside, 68 mg of dulcoside A, and 172 mg of rebaudioside A, three major constituents of the initial extract were obtained from 1 g of the initial mixture at purities of 81%, 83%, and 99%, respectively. The productivity was further improved by intensifying the procedure on the 231 partition cell centrifugal partition chromatography column of 303 mL with the sample mass loading increased up to 5 g, resulting in the recovery of 1.2 g of stevioside, 100 mg of dulcoside A, and 1.1 g of rebaudioside A at purities of 79%, 62%, and 98%, respectively. The structures of the isolated compounds were validated by HPLC-UV, ESI-MS, (1)H, and (13)C NMR analyses. Altogether, the results demonstrate that the column design (i.e., the partition cell number) is an important aspect to be considered for a larger scale centrifugal partition chromatography

Separation of cannabinoids on three different mixed-mode columns containing carbon/nanodiamond/amine-polymer superficially porous particles.

PubMed

Hung, Chuan-Hsi; Zukowski, Janusz; Jensen, David S; Miles, Andrew J; Sulak, Clayton; Dadson, Andrew E; Linford, Matthew R

2015-09-01

Three mixed-mode high-performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine-polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C18 ), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed-mode column (C18 ) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed-mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C18 ) mixed-mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isoflavone-free soy protein prepared by column chromatography reduces plasma cholesterol in rats.

PubMed

Fukui, Kensuke; Tachibana, Nobuhiko; Wanezaki, Satoshi; Tsuzaki, Shinichi; Takamatsu, Kiyoharu; Yamamoto, Takashi; Hashimoto, Yukio; Shimoda, Tadahisa

2002-09-25

To know whether isoflavones are responsible for the hypocholesterolemic effect of soy protein, the effect on plasma cholesterol of isoflavone-free soy protein prepared by column chromatography was examined in rats. Five-week-old male Sprague-Dawley rats were fed cholesterol-enriched AIN-93G diets containing either 20% casein (CAS), 20% soy protein isolate (SPI), 20% isoflavone-free SPI (IF-SPI), 19.7% IF-SPI + 0.3% isoflavone-rich fraction (isoflavone concentrate, IC), or 20% CAS + 0.3% IC for 2 weeks. Plasma total cholesterol concentrations of rats fed SPI and IF-SPI were comparable and were significantly lower than that of rats fed CAS. The addition of IC to the CAS and IF-SPI did not influence plasma cholesterol level. Fecal steroid excretion of the three SPI groups was higher than that of the two CAS groups, whereas the addition of IC showed no effect. Thus, a significant fraction of the cholesterol-lowering effect of SPI in rats can be attributed to the protein content, but the isoflavones and other minor constituents may also play a role.

ENANTIOMER SEPARATION OF POLYCHLORINATED BIPHENYL ATROPISOMERS AND POLYCHLORINATED BIPHENYL RETENTION BEHAVIOR ON MODIFIED CYCLODEXTRIN CAPILLARY GAS CHROMATOGRAPHY COLUMNS

EPA Science Inventory

Seven commercially-available chiral capillary gas chromatography columns containing modified cyclodextrins were evaluated for their ability to separate enantiomers of the 19 stable chiral polychlorinated biphenyl (PCB) atropisomers, and for their ability to separate these enantio...

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

2017-07-01

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.

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Methods for studying reaction kinetics in gas chromatography, exemplified by using the 1-chloro-2,2-dimethylaziridine interconversion reaction.

PubMed

Krupcík, J; Mydlová, J; Májek, P; Simon, P; Armstrong, D W

2008-04-04

the calculation of apparent rate constants for both the interconversion of the first eluted (k (A-->B)(app)) as well as the second eluted (k(B-->A)(app)) enantiomer. The mean value for all the rate constants (from all the reviewed methods) was found for 1-chloro-2,2-dimethylaziridine A-->B enantiomer interconversion at 100 degrees C: k (A-->B)(app)=21.2 x 10(-4)s(-1) with a standard deviation sigma=10.7 x 10(-4). Evaluating data for reaction chromatography at 100 degrees C {k (app)=k(A-->B)(app)=k(B-->A)(app)=13.9 x 10(-4)s(-1), sigma=3.0 x 10(-4)s(-1)} shows that differences between k(A-->B)(app) and k(B-->A)(app) are the same within experimental error. It was shown both theoretically and experimentally that the Arrhenius activation energy (E(a)) calculated from Arrhenius plots (lnk(app) versus 1/T) is proportional to the enthalpy of activation {E(a)=DeltaH+RT}. Statistical treatment of Gibbs activation energy values gave: DeltaG (app)=110.5kJmol(-1), sigma=2.4kJmol(-1), DeltaG (A-->B)(app)=110.5kJmol(-1), sigma=2.2kJmol(-1), DeltaG (B-->A)(app)=110.3kJmol(-1), sigma=2.8kJmol(-1). This shows that the apparent Gibbs energy barriers for the interconversion of 1-chloro-2,2-dimethylaziridine enantiomers are equal DeltaG (app)=DeltaG(A-->B)(app)=DeltaG(B-->A)(app) and within the given precision of measurement independent of the experimental method used.

Polar and nonpolar organic polymer-based monolithic columns for capillary electrochromatography and high-performance liquid chromatography.

PubMed

Rathnasekara, Renuka; Khadka, Shantipriya; Jonnada, Murthy; El Rassi, Ziad

2017-01-01

This review article is a continuation of the previous reviews on the area of monolithic columns covering the progress made in the field over the last couple of years from the beginning of the second half of 2014 until the end of the first half of 2016. It summarizes and evaluates the evolvement of both polar and nonpolar organic monolithic columns and their use in hydrophilic interaction LC and CEC and reversed-phase chromatography and RP-CEC. The review article discusses the results reported in a total of 62 references. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Isolation of two new prenylated flavonoids from Sinopodophyllum emodi fruit by silica gel column and high-speed counter-current chromatography.

PubMed

Sun, Yanjun; Sun, Yinshi; Chen, Hui; Hao, Zhiyou; Wang, Junmin; Guan, Yanbin; Zhang, Yanli; Feng, Weisheng; Zheng, Xiaoke

2014-10-15

Two new prenylated flavonoids, sinoflavonoids A-B, were isolated from the dried fruits of Sinopodophyllum emodi by silica gel column chromatography (SGCC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in water, respectively. The ethyl acetate fraction was pre-separated by SGCC with a petroleum ether-acetone gradient. The eluates containing target compounds were further separated by HSCCC with n-hexane-ethyl acetate-methanol-water (4:6:4:4, v/v). Finally, 17.3mg of sinoflavonoid A and 25.9mg of sinoflavonoid B were obtained from 100mg of the pretreated concentrate. The purities of sinoflavonoid A and sinoflavonoid B were 98.47% and 99.38%, respectively, as determined by HPLC. Their structures were elucidated on the basis of spectroscopic evidences (HR-ESI-MS, (1)H-NMR, (13)C-NMR, HSQC, HMBC). The separation procedures proved to be efficient, especially for trace prenylated flavonoids. Copyright © 2014 Elsevier B.V. All rights reserved.

Separation of amino acids and antibiotics by narrow-bore and normal-bore high-performance liquid chromatography with pre-column derivatization.

PubMed

Fiedler, H P; Plaga, A

1987-01-16

The selectivity, efficiency and lifetime of normal- and narrow-bore columns for high-performance liquid chromatography were investigated for the separation and quantification of amino acids and the amino acid-like antibiotics phosphinothricin and phosphinothricylalanylalanine in biological samples. These compounds were determined by an automated pre-column derivatization with o-phthalaldehyde-2-mercaptoethanol reagent and UV detection at 338 nm.

Direct coupling of microbore HPLC columns to MS systems

NASA Technical Reports Server (NTRS)

Mcnair, H. M.

1985-01-01

A detailed investigation using electron microscopy was conducted which examined the conditions of materials used in the construction of stable, high performance microbore liquid chromatography (LC) columns. Small details proved to be important. The effects of temperature on the elution of several homologous series used as probe compounds was examined in reverse phase systems. They showed that accessible temperature changes provide roughly half the increase in solvent strength that would be obtained going from a 100% aqueous to a 100% organic mobile phase, which is sufficient to warrant their use in many analyses requiring the use of gradients. Air circulation temperature control systems provide the easiest means of obtaining rapid, wide range changes in column temperature. However, slow heat transfer from the gas leads to thermal nonuniformity in the column and a decrease in resolution as the temperature program progresses.

Determination of erythromycin in human plasma, using column liquid chromatography with a polymeric packing material, alkaline mobile phase and amperometric detection.

PubMed

Nilsson, L G; Walldorf, B; Paulsen, O

1987-12-25

A method based on column liquid chromatography was developed for determination of plasma concentrations of erythromycin. PRP-1, a polymeric type of packing material suitable for chromatography and amperometric detection at high pH, was used. The effect of pH on the column performance and on the electrochemical response was studied. A pH of ca. 10 was found to be optimal. After extraction with tert.-butyl methyl ether, plasma concentrations down to 0.2 mumol/l could be measured, using automated sample injection. Oleandomycin was used as internal standard. The method was used for determination of plasma concentrations in a pharmacokinetic study under steady-state conditions.

Liquid Crystals in Chromatography

NASA Astrophysics Data System (ADS)

Witkiewicz, Zygfryd

The following sections are included: * INTRODUCTION * LIQUID CRYSTALS SUITABLE FOR GAS CHROMATOGRAPHY * Monomeric Liquid Crystal Stationary Phases * Polymeric Liquid Crystal Stationary Phases * Polymeric Liquid Crystal Stationary Phases * Conventional Analytical Columns * Capillary Columns * FACTORS AFFECTING THE CHROMATOGRAPHIC SEPARATIONS ON LIQUID CRYSTAL STATIONARY PHASES * Kind of Mesophase of the Liquid Crystal * Molecular Structure of the Liquid Crystals and of the Chromatographed Substances * Substrate on which the Liquid Crystal is Deposited * ANALYTICAL APPLICATIONS OF LIQUID CRYSTAL STATIONARY PHASES IN GAS CHROMATOGRAPHY * Separation of Isomers of Benzene and Naphthalene Derivatives * Separation of Alkane and Alkene Isomers * Separation of Mixtures of Benzene and Aliphatic Hydrocarbon Derivatives Containing Heteroatoms * Separation of Polynuclear Hydrocarbons * INVESTIGATION OF THE PROPERTIES OF LIQUID CRYSTALS BY GAS CHROMATOGRAPHY * APPLICATION OF LIQUID CRYSTALS IN LIQUID CHROMATOGRAPHY * Column Chromatography * Thin-Layer Chromatography * APPLICATION OF LIQUID CRYSTAL STATIONARY PHASES IN SUPERCRITICAL FLUID CHROMATOGRAPHY * FINAL REMARKS * References

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

PubMed Central

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–MSn 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 in A. pendulum. Among them, 38 compounds were identified and 29 alkaloids were reported for the first time for this herb. Therefore, this means that HPLC–MSn combined with column chromatography could work as an effective and reliable tool for rapid identification of the chemical components of herbal medicine. PMID:26896350

Enantioselective Analytical- and Preparative-Scale Separation of Hexabromocyclododecane Stereoisomers Using Packed Column Supercritical Fluid Chromatography.

PubMed

Riddell, Nicole; Mullin, Lauren Gayle; van Bavel, Bert; Ericson Jogsten, Ingrid; McAlees, Alan; Brazeau, Allison; Synnott, Scott; Lough, Alan; McCrindle, Robert; Chittim, Brock

2016-11-10

Hexabromocyclododecane (HBCDD) is an additive brominated flame retardant which has been listed in Annex A of the Stockholm Convention for elimination of production and use. It has been reported to persist in the environment and has the potential for enantiomer-specific degradation, accumulation, or both, making enantioselective analyses increasingly important. The six main stereoisomers of technical HBCDD (i.e., the (+) and (-) enantiomers of α-, β-, and γ-HBCDD) were separated and isolated for the first time using enantioselective packed column supercritical fluid chromatography (pSFC) separation methods on a preparative scale. Characterization was completed using published chiral liquid chromatography (LC) methods and elution profiles, as well as X-ray crystallography, and the isolated fractions were definitively identified. Additionally, the resolution of the enantiomers, along with two minor components of the technical product (δ- and ε-HBCDD), was investigated on an analytical scale using both LC and pSFC separation techniques, and changes in elution order were highlighted. Baseline separation of all HBCDD enantiomers was achieved by pSFC on an analytical scale using a cellulose-based column. The described method emphasizes the potential associated with pSFC as a green method of isolating and analyzing environmental contaminants of concern.

Implementation of high slurry concentration and sonication to pack high-efficiency, meter-long capillary ultrahigh pressure liquid chromatography columns.

PubMed

Godinho, Justin M; Reising, Arved E; Tallarek, Ulrich; Jorgenson, James W

2016-09-02

Slurry packing capillary columns for ultrahigh pressure liquid chromatography is complicated by many interdependent experimental variables. Previous results have suggested that combination of high slurry concentration and sonication during packing would create homogeneous bed microstructures and yield highly efficient capillary columns. Herein, the effect of sonication while packing very high slurry concentrations is presented. A series of six, 1m×75μm internal diameter columns were packed with 200mg/mL slurries of 2.02μm bridged-ethyl hybrid silica particles. Three of the columns underwent sonication during packing and yielded highly efficient separations with reduced plate heights as low as 1.05. Copyright © 2016 Elsevier B.V. All rights reserved.

Excretion of common neutral steroids in healthy subjects as estimated by multi-column chromatography.

PubMed

Vestergaard, P

1978-01-01

Excretion data for common neutral urinary steroids from a total of 330 healthy subjects from different parts of the world and of different sex and age are given. The estimations, which have been performed by multi-column liquid chromatography, include 24 h excretion values for both common 17-oxosteroids and the common metabolites of cortisol, including the cortolones and the cortols. Comparisons are made with values from the world literature and with isotope experiments.

Study on camel IgG purification

PubMed Central

Khamehchian, Sedigheh; Zolfagharian, Hossein; Dounighi, Naser Mohammadpour; Tebianian, Majid; Madani, Rasool

2014-01-01

A combined process of ammonium sulfate precipitation (salting out) and ion-exchange chromatography on DEAE-Sepharose CL-6B was used to prepare camel antivenom (IgG) against Naja Naja Oxiana for therapy. In the ammonium sulfate precipitation, the best condition for fractionation of IgG from the other proteins in camel serum was 55% precipitate. The camel IgG presented as 2 bands with molecular masses of 250 and 100 kDa, the latter corresponding to heavy chain IgG, on 10% gel electrophoresis. A trace amount of non-IgG proteins was not isolated and remained in this precipitate. Therefore in order to effectively separate albumin and the other nonspecific proteins from the IgG, the 25% precipitate of ammonium sulfate precipitation of serum was subjected to DEAE-Sepharose CL-6B column chromatography. A peak of antibody (IgG) could be obtained by elution with sodium phosphate buffer. In this stage, 2 bands of molecular masses of 150 and 75 kDa were observed on 7% gel electrophoresis. A comparative study was performed between camel IgG and conventional horse F(ab)2 antivenoms in term of potency (serum neutralization test and ELISA). Our results showed that the potency of camel antivenom was 4-fold higher than that of horse. It is suggested the combined ammonium sulfate precipitation and ion-exchange chromatography process effectively removed residual proteins in the final camel IgG preparation and can be a suitable method for large-scale refinement of therapeutic camel antivenoms. PMID:24642472

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry.

PubMed

Poggi, Josiane Cristófani; Da Silva, Flávia Garcez; Coelho, Eduardo Barbosa; Marques, Maria Paula; Bertucci, Carlo; Lanchote, Vera Lucia

2012-03-01

Carvedilol is an antihypertensive drug available as a racemic mixture. (-)-(S)-carvedilol is responsible for the nonselective β-blocker activity but both enantiomers present similar activity on α(1)-adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H](+) and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml(-1) for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)-(R)-carvedilol (AUC(0-∞) 205.52 vs. 82.61 (ng h) ml(-1)), with an enantiomer ratio of 2.48. Copyright © 2012 Wiley Periodicals, Inc.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography-triple quadrupole mass spectrometry with novel shell-type column.

PubMed

Du, Gang; Zhao, Haiyu; Song, Yuelin; Zhang, Qingwen; Wang, Yitao

2011-10-01

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones.

PubMed

Carnes, Stephanie; O'Brien, Stacey; Szewczak, Angelica; Tremeau-Cayel, Lauriane; Rowe, Walter F; McCord, Bruce; Lurie, Ira S

2017-09-01

A comparison of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography for the separation of synthetic cathinones has been conducted. Nine different mixtures of bath salts were analyzed in this study. The three different chromatographic techniques were examined using a general set of controlled synthetic cathinones as well as a variety of other synthetic cathinones that exist as positional isomers. Overall 35 different synthetic cathinones were analyzed. A variety of column types and chromatographic modes were examined for developing each separation. For the ultra high performance supercritical fluid chromatography separations, analyses were performed using a series of Torus and Trefoil columns with either ammonium formate or ammonium hydroxide as additives, and methanol, ethanol or isopropanol organic solvents as modifiers. Ultra high performance liquid chromatographic separations were performed in both reversed phase and hydrophilic interaction chromatographic modes using SPP C18 and SPP HILIC columns. Gas chromatography separations were performed using an Elite-5MS capillary column. The orthogonality of ultra high performance supercritical fluid chromatography, ultra high performance liquid chromatography, and gas chromatography was examined using principal component analysis. For the best overall separation of synthetic cathinones, the use of ultra high performance supercritical fluid chromatography in combination with gas chromatography is recommended. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of three different C18 HPLC columns with different particle sizes for the optimization of aflatoxins analysis.

PubMed

Medina, A; Magan, N

2012-03-15

In this work we compared the performance of chromatography columns with particles of 5 and 3 μm with the new 2.7 μm solid core particles for the analysis of aflatoxins B1, G1, B2, and G2 using trifluoroacetic acid pre-column derivatization. Three different columns have been used and chromatographic parameters as retention time, resolution, limit of detection (LOD), limit of quantification (LOQ) were obtained from all of them and compared. The results show that comparing with the traditional columns, shorter columns (100 mm × 4.6 mm) with the new solid core particles are suitable for the analysis of these mycotoxins and allowed the reduction of the analysis time by 45.5% and 33.3% with respect to columns with particle size 5 μm (150 mm × 4.6 mm) and 3 μm (150 mm × 4.6 mm) respectively, without any detrimental effect on performance. This leads to the reduction of the analysis costs by saving on organic solvents and increasing the total number of analyses per day. The capability of these columns for analyzing samples, in different culture media, was assessed by analyzing different samples from: yeasts extract sucrose medium, corn meal agar medium and fresh hazelnut media. Copyright © 2012 Elsevier B.V. All rights reserved.

Preparation of goat and rabbit anti-camel immunoglobulin G whole molecule labeled with horseradish peroxidase

PubMed Central

Abdel-Rahman, Eman Hussein; El-Jakee, Jakeen Kamal; Hatem, Mahmoud Essam; Ata, Nagwa Sayed; Fouad, Ehab Ali

2017-01-01

Aim: As the labeled anti-camel immunoglobulins (Igs) with enzymes for enzyme-linked immunosorbent assay (ELISA) are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP) to save hard curacy. Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA. Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at −20°C as proved by ELISA. Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at −20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for preparation of IgG

Simultaneous determination of aflatoxin B₁, B₂, G₁, and G₂ in corn powder, edible oil, peanut butter, and soy sauce by liquid chromatography with tandem mass spectrometry utilizing turbulent flow chromatography.

PubMed

Fan, Sufang; Li, Qiang; Zhang, Xiaoguang; Cui, Xiaobin; Zhang, Dongsheng; Zhang, Yan

2015-05-01

A novel fully automated method based on dual column switching using turbulent flow chromatography followed by liquid chromatography with tandem mass spectrometry was developed for the determination of aflatoxin B1 , B2 , G1 , and G2 in corn powder, edible oil, peanut butter, and soy sauce samples. After ultrasound-assisted extraction, samples were directly injected to the chromatographic system and the analytes were concentrated into the clean-up loading column. Through purge switching, the analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlow(TM) columns, transfer flow rate, transfer time were optimized. The limits of detection and quantification of this method ranged between 0.2-2.0 and 0.5-4.0 μg/kg for aflatoxins in different matrixes, respectively. Recoveries of aflatoxins were in range of 83-108.1% for all samples, matrix effects were in range of 34.1-104.7%. The developed method has been successfully applied in the analysis of aflatoxin B1 , B2 , G1 , and G2 in real samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitation of acrylamide in food products by liquid chromatography/mass spectrometry.

PubMed

Eberhart, B Loye; Ewald, Deborah K; Sanders, Robert A; Tallmadge, Daniel H; Zyzak, David V; Strothers, Melissa A

2005-01-01

A simple and inexpensive liquid chromatography/mass spectrometry (LC/MS) method was developed for the quantitation of acrylamide in various food products. The method involved spiking the isotope-substituted internal standard (1-C13 acrylamide) onto 6.00 g of the food product, adding 40 mL distilled/deionized water, and heating at 65 degrees C for 30 min. Afterwards, 10 mL ethylene dichloride was added and the mixture was homogenized for 30 s and centrifuged at 2700 x g for 30 min, and then 8 g supernatant was extracted with 10, 5, and 5 mL portions of ethyl acetate. The extracts were combined, dried with sodium sulfate, and concentrated to 100-200 microL. Acrylamide was determined by analysis of the final extract on a single quadrupole, bench-top mass spectrometer with electrospray ionization, using a 2 mm id C18 column and monitoring m/z = 72 (acrylamide) and m/z = 73 (internal standard). For difficult food matrixes, such as coffee and cocoa, a solid-phase extraction cleanup step was incorporated to improve both chromatography and column lifetime. The method had a limit of quantitation of 10 ppb, and coefficients of determination (r2) for calibration curves were typically better than 0.998. Acceptable spike recovery results were achieved in 11 different food matrixes. Precision in potato chip analyses was 5-8% (relative standard deviation). This method provides an LC/MS alternative to the current LC/MS/MS methods and derivatization gas chromatography/mass spectrometry methods, and is applicable to difficult food products such as coffee, cocoa, and high-salt foods.

Long-chain alkylimidazolium ionic liquids, a new class of cationic surfactants coated on ODS columns for anion-exchange chromatography.

PubMed

Qiu, Hongdeng; Zhang, Qinghua; Chen, Limei; Liu, Xia; Jiang, Shengxiang

2008-08-01

Separations of common inorganic anions were carried out on ODS columns coated with two long-chain alkylimidazolium ionic liquids ([C(12)MIm]Br and [C(14)MIm]Br) as new cationic surfactants for ion chromatography. With phthalate buffer solution as the mobile phases and non-suppressed conductivity detection, high column efficiencies and excellent selectivity were obtained in the separation of inorganic anions. Chromatographic parameters are calculated and the results show that the coated column possesses significant potential for the analysis of some inorganic anions such as CH(3)COO(-), IO(3)(-), Cl(-), BrO(3)(-), NO(2)(-), Br(-), NO(3)(-), SO(4)(2-), I(-), BF(4)(-), and SCN(-). The effect of eluent pH values on the separation of anions has been studied on the column coated with [C(12)MIm]Br. The stability of the coated columns was also examined.

Estimations of temperature deviations in chromatographic columns using isenthalpic plots. I. Theory for isocratic systems.

PubMed

Tarafder, Abhijit; Iraneta, Pamela; Guiochon, Georges; Kaczmarski, Krzysztof; Poe, Donald P

2014-10-31

We propose to use constant enthalpy or isenthalpic diagrams as a tool to estimate the extent of the temperature variations caused by the mobile phase pressure drop along a chromatographic column, e.g. of its cooling in supercritical fluid and its heating in ultra-performance liquid chromatography. Temperature strongly affects chromatographic phenomena. Any of its variations inside the column, whether intended or not, can lead to significant changes in separation performance. Although instruments use column ovens in order to keep constant the column temperature, operating conditions leading to a high pressure drop may cause significant variations of the column temperature, both in the axial and the radial directions, from the set value. Different ways of measuring these temperature variations are available but they are too inconvenient to be employed in many practical situations. In contrast, the thermodynamic plot-based method that we describe here can easily be used with only a ruler and a pencil. They should be helpful in developing methods or in analyzing results in analytical laboratories. Although the most effective application area for this approach should be SFC (supercritical fluid chromatography), it can be applied to any chromatographic conditions in which temperature variations take place along the column due to the pressure drop, e.g. in ultra-high pressure liquid chromatography (UHPLC). The method proposed here is applicable to isocractic conditions only. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins.

PubMed

Ishihara, Takashi; Kadoya, Toshihiko; Yamamoto, Shuichi

2007-08-24

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.

Hydrodynamic chromatography of polystyrene microparticles in micropillar array columns.

PubMed

Op de Beeck, Jeff; De Malsche, Wim; Vangelooven, Joris; Gardeniers, Han; Desmet, Gert

2010-09-24

We report on the possibility to perform HDC in micropillar array columns and the potential advantages of such a system. The HDC performance of a pillar array column with pillar diameter = 5 microm and an interpillar distance of 2.5 microm has been characterized using both a low MW tracer (FITC) and differently sized polystyrene bead samples (100, 200 and 500 nm). The reduced plate height curves that were obtained for the different investigated markers all overlapped very well, and attained a minimum value of about h(min)=0.3 (reduction based on the pillar diameter), corresponding to 1.6 microm in absolute value and giving good prospects for high efficiency separations. The obtained reduced retention time values were in fair agreement with that predicted by the Di Marzio and Guttman model for a flow between flat plates, using the minimal interpillar distance as characteristic interplate distance. Copyright 2010 Elsevier B.V. All rights reserved.

Improved purification of immunoglobulin G from plasma by mixed-mode chromatography.

PubMed

Chai, Dong-Sheng; Sun, Yan; Wang, Xiao-Ning; Shi, Qing-Hong

2014-12-01

Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct enantioseparation of nitrogen-heterocyclic pesticides on cellulose-based chiral column by high-performance liquid chromatography.

PubMed

Chai, Tingting; Yang, Wenwen; Qiu, Jing; Hou, Shicong

2015-01-01

The enantiomeric separation of eight pesticides including bitertanol (), diclobutrazol (), fenbuconazole (), triticonazole (), imazalil (), triapenthenol (), ancymidol (), and carfentrazone-ethyl () was achieved, using normal-phase high-performance liquid chromatography on two cellulosed-based chiral columns. The effects of isopropanol composition from 2% to 30% in the mobile phase and column temperature from 5 to 40 °C were investigated. Satisfactory resolutions were obtained for bitertanol (), triticonazole (), imazalil () with the (+)-enantiomer eluted first and fenbuconazole () with the (-)-enantiomer eluted first on Lux Cellulose-2 and Lux Cellulose-3. (+)-Enantiomers of diclobutrazol () and triapenthenol () were first eluted on Lux Cellulose-2. (-)-Carfentrazone-ethyl () were eluted first on Lux Cellulose-2 and Lux Cellulose-3 with incomplete separation. Reversed elution orders were obtained for ancymidol (7). (+)-Ancymidol was first eluted on Lux Cellulose-2 while on Lux Cellulose-3 (-)-ancymidol was first eluted. The results of the elution order at different column temperatures suggested that column temperature did not affect the optical signals of the enantiomers. These results will be helpful to prepare and analyze individual enantiomers of chiral pesticides. © 2014 Wiley Periodicals, Inc.

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns.

PubMed

Rocco, Anna; Maruška, Audrius; Fanali, Salvatore

2012-03-01

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin (TM-β-CD) or hydroxylpropyl-β-cyclodextrin (HP-β-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 μm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-β-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-β-CD was chosen as an alternative to TM-β-CD. Employing the monolithic column, the effects of different parameters such as HP-β-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R(s) = 1.80 for naproxen to R(s) = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-β-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R(s) value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R(s) = 0.89).

Sequential Injection Chromatography with an Ultra-short Monolithic Column for the Low-Pressure Separation of α-Tocopherol and γ-Oryzanol in Vegetable Oils and Nutrition Supplements.

PubMed

Thaithet, Sujitra; Kradtap Hartwell, Supaporn; Lapanantnoppakhun, Somchai

2017-01-01

A low-pressure separation procedure of α-tocopherol and γ-oryzanol was developed based on a sequential injection chromatography (SIC) system coupled with an ultra-short (5 mm) C-18 monolithic column, as a lower cost and more compact alternative to the HPLC system. A green sample preparation, dilution with a small amount of hexane followed by liquid-liquid extraction with 80% ethanol, was proposed. Very good separation resolution (R s = 3.26), a satisfactory separation time (10 min) and a total run time including column equilibration (16 min) were achieved. The linear working range was found to be 0.4 - 40 μg with R 2 being more than 0.99. The detection limits of both analytes were 0.28 μg with the repeatability within 5% RSD (n = 7). Quantitative analyses of the two analytes in vegetable oil and nutrition supplement samples, using the proposed SIC method, agree well with the results from HPLC.

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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Chromatographic test methods for characterizing alkylsiloxane-bonded silica columns for reversed-phase liquid chromatography.

PubMed

Poole, Colin F

2018-06-07

Major obstacles to formulating a simple retention mechanism for reversed-phase liquid chromatography have a direct impact on the development of experimental methods for column characterization as they limit our capability to understand observed differences in retention at a system level. These problems arise from the heterogeneous composition of the stationary phase, the difficulty of providing a working definition for the phase ratio, and uncertainty as to whether the distribution mechanism for varied compounds is a partition, adsorption or mixed (combination) of these models. Retention factor and separation factor measurements offer little guidance as they represent an average of various and variable contributing factors that can only be interpreted by assuming a specific model. Column characterization methods have tended to ignore these difficulties by inventing a series of terms to describe column properties, such as hydrophobicity, hydrophilicity, silanol activity, steric resistance, etc., without proper definition. This has allowed multiple scales to be proposed for the same property which generally are only weakly correlated. Against this background we review the major approaches for the characterization of alkylsiloxane-bonded silica stationary phases employing prototypical compounds, the hydrophobic-subtraction model and the solvation parameter model. Those methods using prototypical compounds are limited by the lack of compounds with a singular dominant interaction. The multivariate approaches that extract column characteristic properties from the retention of varied compounds are more hopeful but it is important to be more precise in defining the characteristic column properties and cognizant that general interpretation of these properties for varied columns cannot escape the problem of a poor understanding of the distribution mechanism. Copyright © 2018 Elsevier B.V. All rights reserved.

Flow-switching device for comprehensive two-dimensional gas chromatography.

PubMed

Bueno, Pedro A; Seeley, John V

2004-02-20

A simple flow-switching device has been developed as a differential flow modulator for comprehensive two-dimensional gas chromatography (GC x GC). The device is assembled from tubing, four tee unions, and a solenoid valve. The solenoid valve is located outside the oven of the gas chromatograph and is not in the sample path. The modulation technique has no inherent temperature restrictions and passes 100% of the primary column effluent to the secondary column(s). Secondary peaks are produced with widths at half maximum less than 100 ms when operating in GC x 2GC mode with a 2.0 s modulation period. The efficacy of this approach is demonstrated through the analysis of a standard mixture of volatile organic compounds (VOCs) and diesel fuel.

Cold column trapping-cloud point extraction coupled to high performance liquid chromatography for preconcentration and determination of curcumin in human urine.

PubMed

Rahimi, Marzieh; Hashemi, Payman; Nazari, Fariba

2014-05-15

A cold column trapping-cloud point extraction (CCT-CPE) method coupled to high performance liquid chromatography (HPLC) was developed for preconcentration and determination of curcumin in human urine. A nonionic surfactant, Triton X-100, was used as the extraction medium. In the proposed method, a low surfactant concentration of 0.4% v/v and a short heating time of only 2min at 70°C were sufficient for quantitative extraction of the analyte. For the separation of the extraction phase, the resulted cloudy solution was passed through a packed trapping column that was cooled to 0 °C. The temperature of the CCT column was then increased to 25°C and the surfactant rich phase was desorbed with 400μL ethanol to be directly injected into HPLC for the analysis. The effects of different variables such as pH, surfactant concentration, cloud point temperature and time were investigated and optimum conditions were established by a central composite design (response surface) method. A limit of detection of 0.066mgL(-1) curcumin and a linear range of 0.22-100mgL(-1) with a determination coefficient of 0.9998 were obtained for the method. The average recovery and relative standard deviation for six replicated analysis were 101.0% and 2.77%, respectively. The CCT-CPE technique was faster than a conventional CPE method requiring a lower concentration of the surfactant and lower temperatures with no need for the centrifugation. The proposed method was successfully applied to the analysis of curcumin in human urine samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization and purification of anthocyanins from black peanut (Arachis hypogaea L.) skin by combined column chromatography.

PubMed

Zhao, Zhenlei; Wu, Min; Zhan, Yali; Zhan, Kanghua; Chang, Xiulian; Yang, Hongshun; Li, Zhanming

2017-10-13

Black peanut skins as a byproduct from peanut industry contain abundant anthocyanins, evaluated as 8.61±0.27mg/g dry black peanut skins, are currently poorly exploited. In this work, four anthocyanins and three major flavonols were detected and identified by HPLC-PDA-ESI-MS/MS from the acidified water extract of black peanut skins of Arachis hypogaea L. After preliminary removal of flavonols by ethyl acetate (EtOAc), further purification of the anthocyanins was conducted using a combination of Amberlite XAD-7HP and ODS-AQ-HG column chromatography methods. Two most abundant monomeric anthocyanins cyanidin-3-O-sophoroside (5.77±0.42mg) and cyanidin-3-O-sambubioside (4.10±0.17mg) were eventually obtained from 2g dry black peanut skins, and their purities were determined by HPLC-PDA as 97.29% and 98.28% at the yields of 87.47% and 64.27% on the basis of their total amount in the crude extracts, respectively. These sequential treatments can be easily adapted to large-scale fractionation of pure anthocyanin monomers. Copyright © 2017. Published by Elsevier B.V.

Analysis of 100 pharmaceuticals and their degradates in water samples by liquid chromatography/quadrupole time-of-flight mass spectrometry.

PubMed

Ferrer, Imma; Thurman, E Michael

2012-10-12

A straightforward methodology for the chromatographic separation and accurate mass identification of 100 pharmaceuticals including some of their degradation products was developed using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF-MS). A table compiling the protonated or deprotonated exact masses for all compounds, as well as the exact mass of several fragment ions obtained by MS-MS is included. Excellent chromatographic separation was achieved by using 3.5 μm particle size columns and a slow and generic 30-min gradient. Isobaric and isomeric compounds (same nominal mass and same exact mass, respectively) were distinguished by various methods, including chromatography separation, MS-MS fragmentation, and isotopic signal identification. Method reporting limits of detection ranged from 1 to 1000 ng/L, after solid-phase extraction of 100mL aqueous samples. The methodology was successfully applied to the analysis of surface water impacted by wastewater effluent by identifying many of the pharmaceuticals and metabolites included in the list. Examples are given for some of the most unusual findings in environmental samples. This paper is meant to serve as a guide for those doing analysis of pharmaceuticals in environmental samples, by providing exact mass measurements of several well known, as well as newly identified and environmentally relevant pharmaceuticals in water samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Determination of citrus limonoid glucosides by high performance liquid chromatography coupled to post-column reaction with Ehrlich’s Reagent

USDA-ARS?s Scientific Manuscript database

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 ...

Separation techniques: Chromatography

PubMed Central

Coskun, Ozlem

2016-01-01

Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. Proteins can be purified based on characteristics such as size and shape, total charge, hydrophobic groups present on the surface, and binding capacity with the stationary phase. Four separation techniques based on molecular characteristics and interaction type use mechanisms of ion exchange, surface adsorption, partition, and size exclusion. Other chromatography techniques are based on the stationary bed, including column, thin layer, and paper chromatography. Column chromatography is one of the most common methods of protein purification. PMID:28058406

Ultrafast UPLC-ESI-MS and HPLC with monolithic column for determination of principal flavor compounds in vanilla pods.

PubMed

Sharma, Upendra K; Sharma, Nandini; Sinha, Arun K; Kumar, Neeraj; Gupta, Ajai P

2009-10-01

In this study, two novel chromatographic methods based on monolithic column high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC) were developed for the ultrafast determination of principal flavor compounds namely vanillin, vanillic acid, p-hydroxybenzoic acid, and p-hydroxybenzaldehyde in ethanolic extracts of Vanilla planifolia pods. Good separation was achieved within 2.5 min using Chromolith RP18e column (100 mm x 4.6 mm) for HPLC and Acquity BEH C-18 (100 mm x 2.1 mm, 1.7 microm) column for UPLC. Both methods were compared in terms of total analysis time, mobile phase consumption, sensitivity, and validation parameters like precision, accuracy, LOD, and LOQ. Further, system suitability test data including resolution, capacity factor, theoretical plates, and tailing factor was determined for both the methods by ten replicate injections. Monolithic column based HPLC gave better results for most of the selected parameters while UPLC was found to be more eco-friendly with low mobile phase consumption and better sensitivity. Both methods may be used conveniently for the high throughput analysis of large number of samples in comparison to traditional particulate column.

Extending the upper temperature range of gas chromatography with all-silicon microchip columns using a heater/clamp assembly.

PubMed

Ghosh, Abhijit; Johnson, Jacob E; Nuss, Johnathan G; Stark, Brittany A; Hawkins, Aaron R; Tolley, Luke T; Iverson, Brian D; Tolley, H Dennis; Lee, Milton L

2017-09-29

Miniaturization of gas chromatography (GC) instrumentation is of interest because it addresses current and future issues relating to compactness, portability and field application. While incremental advancements continue to be reported in GC with columns fabricated in microchips (referred to in this paper as "microchip columns"), the current performance is far from acceptable. This lower performance compared to conventional GC is due to factors such as pooling of the stationary phase in corners of non-cylindrical channels, adsorption of sensitive compounds on incompletely deactivated surfaces, shorter column lengths and less than optimum interfacing to injector and detector. In this work, a GC system utilizing microchip columns was developed that solves the latter challenge, i.e., microchip interfacing to injector and detector. A microchip compression clamp was constructed to heat the microchip (i.e., primary heater), and seal the injector and detector fused silica interface tubing to the inlet and outlet ports of the microchip channels with minimum extra-column dead volume. This clamp allowed occasional operation up to 375°C and routine operation up to 300°C. The compression clamp was constructed of a low expansion alloy, Kovar™, to minimize leaking due to thermal expansion mismatch at the interface during repeated thermal cycling, and it was tested over several months for more than one hundred injections without forming leaks. A 5.9m long microcolumn with rectangular cross section of 158μm×80μm, which approximately matches a 100μm i.d. cylindrical fused silica column, was fabricated in a silicon wafer using deep reactive ion etching (DRIE) and high temperature fusion bonding; finally, the channel was coated statically with a 1% vinyl, 5% phenyl, 94% methylpolysiloxane stationary phase. High temperature separations of C10-C40 n-alkanes and a commercial diesel sample were demonstrated using the system under both temperature programmed GC (TPGC) and thermal

Achieving high peak capacity production for gas chromatography and comprehensive two-dimensional gas chromatography by minimizing off-column peak broadening.

PubMed

Wilson, Ryan B; Siegler, W Christopher; Hoggard, Jamin C; Fitz, Brian D; Nadeau, Jeremy S; Synovec, Robert E

2011-05-27

By taking into consideration band broadening theory and using those results to select experimental conditions, and also by reducing the injection pulse width, peak capacity production (i.e., peak capacity per separation time) is substantially improved for one dimensional (1D-GC) and comprehensive two dimensional (GC×GC) gas chromatography. A theoretical framework for determining the optimal linear gas velocity (the linear gas velocity producing the minimum H), from experimental parameters provides an in-depth understanding of the potential for GC separations in the absence of extra-column band broadening. The extra-column band broadening is referred to herein as off-column band broadening since it is additional band broadening not due to the on-column separation processes. The theory provides the basis to experimentally evaluate and improve temperature programmed 1D-GC separations, but in order to do so with a commercial 1D-GC instrument platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a resistively heated transfer line is coupled to a high-speed diaphragm valve to provide a suitable injection pulse width (referred to herein as modified injection). Additionally, flame ionization detection (FID) was modified to provide a data collection rate of 5kHz. The use of long, relatively narrow open tubular capillary columns and a 40°C/min programming rate were explored for 1D-GC, specifically a 40m, 180μm i.d. capillary column operated at or above the optimal average linear gas velocity. Injection using standard auto-injection with a 1:400 split resulted in an average peak width of ∼1.5s, hence a peak capacity production of 40peaks/min. In contrast, use of modified injection produced ∼500ms peak widths for 1D-GC, i.e., a peak capacity production of 120peaks/min (a 3-fold improvement over standard auto-injection). Implementation of modified injection resulted in retention time, peak width

Application of a simple column-switching ion chromatography technique for removal of matrix interferences and sensitive fluorescence determination of acidic compounds (pharmaceutical drugs) in complex samples.

PubMed

Muhammad, Nadeem; Subhani, Qamar; Wang, Fenglian; Guo, Dandan; Zhao, Qiming; Wu, Shuchao; Zhu, Yan

2017-09-15

This work illustrates the introduction of a simple, rugged and flexible column-switching ion chromatography (IC) technique for an automated on-line QuEChERS extracted samples extracts washing followed by sensitive fluorescence (FLD) determination of five acidic pharmaceutical drugs namely; clofibric acid (CLO), ibuprofen (IBU), aspirin (ASP), naproxen (NAP) and flurobrofen (FLU) in three complex samples (spinach, apple and hospital sewage sludge). An old anion exchange column IonPac ® AS11-HC was utilized as a pre-treatment column for on-line washing of inorganic and organic interferences followed by isocratic separation of five acidic drugs with another anion exchange IonPac ® AS12A analytical column by exploiting the column-switching technique. This novel method exhibited good linearity with correlation coefficients (r 2 ) for all drugs were in the range 0.976-0.996. The limit of detection and quantification of all five acidic drugs were in the range 0.024μg/kg to 8.70μg/kg and 0.082μg/kg to 0.029mg/kg, respectively, and better recoveries in the range 81.17-112.5% with percentage relative standard deviations (RSDs) less than 17.8% were obtained. This on-line sample pre-treatment method showed minimum matrix effect in the range of 0.87-1.25 except for aspirin. This simple rugged and flexible column-switching system required only 28min for maximum elimination of matrices and interferences in three complex samples extracts, isocratic separation of five acidic drugs and for the continuous regeneration of pre-treatment column prior to every subsequent analysis. Finally, this simple automated IC system was appeared so rugged and flexible, which can eliminate and wash out most of interference, impurities and matrices in complex samples, simply by adjusting the NaOH and acetonitrile concentration in washing mobile phase with maximum recoveries of acidic analytes of interest. Copyright © 2017. Published by Elsevier B.V.

Simultaneous analysis for water- and fat-soluble vitamins by a novel single chromatography technique unifying supercritical fluid chromatography and liquid chromatography.

PubMed

Taguchi, Kaori; Fukusaki, Eiichiro; Bamba, Takeshi

2014-10-03

Chromatography techniques usually use a single state in the mobile phase, such as liquid, gas, or supercritical fluid. Chromatographers manage one of these techniques for their purpose but are sometimes required to use multiple methods, or even worse, multiple techniques when the target compounds have a wide range of chemical properties. To overcome this challenge, we developed a single method covering a diverse compound range by means of a "unified" chromatography which completely bridges supercritical fluid chromatography and liquid chromatography. In our method, the phase state was continuously changed in the following order; supercritical, subcritical and liquid. Moreover, the gradient of the mobile phase starting at almost 100% CO2 was replaced with 100% methanol at the end completely. As a result, this approach achieved further extension of the polarity range of the mobile phase in a single run, and successfully enabled the simultaneous analysis of fat- and water-soluble vitamins with a wide logP range of -2.11 to 10.12. Furthermore, the 17 vitamins were exceptionally separated in 4min. Our results indicated that the use of dense CO2 and the replacement of CO2 by methanol are practical approaches in unified chromatography covering diverse compounds. Additionally, this is a first report to apply the novel approach to unified chromatography, and can open another door for diverse compound analysis in a single chromatographic technique with single injection, single column and single system. Copyright © 2014. Published by Elsevier B.V.

3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography.

PubMed

Gupta, Vipul; Talebi, Mohammad; Deverell, Jeremy; Sandron, Sara; Nesterenko, Pavel N; Heery, Brendan; Thompson, Fletcher; Beirne, Stephen; Wallace, Gordon G; Paull, Brett

2016-03-03

The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm. Copyright © 2016 Elsevier B.V. All rights reserved.

Instrumentation: Ion Chromatography.

ERIC Educational Resources Information Center

Fritz, James S.

1987-01-01

Discusses the importance of ion chromatography in separating and measuring anions. The principles of ion exchange are presented, along with some applications of ion chromatography in industry. Ion chromatography systems are described, as well as ion pair and ion exclusion chromatography, column packings, detectors, and programming. (TW)

A rapid HPLC column switching method for sample preparation and determination of β-carotene in food supplements.

PubMed

Brabcová, Ivana; Hlaváčková, Markéta; Satínský, Dalibor; Solich, Petr

2013-11-15

A simple and automated HPLC column-switching method with rapid sample pretreatment has been developed for quantitative determination of β-carotene in food supplements. Commercially samples of food supplements were dissolved in chloroform with help of saponification with 1M solution of sodium hydroxide in ultrasound bath. A 20-min sample dissolution/extraction step was necessary before chromatography analysis to transfer β-carotene from solid state of food supplements preparations (capsules,tablets) to chloroform solution. Sample volume - 3μL of chloroform phase was directly injected into the HPLC system. Next on-line sample clean-up was achieved on the pretreatment precolumn Chromolith Guard Cartridge RP-18e (Merck), 10×4.6mm, with a washing mobile phase (methanol:water, 92:8, (v/v)) at a flow rate of 1.5mL/min. Valve switch to analytical column was set at 2.5min in a back-flush mode. After column switching to the analytical column Ascentis Express C-18, 30×4.6mm, particle size 2.7μm (Sigma Aldrich), the separation and determination of β-carotene in food supplements was performed using a mobile phase consisting of 100% methanol, column temperature at 60°C and flow rate 1.5mL/min. The detector was set at 450nm. Under the optimum chromatographic conditions standard calibration curve was measured with good linearity - correlation coefficient for β-carotene (r(2)=0.999014; n=6) between the peak areas and concentration of β-carotene 20-200μg/mL. Accuracy of the method defined as a mean recovery was in the range 96.66-102.40%. The intraday method precision was satisfactory at three concentration levels 20, 125 and 200μg/mL and relative standard deviations were in the range 0.90-1.02%. The chromatography method has shown high sample throughput during column-switching pretreatment process and analysis in one step in short time (6min) of the whole chromatographic analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Determination of 11 industrial antioxidants in the aqueous simulants by ultra-performance liquid chromatography-tandem mass spectrometry].

PubMed

Fan, Sai; Zou, Jianhong; Li, Liping; Zhang, Nan; Liu, Wei; Li, Bing; Zhao, Xudong; Wu, Guohua; Xue, Ying; Zhao, Rong

2014-09-01

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed to identify and determine 11 industrial antioxidants in the aqueous simulants. A ProElut PLS SPE column was used for the enrichment, and an ACQUITY UPLC BEH C18 UPLC column (100 mm x 2.1 mm, 1.7 μm) was used for separation by the gradient elution with pure water and acetonitrile as the mobile phases. The MS/MS detection was performed with an electrospray ionization (ESI) source in negative mode. The external standard method was used for quantitation in the present study. The linear ranges of the 11 analytes were from 5.0 to 100 μg/L. The coefficients of correlation were greater than 0.995. The recoveries of blank aqueous simulants fortified with the 11 analytes at the levels of 5.0, 10.0 and 20.0 μg/L were 61.4% to 109.4% with the relative standard deviations varied from 3.9% to 18.2% (n = 6). The LODs and LOQs of the 11 analytes in aqueous simulants were 0.2-1.0 μg/L and 0.5-3.0 μg/L, respectively. This method is highly sensitive and accurate, and can be applied to the determination of the 11 trace industrial antioxidants in the aqueous simulants.

Optimization of chromatographic conditions for determination of aflatoxin B1, B2, G1 and G2 by using liquid chromatography-mass Spectrometry

NASA Astrophysics Data System (ADS)

Ramadhaningtyas, Dillani Putri; Aryana, Nurhani; Aristiawan, Yosi; Styarini, Dyah

2017-11-01

The optimization of instrument condition and chromatographic separation for analysis of aflatoxin B1, B2, G1 and G2 using liquid chromatography tandem with mass spectrometer detector was conducted in the aim to provide more accurate and reliable analysis results. The aflatoxin known to be serious threat for human health as it is classified as the carcinogenic compounds. The aflatoxin B1, B2, G1 and G2 were selected due to its extensive contamination in various agricultural commodities. The best chromatographic separation was obtained using C-18 column with gradient elution of solvent 5 mM ammonium acetate and 0.1% formic acid in methanol at 7 minutes runtime analysis. The linearity of the detector showed satisfied results as the coefficient determination found to be 0.9994, 0.9996, 0.9998 and 0.9987 for aflatoxin B1, G1, B2, and G2 respectively in the range concentration from 1 to 20 ng/g. The quantifier ion selected for the aflatoxin B1, B2, G1 and G2 was m/z 285.1, 259, 243 and 313 respectively. The instrument precision at these quantifier ions also showed satisfied result with %RSD was around 3.4 to 6.8%. The optimized method present in this study can be used for further sample analysis.

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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

PubMed

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

An Efficient Protocol for Preparation of Gallic Acid from Terminalia bellirica (Gaertn.) Roxb by Combination of Macroporous Resin and Preparative High-Performance Liquid Chromatography.

PubMed

Zou, Denglang; Chen, Tao; Chen, Chen; Li, Hongmei; Liu, Yongling; Li, Yulin

2016-08-01

In this article, macroporous resin column chromatography and preparative high-performance liquid chromatography were applied for preparation of gallic acid from Terminalia bellirica (Gaertn.) Roxb. In the first step, six kinds of resins were investigated by adsorption and desorption tests and AB-8 macroporous resin was selected for the enrichment of gallic acid. As a result, 20 g of gallic acid at a purity of 71% could be separated from 100 g of crude extract in which the content of gallic acid was 16.7% and the recovery of gallic acid reached 85.0%. In the second step, preparative high-performance liquid chromatography was selected to purify gallic acid. As a result, 640 mg of gallic acid at a purity of 99.1% was obtained from 1 g of sample in 35 min. The results demonstrated that macroporous resin coupled with preparative high-performance liquid chromatography was suitable for preparation of gallic acid from T. bellirica (Gaertn.) Roxb. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Combination of COFRADIC and high temperature-extended column length conventional liquid chromatography: a very efficient way to tackle complex protein samples, such as serum.

PubMed

Sandra, Koen; Verleysen, Katleen; Labeur, Christine; Vanneste, Lies; D'Hondt, Filip; Thomas, Grégoire; Kas, Koen; Gevaert, Kris; Vandekerckhove, Joël; Sandra, Pat

2007-03-01

The previously reported COmbined FRActional DIagonal Chromatography (COFRA-DIC) methodology, in which a subset of peptides representative for their parent proteins are sorted, is particularly powerful for whole proteome analysis. This peptide-centric technology is built around diagonal chromatography, where peptide separations are crucial. This paper presents high efficiency peptide separations, in which four 250 x 2.1 mm, 5 microm Zorbax 300SB-C18 columns (total length 1 m) were coupled at operating temperatures of 60'C using a dedicated LC oven and conventional LC equipment. The high efficiency separations were combined with the COFRADIC procedure. This extremely powerful combination resulted, for the analysis of serum, in an increase in the uniquely identified peptide sequences by a factor of 2.6, compared to the COFRADIC procedure on a 25 cm column. This is a reflection of the increased peak capacity obtained on the 1 m column, which was calculated to be a factor 2.7 higher than on the 25 cm column. Besides more efficient sorting, less ion suppression was noticed.

Determination of aflatoxins B1, B2, G1 and G2 in spices using a multifunctional column clean-up.

PubMed

Akiyama, H; Goda, Y; Tanaka, T; Toyoda, M

2001-10-12

A rapid and simple method using a multifunctional column, which contains lipophilic and charged active sites, was developed to analyse aflatoxins B1, B2, G1 and G2 in various spices, such as red pepper and nutmeg. After extraction by acetonitrile:water (9:1) and clean-up using MultiSep #228 column, the aflatoxins and aflatoxin-TFA derivatives are determined using LC with fluorescence detection. Recoveries of each aflatoxin B1, B2, G1 and G2 spiked to red pepper, white pepper, black pepper, nutmeg and tear grass at the level of 10 ng/g were over 80-85% in all instances. The minimum detectable concentration for aflatoxins in red pepper was 0.5 ng/g.

Freeze drying for gas chromatography stationary phase deposition

DOEpatents

Sylwester, Alan P [Livermore, CA

2007-01-02

The present disclosure relates to methods for deposition of gas chromatography (GC) stationary phases into chromatography columns, for example gas chromatography columns. A chromatographic medium is dissolved or suspended in a solvent to form a composition. The composition may be inserted into a chromatographic column. Alternatively, portions of the chromatographic column may be exposed or filled with the composition. The composition is permitted to solidify, and at least a portion of the solvent is removed by vacuum sublimation.

Pressure-dependent boron isotopic fractionation observed by column chromatography

NASA Astrophysics Data System (ADS)

Musashi, M.; Oi, T.; Matsuo, M.; Nomura, M.

2007-12-01

Boron isotopic fractionation factor ( S ) between boron taken up in strongly basic anion exchange resin and boron in aqueous solution was determined by breakthrough column chromatography at 5 and 17 MPa at 25°C, using 0.1 mmol/L boric acid solution as feed solution. The S values obtained were 1.018 and 1.012, respectively, which were smaller than the value reported by using the same chromatographic method at atmospheric pressure at 25°C with the boron concentration of 10 mmol/L, but were larger than the values at the same condition with much higher concentration of 100 and 501 mmol/L, indicating that borate-polymerization reducing the isotopic fractionation was negligible. However, calculations based on the theory of isotope distribution between two phases estimated that 21% (5MPa) and 47% (17MPa) of boron taken up in the resin phase was in the three-coordinated B(OH)3-form, instead of in the four-coordinated B(OH)4--form, at high pressures even with the very diluted solution. We discussed this discrepancy by introducing (1) hydration or (2) a partial molar volume difference between isotopic molecules. It was inferred that borate ions were partially dehydrated upon transfer from the solution phase to the resin phase at high pressures, which resulted in smaller S values compared with those at the atmospheric pressure. Alternatively, it was likely that the S value decreased with increasing pressure, because the difference of the partial isotopic molar volumes between 10B(OH)3 and 11B(OH)3 was larger than that between 10B(OH)4- and 11B(OH)4-. If either will be the case, the influence of a pressure upon the isotope effect may not be negligible for boron isotopic exchange equilibrium. This knowledge is crucial for the principle of the boron isotopic pH-metry reconstructing a chemical variation at the paleo-deep oceanic environment where the early life may have been evolved.

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.

Approximate transient and long time limit solutions for the band broadening induced by the thin sidewall-layer in liquid chromatography columns.

PubMed

Broeckhoven, Ken; Desmet, Gert

2007-11-16

Using a combination of both analytical and numerical techniques, approximate analytical expressions have been established for the transient and long time limit band broadening, originating from the presence of a thin disturbed sidewall layer in liquid chromatography columns, including packed, monolithic as well as microfabricated columns. The established expressions can be used to compare the importance of a thin disturbed sidewall layer with that of other radial heterogeneity effects (such as transcolumn packing density variations due to the relief of packing stresses). The expressions are independent of the actual velocity profile inside the layer as long as the disturbed sidewall layer occupies less than 2.5% of the column width.

High-throughput sample preparation and simultaneous column regeneration liquid chromatography-tandem mass spectrometry method for determination of nitrogen mustard metabolites in human urine.

PubMed

Reddy, Muntha K; Mills, Grier; Nixon, Christopher; Wyatt, Shane A; Croley, Timothy R

2011-08-15

Nitrogen mustards (NMs) are known to have DNA alkylation and strong vesicant properties. Their availability to terrorist organizations makes them a potential choice for chemical attacks on civilian populations. After an exposure, it is difficult to measure NMs directly because of their rapid metabolism in the human body. Therefore to determine an individual's level of exposure to NMs, it is necessary to analyze for NM metabolites being excreted by the body. The metabolites of NMs are generated by a hydrolysis reaction, and are easily detectable by liquid chromatography tandem mass spectrometry (LC-MS/MS). This work is focused on the development of a high-throughput assay for the quantitation of N-ethyldiethanolamine (EDEA) and N-methyldiethanolamine (MDEA) metabolites of bis (2-chloroethyl) ethylethanamine (HN1) and bis (2-chloroethyl) methylethanamine (HN2), respectively. The method uses automated 96-well plate sample preparation of human urine samples and a 2-position 10-port switching valve to allow for simultaneous regeneration of the liquid chromatography (LC) columns. Using this method, over 18 h was saved through the reduction of sample preparation and analysis time when compared to a conventional method for 96 samples. The validated method provided excellent accuracy for both EDEA (100.9%) and MDEA (100.6%) with precision better than 5.27% for each analyte. Copyright © 2011 Elsevier B.V. All rights reserved.

[Rapid determination of trace iodate using monolithic column ion-pair chromatography coupled with direct conductivity detection].

PubMed

Liu, Yuzhen; Yu, Hong; Li, Siwen

2011-10-01

A method was developed on a monolithic column for the fast determination of trace iodate (IO(3)- ) by ion-pair chromatography with direct conductivity detection. The analytes were separated using a mobile phase of tetrabutylammonium hydroxide (TBA)-phthalic acid-acetonitrile on a reversed-phase silica-based monolithic column. The effects of eluent, flow rate and column temperature on the retention of iodate were investigated. The optimized chromatographic conditions for the determination of the anion were as follows: 0. 25 mmol/L TBA-0. 18 mmol/L phthalic acid-3% acetonitrile (pH 5.5) as mobile phase, a flow rate of 4.0 mL/min and a column temperature of 30 degrees C. Under the optimal conditions, retention time of iodate was less than 0. 5 min and the baseline separation of iodate was achieved without any interference by other anions (Cl-, NO , SO4(2)-, I- ). The detection limit (S/N= 3) was 0.36 mg/L for IO(3)- . Relative standard deviation (RSD, n = 5) of chromatographic peak area and retention time were 0. 35% and 0. 28%, respectively. The proposed method was applied to the determination of trace iodate in iodized medicine. The spiked recovery of iodate was 96. 4%. The method is rapid, simple, accurate, reliable, and practical.

Evaluation of ionic liquid stationary phases for one dimensional gas chromatography-mass spectrometry and comprehensive two dimensional gas chromatographic analyses of fatty acids in marine biota.

PubMed

Gu, Qun; David, Frank; Lynen, Frédéric; Vanormelingen, Pieter; Vyverman, Wim; Rumpel, Klaus; Xu, Guowang; Sandra, Pat

2011-05-20

Ionic liquid stationary phases were tested for one dimensional gas chromatography-mass spectrometry (GC-MS) and comprehensive two dimensional gas chromatography (GC×GC) of fatty acid methyl esters from algae. In comparison with polyethylene glycol and cyanopropyl substituted polar stationary phases, ionic liquid stationary phases SLB-IL 82 and SLB-IL 100 showed comparable resolution, but lower column bleeding with MS detection, resulting in better sensitivity. The selectivity and polarity of the ionic liquid phases are similar to a highly polar biscyanopropyl-silicone phase (e.g. HP-88). In GC×GC, using an apolar polydimethyl siloxane×polar ionic liquid column combination, an excellent group-type separation of fatty acids with different carbon numbers and number of unsaturations was obtained, providing information that is complementary to GC-MS identification. Copyright © 2011 Elsevier B.V. All rights reserved.

Simple determination of aflatoxins in rice by ultra-high performance liquid chromatography coupled to chemical post-column derivatization and fluorescence detection.

PubMed

Huertas-Pérez, José Fernando; Arroyo-Manzanares, Natalia; Hitzler, Dominik; Castro-Guerrero, Francisco Germán; Gámiz-Gracia, Laura; García-Campaña, Ana M

2018-04-15

A fast and simple analytical method was developed and characterized for the determination of aflatoxins (B 1 , B 2 , G 1 and G 2 ) in rice. The procedure is based on a simple solid-liquid extraction without further clean-up, and analysis by ultra-high performance liquid chromatography coupled with fluorescence detection. Fluorescence emission of aflatoxins B 1 and G 1 was enhanced by post-column chemical derivatization using pyridinium bromide perbromide. The analytical method was satisfactorily characterized in white and brown rice. Under optimum conditions, external calibration in solvent could be used for quantification purposes and limits of quantification were below the maximum contents established by the European Union regulation for these contaminants/commodity group combination (0.07-0.14 µg/kg for white rice and 0.20-0.28 µg/kg for brown rice). Recovery studies carried out at three different concentration levels (0.5, 2 and 5 µg/kg) showed values in the range of 84.5-105.3%, and RSDs ≤ 5%. Copyright © 2017 Elsevier Ltd. All rights reserved.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

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…

Rapid amino acid quantitation with pre-column derivatization; ultra-performance reverse phase liquid chromatography and single quadrupole mass spectrometry.

PubMed

Pretorius, Carel J; McWhinney, Brett C; Sipinkoski, Bilyana; Wilce, Alice; Cox, David; McWhinney, Avis; Ungerer, Jacobus P J

2018-03-01

We optimized a quantitative amino acid method with pre-column derivatization, norvaline (nva) internal standard and reverse phase ultra-performance liquid chromatography by replacing the ultraviolet detector with a single quadrupole mass spectrometer (MS nva ). We used 13 C 15 N isotopically labeled amino acid internal standards and a C18 column with 1.6μm particles to optimize the chromatography and to confirm separation of isobaric compounds (MS lis ). We compared the analytical performance of MS nva with MS lis and the original method (UV nva ) with clinical samples. The chromatography time per sample of MS nva was 8min, detection capabilities were <1μmol/L for most components, intermediate imprecisions at low concentrations were <10% and there was negligible carryover. MS nva was linear up to a total amino acid concentration in a sample of approximately 9500μmol/L. The agreements between most individual amino acids were satisfactory compared to UV nva with the latter prone to outliers and suboptimal quantitation of urinary arginine, aspartate, glutamate and methionine. MS nva reliably detected argnininosuccinate, β-alanine, citrulline and cysteine-s-sulfate. MS nva resulted in a more than fivefold increase in operational efficiency with accurate detection of amino acids and metabolic intermediates in clinical samples. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Bonded-phase extraction column isolation of organic compounds in groundwater at a hazardous waste site

USGS Publications Warehouse

Rostad, C.E.; Pereira, W.E.; Ratcliff, S.M.

1984-01-01

A procedure for isolation of hazardous organic compounds from water for gas chromatography/mass spectrometry analysis Is presented and applied to creosote- and pentachlorophenol-contaminated groundwater resulting from wood-treatment processes. This simple procedure involved passing a 50-100-mL sample through a bonded-phase extraction column, eluting the trapped organic compounds from the column with 2-4 mL of solvent, and evaporating the sample to 100 ??L with a stream of dry nitrogen, after which the sample was ready for gas chromatography/mass spectrometry analysis. Representative compounds indicative of creosote contamination were used for recovery and precision studies from the cyclohexyl-bonded phase. Recovery of these compounds from n-octyl-, n-octadecyl-, cyclohexyl-, and phenyl-bonded phases was compared. The bonded phase that exhibited the best recovery and least bias toward acidic or basic cmpounds was the n-octadecyl phase. Detailed compound Identification Is given for compounds Isolated from creosote- and pentachlorophenol-contaminated groundwater using the cyclohexyl-bonded phase.

Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column, and nanoelectrospray tip.

PubMed

Yin, Hongfeng; Killeen, Kevin; Brennen, Reid; Sobek, Dan; Werlich, Mark; van de Goor, Tom

2005-01-15

Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient.

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

PubMed

Bereli, Nilay; Ertürk, Gizem; Tümer, M Aşkin; Say, Ridvan; Denizli, Adil

2013-05-01

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.

The design of a new concept chromatography column.

PubMed

Camenzuli, Michelle; Ritchie, Harald J; Ladine, James R; Shalliker, R Andrew

2011-12-21

Active Flow Management is a new separation technique whereby the flow of mobile phase and the injection of sample are introduced to the column in a manner that allows migration according to the principles of the infinite diameter column. A segmented flow outlet fitting allows for the separation of solvent or solute that elutes along the central radial section of the column from that of the sample or solvent that elutes along the wall region of the column. Separation efficiency on the analytical scale is increased by 25% with an increase in sensitivity by as much as 52% compared to conventional separations.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Methods of analysis by the U. S. Geological Survey National Water Quality Laboratory - determination of organonitrogen herbicides in water by solid-phase extraction and capillary-column gas chromatography/mass spectrometry with selected-ion monitoring

USGS Publications Warehouse

Sandstrom, Mark W.; Wydoski, Duane S.; Schroeder, Michael P.; Zamboni, Jana L.; Foreman, William T.

1992-01-01

A method for the isolation of organonitrogen herbicides from natural water samples using solid-phase extraction and analysis by capillary-column gas chromatography/mass spectrometry with selected-ion monitoring is described. Water samples are filtered to remove suspended particulate matter and then are pumped through disposable solid-phase extraction cartridges containing octadecyl-bonded porous silica to remove the herbicides. The cartridges are dried using carbon dioxide, and adsorbed herbicides are removed from the cartridges by elution with 1.8 milliliters of hexaneisopropanol (3:1). Extracts of the eluants are analyzed by capillary-column gas chromatography/mass spectrometry with selected-ion monitoring of at least three characteristic ions. The method detection limits are dependent on sample matrix and each particular herbicide. The method detection limits, based on a 100-milliliter sample size, range from 0.02 to 0.25 microgram per liter. Recoveries averaged 80 to 115 percent for the 23 herbicides and 2 metabolites in 1 reagent-water and 2 natural-water samples fortified at levels of 0.2 and 2.0 micrograms per liter.

An integrated process for the recovery of high added-value compounds from olive oil using solid support free liquid-liquid extraction and chromatography techniques.

PubMed

Angelis, Apostolis; Hamzaoui, Mahmoud; Aligiannis, Nektarios; Nikou, Theodora; Michailidis, Dimitris; Gerolimatos, Panagiotis; Termentzi, Aikaterini; Hubert, Jane; Halabalaki, Maria; Renault, Jean-Hugues; Skaltsounis, Alexios-Léandros

2017-03-31

An integrated extraction and purification process for the direct recovery of high added value compounds from extra virgin olive oil (EVOO) is proposed by using solid support free liquid-liquid extraction and chromatography techniques. Two different extraction methods were developed on a laboratory-scale Centrifugal Partition Extractor (CPE): a sequential strategy consisting of several "extraction-recovery" cycles and a continuous strategy based on stationary phase co-current elution. In both cases, EVOO was used as mobile phase diluted in food grade n-hexane (feed mobile phase) and the required biphasic system was obtained by adding ethanol and water as polar solvents. For the sequential process, 17.5L of feed EVOO containing organic phase (i.e. 7L of EVOO treated) were extracted yielding 9.5g of total phenolic fraction corresponding to a productivity of 5.8g/h/L of CPE column. Regarding the second approach, the co-current process, 2L of the feed oil phase (containing to 0.8L of EVOO) were treated at 100mL/min yielding 1.03g of total phenolic fraction corresponding to a productivity of 8.9g/h/L of CPE column. The total phenolic fraction was then fractionated by using stepwise gradient elution Centrifugal Partition Chromatography (CPC). The biphasic solvent systems were composed of n-hexane, ethyl acetate, ethanol and water in different proportions (X/Y/2/3, v/v). In a single run of 4h on a column with a capacity of 1L, 910mg of oleocanthal, 882mg of oleacein, 104mg of hydroxytyrosol were successfully recovered from 5g of phenolic extract with purities of 85%, 92% and 90%, respectively. CPC fractions were then submitted to orthogonal chromatographic steps (adsorption on silica gel or size exclusion chromatography) leading to the isolation of additional eleven compounds belonging to triterpens, phenolic compounds and secoiridoids. Among them, elenolic acid ethylester was found to be new compound. Thin Layer Chromatography (TLC), Nuclear magnetic Resonance (NMR) and

Estimation of soil organic partition coefficients: from retention factors measured by soil column chromatography with water as eluent.

PubMed

Xu, Feng; Liang, Xinmiao; Lin, Bingcheng; Schramm, Karl-Werner; Kettrup, Antonius

2002-08-30

The retention factors (k) of 104 hydrophobic organic chemicals (HOCs) were measured in soil column chromatography (SCC) over columns filled with three naturally occurring reference soils and eluted with Milli-Q water. A novel method for the estimation of soil organic partition coefficient (Koc) was developed based on correlations with k in soil/water systems. Strong log Koc versus log k correlations (r>0.96) were found. The estimated Koc values were in accordance with the literature values with a maximum deviation of less than 0.4 log units. All estimated Koc values from three soils were consistent with each other. The SCC approach is promising for fast screening of a large number of chemicals in their environmental applications.

A narrow open tubular column for high efficiency liquid chromatographic separation

DOE PAGES

Chen, Huang; Yang, Yu; Qiao, Zhenzhen; ...

2018-01-01

We report a great feature of open tubular liquid chromatography when it is run using an extremely narrow ( e.g. , 2 μm inner diameter) open tubular column: more than 10 million plates per meter can be achieved in less than 10 min and under an elution pressure of ca. 20 bar.

A narrow open tubular column for high efficiency liquid chromatographic separation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Huang; Yang, Yu; Qiao, Zhenzhen

We report a great feature of open tubular liquid chromatography when it is run using an extremely narrow ( e.g. , 2 μm inner diameter) open tubular column: more than 10 million plates per meter can be achieved in less than 10 min and under an elution pressure of ca. 20 bar.

Simple determination of L-hydroxyproline in idiopathic pulmonary fibrosis lung tissues of rats using non-extractive high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization with novel synthetic 9-acetylimidazol-carbazole.

PubMed

Ren, Yan; Zhao, Juanjuan; Shi, Yanan; Chen, Caiyun; Chen, Xiangming; Lv, Changjun

2017-08-05

L-Hydroxyproline (L-Hyp) is an important biomarker for idiopathic pulmonary fibrosis (IPF). The quantitative methods based on high-performance liquid chromatography coupled with fluorescence detection after pre-column derivatization typically requires complicated derivatization conditions and obtains unstable derivatives. Here, a novel derivatization reagent, 9-acetylimidazol-carbazole, was synthesized for the first time to efficiently and rapidly label the amino groups of L-Hyp. The high-performance liquid chromatography method with pre-column derivatization was performed on an Agilent ZORBAX SB-C 18 column (4.6×250mm, 5μm). The product was measured using fluorescence detection at excitation and emission wavelengths of 232 and 370nm, respectively. The method was validated in specificity, linearity, limit of detection (66.7 fmol), limit of quantification (333.3fmol), intra-day precision (0.75%), inter-day precision (3.82%), stability (3.15%), and recovery (90.7-109.4%). The validated method was successfully applied to the determination of L-Hyp in the lung tissues of healthy and IPF rats. The results showed that the concentration of L-Hyp (3.64mg/g) in the IPF model was significantly higher than the concentration (2.33mg/g) in the healthy control group with P<0.01. This is a new method for the determination of L-Hyp and can be used for other amino acid-related studies in the future. Copyright © 2017. Published by Elsevier B.V.

A simple subcritical chromatographic test for an extended ODS high performance liquid chromatography column classification.

PubMed

Lesellier, Eric; Tchapla, Alain

2005-12-23

This paper describes a new test designed in subcritical fluid chromatography (SFC) to compare the commercial C18 stationary phase properties. This test provides, from a single analysis of carotenoid pigments, the absolute hydrophobicity, the silanol activity and the steric separation factor of the ODS stationary phases. Both the choice of the analytical conditions and the validation of the information obtained from the chromatographic measurements are detailed. Correlations of the carotenoid test results with results obtained from other tests (Tanaka, Engelhard, Sander and Wise) performed both in SFC and HPLC are discussed. Two separation factors, calculated from the retention of carotenoid pigments used as probe, allowed to draw a first classification diagram. Columns, which present identical chromatographic behaviors are located in the same area on this diagram. This location can be related to the stationary phase properties: endcapping treatments, bonding density, linkage functionality, specific area or silica pore diameter. From the first classification, eight groups of columns are distinguished. One group of polymer coated silica, three groups of polymeric octadecyl phases, depending on the pore size and the endcapping treatment, and four groups of monomeric stationary phases. An additional classification of the four monomeric groups allows the comparison of these stationary phases inside each group by using the total hydrophobicity. One hundred and twenty-nine columns were analysed by this simple and rapid test, which allows a comparison of columns with the aim of helping along their choice in HPLC.

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...

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

Determination of phenolic acids and flavonoids in Taraxacum formosanum Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization technique.

PubMed

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 C(18) cartridge. A total of 29 compounds were separated within 68 min by employing a Gemini C(18) 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.

An improved liquid chromatography/tandem mass spectrometry method for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA samples using immunoaffinity column purification.

PubMed

Singh, Rajinder; McEwan, Michael; Lamb, John H; Santella, Regina M; Farmer, Peter B

2003-01-01

The analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents an important biomarker of oxidative stress. A sensitive method for the detection of 8-oxodG in DNA samples has been developed that utilizes immunoaffinity column purification of 8-oxodG followed by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) multiple reaction monitoring (MRM) mode analysis. An internal standard of stable-isotopically labelled 8-oxodG containing [(15)N(5)] was added prior to the enzymatic digestion of DNA to deoxynucleosides, which was then subjected to immunoaffinity column purification followed by microbore positive ion LC/MS/MS MRM. The 8-oxo-7,8-dihydroguanine (8-oxoG) base product ion at m/z 168 was monitored following cleavage of the glycosidic bond of the 8-oxodG [M+H](+) ion at m/z 284. Similar determinations were made for [(15)N(5)]8-oxodG by monitoring the [(15)N(5)]8-oxoG base product ion at m/z 173 formed from the [M+H](+) ion at m/z 289. The introduction of the immunoaffinity column purification step into the method represents a significant improvement for the accurate determination of 8-oxodG since all artefactual peaks that are observed following the direct injection of digested DNA onto the LC/MS/MS system are removed. The identity of these artefactual peaks has been confirmed to be 2'-deoxyguanosine (dG), thymidine (dT) and 2'-deoxyadenosine (dA). The presence of these artefactual peaks in MRM mode analysis can be explained as a consequence of a concentration effect due to their considerably higher relative abundance in DNA compared to 8-oxodG. The highest signal intensity was observed for the artefactual peak for dA due to the fact that the adenine base formed an adduct with methanol, which is a constituent of the mobile phase. The resulting [M+H](+) ion at m/z 284 (dA m/z 252 + CH(3)OH m/z 32) gave rise to a product ion at m/z 168 following the loss of deoxyribose in MRM mode analysis. Control calf thymus DNA was digested to

Preparative isolation of alkaloids from Dactylicapnos scandens using pH-zone-refining counter-current chromatography by changing the length of the separation column.

PubMed

Wang, Xiao; Dong, Hongjing; Yang, Bin; Liu, Dahui; Duan, Wenjuan; Huang, Luqi

2011-12-01

pH-Zone-refining counter-current chromatography was successfully applied for the preparative separation of alkaloids from Dactylicapnos scandens. The two-phase solvent system was composed of petroleum ether-ethyl acetate-methanol-water (3:7:1:9, v/v), where 20 mM of triethylamine (TEA) was added to the upper phase as a retainer and 5 mM of hydrochloric acid (HCl) to the aqueous phase as an eluter. In this experiment, the apparatus with an adjustable length of the separation column was used for the separation of alkaloids from D. scandens and the resolution of the compounds can be remarkably improved by increasing the length of the separation column. As a result, 70 mg protopin, 30 mg (+) corydine, 120 mg (+) isocorydine and 40 mg (+) glaucine were obtained from 1.0 g of the crude extracts and each with 99.2%, 96.5%, 99.3%, 99.5% purity as determined by HPLC. The chemical structures of these compounds were confirmed by positive ESI-MS and (1)H NMR. Copyright © 2011 Elsevier B.V. All rights reserved.

Efficiency for unretained solutes in packed column supercritical fluid chromatography. I. Theory for isothermal conditions and correction factors for carbon dioxide.

PubMed

Poe, Donald P

2005-06-17

A general theory for efficiency of nonuniform columns with compressible mobile phase fluids is applied to the elution of an unretained solute in packed-column supercritical fluid chromatography (pSFC). The theoretical apparent plate height under isothermal conditions is given by the Knox equation multiplied by a compressibility correction factor f1, which is equal to the ratio of the temporal-to-spatial average densities of the mobile phase. If isothermal conditions are maintained, large pressure drops in pSFC should not result in excessive efficiency losses for elution of unretained solutes.

Determination of pesticides in composite dietary samples by gas chromatography/mass spectrometry in the selected ion monitoring mode by using a temperature-programmable large volume injector with preseparation column.

PubMed

Rosenblum, L; Hieber, T; Morgan, J

2001-01-01

Use of a temperature-programmable preseparation column in the gas chromatographic (GC) injection port permits determination of a wide range of semi-volatile pesticides including organochlorines, organophosphates, triazines, and anilines in fatty composite dietary samples while reducing sample preparation time and solvent consumption. Dietary samples are mixed with diatomaceous earth and are Soxhlet-extracted with an azeotropic solution of hexane and acetone. Sample preparation uses liquid-liquid partitioning over diatomaceous earth followed by normal phase chromatography over partially deactivated alumina. The final cleanup step occurs in a preseparation column in the GC injector, which is able to perform splitless transfer of the analytes to the analytical column and purge 99% of the high molecular weight residue. Detection is performed by GC/mass spectrometry (MS) in the selected ion monitoring mode. Method detection limits were at or below 2 ng/g for 24 of 35 pesticides studied, with recovery between 70 and 125% for 27 pesticides in samples fortified at 10 ng/g. Recovery was not dependent on fat content when measured in laboratory fortified samples containing 1, 5, and 10% fat by weight. Precision over multiple injections was acceptable, with a relative standard deviation of 2.6-15% for 25 analytes.

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.

Generating multiple independent retention index data in dual-secondary column comprehensive two-dimensional gas chromatography.

PubMed

Bieri, Stefan; Marriott, Philip J

2006-12-01

A method producing simultaneously three retention indexes for compounds has been developed for comprehensive two-dimensional gas chromatography by using a dual secondary column approach (GC x 2GC). For this purpose, the primary flow of the first dimension column was equally diverted into two secondary microbore columns of identical geometry by means of a three-way flow splitter positioned after the longitudinally modulated cryogenic system. This configuration produced a pair of comprehensive two-dimensional chromatograms and generated retention data on three different stationary phases in a single run. First dimension retention indexes were determined on a polar SolGel-Wax column under linear programmed-temperature conditions according to the van den Dool approach using primary alcohol homologues as the reference scale. Calculation of pseudoisothermal retention indexes in both second dimensions was performed on low-polarity 5% phenyl equivalent polysilphenylene/siloxane (BPX5) and 14% cyanopropylphenyl/86% dimethylpolysiloxane (BP10) columns. To construct a retention correlation map in the second dimension separation space upon which KovAts indexes can be derived, two methods exploiting "isovolatility" relationships of alkanes were developed. The first involved 15 sequential headspace samplings of selected n-alkanes by solid-phase microextraction (SPME), with each sampling followed by their injection into the GC at predetermined times during the chromatographic run. The second method extended the second dimension retention map and consisted of repetitive introduction of SPME-sampled alkane mixtures at various isothermal conditions incremented over the temperature program range. Calculated second dimension retention indexes were compared with experimental values obtained in conventional one-dimensional GC. A case study mixture including 24 suspected allergens (i.e., fragrance ingredients) was used to demonstrate the feasibility and potential of retention index

Ion chromatography for determination of nitrite and nitrate in seawater using monolithic ODS columns.

PubMed

Ito, Kazuaki; Takayama, Yohichi; Makabe, Nobuyuki; Mitsui, Ryo; Hirokawa, Takeshi

2005-08-12

A fast and highly sensitive ion chromatographic method using monolithic ODS columns was developed for the determination of nitrite (NO2-) and nitrate (NO3-) in seawater. Two monolithic ODS columns (50 mm x 4.6 mm i.d. + 100 mm x 4.6 mm i.d.) connected in series were coated and equilibrated with 5 mM cetyltrimethylammonium chloride (CTAC) aqueous solution. The column efficiency with 0.5 M NaCl as the mobile phase did not decrease in spite of the increase in flow rate of the mobile phase. Thus, good chromatograms were obtained within 3 minutes for NO2- and NO3 in artificial seawater without interferences by coexisting ions. The detection limit (S/N = 3) with UV detection at 225 nm was 0.8 and 1.6 microg/L for NO2- and NO3-, respectively. The characteristics of the monolithic CTA(+)-coated ODS columns were discussed. The present method was successfully applied to the fast and sensitive determination of NO2- and NO3- in real seawater samples.

Plasma L-ergothioneine measurement by high-performance liquid chromatography and capillary electrophoresis after a pre-column derivatization with 5-iodoacetamidofluorescein (5-IAF) and fluorescence detection.

PubMed

Sotgia, Salvatore; Pisanu, Elisabetta; Pintus, Gianfranco; Erre, Gian Luca; Pinna, Gerard Aime; Deiana, Luca; Carru, Ciriaco; Zinellu, Angelo

2013-01-01

Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%).

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.

A new method for rapid determination of indole-3-carbinol and its condensation products in nutraceuticals using core-shell column chromatography method.

PubMed

Fibigr, Jakub; Šatínský, Dalibor; Havlíková, Lucie; Solich, Petr

2016-02-20

Indole-3-carbinol is a natural glucosinolate known for prevention of human breast, prostate and other types of cancer and it started to be used in commercial preparations, as food supplements. However no analytical method has been proposed for quality control of nutraceuticals with this substance yet. In this paper a new high-performance liquid chromatography (HPLC) method using core-shell column for separation of indole-3-carbinol and its condensation/degradation products was developed and used for the quantitative determination of indole-3-carbinol in nutraceuticals. Separation of indole-3-carbinol, its condensation/degradation products and internal standard ethylparaben was performed on the core-shell column Kinetex 5μ XB-C18 100A (100×4.6mm), particle size 5.0μm, with mobile phase acetonitrile/water according to the gradient program at a flow rate of 1.25mLmin(-1) and at temperature 50°C. The detection wavelength was set at 270nm. Under the optimal chromatographic conditions good linearity of determination was achieved. Available commercial samples of nutraceuticals were extracted with 100% methanol using ultrasound bath. A 5-μL sample volume of the supernatant was directly injected into the HPLC system. The developed method provided rapid and accurate tool for quality control of nutraceuticals based on cruciferous vegetable extracts with indole-3-carbinol content. The presented study showed that the declared content of indole-3-carbinol significantly varied in the different nutraceuticals available on the market. Two analyzed preparations showed the presence of condensation/degradation products of indole-3-carbinol which were not officially declared by the manufacturer. Moreover, further two analyzed nutraceutical preparations showed absolutely no content of declared amount of indole-3-carbinol. Copyright © 2015 Elsevier B.V. All rights reserved.

Counter-current motion in counter-current chromatography.

PubMed

Ito, Yoichiro

2014-12-12

After the CCC2012 meeting, I have received an e-mail regarding the terminology of "Countercurrent Chromatography". It stated that the term "Countercurrent" is a misnomer, because its stationary phase is motionless in the column and that the method should be renamed as liquid-liquid separations or centrifugal separations. However, it was found that these names are already used for various other techniques as found via Google search. The term "Countercurrent Chromatography" was originally made after two preparative methods of Countercurrent distribution and liquid Chromatography, both having no countercurrent motion in the column. However, it is surprising to find that this F1 hybrid method "Countercurrent Chromatography" can clearly exhibit countercurrent motion within the separation column in both hydrodynamic and hydrostatic equilibrium systems. This justifies that "Countercurrent Chromatography" is a proper term for this chromatographic method. Published by Elsevier B.V.

Improved Thermal Modulator for Gas Chromatography

NASA Technical Reports Server (NTRS)

Hasselbrink, Ernest Frederick, Jr.; Hunt, Patrick J.; Sacks, Richard D.

2008-01-01

An improved thermal modulator has been invented for use in a variant of gas chromatography (GC). The variant in question denoted as two-dimensional gas chromatography (2DGC) or GC-GC involves the use of three series-connected chromatographic columns, in the form of capillary tubes coated interiorly with suitable stationary phases (compounds for which different analytes exhibit different degrees of affinity). The two end columns are relatively long and are used as standard GC columns. The thermal modulator includes the middle column, which is relatively short and is not used as a standard GC column: instead, its temperature is modulated to affect timed adsorption and desorption of analyte gases between the two end columns in accordance with a 2DGC protocol.

Boron isotope fractionation in liquid chromatography with boron-specific resins as column packing material

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oi, Takao; Shimazaki, Hiromi; Ishii, Reiko

1997-07-01

Boron-specific resins with n-methyl glucamine as the functional group were used as column packing material of liquid chromatography for boron isotope separation. The shapes of chromatograms in reverse breakthrough experiments were heavily dependent on the pH of the eluents, and there existed a pH value at which a chromatogram of the displacement type was realized nearly ideally. The value of the single-stage separation factor for the boron isotopes varied between 1.010 and 1.022, depending on the temperature and the form of the resins. The existence of the three-coordinate boron species in addition to the four-coordinate species in the resin phasemore » is suggested.« less

Mass: 100 mg, 10 g, 100 g, 1 kg, 10 kg and 50 kg

NASA Astrophysics Data System (ADS)

Kaçmaz, Sevda; Davidson, Stuart

2017-01-01

Bilateral mass comparison of mass standards of 100 mg, 2 g, 20 g, 500 g and 10 kg was carried out in 2011 to 2012 between UME and NPL. UME was the pilot laboratory. For all mass standards, the results show adequate agreement between the laboratories. Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

Development and application of a high-performance liquid chromatography method using monolithic columns for the analysis of ecstasy tablets.

PubMed

Mc Fadden, Kim; Gillespie, John; Carney, Brian; O'Driscoll, Daniel

2006-07-07

A rapid and selective HPLC method using monolithic columns was developed for the separation and quantification of the principal amphetamines in ecstasy tablets. Three monolithic (Chromolith RP18e) columns of different lengths (25, 50 and 100 mm) were assessed. Validation studies including linearity, selectivity, precision, accuracy and limit of detection and quantification were carried out using the Chromolith SpeedROD, RP-18e, 50 mm x 4.6 mm column. Column backpressure and van Deemter plots demonstrated that monolithic columns provide higher efficiency at higher flow rates when compared to particulate columns without the loss of peak resolution. Application of the monolithic column to a large number of ecstasy tablets seized in Ireland ensured its suitability for the routine analysis of ecstasy tablets.

Ultrahigh performance supercritical fluid chromatography of lipophilic compounds with application to synthetic and commercial biodiesel.

PubMed

Ashraf-Khorassani, M; Yang, J; Rainville, P; Jones, M D; Fountain, K J; Isaac, G; Taylor, L T

2015-03-01

Ultrahigh performance supercritical fluid chromatography (UHPSFC) in combination with sub-2μm particles and either diode array ultraviolet (UV), evaporative light scattering, (ELSD), or mass spectrometric (MS) detection has been shown to be a valuable technique for the determination of acylglycerols in soybean, corn, sesame, and tobacco seed oils. Excellent resolution on an un-endcapped single C18 column (3.0mm×150mm) with a mobile phase gradient of acetonitrile and carbon dioxide in as little as 10min served greatly as an improvement on first generation packed column SFC instrumentation. Unlike high resolution gas chromatography and high performance liquid chromatography with mass spectrometric detection, UHPSFC/MS was determined to be a superior analytical tool for both separation and detection of mono-, di-, and tri-acylglycerols as well as free glycerol itself in biodiesel without derivatization. Baseline separation of residual tri-, di-, and mono-acylglycerols alongside glycerol at 0.05% (w/w) was easily obtained employing packed column SFC. The new analytical methodology was applied to both commercial B100 biodiesel (i.e. fatty acid methyl esters) derived from vegetable oil and to an "in-house" synthetic biodiesel (i.e. fatty acid ethyl esters) derived from tobacco seed oil and ethanol both before and after purification via column chromatography on bare silica. Copyright © 2014 Elsevier B.V. All rights reserved.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Preparation, characterization and application of a reversed phase liquid chromatography/hydrophilic interaction chromatography mixed-mode C18-DTT stationary phase.

PubMed

Wang, Qing; Long, Yao; Yao, Lin; Xu, Li; Shi, Zhi-Guo; Xu, Lanying

2016-01-01

A mixed-mode chromatographic stationary phase, C18-DTT (dithiothreitol) silica (SiO2) was prepared through "thiol-ene" click chemistry. The obtained material was characterized by fourier transform infrared spectroscope, nitrogen adsorption analysis and contact angle analysis. Chromatographic performance of the C18-DTT was systemically evaluated by studying the effect of acetonitrile content, pH, buffer concentration of the mobile phase and column temperature. It was demonstrated that the novel stationary phase possessed reversed phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode property. The stop-flow test revealed that C18-DTT exhibited excellent compatibility with 100% aqueous mobile phase. Additionally, the stability and column-to-column reproducibility of the C18-DTT material were satisfactory, with relative standard deviations of retention factor of the tested analytes (verapamil, fenbufen, guanine, tetrandrine and nicotinic acid) in the range of 1.82-3.72% and 0.85-1.93%, respectively. Finally, the application of C18-DTT column was demonstrated in the separation of non-steroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo-analytes and polycyclic aromatic hydrocarbons. It had great resolving power in the analysis of various compounds in HILIC and RPLC chromatographic conditions and was a promising RPLC/HILIC mixed-mode stationary phase. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparison between the intra-particle diffusivity in the hydrophilic interaction chromatography and reversed phase liquid chromatography modes. Impact on the column efficiency.

PubMed

Gritti, Fabrice; Guiochon, Georges

2013-07-05

The effective diffusion coefficients of five low molecular weigh compounds (naphthalene, uracil, uridine, adenosine, and cytosine) were measured at room temperature in a 4.6mm×100mm column packed with 3.5μm XBridge HILIC particles. The mobile phase was an acetonitrile-water mixture (92.5/7.5, v/v) containing 10mM ammonium acetate and 0.02% acetic acid. Using a physically reliable model of effective diffusion in binary composite media (Torquato's model), accurate estimates of the intra-particle diffusivities in the HILIC particles were obtained as a function of the retention of these analytes. The HILIC diffusion coefficients were compared to those previously obtained for endcapped RPLC-C18 particles (5.0μm Gemini-C18). The experimental results confirm that adsorption sites are not localized in RPLC whereas they are so in the HILIC mode. In contrast to RPLC columns, HILIC columns provide longitudinal diffusion B/u terms that increase very little with increasing retention factors. This confirms the absence of surface diffusion in HILIC. The impact of intra-particle diffusivity on the column efficiency was projected in HILIC and RPLC on the basis of the measured intra-particle diffusivities and on the well established theory of band broadening in particulate columns. Accordingly, RPLC columns generate short-range eddy dispersion and solid-liquid mass transfer resistance Cu terms that increase less than do HILIC column with increasing retention factors. The HETP contribution caused by the trans-column structure heterogeneity is smaller in the HILIC than in the RPLC modes because the transverse excursion length is smaller in HILIC. Even though the overall column efficiencies are comparable in HILIC and RPLC, this study shows that the individual mass transfer phenomena are inherently different in the HILIC and the RPLC retention modes. Copyright © 2013 Elsevier B.V. All rights reserved.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

Ultra high-performance liquid chromatography of porphyrins in clinical materials: column and mobile phase selection and optimisation.

PubMed

Benton, Christopher M; Lim, Chang Kee; Moniz, Caje; Jones, Donald J L

2012-06-01

Ultra high-performance liquid chromatographic (UHPLC) systems on columns packed with materials ranging from 1.9 to 2.7 µm average particle size were assessed for the fast and sensitive analysis of porphyrins in clinical materials. The fastest separation was achieved on an Agilent Poroshell C(18) column (2.7 µm particle size, 50 × 4.6 mm i.d.), followed by a Thermo Hypersil Gold C(18) column (1.9 µm particle size, 50 × 2.1 mm i.d.) and the Thermo Hypersil BDS C(18) column (2.4 µm particle size, 100 × 2.1 mm i.d.). All columns required a mobile phase containing 1 m ammonium acetate buffer, pH 5.16, with a mixture of acetonitrile and methanol as the organic modifiers for optimum resolution of the type I and III isomers, particularly for uroporphyrin I and III isomers. All UHPLC columns were suitable and superior to conventional HPLC columns packed with 5 µm average particle size materials for clinical sample analysis. Copyright © 2011 John Wiley & Sons, Ltd.

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 Central

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. PMID:23894273

Separation of polychlorinated biphenyls by fast gas chromatography.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alvarado, J. S.; Silzer, J.; Lemley, F.

1997-12-01

The separation of commercially available polychlorinated biphenyls (PCBs) by fast gas chromatography (fast GC) has been studied. Aroclor 1254 was separated by using two column types: DB-1 and SPB-608. The fast GC used a split-splitless injector to introduce the sample, followed by a cold trap at -90 C to focus the sample. Rapid heating was used to introduce the sample into the short chromatographic column to decrease band broadening. Hydrogen was the carrier gas at velocities of 100 to 125 cm s-1. Analyses were performed by using an electron capture detector (ECD). Separation was achieved with both columns in lessmore » than 6 min. With the greatly shortened run times, reproducibility can be tested quickly and consequently with low cost.« less

Broad-spectrum immunoaffinity cleanup for the determination of aflatoxins B1, B2, G1, G2, M1, M2 in Ophiocordyceps sinensis and its pharmaceutical preparations by ultra performance liquid chromatography tandem mass spectrometry.

PubMed

Sun, Shujuan; Xie, Jie; Peng, Tao; Shao, Bing; Zhu, Kui; Sun, Yuanze; Yao, Kai; Gu, Qiang; Zhang, Jing; Fan, Chunlin; Chen, Ying; Jiang, Haiyang

2017-11-15

An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of aflatoxins B 1 , B 2 , G 1 , G 2 , M 1 and M 2 (AFB 1 , AFB 2 , AFG 1 , AFG 2 , AFM 1 and AFM 2 ) in Ophiocordyceps sinensis and its pharmaceutical preparations. A rapid and reliable immunoaffinity column containing a broad-spectrum monoclonal antibody for six aflatoxins was used for sample cleanup. Under the optimized conditions, the home-made immunoaffinity column capacity were about 315, 319, 292, 102, 444 and 369ng/mL gel for AFB 1 , AFB 2 , AFG 1 , AFG 2 , AFM 1 and AFM 2 , respectively. Recoveries for all tested aflatoxins ranged from 79.28% to 103.42% with relative standard deviation less than 8%. The limits of quantitation were in the range of 0.008-0.045μg/kg. Among 31 real samples analyzed, one sample was contaminated with AFB 1 , AFB 2 and AFM 1 at levels of 0.483, 0.068 and 0.104μg/kg, respectively. The established method is simple, accurate, and can be effectively used to determine the aflatoxins in Ophiocordyceps sinensis and its pharmaceutical preparations. Copyright © 2017 Elsevier B.V. All rights reserved.

Supercritical Fluid Chromatography of Drugs: Parallel Factor Analysis for Column Testing in a Wide Range of Operational Conditions

PubMed Central

Al-Degs, Yahya; Andri, Bertyl; Thiébaut, Didier; Vial, Jérôme

2017-01-01

Retention mechanisms involved in supercritical fluid chromatography (SFC) are influenced by interdependent parameters (temperature, pressure, chemistry of the mobile phase, and nature of the stationary phase), a complexity which makes the selection of a proper stationary phase for a given separation a challenging step. For the first time in SFC studies, Parallel Factor Analysis (PARAFAC) was employed to evaluate the chromatographic behavior of eight different stationary phases in a wide range of chromatographic conditions (temperature, pressure, and gradient elution composition). Design of Experiment was used to optimize experiments involving 14 pharmaceutical compounds present in biological and/or environmental samples and with dissimilar physicochemical properties. The results showed the superiority of PARAFAC for the analysis of the three-way (column × drug × condition) data array over unfolding the multiway array to matrices and performing several classical principal component analyses. Thanks to the PARAFAC components, similarity in columns' function, chromatographic trend of drugs, and correlation between separation conditions could be simply depicted: columns were grouped according to their H-bonding forces, while gradient composition was dominating for condition classification. Also, the number of drugs could be efficiently reduced for columns classification as some of them exhibited a similar behavior, as shown by hierarchical clustering based on PARAFAC components. PMID:28695040

Simultaneous analysis of tert-butylhydroquinone, tert-butylquinone, butylated hydroxytoluene, 2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, α-tocopherol, γ-tocopherol, and δ-tocopherol in edible oils by normal-phase high performance liquid chromatography.

PubMed

Li, Jun; Bi, Yanlan; Sun, Shangde; Peng, Dan

2017-11-01

A normal-phase high performance liquid chromatography method for the simultaneous determination of tert-butylhydroquinone, tert-butylquinone, butylated hydroxytoluene, 2-tert-butyl-4-hydroxyanisole, 3-tert-butyl-4-hydroxyanisole, α-tocopherol, γ-tocopherol, and δ-tocopherol in edible oils was investigated. A silica column was used to separate the analytes with the gradient elution. An ultraviolet-visible detector was set at dual wavelengths mode (280 and 310nm). The column temperature was 30°C. The analytes were directly extracted with methanol. Results showed that the normal-phase high performance liquid chromatography method performed well with wide liner ranges (0.10∼500.00μg/mL, R 2 >0.9998), low limits of detection and quantitation (below 0.40 and 1.21μg/mL, respectively), and good recoveries (81.38∼102.34% in soybean oils and 83.03∼100.79% in lard, respectively). The reduction of tert-butylquinone caused by the reverse-phase high performance liquid chromatography during the injection was avoided with the current normal-phase method. The two isomers of butylated hydroxyanisole can also be separated with good resolution. Copyright © 2017 Elsevier Ltd. All rights reserved.

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…

Methods of analysis by the U.S. Geological Survey National Water Quality Laboratory; determination of semivolatile organic compounds in bottom sediment by solvent extraction, gel permeation chromatographic fractionation, and capillary-column gas chromatography/mass spectrometry

USGS Publications Warehouse

Furlong, E.T.; Vaught, D.G.; Merten, L.M.; Foreman, W.T.; Gates, Paul M.

1996-01-01

A method for the determination of 79 semivolatile organic compounds (SOCs) and 4 surrogate compounds in soils and bottom sediment is described. The SOCs are extracted from bottom sediment by solvent extraction, followed by partial isolation using high-performance gel permeation chromatography (GPC). The SOCs then are qualitatively identified and quantitative concentrations determined by capillary-column gas chromatography/mass spectrometry (GC/MS). This method also is designed for an optional simultaneous isolation of polychlorinated biphenyls (PCBs) and organochlorine (OC) insecticides, including toxaphene. When OCs and PCBs are determined, an additional alumina- over-silica column chromatography step follows GPC cleanup, and quantitation is by dual capillary- column gas chromatography with electron-capture detection (GC/ECD). Bottom-sediment samples are centrifuged to remove excess water and extracted overnight with dichloromethane. The extract is concentrated, centrifuged, and then filtered through a 0.2-micrometer polytetrafluoro-ethylene syringe filter. Two aliquots of the sample extract then are quantitatively injected onto two polystyrene- divinylbenzene GPC columns connected in series. The SOCs are eluted with dichloromethane, a fraction containing the SOCs is collected, and some coextracted interferences, including elemental sulfur, are separated and discarded. The SOC-containing GPC fraction then is analyzed by GC/MS. When desired, a second aliquot from GPC is further processed for OCs and PCBs by combined alumina-over-silica column chromatography. The two fractions produced in this cleanup then are analyzed by GC/ECD. This report fully describes and is limited to the determination of SOCs by GC/MS.

Acaricidal activity of petroleum ether extract of neem (Azadirachta indica) oil and its four fractions separated by column chromatography against Sarcoptes scabiei var. cuniculi larvae in vitro.

PubMed

Deng, Yunxia; Shi, Dongxia; Yin, Zhongqiong; Guo, Jianhong; Jia, Renyong; Xu, Jiao; Song, Xu; Lv, Cheng; Fan, Qiaojia; Liang, Xiaoxia; Shi, Fei; Ye, Gang; Zhang, Wei

2012-04-01

The petroleum ether extract of neem oil and its four fractions separated by column chromatography was diluted at different concentrations with liquid paraffin. The acaricidal bioassay was conducted using a dipping method. The results indicated that the median lethal concentration (LC50) of the petroleum ether extract (at the concentration of 500.0ml/l) was 70.9ml/l, 24h after treatment. At concentrations of 500.0, 250.0, 125.0, 62.5 and 31.2ml/l, the median lethal times (LT50) of the petroleum ether extract were 8.7, 8.8, 10.8, 11.5 and 13.1h, respectively. Thin-layer chromatography (TLC) showed that the petroleum ether extract of neem oil separated into four fractions (F1-F4). Acaricidal activity of 68.3% and 100.0% in the F2 and F4 was confirmed. These results suggest that petroleum ether extracts of neem oil and its four fractions possess useful acaricidal activity in vitro. Copyright © 2012 Elsevier Inc. All rights reserved.

Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection

PubMed Central

Sotgia, Salvatore; Pisanu, Elisabetta; Pintus, Gianfranco; Erre, Gian Luca; Pinna, Gerard Aime; Deiana, Luca; Carru, Ciriaco; Zinellu, Angelo

2013-01-01

Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%). PMID:23922985

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. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

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. 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. 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 development other of HIC-compatible protein

Rapid quantification of iodopropynyl butylcarbamate as the preservative in cosmetic formulations using high-performance liquid chromatography-electrospray mass spectrometry.

PubMed

Frauen, M; Steinhart, H; Rapp, C; Hintze, U

2001-07-01

A simple, rapid and reproducible method for identification and quantification of iodopropynyl butylcarbamate (IPBC) in different cosmetic formulations is presented. The determination was carried out using a high-performance liquid chromatography (HPLC) procedure on a reversed phase column coupled to a single quadrupole mass spectrometer (MS) via an electrospray ionization (ESI) interface. Detection was performed in the positive selected ion-monitoring mode. In methanol/water extracts from different cosmetic formulations a detection limit between 50 and 100 ng/g could be achieved. A routine analytical procedure could be set up with good quantification reliability (relative standard deviation between 0.9 and 2.9%).

Preparation and evaluation of a mixed-bed immunoaffinity column for selective purification of sixteen sulfonamides in pork muscle.

PubMed

Li, Yingguo; Chen, Yiqiang; Li, Zhengguo; Zhang, Lei; Li, Xianliang; Xi, Cunxian; Wang, Guomin; Wang, Xiong; Guo, Qi; Li, Na

2012-03-01

This paper describes the preparation of a novel mixed-bed immunoaffinity chromatography (IAC) column by coupling four monoclonal antibodies against different sulfonamides (SAs) to Sepharose 4B. The IAC column can be used to simultaneously extract and purify 16 SAs in pork muscle. The dynamic column capacities for all SAs in mixed standard solution were between 312 and 479 ng/mL gel. After simple extraction and IAC cleanup, the sample solution can be directly injected for liquid chromatography-ultraviolet analysis. The recoveries of SAs from spiked samples at levels of 25, 50 and 100 µg/kg ranged from 83.3 to 103.1% with variation coefficient less than 8.6%. The comparison of IAC with liquid-liquid extraction and solid phase extraction indicated that IAC has better purification effect and needs less organic solution than conventional methods, thus it would be an ideal method for selective purification of SAs in pork muscle.

[Determination of residual solvents in 7-amino-3-chloro cephalosporanic acid by gas chromatography].

PubMed

Ma, Li; Yao, Tong-wei

2011-01-01

To develop a gas chromatography method for determination of residual solvents in 7-amino-3-chloro cephalosporanic acid (7-ACCA). The residual levels of acetone, methanol, dichloromethane, ethyl acetate, isobutanol, pyridine and toluene in 7-ACCA were measured by gas chromatography using Agilent INNOWAX capillary column (30 m × 0.32 mm,0.5 μm). The initial column temperature was 70° maintained for 6 min and then raised (10°C/min) to 160°C for 1 min. Nitrogen gas was used as carrier and FID as detector. The flow of carrier was 1.0 ml/min, the temperature of injection port and detector was 200°C and 250°C, respectively. The limits of detection for acetone, methanol, dichloromethane, ethyl acetate, isobutanol, pyridine, toluene in 7-ACCA were 2.5 μg/ml, 1.5 μg/ml, 15 μg/ml, 2.5 μg/ml, 2.5 μg/ml, 2.5 μg/ml and 11 μg/ml, respectively. Only acetone was detected in the sample, and was less than the limits of Ch.P. The method can effectively detect the residual solvents in 7-ACCA.

The non-modulated transfer of total effluent for serially coupled columns in gas chromatography.

PubMed

Krupcík, Ján; Mydlová-Memersheimerová, Janka; Májek, Pavel; Zapadlo, Michal; Sandra, Pat

2010-03-12

The non-modulated transfer (NMT) of the total effluent of the separation of polychlorinated biphenyls (PCBs) on two columns coupled in series has been studied. A non-polar poly(5%-phenyl-95%-methyl)siloxane column (40 m x 100 microm x 0.1 microm) and a polar 70%-cyanopropyl-polysilphenylene-siloxane column (4 m x 0.1 mm x 0.1 microm) were used as (1)D and (2)D columns, respectively. The effluents from both the (1)D column and the (1)D+(2)D column series were monitored independently by two FIDs. Data from the (1)D and (1)D+(2)D integration reports were used to evaluate the NMT experiment. (1)D retention times, t(R,i,D1), were directly accessible from (1)D integration report while (2)D retention times, t(R,i,B), were calculated for all corresponding peak pairs from (1)D and (1)D+(2)D integration reports as a difference t(R,i,D2)=t(R,i,D1+D2)-t(R,i,D1). Search for corresponding peaks of PCB congeners in the (1)D and (1)D+(2)D chromatograms is elucidated in detail on standard PCB samples and on PCB congeners present in the technical formulation Arochlor 1242. Both retention times (t(R,i,D1) and t(R,i,D2)) as well as peak widths at half height (w(h,i)) and peak heights (h(i)) obtained from integration reports were used to construct 2D and 3D images for PCB NMT separations on serially coupled columns. The performance of the NMT procedure is illustrated by the separation of (i) standard PCB solutions, (ii) a mixture of the 209 PCBs, and (iii) a mixture of Arochlor 1242 and hydrocarbons on the DB-5+BPX-70 column series. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Efficiency gain limits of the parallel segmented inlet and outlet flow concept in analytical liquid chromatography columns suffering from radial transcolumn packing density gradients.

PubMed

Broeckhoven, Ken; Desmet, Gert

2012-10-05

The maximal gain in efficiency that can be expected from the use of the segmented column end fittings that were recently introduced to alleviate the effect of transcolumn packing density gradients has been quantified and generalized using numerical computations of the band broadening process. It was found that, for an unretained compound in a column with a parabolic packing density gradient, the use of a segmented inlet or a segmented outlet allows to eliminate about 60-100% of the plate height contribution (H(tc)) originating from a parabolic transcolumn velocity gradient in a d(c)=4.6 mm column. In a d(c)=2.1 mm column, these percentages change from 10 to 100%. Using a combined segmented in- and outlet, H(tc) can be reduced by about 90-100% (d(c)=4.6 mm column) or 20-100% (d(c)=2.1 mm column). The strong variation of these gain percentages is due to fact that they depend very strongly on the column length and the flow rate. Dimensionless graphs have been established that allow to directly quantify the effect for each specific case. It was also found that, in agreement with one's physical intuition, trans-column velocity profiles that are more flat in the central region benefit more from the concept than sharp, parabolic-like profiles. The gain margins furthermore tend to become smaller with increasing retention and increasing diffusion coefficient. Copyright © 2012 Elsevier B.V. All rights reserved.

Validation of column-based chromatography processes for the purification of proteins. Technical report No. 14.

PubMed

2008-01-01

PDA Technical Report No. 14 has been written to provide current best practices, such as application of risk-based decision making, based in sound science to provide a foundation for the validation of column-based chromatography processes and to expand upon information provided in Technical Report No. 42, Process Validation of Protein Manufacturing. The intent of this technical report is to provide an integrated validation life-cycle approach that begins with the use of process development data for the definition of operational parameters as a basis for validation, confirmation, and/or minor adjustment to these parameters at manufacturing scale during production of conformance batches and maintenance of the validated state throughout the product's life cycle.

Radial Chromatography for the Separation of Nitroaniline Isomers

ERIC Educational Resources Information Center

Miller, Robert B.; Case, William S.

2011-01-01

Separation techniques are usually presented in the undergraduate organic laboratory to teach students how to purify and isolate compounds. Often the concept of liquid chromatography is introduced by having students create "silica gel columns" to separate components of a reaction mixture. Although useful, column chromatography can be a laborious…

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. Copyright © 2013 Elsevier B.V. All rights reserved.

[Determination of aflatoxin B1, B2, G1, G2 in armeniacae semen amarum by high-performance liquid chromatography-tandem mass spectrometry].

PubMed

Zheng, Run-Sheng; Xu, Hui; Wang, Wen-Li; Zhan, Ruo-Ting; Chen, Wei-Wen

2013-10-01

A simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in Armeniacae Semen Amarum and the application was performance in 11 samples collected from different markets, medical stores and hospitals. The sample was extracted with 84% acetonitrile/water and 250 microL extraction was directly injected into a LC-MS/MS system without further purification procedure after being redissolved with methanol. The LC separation was performed on a C18 column with a linear gradient elution program of 4 mmol x L(-1) NH4 Ac-0.1% formic acid solution and menthol as the mobile phase. Selected reaction monitoring (SRM) was used for selective determination of the four aflatoxins on a triple quadruple mass spectrometer, which was operated in positive ionization modes. All the four aflatoxins showed a good linear relationship with r > 0.999 0, the average recoveries were between 87.88% and 102.9% and the matrix effect was ranged from 90.71% to 99.30% in low, intermediate and high levels. Furthermore, the higher recovery was obtained by the method reported in this study, comparing to the cleanup procedure with the Mycosep 226 purification column. Eleven samples collected were detected and the contamination levels of the AFB1 were between 1.590-2.340 microg x kg(-1) and the AF (B1 + B2 + G1 + G2) was ranged from 2.340 to 3.340 microg x kg(-1). In summary, the developed method was suitable to detect and screen AFB1, AFB2, AFG1, AFG2 in Armeniacae Semen Amarum.

Chromatographic performance of synthetic polycrystalline diamond as a stationary phase in normal phase high performance liquid chromatography.

PubMed

Peristyy, Anton; Paull, Brett; Nesterenko, Pavel N

2015-04-24

The chromatographic properties of high pressure high temperature synthesised diamond (HPHT) are investigated in normal phase mode of high performance liquid chromatography. Purified nonporous irregular shape particles of average particles size 1.2 μm and specific surface area 5.1 m(2) g(-1) were used for packing 100×4.6 mm ID or 50×4.6 mm ID stainless steel columns. The retention behaviour of several classes of compounds including alkyl benzenes, polyaromatic hydrocarbons (PAH), alkylphenylketones, phenols, aromatic acids and bases were studied using n-hexane-2-propanol mixtures as mobile phase. The results are compared with those observed for microdispersed sintered detonation nanodiamond (MSDN) and porous graphitic carbon (PGC). HPHT diamond revealed distinctive separation selectivity, which is orthogonal to that observed for porous graphitic carbon; while selectivities of HPHT diamond and microdispersed sintered detonation nanodiamonds are similar. Owing to non-porous particle nature, columns packed with high pressure high temperature diamond exhibited excellent mass transfer and produce separations with maximum column efficiency of 128,200 theoretical plates per meter. Copyright © 2015 Elsevier B.V. All rights reserved.

Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium.

PubMed

Qu, Huihua; Zhang, Yue; Qu, Baoping; Cheng, Jinjun; Liu, Shuchen; Feng, Shenglan; Wang, Qingguo; Zhao, Yan

2016-04-01

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme-linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti-naringin monoclonal antibodies to CNBr-activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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. Copyright © 2016 Elsevier B.V. All rights reserved.

Monolithic metal-organic framework MIL-53(Al)-polymethacrylate composite column for the reversed-phase capillary liquid chromatography separation of small aromatics.

PubMed

Yusuf, Kareem; Badjah-Hadj-Ahmed, Ahmed Yacine; Aqel, Ahmad; ALOthman, Zeid Abdullah

2016-03-01

A monolithic capillary column containing a composite of metal-organic framework MIL-53(Al) incorporated into hexyl methacrylate-co-ethylene dimethacrylate was prepared to enhance the separation of mixtures of small aromatic compounds by using capillary liquid chromatography. The addition of 10 mg/mL MIL-53(Al) microparticles increased the micropore content in the monolithic matrix and increased the Brunauer-Emmett-Teller surface area from 26.92 to 85.12 m(2) /g. The presence of 1,4-benzenedicarboxylate moieties within the structure of MIL-53(Al) as an organic linker greatly influenced the separation of aromatic mixtures through π-π interactions. High-resolution separation was obtained for a series of alkylbenzenes (with resolution factors in the range 0.96-1.75) in less than 8 min, with 14 710 plates/m efficiency for propylbenzene, using a binary polar mobile phase of water/acetonitrile in isocratic mode. A reversed-phase separation mechanism was indicated by the increased retention factor and resolution as the water percentage in the mobile phase increased. A stability study on the composite column showed excellent mechanical stability under various conditions. The higher resolution and faster separation observed at increased temperature indicated an exothermic separation, whereas the negative values for the free energy change of transfer indicated a spontaneous process. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation.

PubMed Central

Onishi, S; Itoh, S; Ishida, Y

1982-01-01

An accurate and sensitive method that involves the group separations of serum bile acids (i.e. free and glycine- and taurine-conjugated bile acid fractions) by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 is described. Each group was then analysed by high-pressure liquid chromatography by using the post-column reaction technique with immobilized 3 alpha-hydroxy steroid dehydrogenase. The bile acid patterns in the umbilical venous serum samples were analysed by this method. Taurochenodeoxycholate predominated in the umbilical blood. PMID:6956336

Crocheted ETFE-reactor for on-line post-column photoderivatization of diclofenac in high-performance liquid chromatography.

PubMed

Kuhlmann, O; Krauss, G J

1997-12-01

A sensitive and selective bioanalytical method for diclofenac using reversed-phase HPLC and fluorescence detection is described. Diclofenac was detected as its fluorescent derivative after on-line post-column photoderivatization. Irradiation with UV light of diclofenac in aqueous solutions leads to the sequential loss of both chlorine substituents and ring closure. The major product, carbazole-1-acetic acid, was detected by a fluorescence detector using an excitation wavelength of 286 nm and an emission wavelength of 360 nm. The self-made reactor was a crocheted ethylene and tetrafluoroethylene (ETFE, named TEFZEL) capillary, 20 m in length, wound directly around a 253.7 nm UV lamp. The capillary was crocheted in order to overcome peak widening. Chromatographic separation was achieved by using a Regis SPS 100 RP-8 column (5 microm; 150 mm x 4.6 mm i.d.) and a LiChrospher 100 RP-18 (5 microm) guard column from E. Merck. The detection limit was 1 ng ml(-1) at an injection volume of 20 microl. Daily relative standard deviations (RSD) were 5.5%, (73 ng diclofenac/ml, n = 9), and 5.1% (405 ng diclofenac/ml, n = 6), respectively. Chromatograms of human aqueous humor and human serum containing diclofenac, and figures showing the time dependent increase/decrease of the photoderivatization product, are shown.

Determination of gas-liquid partition coefficients of several organic solutes in trihexyl(tetradecyl)phosphonium bromide using capillary gas chromatography columns.

PubMed

Ronco, Nicolás R; Menestrina, Fiorella; Romero, Lílian M; Castells, Cecilia B

2017-06-09

In this paper, we report gas-liquid partition constants for thirty-five volatile organic solutes in the room temperature ionic liquid trihexyl(tetradecyl)phosphonium bromide measured by gas-liquid chromatography using capillary columns. The relative contribution of gas-liquid partition and interfacial adsorption to retention was evaluated through the use of columns with different the phase ratio. Four capillary columns with exactly known phase ratios were constructed and employed to measure the solute retention factors at four temperatures between 313.15 and 343.15K. The partition coefficients were calculated from the slopes of the linear regression between solute retention factors and the reciprocal of phase ratio at a given temperature according to the gas-liquid chromatographic theory. Gas-liquid interfacial adsorption was detected for a few solutes and it has been considered for the calculations of partition coefficient. Reliable solute's infinite dilution activity coefficients can be obtained when retention data are determined by a unique partitioning mechanism. The partial molar excess enthalpies at infinite dilution have been estimated from the dependence of experimental values of solute activity coefficients with the column temperature. A thorough discussion of the uncertainties of the experimental measurements and the main advantages of the use of capillary columns to acquire the aforementioned relevant thermodynamic information was performed. Copyright © 2017 Elsevier B.V. All rights reserved.

[Determination of fumonisins B1 and B2 in corn by high performance liquid chromatography with post-column derivatization method].

PubMed

Zhang, Xiaoxu; Xiao, Zhiyong; Zhang, Hongyan; Yang, Lili; Ma, Liyan

2012-08-01

A high performance liquid chromatography-fluorescence detection with post-column derivatization method was developed to detect fumonisin B1 (FB1) and fumonisin B2 (FB2) in corn. Several factors, such as the pH of derivatization buffer, concentration and flow rate of derivatization reagents, excitation wavelength, emission wavelength, which affected the detection of fumonisins were optimized. The separation was performed on a ZORBAX SB C18 column operated at 40 degrees C with the gradient elution by two mobile phases of 0.1 mol/L sodium dihydrogen phosphate solution (pH 3.3) and methanol at a flow rate of 0.8 mL/min. The derivatization was performed at ambient temperature. The o-phthalaldehyde (OPA) flow rate was 0.4 mL/min. The results showed that the optimum conditions were pH 10.5 of the derivatization reagent, OPA concentration at 2 g/L, and excitation wavelength of 335 nm, emission wavelength of 440 nm. The linear plots of FB1 and FB2 were obtained between 0.2 to 20 mg/L, with the correlation coefficients above 0.999 for both FB1 and FB2. The limits of detection of fumonisins B1 and B2 were 0.02 mg/kg. The mean recoveries at the three spiked levels of 0.1 - 4.0 mg/kg were 82.5% - 89.8%. This method is accurate, simple, rapid and suitable for the determination of fumonisins B1 and B2 in corn.

Chromatographic column evaluation for the untargeted profiling of glucosinolates in cauliflower by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry.

PubMed

Capriotti, Anna Laura; Cavaliere, Chiara; La Barbera, Giorgia; Montone, Carmela Maria; Piovesana, Susy; Zenezini Chiozzi, Riccardo; Laganà, Aldo

2018-03-01

The untargeted profiling is a promising approach for the characterization of secondary metabolites in biological matrices. Thanks to the recent rapid development of high-resolution mass spectrometry (HRMS) instrumentations, the number of applications by untargeted approaches for biological samples profiling has widely increased in the recent years. Despite the high potentialities of HRMS, however, a major issue in natural products analysis often arises in the upstream process of compounds separation. A separation technique is necessary to avoid phenomena such as signal suppression, and it is especially needed in the presence of isomeric metabolites, which are otherwise indistinguishable. Glucosinolates (GLSs), a group of secondary metabolites widely distributed among plants, resulted to be associated to the prevention of some serious diseases, such as cancer. This led to the development of several methods for the analysis of GLSs in vegetables tissues. The issue of GLSs chromatographic separation has been widely studied in the past because of the difficulty in the analysis of this highly polar and variable class of compounds. Several alternatives to reversed phase (RP) chromatography, sometimes not compatible with the coupling of liquid chromatography with mass spectrometry, have been tested for the analysis of intact GLSs. However, the availability of new stationary phases, in the last years, could allow the re-evaluation of RP chromatography for the analysis of intact GLSs. In this work, a thorough evaluation of four RP chromatographic columns for the analysis of GLSs in cauliflower (Brassica oleracea L. var. botrytis) extracts by an ultra-high performance liquid chromatographic system coupled via electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer is presented. The columns tested were the following: one column Luna Omega polar C 18 , one column Kinetex Biphenyl, one column Kinetex core-shell XB-C 18 , two columns Kinetex core-shell XB-C 18

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. Copyright © 2014 Elsevier B.V. All rights reserved.

HPLC column-switching technique for sample preparation and fluorescence determination of propranolol in urine using fused-core columns in both dimensions.

PubMed

Satínský, Dalibor; Havlíková, Lucie; Solich, Petr

2013-08-01

A new and fast high-performance liquid chromatography (HPLC) column-switching method using fused-core columns in both dimensions for sample preconcentration and determination of propranolol in human urine has been developed. On-line sample pretreatment and propranolol preconcentration were performed on an Ascentis Express RP-C-18 guard column (5 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water (5:95, v/v) at a flow rate of 2.0 mL min(-1) and at a temperature of 50 °C. Valve switch from pretreatment column to analytical column was set at 4.0 min in a back-flush mode. Separation of propranolol from other endogenous urine compounds was achieved on the fused-core column Ascentis Express RP-Amide (100 × 4.6 mm), particle size, 2.7 μm, with mobile phase acetonitrile/water solution of 0.5% triethylamine, pH adjusted to 4.5 by means of glacial acetic acid (25:75, v/v), at a flow rate of 1.0 mL min(-1) and at a temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 229/338 nm. A volume of 1,500 μL of filtered urine sample solution was injected directly into the column-switching HPLC system. The total analysis time including on-line sample pretreatment was less than 8 min. The experimentally determined limit of detection of the method was found to be 0.015 ng mL(-1).

[High-performance liquid-liquid chromatography in beverage analysis].

PubMed

Bricout, J; Koziet, Y; de Carpentrie, B

1978-01-01

Liquid liquid chromatography was performed with columns packed with stationary phases chemically bonded to silica microparticules. These columns show a high efficiency and are used very easily. Flavouring compounds like aromatic aldehydes which have a low volatility were analyzed in brandy using a polar phase alkylnitrile. Sapid substances like amarogentin in Gentiana lutea or glyryrrhizin in Glycyrrhiza glabra were determined by reversed phase chromatography. Finally ionizable substances like synthetic dyes can be analyzed by paired ion chromatography witha non polar stationary phase.

Long-term behavior of passively aerated compost methanotrophic biofilter columns.

PubMed

Wilshusen, J H; Hettiaratchi, J P A; Stein, V B

2004-01-01

The methane oxidation potential of several types of compost methanotrophic biofilter columns were compared in the laboratory over a period of 220 days. The results indicate an increase in methanotrophic activity over a period of about 100 days, up to a maximum of 400 g m(-2) day(-1), and a gradual decline to about 100 g m(-2) day(-1) within the next 120 days. High methane oxidation rates appear to be restricted to a small area of the column, 10-15 cm thick. Based on the laboratory investigations carried out to determine the cause for the decline in methane oxidation rate, it was concluded that the formation of exopolymeric substances (EPS), at the zones of maximum methane oxidation, was responsible for this decline. In monitoring methane oxidation in a column for up to 600 days, it was observed that mixing of the medium after formation of EPS enabled the column to temporarily recover high performance. The results suggest that stable, homogenous compost, with a low C/N and low ammonium content, mixed on a regular basis, could achieve and maintain high methane oxidation efficiencies. Copyright 2004 Elsevier Ltd.

Novel dispersive micro-solid-phase extraction combined with ultrahigh-performance liquid chromatography-high-resolution mass spectrometry to determine morpholine residues in citrus and apples.

PubMed

Chen, Dawei; Miao, Hong; Zou, Jianhong; Cao, Pei; Ma, Ning; Zhao, Yunfeng; Wu, Yongning

2015-01-21

This paper presents a new analytical method for the determination of morpholine residues in citrus and apples using a novel dispersive micro-solid-phase extraction (DMSPE), followed by ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS). Samples were extracted with 1% formic acid in acetonitrile/water (1:1, v/v) and then cleaned up using the DMSPE procedure. Morpholine from the extract was adsorbed to a polymer cation exchange sorbent and eluted with ammonium hydroxide/acetonitrile (3:97, v/v) through a 1 mL syringe with a 0.22 μm nylon syringe filter. All of the samples were analyzed by UHPLC-HRMS/MS on a Waters Acquity BEH hydrophilic interaction chromatography column using 0.1% formic acid and 4 mM ammonium formate in water/acetonitrile as the mobile phase with gradient elution. The method showed good linearity (R(2) > 0.999) in the range of 1-100 μg/L for the analyte. The limit of detection and limit of quantitation values of morpholine were 2 and 5 μg/kg, respectively. The average recoveries of morpholine from the citrus and apple samples spiked at three different concentrations (5, 20, and 100 μg/kg) were in a range from 78.4 to 102.7%.

A narrow open tubular column for high efficiency liquid chromatographic separation.

PubMed

Chen, Huang; Yang, Yu; Qiao, Zhenzhen; Xiang, Piliang; Ren, Jiangtao; Meng, Yunzhu; Zhang, Kaiqi; Juan Lu, Joann; Liu, Shaorong

2018-04-30

We report a great feature of open tubular liquid chromatography when it is run using an extremely narrow (e.g., 2 μm inner diameter) open tubular column: more than 10 million plates per meter can be achieved in less than 10 min and under an elution pressure of ca. 20 bar. The column is coated with octadecylsilane and both isocratic and gradient separations are performed. We reveal a focusing effect that may be used to interpret the efficiency enhancement. We also demonstrate the feasibility of using this technique for separating complex peptide samples. This high-resolution and fast separation technique is promising and can lead to a powerful tool for trace sample analysis.

Highly sensitive determination of 2,4,6-trinitrotoluene and related byproducts using a diol functionalized column for high performance liquid chromatography.

PubMed

Gumuscu, Burcu; Erdogan, Zeynep; Guler, Mustafa O; Tekinay, Turgay

2014-01-01

In this work, a new detection method for complete separation of 2,4,6-trinitrotoluene (TNT); 2,4-dinitrotoluene (2,4-DNT); 2,6-dinitrotoluene (2,6-DNT); 2-aminodinitrotoluene (2-ADNT) and 4-aminodinitrotoluene (4-ADNT) molecules in high-performance liquid-chromatography (HPLC) with UV sensor has been developed using diol column. This approach improves on cost, time, and sensitivity over the existing methods, providing a simple and effective alternative. Total analysis time was less than 13 minutes including column re-equilibration between runs, in which water and acetonitrile were used as gradient elution solvents. Under optimized conditions, the minimum resolution between 2,4-DNT and 2,6-DNT peaks was 2.06. The recovery rates for spiked environmental samples were between 95-98%. The detection limits for diol column ranged from 0.78 to 1.17 µg/L for TNT and its byproducts. While the solvent consumption was 26.4 mL/min for two-phase EPA and 30 mL/min for EPA 8330 methods, it was only 8.8 mL/min for diol column. The resolution was improved up to 49% respect to two-phase EPA and EPA 8330 methods. When compared to C-18 and phenyl-3 columns, solvent usage was reduced up to 64% using diol column and resolution was enhanced approximately two-fold. The sensitivity of diol column was afforded by the hydroxyl groups on polyol layer, joining the formation of charge-transfer complexes with nitroaromatic compounds according to acceptor-donor interactions. Having compliance with current requirements, the proposed method demonstrates sensitive and robust separation.

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. Copyright © 2014 Elsevier B.V. All rights reserved.

High-sensitivity direct analysis of aflatoxins in peanuts and cereal matrices by ultra-performance liquid chromatography with fluorescence detection involving a large volume flow cell.

PubMed

Oulkar, Dasharath; Goon, Arnab; Dhanshetty, Manisha; Khan, Zareen; Satav, Sagar; Banerjee, Kaushik

2018-04-03

This paper reports a sensitive and cost effective method of analysis for aflatoxins B1, B2, G1 and G2. The sample preparation method was primarily optimised in peanuts, followed by its validation in a range of peanut-processed products and cereal (rice, corn, millets) matrices. Peanut slurry [12.5 g peanut + 12.5 mL water] was extracted with methanol: water (8:2, 100 mL), cleaned through an immunoaffinity column and thereafter measured directly by ultra-performance liquid chromatography-fluorescence (UPLC-FLD) detection, within a chromatographic runtime of 5 minutes. The use of a large volume flow cell in the FLD nullified the requirement of any post-column derivatisation and provided the lowest ever reported limits of quantification of 0.025 for B1 and G1 and 0.01 μg/kg for B2 and G2. The single laboratory validation of the method provided acceptable selectivity, linearity, recovery and precision for reliable quantifications in all the test matrices as well as demonstrated compliance with the EC 401/2006 guidelines for analytical quality control of aflatoxins in foodstuffs.

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.

Maximizing performance in supercritical fluid chromatography using low-density mobile phases.

PubMed

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

2016-10-14

The performance of a 3.0mm×150mm column packed with 1.8μm fully porous HSS-SB-C 18 particles was investigated in supercritical fluid chromatography (SFC) with low-density, highly expansible carbon dioxide. These conditions are selected for the analysis of semi-volatile compounds. Elevated temperatures (>100°C) were then combined with low column back pressures (<100bar). In this work, the inlet temperature of pure carbon dioxide was set at 107°C, the active back pressure regulator (ABPR) pressure was fixed at 100bar, and the flow rate was set at 2.1mL/min at 12°C (liquefied carbon dioxide) and at an inlet column pressure close to 300bar. Nine n-alkylbenzenes (from benzene to octadecylbenzene) were injected under linear (no sample overload) conditions. The severe steepness of the temperature gradients across the column diameter were predicted from a simplified heat transfer model. Such conditions dramatically lower the column performance by affecting the symmetry of the peak shape. In order to cope with this problem, three different approaches were experimentally tested. They include (1) the decoupling and the proper selection of the inlet eluent temperature with respect to the oven temperature, (2) the partial thermal insulation of the column using polyethylene aerogel, and (3) the application of a high vacuum (10 -5 Torr provided by a turbo-molecular pump) in a housing chamber surrounding the whole column body. The results reveal that (1) the column efficiency can be maximized by properly selecting the difference between the eluent and the oven temperatures, (2) the mere wrapping of the column with an excellent insulating material is insufficient to fully eliminate heat exchanges by conduction and the undesirable radial density gradients across the column i.d., and (3) the complete thermal insulation of the SFC column under high vacuum allows to maximize the column efficiency by maintaining the integrity of the peak shape. Copyright © 2016 Elsevier B.V. All

Chromatographic efficiency of polar capillary columns applied for the analysis of fatty acid methyl esters by gas chromatography.

PubMed

Waktola, Habtewold D; Mjøs, Svein A

2018-04-01

The chromatographic efficiency that could be achieved in temperature-programmed gas chromatography was compared for four capillary columns that are typically applied for analysis of fatty acid methyl esters (FAME). Three different carrier gases, hydrogen, helium and nitrogen, were applied. For each experiment, the carrier gas velocities and the temperature rates were varied with a full 9 × 3 design, with nine levels on the carrier gas velocity and temperature rates of 1, 2 or 3°C/min. Response surface methodology was used to create models of chromatographic efficiency as a function of temperature rate and carrier gas velocity. The chromatographic efficiency was defined as the inverse of peak widths measured in retention index units. The final results were standardized so that the efficiencies that could be achieved within a certain time frame, defined by the retention time of the last compound in the chromatogram, could be compared. The results show that there were clear differences in the efficiencies that could be achieved with the different columns and that the efficiency decreased with increasing polarity of the stationary phase. The differences can be explained by higher resistance to mass transfer in the stationary phase in the most polar columns. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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. Copyright © 2012 Elsevier B.V. All rights reserved.

Occurrence of turbulent flow conditions in supercritical fluid chromatography.

PubMed

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

2014-09-26

Having similar densities as liquids but with viscosities up to 20 times lower (higher diffusion coefficients), supercritical CO2 is the ideal (co-)solvent for fast and/or highly efficient separations without mass-transfer limitations or excessive column pressure drops. Whereas in liquid chromatography the flow remains laminar in both the packed bed and tubing, except in extreme cases (e.g. in a 75 μm tubing, pure acetonitrile at 5 ml/min), a supercritical fluid can experience a transition from laminar to turbulent flow in more typical operation modes. Due to the significant lower viscosity, this transition for example already occurs at 1.3 ml/min for neat CO2 when using connection tubing with an ID of 127 μm. By calculating the Darcy friction factor, which can be plotted versus the Reynolds number in a so-called Moody chart, typically used in fluid dynamics, higher values are found for stainless steel than PEEK tubing, in agreement with their expected higher surface roughness. As a result turbulent effects are more pronounced when using stainless steel tubing. The higher than expected extra-column pressure drop limits the kinetic performance of supercritical fluid chromatography and complicates the optimization of tubing ID, which is based on a trade-off between extra-column band broadening and pressure drop. One of the most important practical consequences is the non-linear increase in extra-column pressure drop over the tubing downstream of the column which leads to an unexpected increase in average column pressure and mobile phase density, and thus decrease in retention. For close eluting components with a significantly different dependence of retention on density, the selectivity can significantly be affected by this increase in average pressure. In addition, the occurrence of turbulent flow is also observed in the detector cell and connection tubing. This results in a noise-increase by a factor of four when going from laminar to turbulent flow (e.g. going

Spiral Countercurrent Chromatography

PubMed Central

Ito, Yoichiro; Knight, Martha; Finn, Thomas M.

2013-01-01

For many years, high-speed countercurrent chromatography conducted in open tubing coils has been widely used for the separation of natural and synthetic compounds. In this method, the retention of the stationary phase is solely provided by the Archimedean screw effect by rotating the coiled column in the centrifugal force field. However, the system fails to retain enough of the stationary phase for polar solvent systems such as the aqueous–aqueous polymer phase systems. To address this problem, the geometry of the coiled channel was modified to a spiral configuration so that the system could utilize the radially acting centrifugal force. This successfully improved the retention of the stationary phase. Two different types of spiral columns were fabricated: the spiral disk assembly, made by stacking multiple plastic disks with single or four interwoven spiral channels connected in series, and the spiral tube assembly, made by inserting the tetrafluoroethylene tubing into a spiral frame (spiral tube support). The capabilities of these column assemblies were successfully demonstrated by separations of peptides and proteins with polar two-phase solvent systems whose stationary phases had not been well retained in the earlier multilayer coil separation column for high-speed countercurrent chromatography. PMID:23833207

Prediction of soil organic carbon partition coefficients by soil column liquid chromatography.

PubMed

Guo, Rongbo; Liang, Xinmiao; Chen, Jiping; Wu, Wenzhong; Zhang, Qing; Martens, Dieter; Kettrup, Antonius

2004-04-30

To avoid the limitation of the widely used prediction methods of soil organic carbon partition coefficients (KOC) from hydrophobic parameters, e.g., the n-octanol/water partition coefficients (KOW) and the reversed phase high performance liquid chromatographic (RP-HPLC) retention factors, the soil column liquid chromatographic (SCLC) method was developed for KOC prediction. The real soils were used as the packing materials of RP-HPLC columns, and the correlations between the retention factors of organic compounds on soil columns (ksoil) and KOC measured by batch equilibrium method were studied. Good correlations were achieved between ksoil and KOC for three types of soils with different properties. All the square of the correlation coefficients (R2) of the linear regression between log ksoil and log KOC were higher than 0.89 with standard deviations of less than 0.21. In addition, the prediction of KOC from KOW and the RP-HPLC retention factors on cyanopropyl (CN) stationary phase (kCN) was comparatively evaluated for the three types of soils. The results show that the prediction of KOC from kCN and KOW is only applicable to some specific types of soils. The results obtained in the present study proved that the SCLC method is appropriate for the KOC prediction for different types of soils, however the applicability of using hydrophobic parameters to predict KOC largely depends on the properties of soil concerned.

Column Selection for Biomedical Analysis Supported by Column Classification Based on Four Test Parameters.

PubMed

Plenis, Alina; Rekowska, Natalia; Bączek, Tomasz

2016-01-21

This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the FKUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the FKUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the FKUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the FKUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis.

[Rapid identification of micro-constituents in monoammonium glycyrrhizinate raw materials by high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry].

PubMed

Yang, Xue-Dong; Tang, Xu-Yan; Sang, Lin

2012-11-01

To establish a method for rapid identification of micro-constituents in monoammonium glycyrrhizinate by high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry. HPLC preparative chromatograph was adopted for determining the optimal method for high-pressure solid phase extraction under optimal conditions. 5C18-MS-II column (20.0 mm x 20.0 mm) was used as the extraction column, with 35% acetonitrile-acetic acid solution (pH 2. 20) as eluent at the speed of 16 mL x min(-1). The sample size was 0.5 mL, and the extraction cycle was 4.5 min. Then, extract liquid was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) after being concentrated by 100 times. Under the optimal condition of high-pressure solid phase extraction-high performance liquid chromatography-mass spectrometry, 10 components were rapidly identified from monoammonium glycyrrhizinate raw materials. Among them, the chemical structures of six micro-constituents were identified as 3-O-[beta-D-glucuronopyranosyl-beta-D-glucuronopyranosyl]-30-0-beta-D-apiopyranosylglycyrrhetic/3-O- [P-D-glucuronopyranosyl-beta-D-glucuronopyranosyl]-30-O-beta-D-arabinopyranosylglycyrrhetic, glycyrrhizic saponin F3, 22-hydroxyglycyrrhizin/18alpha-glycyrrhizic saponin G2, 3-O-[beta-D-rhamnopyranosyl]-24-hydroxyglycyrrhizin, glycyrrhizic saponin J2, and glycyrrhizic saponin B2 by MS(n) spectra analysis and reference to literatures. Four main chemical components were identified as glycyrrhizic saponin G2, 18beta-glycyrrhizic acid, uralglycyrrhizic saponin B and 18alpha-glycyrrhizic acid by liquid chromatography, MS(n) and ultraviolet spectra information and comparison with reference substances. The method can be used to identify chemical constituents in monoammonium glycyrrhizinate quickly and effectively, without any reference substance, which provides basis for quality control and safe application of monoammonium glycyrrhizinate-related products.

Determination of azithromycin residue in pork using a molecularly imprinted monolithic microcolumn coupled to liquid chromatography with tandem mass spectrometry.

PubMed

Zhou, Tong; Yang, Haicui; Jin, Zhen; Liu, Qingying; Song, Xuqin; He, Limin; Fang, Binghu; Meng, Chenying

2016-04-01

Using spiramycin as a dummy template, a molecularly imprinted polymer monolithic micro-column with high selection to azithromycin was prepared in a micropipette tip. The imprinting factor of the monolithic micro-column prepared was approximately 2.67 and the morphological structure of the polymers was characterized by scanning electron microscopy. A simple, sensitive, and reproducible method based on the imprinted monolithic micro-column coupled to liquid chromatography with tandem mass spectrometry was developed for determining the residues of azithromycin in pork. Pork samples were extracted with acetonitrile, cleaned up under the optimal monolithic micro-column conditions, and analyzed using liquid chromatography with tandem mass spectrometry in the multiple reaction monitoring mode. The assay exhibited a linear dynamic range of 0.50-50 μg/L with the correlation coefficient (r(2) ) above 0.99. In the three spiking levels of 0.50, 1.0, and 10 μg/kg, the average recoveries of azithromycin from pork samples were between 85.8 and 96.5% with a relative standard deviation below 10%. The limit of detection and limit of quantitation were 0.03 and 0.1 μg/kg, respectively. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments.

PubMed

Gritti, Fabrice; Sanchez, Carl A; Farkas, Tivadar; Guiochon, Georges

2010-04-30

A series of experiments and measurements demonstrate the importance of minimizing the extra-column band broadening contribution of the instrument used. The combination of several measures allowed the achievement of the full potential efficiency of three Kinetex-C(18) columns, using a conventional liquid chromatograph. The first measure consists in minimizing the extra-column volume of the instrument, without increasing much its back pressure contribution, by changing the needle seat volume, the inner diameter and length of the capillary connectors, and the volume of the detector cell of a standard instrument (Agilent 1100). The second measure consists in injecting a volume of weak eluent (less than half the elution strength of the mobile phase) right after the sample, before the sample had time to reach the column. Experimental results show that these changes could provide most of the resolution expected from the true column performance. After the changes were made, the resolutions of the 2.1 mm x 50 mm, 4.6 mm x 50 mm, and 4.6 mm x 100 mm Kinetex-C(18) columns for compounds having retention factors close to 1 were increased by about 180, 35, and 30%, respectively. The resolutions obtained are then similar to those measured with advanced instruments like the Agilent 1200, the Agilent 1290 Infinity HPLC, and the Acquity chromatographs. 2010 Elsevier B.V. All rights reserved.

Speed-resolution advantage of turbulent supercritical fluid chromatography in open tubular columns: II - Theoretical and experimental evidences.

PubMed

Gritti, Fabrice; Fogwill, Michael

2017-06-09

The potential advantage of turbulent supercritical fluid chromatography (TSFC) in open tubular columns (OTC) was evaluated on both theoretical and practical viewpoints. First, the dispersion model derived by Golay in 1958 and recently extended from laminar to turbulent flow regime is used for the predictions of the speed-resolution performance in TSFC. The average dispersion coefficient of matter in the turbulent flow regime was taken from the available experimental data over a range of Reynolds number from 2000 to 6000. Kinetic plots are built at constant pressure drop (ΔP=4500psi) and Schmidt number (Sc=15) for four inner diameters (10, 30, 100, and 300μm) of the OTC and for three retention factors (0, 1, and 10). Accordingly, in turbulent flow regime, for a Reynolds number of 4000 and a retention factor of 1 (the stationary film thickness is assumed to be negligible with respect to the OTC diameter), the theory projects that a 300μm i.d. OTC has the same speed-resolution power (200,000 theoretical plates; 2.4min hold-up time) as that of a 10μm i.d. OTC operated in laminar flow regime. Secondly, the experimental plate heights of n-butylbenzene are measured in laminar and turbulent flow regimes for a 180μm×4.8m fused silica capillary column using pure carbon dioxide as the mobile phase. The back pressure regulator was set at 1500psi, the temperature was uniform at 297K, and the flow rate was increased step-wise from 0.50 to 3.60mL/min so that the experimental Reynolds number increases from 700 to 5400. The experiments are in good agreement with the plate heights projected in TSFC at high flow rates and with those expected at low flow rates in a laminar flow regime. Copyright © 2017 Elsevier B.V. All rights reserved.

Automation of nanoflow liquid chromatography-tandem mass spectrometry for proteome analysis by using a strong cation exchange trap column.

PubMed

Jiang, Xiaogang; Feng, Shun; Tian, Ruijun; Han, Guanghui; Jiang, Xinning; Ye, Mingliang; Zou, Hanfa

2007-02-01

An approach was developed to automate sample introduction for nanoflow LC-MS/MS (microLC-MS/MS) analysis using a strong cation exchange (SCX) trap column. The system consisted of a 100 microm id x 2 cm SCX trap column and a 75 microm id x 12 cm C18 RP analytical column. During the sample loading step, the flow passing through the SCX trap column was directed to waste for loading a large volume of sample at high flow rate. Then the peptides bound on the SCX trap column were eluted onto the RP analytical column by a high salt buffer followed by RP chromatographic separation of the peptides at nanoliter flow rate. It was observed that higher performance of separation could be achieved with the system using SCX trap column than with the system using C18 trap column. The high proteomic coverage using this approach was demonstrated in the analysis of tryptic digest of BSA and yeast cell lysate. In addition, this system was also applied to two-dimensional separation of tryptic digest of human hepatocellular carcinoma cell line SMMC-7721 for large scale proteome analysis. This system was fully automated and required minimum changes on current microLC-MS/MS system. This system represented a promising platform for routine proteome analysis.

Highly Sensitive Determination of 2,4,6-Trinitrotoluene and Related Byproducts Using a Diol Functionalized Column for High Performance Liquid Chromatography

PubMed Central

Gumuscu, Burcu; Erdogan, Zeynep; Guler, Mustafa O.; Tekinay, Turgay

2014-01-01

In this work, a new detection method for complete separation of 2,4,6-trinitrotoluene (TNT); 2,4-dinitrotoluene (2,4-DNT); 2,6-dinitrotoluene (2,6-DNT); 2-aminodinitrotoluene (2-ADNT) and 4-aminodinitrotoluene (4-ADNT) molecules in high-performance liquid-chromatography (HPLC) with UV sensor has been developed using diol column. This approach improves on cost, time, and sensitivity over the existing methods, providing a simple and effective alternative. Total analysis time was less than 13 minutes including column re-equilibration between runs, in which water and acetonitrile were used as gradient elution solvents. Under optimized conditions, the minimum resolution between 2,4-DNT and 2,6-DNT peaks was 2.06. The recovery rates for spiked environmental samples were between 95–98%. The detection limits for diol column ranged from 0.78 to 1.17 µg/L for TNT and its byproducts. While the solvent consumption was 26.4 mL/min for two-phase EPA and 30 mL/min for EPA 8330 methods, it was only 8.8 mL/min for diol column. The resolution was improved up to 49% respect to two-phase EPA and EPA 8330 methods. When compared to C-18 and phenyl-3 columns, solvent usage was reduced up to 64% using diol column and resolution was enhanced approximately two-fold. The sensitivity of diol column was afforded by the hydroxyl groups on polyol layer, joining the formation of charge-transfer complexes with nitroaromatic compounds according to acceptor-donor interactions. Having compliance with current requirements, the proposed method demonstrates sensitive and robust separation. PMID:24905826

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. Copyright © 2016 Elsevier B.V. All rights reserved.

[Comparison of acetonitrile, ethanol and chromatographic column to eliminate high-abundance proteins in human serum].

PubMed

Li, Yin; Liao, Ming; He, Xiao; Zhou, Yi; Luo, Rong; Li, Hongtao; Wang, Yun; He, Min

2012-11-01

To compare the effects of acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal to eliminate high-abundance proteins in human serum. Elimination of serum high-abundance proteins performed with acetonitrile precipitation, ethanol precipitation and multiple affinity chromatography column Human 14 removal. Bis-Tris Mini Gels electrophoresis and two-dimensional gel electrophoresis to detect the effect. Grey value analysis from 1-DE figure showed that after serum processed by acetonitrile method, multiple affinity chromatography column Human 14 removal method and ethanol method, the grey value of albumin changed into 157.2, 40.8 and 8.2 respectively from the original value of 19. 2-DE analysis results indicated that using multiple affinity chromatography column Human 14 method, the protein points noticeable increased by 137 compared to the original serum. After processed by acetonitrile method and ethanol method, the protein point reduced, but the low abundance protein point emerged. The acetonitrile precipitation could eliminate the vast majority of high abundance proteins in serum and gain more proteins of low molecular weight, ethanol precipitation could eliminate part of high abundance proteins in serum, but low abundance proteins less harvested, and multiple affinity chromatography column Human 14 method could effectively removed the high abundance proteins, and keep a large number of low abundance proteins.

Development of an efficient fractionation method for the preparative separation of sesquiterpenoids from Tussilago farfara by counter-current chromatography.

PubMed

Song, Kwangho; Lee, Kyoung Jin; Kim, Yeong Shik

2017-03-17

A novel application of counter-current chromatography (CCC) to enrich plant extracts using direct and continuous injection (CCC-DCI) was developed to fractionate sesquiterpenoids from the buds of Tussilago farfara L. In this study, an n-hexane-acetonitrile-water (HAcW) solvent system was separately pumped into the CCC column, and an extraction solution (45% acetonitrile) was directly and continuously injected into the CCC column. Since the extraction solution was used as a mobile phase in this method, solvent consumption could be greatly reduced. To enrich the extraction solution (315.9g/5.4L), only 4.2L water, 4.6L acetonitrile, and 1.2L n-hexane were used, including the extraction step. Finally, 6.8g of a sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9g) of Tussilago farfara (1kg) in a single CCC run with a separation time of 8.5h. The sample injection capacity of CCC-DCI was greater than 300g; this amount of sample could not be handled in conventional CCC or other fractionation methods with the same column volume. Moreover, three major sesquiterpenoids (1: tussilagone, 2: 14-acetoxy-7β-(3'-ethyl cis-crotonoyloxy)-1α-(2'-methylburyryloxy)-notonipetranone, and 3: 7β-(3'-ethyl cis-crotonoyloxy)-1α-(2'-methylburyryloxy)-3, 14-dehydro-Z-notonipetranone) were purified from the STE fraction by CCC, and their chemical structures were elucidated by 1 H NMR and 13 C NMR. A quantification study was conducted, and the contents of compounds 1-3 in the CCC-DCI fraction were higher than those of conventional multi-step fractionations performed in series: solvent partitioning and open column chromatography. Furthermore, the average CCC-DCI recoveries were 96.1% (1), 96.9% (2), and 94.6% (3), whereas the open column chromatography recoveries were 77.7% (1), 66.5% (2), and 58.4% (3). The developed method demonstrates that CCC is a useful technique for enriching target components from natural products. Copyright © 2017 Elsevier B.V. All

Determination of active components of Ginkgo biloba in human urine by capillary high-performance liquid chromatography/mass spectrometry with on-line column-switching purification.

PubMed

Ding, Shujing; Dudley, Ed; Chen, Lijuan; Plummer, Sue; Tang, Jiandong; Newton, Russell P; Brenton, A Gareth

2006-01-01

Ginkgo biloba is one of the most popular herbal nutritional supplements, with terpene lactones and flavonoids being the two major active components. An on-line purification high-performance liquid chromatography/mass spectrometry (HPLC/MS) method was successfully developed for the quantitative determination of flavonoids and terpene lactones excreted in human urine after ingesting the herbal supplement. Satisfactory separation was obtained using a C18 capillary column made in-house with sample clean-up and pre-concentration achieved using a C18 pre-column with column switching. High selectivity and limits of detection of 1-18 ng/mL were achieved using a selected ion monitoring (SIM) scan in negative ion mode; the on-line solid-phase extraction (SPE) recovery of the active components in Ginkgo biloba determined in this study was greater than 75%. Copyright 2006 John Wiley & Sons, Ltd.

Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatography-mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides.

PubMed

Eeltink, Sebastiaan; Wouters, Sam; Dores-Sousa, José Luís; Svec, Frantisek

2017-05-19

This review focuses on the preparation of organic polymer-based monolithic stationary phases and their application in the separation of biomolecules, including antibodies, intact proteins and protein isoforms, oligonucleotides, and protein digests. Column and material properties, and the optimization of the macropore structure towards kinetic performance are also discussed. State-of-the-art liquid chromatography-mass spectrometry biomolecule separations are reviewed and practical aspects such as ion-pairing agent selection and carryover are presented. Finally, advances in comprehensive two-dimensional LC separations using monolithic columns, in particular ion-exchange×reversed-phase and reversed-phase×reversed-phase LC separations conducted at high and low pH, are shown. Copyright © 2017 Elsevier B.V. All rights reserved.

Post-column mobile phase adjustment: a strategy to eliminate the contradiction between liquid chromatography and mass spectrometry in the determination of flavonoids in rat plasma.

PubMed

Zheng, Shirui; Ma, Zhiyuan; Han, Haixia; Ye, Jianfeng; Wang, Ruwei; Cai, Sheng; Zhou, Hui; Yu, Lushan; Zeng, Su; Jiang, Huidi

2014-07-01

Flavonoids are a group of important naturally occurring polyphenolic compounds with a wide range of biological effects. In this study, a sensitive liquid chromatography tandem mass spectrometry method was developed to simultaneously determine multiple active flavonoids, including quercetin (Que), kaempferol (Kae), apigenin (Api), isorhamnetin (Iso), luteolin (Lut), and naringenin (Nar), in rat plasma. To achieve a satisfied peak shape and LC separation, formic acid with the concentration between 0.05 and 0.2%, or in some case 5%, was generally used to acidify the LC mobile phase in reported studies. Here we found that even 0.05% formic acid could lead to strong mass signal suppression, and the absence of formic acid could reverse the signal suppression but cause serious peak tailing. There is an irreconcilable contradiction between liquid chromatography (LC) and mass spectrometry (MS). In order to simultaneously satisfy LC and MS, LC mobile phase with 0.00075% formic acid and post column mobile phase adjustment with 0.0677% ammonium solution in isopropanol were applied. Compared with the conventional method with mobile phase containing 0.05% formic acid, the mass signal response of Que, Kae, Api, Iso, Lut, Nar, and Oka increased 26.2, 18.6, 13.6, 23.5, 17.5, 15.6 and 15.4 fold, respectively. In addition, the post column mobile phase addition exhibited the better peak shape for the reduction of analytes longitudinal diffusion. The method has been fully validated according to FDA guidelines within the linear range between 0.328 ng mL⁻¹ and 168 ng mL⁻¹, and successfully applied to a pilot pharmacokinetic study of rats after administering 5.43 g kg⁻¹ Pollen of Brassica campestris. Copyright © 2014 Elsevier B.V. All rights reserved.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

PubMed Central

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

On-column nitrosation of amines observed in liquid chromatography impurity separations employing ammonium hydroxide and acetonitrile as mobile phase.

PubMed

Myers, David P; Hetrick, Evan M; Liang, Zhongming; Hadden, Chad E; Bandy, Steven; Kemp, Craig A; Harris, Thomas M; Baertschi, Steven W

2013-12-06

The availability of high performance liquid chromatography (HPLC) columns capable of operation at pH values up to 12 has allowed a greater selectivity space to be explored for method development in pharmaceutical analysis. Ammonium hydroxide is of particular value in the mobile phase because it is compatible with direct interfacing to electrospray mass spectrometers. This paper reports an unexpected N-nitrosation reaction that occurs with analytes containing primary and secondary amines when ammonium hydroxide is used to achieve the high pH and acetonitrile is used as the organic modifier. The nitrosation reaction has generality. It has been observed on multiple columns from different vendors and with multiple amine-containing analytes. Ammonia was established to be the source of the nitroso nitrogen. The stainless steel column frit and metal ablated from the frit have been shown to be the sites of the reactions. The process is initiated by removal of the chromium oxide protective film from the stainless steel by acetonitrile. It is hypothesized that the highly active, freshly exposed metals catalyze room temperature oxidation of ammonia to NO but that the actual nitrosating agent is likely N(2)O(3). Copyright © 2013 Elsevier B.V. All rights reserved.

Choline in infant formula and adult nutritionals by ion chromatography and suppressed conductivity: First Action 2012.20.

PubMed

Oates, Kassandra; Chen, Lillian; De Borba, Brian; Mohindra, Deepali; Rohrer, Jeffrey; Dowell, Dawn

2013-01-01

Single-laboratory validation (SLV) data from a method for the determination of choline in infant formula and adult nutritionals by ion chromatography (IC) and suppressed conductivity were generated and presented to the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Expert Review Panel (ERP) at the AOAC Annual Meeting held in Las Vegas, NV, during September 30 to October 3, 2012. The ERP reviewed the data and concluded that the data met the standard method performance requirements (SMPRs) established and approved the method as AOAC Official First Action. At the ERP's request, a second, full SLV was performed on 17 SPIFAN matrixes that included fortified and placebo products. Prior to IC analysis, microwave-assisted acid hydrolysis was used to digest and release bound choline from powder and ready-to-feed (RTF) infant formula and adult nutritional samples. Following hydrolysis, separation of choline from common cations was achieved on a Thermo Scientific Dionex IonPac CS19 column followed by suppressed conductivity detection. Total choline was measured and reported as the choline ion in mg/100 g reconstituted material or RTF as-is. The system was calibrated over the analytical range specified in the SMPR (2-250 mg/100 g). Recoveries of spiked samples at 50 and 100% of the fortified choline amounts ranged from 93.1 to 100.7% with RSDs < or = 6.7% for product containing < 2 mg/100 g and < or = 4.1% for product containing 2-100 mg/100 g. Accuracy for the National Institute of Standards and Technology Standard Reference Material 1849a was determined over a 6-day interval and found to be 10.2 +/- 0.2 mg/100 g calculated as the reconstituted powder with an RSD of 1.8%. The LOD was determined to be 0.009, and the LOQ 0.012 mg/100 g, well below the SMPR requirements of 0.7 and 2 mg/100 g, respectively. Repeatability RSDs over the range of the assay (2-200 mg/100 g) ranged from 1.0 to 5.93%

Simultaneous determination of quinolones for veterinary use by high-performance liquid chromatography with electrochemical detection.

PubMed

Rodríguez Cáceres, M I; Guiberteau Cabanillas, A; Galeano Díaz, T; Martínez Cañas, M A

2010-02-01

A selective method based on high-performance liquid chromatography with electrochemical detection (HPLC-ECD) has been developed to enable simultaneous determination of three fluoroquinolones (FQs), namely danofloxacin (DANO), difloxacin (DIFLO) and sarafloxacin (SARA). The fluoroquinolones are separated on a Novapack C-18 column and detected in a high sensitivity amperometric cell at a potential of +0.8 V. Solid-phase extraction was used for the extraction of the analytes in real samples. The range of concentration examined varied from 10 to 150 ng g(-1) for danofloxacin, from 25 to 100 ng g(-1) for sarafloxacin and from 50 to 315 ng g(-1) for difloxacin, respectively. The method presents detection limits under 10 ng g(-1) and recoveries around 90% for the three analytes have been obtained in the experiments with fortified samples. This HPLC-ECD approach can be useful in the routine analysis of antibacterial residues being less expensive and less complicated than other more powerful tools as hyphenated techniques. 2009 Elsevier B.V. All rights reserved.

Liquid-chromatographic analysis for cyclosporine with use of a microbore column and small sample volume.

PubMed

Annesley, T; Matz, K; Balogh, L; Clayton, L; Giacherio, D

1986-07-01

This liquid-chromatographic assay requires 0.2 to 0.5 mL of whole blood, avoids the use of diethyl ether, and consumes only 10 to 20% of the solvents used in prior methods. Sample preparation involves an acidic extraction with methyl-t-butyl ether, performed in a 13 X 100 mm disposable glass tube, then a short second extraction of the organic phase with sodium hydroxide. After evaporation of the methyl-t-butyl ether, chromatography is performed on an "Astec" 2.0-mm (i.d.) octyl column. We compared results by this procedure with those by use of earlier larger-scale extractions and their respective 4.6-mm (i.d.) columns; analytical recoveries of cyclosporins A and D were comparable with previous findings and results for patients' specimens were equivalent, but the microbore columns provided greatly increased resolution and sensitivity.

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.

Acylhydrazone bond dynamic covalent polymer gel monolithic column online coupling to high-performance liquid chromatography for analysis of sulfonamides and fluorescent whitening agents in food.

PubMed

Zhang, Chengjiang; Luo, Xialin; Wei, Tianfu; Hu, Yufei; Li, Gongke; Zhang, Zhuomin

2017-10-13

A new dynamic covalent polymer (DCP) gel was well designed and constructed based on imine chemistry. Polycondensation of 4,4'-biphenyldicarboxaldehyde and 1,3,5-benzenetricarbohydrazide via Schiff-base reaction resulted in an acylhydrazone bond gel (AB-gel) DCP. AB-gel DCP had three-dimensional network of interconnected nanoparticles with hierarchically porous structure. AB-gel DCP was successfully fabricated as a monolithic column by an in-situ chemical bonding method for online enrichment and separation purpose with excellent permeability. AB-gel DCP based monolithic column showed remarkable adsorption affinity towards target analytes including sulfonamides (SAs) and fluorescent whitening agents (FWAs) due to its strong π-π affinity, hydrophobic effect and hydrogen bonding interaction. Then, AB-gel DCP based monolithic column was applied for online separation and analysis of trace SAs and FWAs in food samples coupled with high-performance liquid chromatography (HPLC). Sulfathiazole (ST) and sulfadimidine (SM2) in one positive weever sample were actually found and determined with concentrations of 273.8 and 286.3μg/kg, respectively. 2,5-Bis(5-tert-butyl-2-benzoxazolyl) thiophene (FWA184) was actually quantified in one tea infusion sample with the concentration of 268.5ng/L. The spiked experiments suggested the good recoveries in range of 74.5-110% for SAs in weever and shrimp samples with relative standard deviations (RSDs) less than 9.7% and in range of 74.0-113% for FWAs in milk and tea infusion samples with RSDs less than 9.0%. AB-gel DCP monolithic column was proved to be a promising sample preparation medium for online separation and analysis of trace analytes in food samples with complex matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics.

PubMed

Shen, Yufeng; Tolić, Nikola; Piehowski, Paul D; Shukla, Anil K; Kim, Sangtae; Zhao, Rui; Qu, Yi; Robinson, Errol; Smith, Richard D; Paša-Tolić, Ljiljana

2017-05-19

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. Herein, we report use of long (≥1M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5MPa or 14Kpsi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5-5μm particles were used as packings and long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50kDa. Larger proteoforms (50-110kDa) were chromatographed on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200-400Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ∼900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC-MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Our initial data indicate that MS detection and fragmentation inefficiencies provided by current high

Column Selection for Biomedical Analysis Supported by Column Classification Based on Four Test Parameters

PubMed Central

Plenis, Alina; Rekowska, Natalia; Bączek, Tomasz

2016-01-01

This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the FKUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the FKUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the FKUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the FKUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis. PMID:26805819

Determination of six polyether antibiotic residues in foods of animal origin by solid phase extraction combined with liquid chromatography-tandem mass spectrometry.

PubMed

Ha, Jing; Song, Ge; Ai, Lian-Feng; Li, Jian-Chen

2016-04-01

A new method using solid phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of six polyether antibiotics, including lasalocid, salinomycin, monensin, narasin, madubamycin and nigericin residues, in foods of animal origin. The samples were extracted with acetonitrile and purified by ENVI-Carb SPE columns after comparing the impurity effect and maneuverability of several SPE cartridges. Subsequently, the analytes were separated on a Hypersil Gold column (2.1×150mm, 5μm) and analyzed by MS/MS detection. The limit of quantization (LOQ) for milk and chicken was 0.4μg/kg, and for chicken livers and eggs, it was 1μg/kg. The linearity was satisfactory with a correlation coefficient of >0.9995 at concentrations ranging from 2 to 100μg/L. The average recoveries of the analytes fortified at three levels ranged from 68.2 to 114.3%, and the relative standard deviations ranged from 4.5 to 12.1%. The method was suitable for quantitative analysis and confirmation of polyether antibiotic residues in foods of animal origin. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a novel engineered E. coli host cell line platform with improved column capacity performance for ion-exchange chromatography.

PubMed

Mukherjee, Rudra Palash; Fruchtl, McKinzie S; Beitle, Robert R; Brune, Ellen M

2018-02-01

This article reports on the analysis of an engineered Escherichia coli designed to reduce the host cell protein (HCP) burden on recombinant protein purification by column chromatography. Since downstream purification accounts for a major portion of production costs when using a recombinant platform, minimization of HCPs that are initially captured or otherwise interfere during chromatography will positively impact the entire purification process. Such a strategy, of course, would also require the cell line to grow, and express recombinant proteins, at levels comparable to, or better than, its parent strain. An E. coli strain with a small number of strategic deletions (LTSF06) was transformed to produce three different recombinant biologics to examine growth and expression, and with another model protein to assess growth and the effect of selectively reduced HCPs on target product capture on DEAE ion exchange medium. Cell growth levels were maintained or increased for all constructs, and a significant reduction in HCP adsorption was realized. Indeed, a breakthrough analysis indicated that as a result of reducing adsorption of particular HCPs, a 37% increase in target protein capture was observed. This increase in product capture efficiency was achieved by focusing not on HCPs that co-elute with the recombinant target, but rather on those possessing particular column adsorption and elution characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

Separation analysis of macrolide antibiotics with good performance on a positively charged C18HCE column.

PubMed

Wei, Jie; Shen, Aijin; Yan, Jingyu; Jin, Gaowa; Yang, Bingcheng; Guo, Zhimou; Zhang, Feifang; Liang, Xinmiao

2016-03-01

The separation of basic macrolide antibiotics suffers from peak tailing and poor efficiency on traditional silica-based reversed-phase liquid chromatography columns. In this work, a C18HCE column with positively charged surface was applied to the separation of macrolides. Compared with an Acquity BEH C18 column, the C18HCE column exhibited superior performance in the aspect of peak shape and separation efficiency. The screening of mobile phase additives including formic acid, acetic acid and ammonium formate indicated that formic acid was preferable for providing symmetrical peak shapes. Moreover, the influence of formic acid content was investigated. Analysis speed and mass spectrometry compatibility were also taken into account when optimizing the separation conditions for liquid chromatography coupled with tandem mass spectrometry. The developed method was successfully utilized for the determination of macrolide residues in a honey sample. Azithromycin was chosen as the internal standard for the quantitation of spiramycin and tilmicosin, while roxithromycin was used for erythromycin, tylosin, clarithromycin, josamycin and acetylisovaleryltylosin. Good correlation coefficients (r(2) > 0.9938) for all macrolides were obtained. The intra-day and inter-day recoveries were 73.7-134.7% and 80.7-119.7% with relative standard deviations of 2.5-8.0% and 3.9-16.1%, respectively. Outstanding sensitivity with limits of quantitation (S/N ≥ 10) of 0.02-1 μg/kg and limits of detection (S/N ≥ 3) of 0.01-0.5 μg/kg were achieved. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of Gonyautoxin-4 in Echinoderms and Gastropod Matrices by Conversion to Neosaxitoxin Using 2-Mercaptoethanol and Post-Column Oxidation Liquid Chromatography with Fluorescence Detection

PubMed Central

Silva, Marisa; Rey, Verónica; Botana, Ana; Vasconcelos, Vitor; Botana, Luis

2015-01-01

Paralytic Shellfish Toxin blooms are common worldwide, which makes their monitoring crucial in the prevention of poisoning incidents. These toxins can be monitored by a variety of techniques, including mouse bioassay, receptor binding assay, and liquid chromatography with either mass spectrometric or pre- or post-column fluorescence detection. The post-column oxidation liquid chromatography with fluorescence detection method, used routinely in our laboratory, has been shown to be a reliable method for monitoring paralytic shellfish toxins in mussel, scallop, oyster and clam species. However, due to its high sensitivity to naturally fluorescent matrix interferences, when working with unconventional matrices, there may be problems in identifying toxins because of naturally fluorescent interferences that co-elute with the toxin peaks. This can lead to erroneous identification. In this study, in order to overcome this challenge in echinoderm and gastropod matrices, we optimized the conversion of Gonyautoxins 1 and 4 to Neosaxitoxin with 2-mercaptoethanol. We present a new and less time-consuming method with a good recovery (82.2%, RSD 1.1%, n = 3), requiring only a single reaction step. PMID:26729166

Determination of Flurbiprofen in Human Plasma by High-Performance Liquid Chromatography.

PubMed

Yilmaz, Bilal; Erdem, Ali Fuat

2015-10-01

A simple high-performance liquid chromatography method has been developed for determination of flurbiprofen in human plasma. The method was validated on an Ace C18 column using UV detection. The mobile phase was acetonitrile-0.05 M potassium dihydrogen phosphate solution (60:40, v/v) adjusted to pH 3.5 with phosphoric acid. The calibration curve was linear between the concentration range of 0.10-5.0 μg/mL. Intra- and inter-day precision values for flurbiprofen in plasma were <4.47, and accuracy (relative error) was better than 3.67%. The extraction recoveries of flurbiprofen from human plasma were between 93.0 and 98.9%. The limits of detection and quantification of flurbiprofen were 0.03 and 0.10 μg/mL, respectively. In addition, this assay was applied to determine the pharmacokinetic parameters of flurbiprofen in six healthy Turkish volunteers who had been given 100 mg flurbiprofen. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Determination of alpha-hydroxy acids in cosmetic products by high-performance liquid chromatography with a narrow-bore column.

PubMed

Nicoletti, I; Corradini, C; Cogliandro, E; Cavazza, A

1999-08-01

This paper reports the results of a study carried out to develop a simple, rapid and sensitive method for the separation, identification and quantitative measurement of alpha-hydroxy acids in commercial cosmetics using high-performance liquid chromatography (HPLC). This method is successfully applied to the simultaneous identification and quantitative determination of glycolic, lactic, malic, tartaric and citric acids employing a reversed phase narrow-bore column under isocratic condition and UV detection. The method is validated by determining the precision of replicate analyses and accuracy by analyzing samples with and without adding know amount of the alpha-hydroxy acids. The procedure is suitable for routine analyses of commercial cosmetics.

Characterization and purification of bile salt hydrolase from Lactobacillus sp. strain 100-100

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lundeen, S.G.; Savage, D.C.

1990-08-01

The authors have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving ({sup 14}C)taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in mediummore » free of bile salts. In cell-free extracts, however, the activity was about equal whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Whether the enzyme exists in two forms in the cells remains to be determined.« less

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

Flow-through Column Experiments and Modeling of Microbially Mediated Cr(VI) Reduction at Hanford 100H

NASA Astrophysics Data System (ADS)

Yang, L.; Molins, S.; Beller, H. R.; Brodie, E. L.; Steefel, C.; Nico, P. S.; Han, R.

2010-12-01

Microbially mediated Cr(VI) reduction at the Hanford 100H area was investigated by flow-through column experiments. Three separate experiments were conducted to promote microbial activities associated with denitrification, iron and sulfate reduction, respectively. Replicate columns packed with natural sediments from the site under anaerobic environment were injected with 5mM Lactate as the electron donor and 5 μM Cr(VI) in all experiments. Sulfate and nitrate solutions were added to act as the main electron acceptors in the respective experiments, while iron columns relied on the indigenous sediment iron (and manganese) oxides as electron acceptors. Column effluent solutions were analyzed by IC and ICP-MS to monitor the microbial consumption/conversion of lactate and the associated Cr(VI) reduction. Biogeochemical reactive transport modeling was performed to gain further insights into the reaction mechanisms and Cr(VI) bioreduction rates. All experimental columns showed a reduction of the injected Cr(VI). Columns under denitrifying conditions showed the least Cr(VI) reduction at early stages (<60 days) compared to columns run under other experimental conditions, but became more active over time, and ultimately showed the most consistent Cr(VI) reduction. A strong correlation between denitrification and Cr(VI) reduction processes was observed and was in agreement with the results obtained in batch experiments with a denitrifying bacterium isolated from the Hanford site. The accumulation of nitrite does not appear to have an adverse effect on Cr(VI) reduction rates. Reactive transport simulations indicated that biomass growth completely depleted influent ammonium, and called for an additional source of N to account for the measured reduction rates. Iron columns were the least active with undetectable consumption of the injected lactate, slowest cell growth, and the smallest change in Cr(VI) concentrations during the course of the experiment. In contrast, columns

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

PubMed

Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

2017-01-01

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparative liquid column electrophoresis of T and B lymphocytes at gravity = 1

NASA Technical Reports Server (NTRS)

Van Oss, C. J.; Bigazzi, P. E.; Gillman, C. F.; Allen, R. E.

1974-01-01

Vertical liquid columns containing low-molecular-weight dextran density gradients can be used for preparative lymphocyte electrophoresis on earth, in simulation of zero gravity conditions. Another method that has been tested at 1 g, is the electrophoresis of lymphocytes in an upward direction in vertical columns. By both methods up to 100 million lymphocytes can be separated at one time in a 30-cm glass column of 8-mm inside diameter, at 12 V/cm, in two hours. Due to convection and sedimentation problems, the separation at 1 g is less than ideal, but it is expected that at zero gravity electrophoresis will probe to be a uniquely powerful cell separation tool.

Simultaneous achiral-chiral analysis of pharmaceutical compounds using two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography.

PubMed

Venkatramani, C J; Al-Sayah, Mohammad; Li, Guannan; Goel, Meenakshi; Girotti, James; Zang, Lisa; Wigman, Larry; Yehl, Peter; Chetwyn, Nik

2016-02-01

A new interface was designed to enable the coupling of reversed phase liquid chromatography (RPLC) and supercritical fluid chromatography (SFC). This online two-dimensional chromatographic system utilizing RPLC in the first dimension and SFC in the second was developed to achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The interface consists of an eight-port, dual-position switching valve with small volume C-18 trapping columns. The peaks of interest eluting from the first RPLC dimension column were effectively focused as sharp concentration pulses on small volume C-18 trapping column/s and then injected onto the second dimension SFC column. The first dimension RPLC separation provides the achiral purity result, and the second dimension SFC separation provides the chiral purity result (enantiomeric excess). The results are quantitative enabling simultaneous achiral, chiral analysis of compounds. The interface design and proof of concept demonstration are presented. Additionally, comparative studies to conventional SFC and case studies of the applications of 2D LC-SFC in pharmaceutical analysis is presented. Copyright © 2015 Elsevier B.V. All rights reserved.

21 CFR 862.2250 - Gas liquid chromatography system for clinical use.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Gas liquid chromatography system for clinical use... Instruments § 862.2250 Gas liquid chromatography system for clinical use. (a) Identification. A gas liquid... pressure. The device may include accessories such as columns, gases, column supports, and liquid coating...

21 CFR 862.2250 - Gas liquid chromatography system for clinical use.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Gas liquid chromatography system for clinical use... Instruments § 862.2250 Gas liquid chromatography system for clinical use. (a) Identification. A gas liquid... pressure. The device may include accessories such as columns, gases, column supports, and liquid coating...

21 CFR 862.2250 - Gas liquid chromatography system for clinical use.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Gas liquid chromatography system for clinical use... Instruments § 862.2250 Gas liquid chromatography system for clinical use. (a) Identification. A gas liquid... pressure. The device may include accessories such as columns, gases, column supports, and liquid coating...

21 CFR 862.2250 - Gas liquid chromatography system for clinical use.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gas liquid chromatography system for clinical use... Instruments § 862.2250 Gas liquid chromatography system for clinical use. (a) Identification. A gas liquid... pressure. The device may include accessories such as columns, gases, column supports, and liquid coating...

21 CFR 862.2250 - Gas liquid chromatography system for clinical use.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Gas liquid chromatography system for clinical use... Instruments § 862.2250 Gas liquid chromatography system for clinical use. (a) Identification. A gas liquid... pressure. The device may include accessories such as columns, gases, column supports, and liquid coating...

Ionic liquid-based microwave-assisted extraction for the determination of flavonoid glycosides in pigeon pea leaves by high-performance liquid chromatography-diode array detector with pentafluorophenyl column.

PubMed

Wei, Wei; Fu, Yu-jie; Zu, Yuan-gang; Wang, Wei; Luo, Meng; Zhao, Chun-jian; Li, Chun-ying; Zhang, Lin; Wei, Zuo-fu

2012-11-01

In this study, an ionic liquid-based microwave-assisted extraction (ILMAE) followed by high-performance liquid chromatography-diode array detector with a pentafluorophenyl column for the extraction and quantification of eight flavonoid glycosides in pigeon pea leaves is described. Compared with conventional extraction methods, ILMAE is a more effective and environment friendly method for the extraction of nature compounds from herbal plants. Nine different types of ionic liquids with different cations and anions were investigated. The results suggested that varying the anion and cation had significant effects on the extraction of flavonoid glycosides, and 1.0 M 1-butyl-3-methylimidazolium bromide ([C4MIM]Br) solution was selected as solvent. In addition, the extraction procedures were also optimized using a series of single-factor experiments. The optimum parameters were obtained as follows: extraction temperature 60°C, liquid-solid ratio 20:1 mL/g and extraction time 13 min. Moreover, an HPLC method using pentafluorophenyl column was established and validated. Good linearity was observed with the regression coefficients (r(2)) more than 0.999. The limit of detection (LODs) (S/N = 3) and limit of quantification (LOQs) (S/N = 10) for the components were less than 0.41 and 1.47 μg/mL, respectively. The inter- and intraday precisions that were used to evaluate the reproducibility and relative standard deviation (RSD) values were less than 4.57%. The recoveries were between 97.26 and 102.69%. The method was successfully used for the analysis of samples of pigeon pea leaves. In conclusion, the developed ILMAE-HPLC-diode array detector using pentafluorophenyl column method can be applied for quality control of pigeon pea leaves and related medicinal products. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design and implementation of an array of micro-electrochemical detectors for two-dimensional liquid chromatography--proof of principle.

PubMed

Abia, Jude A; Putnam, Joel; Mriziq, Khaled; Guiochon, Georges A

2010-03-05

Simultaneous two-dimensional liquid chromatography (2D-LC) is an implementation of two-dimensional liquid chromatography which has the potential to provide very fast, yet highly efficient separations. It is based on the use of time x space and space x space separation systems. The basic principle of this instrument has been validated long ago by the success of two-dimensional thin layer chromatography. The construction of a pressurized wide and flat column (100 mm x 100 mm x 1 mm) operated under an inlet pressure of up to 50 bar was described previously. However, to become a modern analytical method, simultaneous 2D-LC requires the development of detectors suitable for the monitoring of the composition of the eluent of this pressurized planar, wide column. An array of five equidistant micro-electrochemical sensors was built for this purpose and tested. Each sensor is a three-electrode system, with the working electrode being a 25 microm polished platinum micro-electrode. The auxiliary electrode is a thin platinum wire and the reference electrode an Ag/AgCl (3M sat. KCl) electrode. In this first implementation, proof of principle is demonstrated, but the final instrument will require a much larger array. 2010 Elsevier B.V. All rights reserved.

Isolation and purification of six iridoid glycosides from gardenia jasminoides fruit by medium-pressure liquid chromatography combined with macroporous resin chromatography.

PubMed

Wang, Yun; Liu, Hui; Shen, Lifeng; Yao, Lan; Ma, Yinlian; Yu, Dingrong; Chen, Jianhong; Li, Puling; Chen, Ying; Zhang, Cun

2015-12-01

Gardeniae fructus is one of the most frequently used herbs in traditional Chinese medicine. In the present study, a process for the enrichment of six iridoid glycosides from Gardeniae fructus was developed using medium-pressure liquid chromatography combined with macroporous resin and reversed-phase chromatography. The purities of different fractions from Gardeniae fructus were assessed using quantitative high-performance liquid chromatography. After fractionation using HPD-100 column chromatography, a 30% ethanol fraction was selected based on high-performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis to separate and purify. Based on the orientation analysis results, six compounds-deacetyl asperulosidic acid methyl ester, gardenoside, ixoroside, scandoside methyl ester, genipin-1-O-β-d-gentiobioside, and geniposide-were successfully isolated and purified in three to four combined steps from Gardeniae fructus. The purities of these compounds were found by high-performance liquid chromatography analysis to be 97.9, 98.1, 95.5, 96.3, 97.1, and 98.7%, respectively. Moreover, their structures were elucidated by NMR spectroscopy and liquid chromatography with tandem mass spectrometry. The separation process was highly efficient, rapid, and accurate, making it a potential approach for the large-scale production of iridoids in the laboratory and providing several marker compounds for quality control. This procedure may be meaningful for the purification of other natural products used in traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of alpidem, an imidazopyridine anxiolytic, and its metabolites by column-switching high-performance liquid chromatography with fluorescence detection.

PubMed

Flaminio, L; Ripamonti, M; Ascalone, V

1994-05-13

Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo[1,2-a]pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed on-line), involves the automatic injection of plasma samples (200 microliters) on to a precolumn filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the precolumn was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 ml min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng ml-1, respectively, in human plasma.

Using 100G Network Technology in Support of Petascale Science

NASA Technical Reports Server (NTRS)

Gary, James P.

2011-01-01

NASA in collaboration with a number of partners conducted a set of individual experiments and demonstrations during SC 10 that collectively were titled "Using 100G Network Technology in Support of Petascale Science". The partners included the iCAIR, Internet2, LAC, MAX, National LambdaRail (NLR), NOAA and SCinet Research Sandbox (SRS) as well as the vendors Ciena, Cisco, ColorChip, cPacket, Extreme Networks, Fusion-io, HP and Panduit who most generously allowed some of their leading edge 40G/100G optical transport, Ethernet switch and Internet Protocol router equipment and file server technologies to be involved. The experiments and demonstrations featured different vendor-provided 40G/100G network technology solutions for full-duplex 40G and 100G LAN data flows across SRS-deployed single-node fiber-pairs among the Exhibit Booths of NASA, the National Center for Data lining, NOAA and the SCinet Network Operations Center, as well as between the NASA Exhibit Booth in New Orleans and the Starlight Communications Exchange facility in Chicago across special SC 10- only 80- and 100-Gbps wide area network links provisioned respectively by the NLR and Internet2, then on to GSFC across a 40-Gbps link. provisioned by the Mid-Atlantic Crossroads. The networks and vendor equipment were load-stressed by sets of NASA/GSFC High End Computer Network Team-built, relatively inexpensive, net-test-workstations that are capable of demonstrating greater than 100Gbps uni-directional nuttcp-enabled memory-to-memory data transfers, greater than 80-Gbps aggregate--bidirectional memory-to-memory data transfers, and near 40-Gbps uni-directional disk-to-disk file copying. This paper will summarize the background context, key accomplishments and some significances of these experiments and demonstrations.

On the feasibility to conduct gradient liquid chromatography separations in narrow-bore columns at pressures up to 2000bar.

PubMed

De Pauw, Ruben; Swier, Tim; Degreef, Bart; Desmet, Gert; Broeckhoven, Ken

2016-11-18

The limits in operating pressures are extended for narrow-bore columns in gradient elution up to 2000bar. As the required pumps for these pressures are incompatible with common chromatographic solvents and are not suitable to apply a mobile phase composition gradient, a mobile phase delivery and injection system is described and experimentally validated which allows to use any possible chromatographic solvent in isocratic and gradient elution. The mobile phase delivery and injection system also allows to perform multiple separations without the need to depressurize the column. This system consists out of 5 dual on/off valves and two large volume loops in which the gradient and equilibration volume of initial mobile phase are loaded by a commercial liquid chromatography pump. The loops are then flushed toward the column at extreme pressures. The mobile phase delivery and injection system is first evaluated in isocratic elution and shows a comparable performance to a state-of-the-art commercial flow-through-needle injector but with twice the pressure rating. Distortion of the loaded gradient by dispersion in the gradient storage loop is studied. The effect of the most important parameters (such as flow rate, pressure and gradient steepness) is experimentally investigated. Different gradient steepnesses and volumes can be applied at different flow rates and operating pressures with a good repeatability. Due to the isobaric operation of the pumps, the gradient is monitored in real-time by a mass flow meter installed at the detector outlet. The chromatograms are then converted from time to volume-base. A separation of a 19-compound sample is performed on a 300×2.1mm column at 1000bar and on a 600×2.1mm column at 2000bar. The peak capacity was found to increase from 141 to 199 and thus scales with L as is predicted by theory. This allows to conclude that the inlet pressure for narrow-bore columns in gradient elution can be increased up to 2000bar without fundamental

A Laboratory Experiment To Measure Henry's Law Constants of Volatile Organic Compounds with a Bubble Column and a Gas Chromatography Flame Ionization Detector (GC-FID)

ERIC Educational Resources Information Center

Lee, Shan-Hu; Mukherjee, Souptik; Brewer, Brittany; Ryan, Raphael; Yu, Huan; Gangoda, Mahinda

2013-01-01

An undergraduate laboratory experiment is described to measure Henry's law constants of organic compounds using a bubble column and gas chromatography flame ionization detector (GC-FID). This experiment is designed for upper-division undergraduate laboratory courses and can be implemented in conjunction with physical chemistry, analytical…

SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry.

PubMed

Lanshoeft, Christian; Heudi, Olivier; Cianférani, Sarah

2016-05-15

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC-MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 μg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents. Copyright © 2016 Elsevier Inc. All rights reserved.

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. Copyright © 2013 Elsevier B.V. All rights reserved.

Purification of anti-Japanese encephalitis virus monoclonal antibody by ceramic hydroxyapatite chromatography without proteins A and G.

PubMed

Saito, Maiko; Kurosawa, Yae; Okuyama, Tsuneo

2012-02-01

Antibody purification using proteins A and G has been a standard method for research and industrial processes. The conventional method, however, includes a three-step process, including buffer exchange, before chromatography. In addition, proteins A and G require low pH elution, which causes antibody aggregation and inactivates the antibody's immunity. This report proposes a two-step method using hydroxyapatite chromatography and membrane filtration, without proteins A and G. This novel method shortens the running time to one-third the conventional method for each cycle. Using our two-step method, 90.2% of the monoclonal antibodies purified were recovered in the elution fraction, the purity achieved was >90%, and most of the antigen-specific activity was retained. This report suggests that the two-step method using hydroxyapatite chromatography and membrane filtration should be considered as an alternative to purification using proteins A and G.

Chelate-modified polymers for atmospheric gas chromatography

NASA Technical Reports Server (NTRS)

Christensen, W. W.; Mayer, L. A.; Woeller, F. H. (Inventor)

1980-01-01

Chromatographic materials were developed to serve as the stationary phase of columns used in the separation of atmospheric gases. These materials consist of a crosslinked porous polymer matrix, e.g., a divinylbenzene polymer, into which has been embedded an inorganic complexed ion such as N,N'-ethylene-bis-(acetylacetoniminato)-cobalt (2). Organic nitrogenous bases, such as pyridine, may be incorporated into the chelate polymer complexes to increase their chromatographic utility. With such materials, the process of gas chromatography is greatly simplified, especially in terms of time and quantity of material needed for a gas separation.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

A strategy for efficient discovery of new natural compounds by integrating orthogonal column chromatography and liquid chromatography/mass spectrometry analysis: Its application in Panax ginseng, Panax quinquefolium and Panax notoginseng to characterize 437 potential new ginsenosides.

PubMed

Yang, Wen-zhi; Ye, Min; Qiao, Xue; Liu, Chun-fang; Miao, Wen-juan; Bo, Tao; Tao, Hai-yan; Guo, De-an

2012-08-20

To discover new natural compounds from herbal medicines tends to be more and more difficult. In this paper, a strategy integrating orthogonal column chromatography and liquid chromatography/mass spectrometry (LC/MS) analysis was proposed, and was applied for rapid discovery of new ginsenosides from Panax ginseng (PG), Panax quinquefolium (PQ), and Panax notoginseng (PN). The ginsenosides extracts were fractionated by MCI gel×silica gel orthogonal column chromatography. The fractions were then separated on a C(18) HPLC column, eluted with a three-component mobile phase (CH(3)CN/CH(3)OH/3mM CH(3)COONH(4)H(2)O), and detected by electrospray ionization tandem mass spectrometry. The structures of unknown ginsenosides were elucidated by analyzing negative and positive ion mass spectra, which provided complementary information on the sapogenins and oligosaccharide chains, respectively. A total of 623 comprising 437 potential new ginsenosides were characterized from the ethanol extracts of PG, PQ and PN. New acylations, diversified saccharide chains and C-17 side chains constituted novelty of the newly identified ginsenosides. An interpretation guideline was proposed for structural characterization of unknown ginsenosides by LC/MS. To confirm reliability of this strategy, two targeted unknown trace ginsenosides were obtained in pure form by LC/MS-guided isolation. Based on extensive NMR spectroscopic analysis and other techniques, they were identified as 3-O-[6-O-(E)-butenoyl-β-D-glucopyranosyl(1,2)-β-D-glucopyranosyl]-20(S)-protopanaxadiol-20-O-β-D-glucopyranosyl(1,6)-β-D-glucopyranoside (named ginsenoside IV) and 3-O-β-D-glucopyranosyl(1,2)-β-D-glucopyranosyl-3β,12β,20(S),24(R)-tetra hydroxy-dammar-25-ene-20-O-β-D-glucopyranosyl(1,6)-β-D-glucopyranoside (ginsenoside V), respectively. The fully established structures were consistent with the MS-oriented structural elucidation. This study expanded our understanding on ginsenosides of Panax species, and the

Enantiomeric separation of triazole fungicides with 3-μm and 5-μml particle chiral columns by reverse-phase high-performance liquid chromatography.

PubMed

Qiu, Jing; Dai, Shouhui; Zheng, Chuangmu; Yang, Shuming; Chai, Tingting; Bie, Mei

2011-07-01

This study used chiral columns packed with 3-μm and 5-μm particles to comparatively separate enantiomers of 9 triazole fungicides, and Lux Cellulose-1 columns with chiral stationary phase of cellulose-tris-(3,5-dimethylphenylcarbamate) were used on reverse-phase high-performance liquid chromatography with flow rates of 0.3 and 1.0 mL min(-1) for 3-μm and 5-μm columns, respectively. The (+)-enantiomers of hexaconazole (1), tetraconazole (4), myclobutanil (7), fenbuconazole (8) and the (-)-enantiomers of flutriafol (2), diniconazole (3), epoxiconazole (5), penconazole (6), triadimefon (9) were firstly eluted from both columns, the elution orders identified with an optical rotation detector didn't change with variety of column particles and mobile phases (acetronitrile/water and methanol/water). The plots of natural logarithms of the selectivity factors (ln α) for all fungicides except penconazole (6) versus the inverse of temperature (1/T) were linear in range of 5-40°C. The thermodynamic parameters (ΔH°, ΔS°, ΔΔH° and ΔΔS°) were calculated using Van't Hoff equations to understand the thermosynamic driving forces for enantioseparation. This work will be very helpful to obtain good enantiomeric separation and establish more efficient analytical method for triazole fungicides. Chirality, 2011. © 2011 Wiley-Liss, Inc. Copyright © 2011 Wiley-Liss, Inc.

Isolation of Methoxyfuranocoumarins From Ammi majus by Centrifugal Partition Chromatography.

PubMed

Bartnik, Magdalena; Mazurek, Anna Katarzyna

2016-01-01

Pure methoxyfuranocoumarins were isolated from Ammi majus L. by use of low-pressure column chromatography (LPCC) followed by centrifugal partition chromatography (CPC). The concentrated petroleum ether extract from fruits of A. majus was fractionated on a silica gel column using a gradient of ethyl acetate in dichloromethane (0-80%, v/v). Coumarin-rich fractions were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography with diode array detection (HPLC/DAD). Xanthotoxin (8-MOP) and isopimpinellin (isoP), structurally similar compounds, were isolated in one fraction (FR6). To avoid multistep and long-lasting TLC preparation, optimization of CPC conditions has been performed. In one run, an effective separation of 8-MOP and isoP was achieved. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10 : 8 : 10 : 9; v/v) in an ascending mode (the aqueous phase was a stationary phase, and the organic phase was a mobile phase), with flow rate 3 mL/min and rotation speed 1,600 r.p.m., was used. The identification and high purities of isolated 8-MOP (98.7%) and isoP (100%) were confirmed by HPLC/DAD assay, when compared with standards. The developed CPC method could be applied to the effective isolation of 8-MOP and isoP from plant extracts. The high purity of obtained compounds makes possible further exploitation of these components in biological studies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fast analysis of capsaicinoids in Naga Jolokia extracts (Capsicum chinense) by high-performance liquid chromatography using fused core columns.

PubMed

Stipcovich, Tea; Barbero, Gerardo F; Ferreiro-González, Marta; Palma, Miguel; Barroso, Carmelo G

2018-01-15

A rapid high-performance liquid chromatography method with a C18 reverse-phase fused-core column has been developed for the determination and quantification of the main capsaicinoids (nornordihydrocapsaicin, nordihydrocapsaicin, capsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) present in Naga Jolokia peppers. A fused-core Kinetex™ C18 column (50×2.1mm i.d.; 2.6μm) was used for the analysis. The chromatographic separation was obtained with a gradient method in which the mobile phase was water (0.1% acetic acid) as solvent A and acetonitrile (0.1% acetic acid) as solvent B. The separation of all compounds was achieved in less than 3min with a total analysis time (sample-to-sample) of 10min. The robustness of the method was evaluated. The method showed excellent repeatability and intermediate precision expressed as coefficient of variance of less than 2%. The developed method was employed for the quantification of the major capsaicinoids present in different peppers and commercial products containing chilli peppers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains.

PubMed

Asghar, Muhammad Asif; Iqbal, Javed; Ahmed, Aftab; Khan, Mobeen Ahmed; Shamsuddin, Zuzzer Ali; Jamil, Khalid

2016-06-01

A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40-63 μm) solid-phase extraction cartridges. AFs were separated using a LiChroCART-RP-18 (5 μm, 250 × 4.0 mm(2)) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min(-1) The fluorescence of each AF was detected at λex = 365 nm and λem = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB1, AFB2, AFG1 and AFG2), linearity (R(2) ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ -5.51), robustness (p < 0.0080), ruggedness (p < 0.0100) and average recoveries (89.2-97.8%). The limits of quantification of AFB1, AFB2, AFG1 and AFG2 were 0.080, 0.073, 0.062 and 0.066 ng g(-1), respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice (n = 20), wheat (n = 15) and maize (n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains. © The Author(s) 2014.

Coupling nanoliter high-performance liquid chromatography to inductively coupled plasma mass spectrometry for arsenic speciation.

PubMed

Cheng, Heyong; Shen, Lihuan; Liu, Jinhua; Xu, Zigang; Wang, Yuanchao

2018-04-01

Nanoliter high-performance liquid chromatography shows low consumption of solvents and samples, offering one of the best choices for arsenic speciation in precious samples in combination with inuctively coupled plasma mass spectrometry. A systematic investigation on coupling nanoliter high-performance liquid chromatography to inductively coupled plasma mass spectrometry from instrument design to injected sample volume and mobile phase was performed in this study. Nanoflow mobile phase was delivered by flow splitting using a conventional high-pressure pump with reuse of mobile phase waste. Dead volume was minimized to 60 nL for the sheathless interface based on the previously developed nanonebulizer. Capillary columns for nanoliter high-performance liquid chromatography were found to be sensitive to sample loading volume. An apparent difference was also found between the mobile phases for nanoliter and conventional high-performance liquid chromatography. Baseline separation of arsenite, arsenate, monomethylarsenic, and dimethylarsenic was achieved within 11 min on a 15 cm C 18 capillary column and within 12 min on a 25 cm strong anion exchange column. Detection limits of 0.9-1.8 μg/L were obtained with precisions variable in the range of 1.6-4.2%. A good agreement between determined and certified values of a certified reference material of human urine (GBW 09115) validated its accuracy along with good recoveries (87-102%). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography

PubMed Central

Dutta, Amit K.; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A.; Zhang, Ada W.; Tustian, Andrew D.; Zydney, Andrew L.; Shinkazh, Oleg

2015-01-01

Recent studies using simple model systems have demonstrated that Continuous Countercurrent Tangential Chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an “after binder” to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (~0.67 g/L) and one with high titer (~6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to that obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. PMID:25747172

Purification of monoclonal antibodies from clarified cell culture fluid using Protein A capture continuous countercurrent tangential chromatography.

PubMed

Dutta, Amit K; Tran, Travis; Napadensky, Boris; Teella, Achyuta; Brookhart, Gary; Ropp, Philip A; Zhang, Ada W; Tustian, Andrew D; Zydney, Andrew L; Shinkazh, Oleg

2015-11-10

Recent studies using simple model systems have demonstrated that continuous countercurrent tangential chromatography (CCTC) has the potential to overcome many of the limitations of conventional Protein A chromatography using packed columns. The objective of this work was to optimize and implement a CCTC system for monoclonal antibody purification from clarified Chinese Hamster Ovary (CHO) cell culture fluid using a commercial Protein A resin. Several improvements were introduced to the previous CCTC system including the use of retentate pumps to maintain stable resin concentrations in the flowing slurry, the elimination of a slurry holding tank to improve productivity, and the introduction of an "after binder" to the binding step to increase antibody recovery. A kinetic binding model was developed to estimate the required residence times in the multi-stage binding step to optimize yield and productivity. Data were obtained by purifying two commercial antibodies from two different manufactures, one with low titer (∼ 0.67 g/L) and one with high titer (∼ 6.9 g/L), demonstrating the versatility of the CCTC system. Host cell protein removal, antibody yields and purities were similar to those obtained with conventional column chromatography; however, the CCTC system showed much higher productivity. These results clearly demonstrate the capabilities of continuous countercurrent tangential chromatography for the commercial purification of monoclonal antibody products. Copyright © 2015 Elsevier B.V. All rights reserved.

Recent trends in ultra-fast HPLC: new generation superficially porous silica columns.

PubMed

Ali, Imran; Al-Othman, Zeid A; Nagae, Norikaju; Gaitonde, Vinay D; Dutta, Kamlesh K

2012-12-01

New generation columns, i.e. packed with superficially porous silica particles are available as trade names with following manufacturers: Halo, Ascentis Express, Proshell 120, Kinetex, Accucore, Sunshell, and Nucleoshell. These provide ultra-fast HPLC separations for a variety of compounds with moderate sample loading capacity and low back pressure. Chemistries of these columns are C(8), C(18), RP-Amide, hydrophilic interaction liquid chromatography, penta fluorophenyl (PFP), F5, and RP-aqua. Normally, the silica gel particles are of 2.7 and 1.7 μm as total and inner solid core diameters, respectively, with 0.5-μm-thick of outer porous layer having 90 Å pore sizes and 150 m(2)/g surface area. This article describes these new generation columns with special emphasis on their textures and chemistries, separations, optimization, and comparison (inter and intra stationary phases). Besides, future perspectives have also been discussed. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

DOE PAGES

Shen, Yufeng; Tolić, Nikola; Piehowski, Paul D.; ...

2017-01-05

Separation of proteoforms for global intact protein analysis (i.e. top-down proteomics) has lagged well behind what is achievable for peptides in traditional bottom-up proteomic approach and is becoming a true bottle neck for top-down proteomics. We report use of long (≥1 M) columns containing short alkyl (C1-C4) bonded phases to achieve high-resolution RPLC for separation of proteoforms. At a specific operation pressure limit (i.e., 96.5 MPa or 14 K psi used in this work), column length was found to be the most important factor for achieving maximal resolution separation of proteins when 1.5–5 μm particles were used as packings andmore » long columns provided peak capacities greater than 400 for proteoforms derived from a global cell lysate with molecular weights below 50 kDa. Furthermore, we chromatographed larger proteoforms (50–110 kDa) on long RPLC columns and detected by MS; however, they cannot be identified yet by tandem mass spectrometry. Our experimental data further demonstrated that long alkyl (e.g., C8 and C18) bonded particles provided high-resolution RPLC for <10 kDa proteoforms, not efficient for separation of global proteoforms. Reversed-phase particles with porous, nonporous, and superficially porous surfaces were systematically investigated for high-resolution RPLC. Pore size (200–400 Å) and the surface structure (porous and superficially porous) of particles was found to have minor influences on high-resolution RPLC of proteoforms. RPLC presented herein enabled confident identification of ~900 proteoforms (1% FDR) for a low-microgram quantity of proteomic samples using a single RPLC–MS/MS analysis. The level of RPLC performance attained in this work is close to that typically realized in bottom-up proteomics, and broadly useful when applying e.g., the single-stage MS accurate mass tag approach, but less effective when combined with current tandem MS. Finally, our initial data indicate that MS detection and fragmentation

Loading properties of porous layered capillary columns with sorbents of different natures

NASA Astrophysics Data System (ADS)

Patrushev, Y. V.; Nikolaeva, O. A.; Sidelnikov, V. N.

2017-04-01

Loading properties are studied for the commercial porous layered capillary columns GASPRO, Rt-Q-BOND, and for columns with porous layers based on the divinylbenzene-vinylimidazole copolymer (DVB-VIm), poly(trimethylsilyl)propyn (PTMSP) and ordered silica of the MCM-41 type. It is shown that the loading capacity of a column based on MCM-41 is 5-10 times higher than in the other considered columns. The loading properties of porous layered columns and columns for gas-liquid chromatography are compared.

Development of a 45kpsi ultrahigh pressure liquid chromatography instrument for gradient separations of peptides using long microcapillary columns and sub-2μm particles.

PubMed

Grinias, Kaitlin M; Godinho, Justin M; Franklin, Edward G; Stobaugh, Jordan T; Jorgenson, James W

2016-10-21

Commercial chromatographic instrumentation for bottom-up proteomics is often inadequate to resolve the number of peptides in many samples. This has inspired a number of complex approaches to increase peak capacity, including various multidimensional approaches, and reliance on advancements in mass spectrometry. One-dimensional reversed phase separations are limited by the pressure capabilities of commercial instruments and prevent the realization of greater separation power in terms of speed and resolution inherent to smaller sorbents and ultrahigh pressure liquid chromatography. Many applications with complex samples could benefit from the increased separation performance of long capillary columns packed with sub-2μm sorbents. Here, we introduce a system that operates at a constant pressure and is capable of separations at pressures up to 45kpsi. The system consists of a commercially available capillary liquid chromatography instrument, for sample management and gradient creation, and is modified with a storage loop and isolated pneumatic amplifier pump for elevated separation pressure. The system's performance is assessed with a complex peptide mixture and a range of microcapillary columns packed with sub-2μm C18 particles. Copyright © 2016 Elsevier B.V. All rights reserved.

Systematic evaluation of commercially available ultra-high performance liquid chromatography columns for drug metabolite profiling: optimization of chromatographic peak capacity.

PubMed

Dubbelman, Anne-Charlotte; Cuyckens, Filip; Dillen, Lieve; Gross, Gerhard; Hankemeier, Thomas; Vreeken, Rob J

2014-12-29

The present study investigated the practical use of modern ultra-high performance liquid chromatography (UHPLC) separation techniques for drug metabolite profiling, aiming to develop a widely applicable, high-throughput, easy-to-use chromatographic method, with a high chromatographic resolution to accommodate simultaneous qualitative and quantitative analysis of small-molecule drugs and metabolites in biological matrices. To this end, first the UHPLC system volume and variance were evaluated. Then, a mixture of 17 drugs and various metabolites (molecular mass of 151-749Da, logP of -1.04 to 6.7), was injected on six sub-2μm particle columns. Five newest generation core shell technology columns were compared and tested against one column packed with porous particles. Two aqueous (pH 2.7 and 6.8) and two organic mobile phases were evaluated, first with the same flow and temperature and subsequently at each column's individual limit of temperature and pressure. The results demonstrated that pre-column dead volume had negligible influence on the peak capacity and shape. In contrast, a decrease in post-column volume of 57% resulted in a substantial (47%) increase in median peak capacity and significantly improved peak shape. When the various combinations of stationary and mobile phases were used at the same flow rate (0.5mL/min) and temperature (45°C), limited differences were observed between the median peak capacities, with a maximum of 26%. At higher flow though (up to 0.9mL/min), a maximum difference of almost 40% in median peak capacity was found between columns. The finally selected combination of solid-core particle column and mobile phase composition was chosen for its selectivity, peak capacity, wide applicability and peak shape. The developed method was applied to rat hepatocyte samples incubated with the drug buspirone and demonstrated to provide a similar chromatographic resolution, but a 6 times higher signal-to-noise ratio than a more traditional UHPLC

Rapid separation of beryllium and lanthanide derivatives by capillary gas chromatography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harvey, Scott D.; Lucke, Richard B.; Douglas, Matt

2012-09-04

Previous studies describe derivatization of metal ions followed by analysis using gas chromatography, usually on packed columns. In many of these studies, stable and volatile derivatives were formed using fluorinated β-diketonate reagents. This paper extends previous work by investigating separations of the derivatives on small-diameter capillary gas chromatography columns and exploring on-fiber, solid-phase microextraction derivatization techniques for beryllium. The β-diketonate used for these studies was 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Derivatization of lanthanides also required addition of a neutral donor, dibutyl sulfoxide, in addition to 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Unoptimized separations on a 100-μm i.d. capillary column proved capable of rapid separations (within 15 min) of lanthanidemore » derivatives that are adjacent to one another in the periodic table. Full-scan mass spectra were obtained from derivatives containing 5 ng of each lanthanide. Studies also developed a simple on-fiber solid-phase microextraction derivatization of beryllium. Beryllium could be analyzed in the presence of other alkali earth elements (Ba(II) and Sr(II)) without interference. Finally, extension of the general approach was demonstrated for several additional elements (i.e. Cu(II), Cr(III), and Ga(III)).« less

Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

PubMed

Tang, Weizhuo; Sun, Baoshan; Zhao, Yuqing

2014-02-01

In this study, the preparative separation and purification of rosmarinic acid (RA) from perilla seed meal (PSM), which is a by-product of edible oil production, was achieved using combined column chromatography over macroporous and polyamide resins. To optimize the RA enrichment process, the performance and separation characteristics of nine selected macroporous resins with different chemical and physical properties were investigated. SP825 resin was the most effective: the content of RA increased from 0.27% in the original extract to 16.58% in the 50% ethanol fraction (a 61.4-fold increase). During further purification treatment on polyamide resin, 90.23% pure RA could be obtained in the 70% ethanol fraction. RA with a higher purity (>95%) could also be easily obtained using one crystallization operation. The proposed method is simple, easily operated, cost-effective, and environmentally friendly and is suitable for both large-scale RA production and waste management. Copyright © 2013 Elsevier B.V. All rights reserved.

An Inexpensive Liquid Chromatography Apparatus for Undergraduate Teaching.

ERIC Educational Resources Information Center

McCamish, Malcolm; And Others

1982-01-01

Describes an inexpensive, low-pressure liquid chromatography pump, slurry filler, stainless steel columns, and injector system suitable for the undergraduate laboratory or routine analysis. Includes sectional diagram of the pump and construction diagram of the preparative columns. (Author/SK)

[Determination and analysis of toluene diisocyanate metabolites in mice using gas chromatography-mass spectrometry].

PubMed

Ji, Yu-Bin; Ji, Chen-Feng; Zou, Xiang; Liu, Hui-Xin

2007-09-01

In the present research we used gas chromatography-mass spectrometry (G C-MS) to determine metabolites of toluene diisocyanate (TDI) in mice and deduce the pathway for toluene diisocyanate metabolism in the organism. Conditions for TDI chromatography: Supelco PTETM-5 chromatographic column (30 mm x 0.25 mm x 0.25 microm); initial column temperature: 90 degrees C, which was maintained for 30 min, then the temperature was increased at a rate of 40 degrees C x min(-1) to 280 degrees C, and maintained for 5.25 min; temperature for the vaporizing chamber: 250 degrees C; carrier gas: helium flowing at 1.0 microL x min(-1). Conditions for chromatography of TDI metabolites in the organism: 94% methyl, 4% ethenyl-bonded-phase fused-silica capillary column (30 + 2 m x 0.25 + 0.02 mm); initial column temperature: 30 degrees C, which was maintained for 5 min, after and then was increased at a rate of 80 degrees C x min(-1) to 280 degrees C, and maintained for 5 min; temperature for the vaporizing chamber: 250 degrees C; carrier gas: helium flowing at 1.0 microL x min(-1). Conditions for mass spectrometry: EI for ionization; 70 eV for ionization energy; 280 degrees C for connecting tube temperature; 35-350 micro for range of scanning; and 1.0 microL for sample size. The results showed that 2 ,4-toluene diisocyanate was metabolized into 2,4-diaminotoluene. Under the conditions selected for GC-MS, TDI metabolites in the organism can be isolated and identified.

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. Copyright © 2015 Elsevier B.V. All rights reserved.

A study of retention characteristics and quality control of nutraceuticals containing resveratrol and polydatin using fused-core column chromatography.

PubMed

Fibigr, Jakub; Šatínský, Dalibor; Solich, Petr

2016-02-20

A new high-performance liquid chromatography method using fused-core column for fast separation of resveratrol and polydatin has been developed and used for quality control of nutraceuticals with resveratrol and polydatin content. Retention characteristics (log k) were studied under different conditions on C-18, RP-Amide C-18, Phenyl-hexyl, Pentafluorophenyl (F5) and Cyano stationary phases for both compounds. The effect of the volume fraction of acetonitrile on a retention factors log k of resveratrol and polydatin were evaluated. The optimal separation conditions for resveratrol, polydatin and internal standard p-nitrophenol were found on the fused-core column Ascentis Express ES-Cyano (100×3.0mm), particle size 2.7μm, with mobile phase acetonitrile/water solution with 0.5% acetic acid pH 3 (20:80, v/v) at a flow rate of 1.0mL/min and at 60°C. The detection wavelength was set at 305nm. Under the optimal chromatographic conditions, good linearity with regression coefficients in the range (r=0.9992-0.9998; n=10) for both compounds was achieved. Commercial samples of nutraceuticals were extracted with methanol using ultrasound bath for 15min. A 5μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery was in the range 83.2-107.3% for both nutraceuticals. The intraday method precision was found satisfactory and relative standard deviations of sample analysis were in the range 0.8-4.7%. The developed method has shown high sample throughput during sample preparation process, modern separation approach, and short time (3min) of analysis. The results of study showed that the declared content of resveratrol and polydatin varied widely in different nutraceuticals according the producers (71.50-115.00% of declared content). Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography.

PubMed

Sadavarte, Rahul; Madadkar, Pedram; Filipe, Carlos Dm; Ghosh, Raja

2018-01-15

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster. Copyright © 2017 Elsevier B.V. All rights reserved.

Ionic liquid stationary phases for gas chromatography.

PubMed

Poole, Colin F; Poole, Salwa K

2011-04-01

This article provides a summary of the development of ionic liquids as stationary phases for gas chromatography beginning with early work on packed columns that established details of the retention mechanism and established working methods to characterize selectivity differences compared with molecular stationary phases through the modern development of multi-centered cation and cross-linked ionic liquids for high-temperature applications in capillary gas chromatography. Since there are many reviews on ionic liquids dealing with all aspects of their chemical and physical properties, the emphasis in this article is placed on the role of gas chromatography played in the design of ionic liquids of low melting point, high thermal stability, high viscosity, and variable selectivity for separations. Ionic liquids provide unprecedented opportunities for extending the selectivity range and temperature-operating range of columns for gas chromatography, an area of separation science that has otherwise been almost stagnant for over a decade. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100.

PubMed

Stanton, J Brett; Goldberg, Andrew F X; Hoppe, George; Marmorstein, Lihua Y; Marmorstein, Alan D

2006-02-01

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (nu) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be approximately 138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.

Semi-micro high-performance liquid chromatographic analysis of tiropramide in human plasma using column-switching.

PubMed

Baek, Soo Kyoung; Lee, Seung Seok; Park, Eun Jeon; Sohn, Dong Hwan; Lee, Hye Suk

2003-02-05

A rapid and sensitive column-switching semi-micro high-performance liquid chromatography method was developed for the direct analysis of tiropramide in human plasma. The plasma sample (100 microl) was directly injected onto Capcell Pak MF Ph-1 precolumn where deproteinization and analyte fractionation occurred. Tiropramide was then eluted into an enrichment column (Capcell Pak UG C(18)) using acetonitrile-potassium phosphate (pH 7.0, 50 mM) (12:88, v/v) and was analyzed on a semi-micro C(18) analytical column using acetonitrile-potassium phosphate (pH 7.0, 10 mM) (50:50, v/v). The method showed excellent sensitivity (limit of quantification 5 ng/ml), and good precision (C.V.

Repeatability of the efficiency of columns packed with sub-3μm core-shell particles: Part I. 2.6μm Kinetex-C(18) particles in 4.6mm and 2.1mm×100mm column formats.

PubMed

Gritti, Fabrice; Guiochon, Georges

2012-08-24

The column-to-column repeatability of the mass transfer mechanism in columns packed with sub-3μm shell particles was investigated. The parameters of this mechanism were measured for twelve columns (six 2.1mm×100mm and six 4.6mm×100mm) packed with the same batch of 2.6μm Kinetex-C(18) particles (Phenomenex, CA, USA). For both series, the manufacturer provided columns at different positions in the efficiency distribution given by the quality test control. Three compounds were used, uracil, naphthalene and insulin. The reduced longitudinal diffusion term was measured with the peak parking (PP) method, the reduced solid-liquid mass transfer resistance term was given by a combination of the PP results and a model of effective diffusion in ternary composite materials (non-porous cores, concentric porous shell, and eluent matrix), validated previously. The overall eddy diffusion term was obtained by subtraction of these two HETP terms from the overall reduced HETP measured by numerical integration of the entire peak profiles. The results demonstrate that the dispersion of the column efficiencies is only due to the random nature of the packing process. At the highest reduced velocity achieved, the relative standard deviations (RSDs) of the eddy diffusion term for the 2.1mm I.D. columns were ca. 7% and 3% for the low molecular weight compounds and for insulin, respectively. For the 4.6mm I.D. columns, these RSDs were 15% and 5%, respectively. The larger RSDs for the 4.6mm I.D. columns is explained by the exceptionally low value of the eddy diffusion term. Copyright © 2012 Elsevier B.V. All rights reserved.

Clean-up of a pesticide-lanolin mixture by gel permeation chromatography.

PubMed

López-Mesas, M; Crespi, M; Brach, J; Mullender, J P

2000-12-01

In this study, the efficiency of a clean-up method by gel permeation chromatography (GPC) for the separation of pesticides from lanolin is analyzed. The pesticides analyzed belong to two different families, organophosphorous and synthetic pyrethroids. Lanolin, a standard mixture of the pesticides, and a lanolin-pesticides mixture are injected in a GPC column. The recoveries and elution times from the GPC column of lanolin (by a gravimetric method) and pesticides (by gas chromatography-electron capture detector) are determined. From this column, a good separation of the lanolin-pesticides mixture is observed.

Fractionation of persistent organic pollutants in fish oil by high-performance liquid chromatography on a 2-(1-pyrenyl)ethyl silica column.

PubMed

Ortiz, X; Martí, R; Montaña, M J; Gasser, M; Margarit, L; Broto, F; Díaz-Ferrero, J

2010-09-01

The analysis of persistent organic pollutants in foodstuffs has become necessary for control of their levels in products for human and animal consumption. These analytical procedures usually require a fractionation step in order to separate the different families of pollutants to avoid interferences during the instrumental determination. In this study the separation was carried out on a 2-(1-pyrenyl)ethyl silica column, where analyte fractionation was based on differences in planarity and aromaticity. The fractionation of several types of persistent organic pollutants found in fish oil samples was studied; the pollutants included polychlorinated dibenzo-p-dioxins and dibenzofurans, polychlorinated biphenyls, polybrominated diphenyl ethers, and some organochlorine pesticides. Fractions were analyzed by high-resolution gas chromatography with electron-capture detection and high-resolution gas chromatography-high resolution mass spectroscopy. Finally, the whole method (including the purification, fractionation, and instrumental determination steps) was validated and successfully applied to the analysis of several samples of fish oil.

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Gas Chromatography

NASA Astrophysics Data System (ADS)

Qian, Michael C.

Gas chromatography (GC) has many applications in the analysis of food products. GC has been used for the determination of fatty acids, triglycerides, cholesterol, gases, water, alcohols, pesticides, flavor compounds, and many more. While GC has been used for other food components such as sugars, oligosaccharides, amino acids, peptides, and vitamins, these substances are more suited to analysis by high performance liquid chromatography. GC is ideally suited to the analysis of volatile substances that are thermally stable. Substances such as pesticides and flavor compounds that meet these criteria can be isolated from a food and directly injected into the GC. For compounds that are thermally unstable, too low in volatility, or yield poor chromatographic separation due to polarity, a derivatization step must be done before GC analysis. The two parts of the experiment described here include the analysis of alcohols that requires no derivatization step, and the analysis of fatty acids which requires derivatization. The experiments specify the use of capillary columns, but the first experiment includes conditions for a packed column.

Rapid determination of nitrite in foods in acidic conditions by high-performance liquid chromatography with fluorescence detection.

PubMed

Wang, Xiao-Fang; Fan, Ji-Cai; Ren, Ren; Jin, Quan; Wang, Jing

2016-06-01

In this study, a simple, rapid, and sensitive method for the determination of nitrite (NO2 (-) ) in food samples by high-performance liquid chromatography with fluorescence detection in acidic conditions had been developed. The derivatization of the nitrite with 2,3-diaminonaphthalene was performed in acidic conditions to yield the highly fluorescent 2,3-naphthotriazole, which was directly analyzed by high-performance liquid chromatography with fluorescence detection without adjusting the solution to alkaline. The analysis column was reversed-phase C8 column. A constant flow rate of 1.0 mL/min was employed using water/acetonitrile as the mobile phase in isocratic mode (70:30, v/v). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. The standard calibration curves were linear for nitrite in different matrixes in the concentration range of 0-100 μg/L, and the correlation coefficients ranged from 0.9978 to 0.9998. The limits of detection and quantification were in the ranges of 0.012-0.060 and 0.040-0.20 mg/kg, respectively. The recoveries of nitrite from samples spiked at three different concentrations were 74.0-113.2%, and the relative standard deviations of the recovery results (n = 6) were 1.67-10.8%. The proposed method has good repeatability and is very sensitive and simple. It has been successfully used to determine nitrite in foods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparative separation of capsaicin and dihydrocapsaicin from Capsicum frutescens by high-speed counter-current chromatography.

PubMed

Peng, Aihua; Ye, Haoyu; Li, Xia; Chen, Lijuan

2009-09-01

Capsaicin and dihydrocapsaicin are two main bioactive components of Capsicum frutescens and are widely used as food additives and drugs in China and India. Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal-phase thin-layer chromatography (TLC) becomes difficult. This study involves separating capsaicin and dihydrocapsaicin with sufficient purity and recovery using high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-ethyl acetate-methanol-water-acetic acid (20:20:20:20:2, v/v/v/v/v). Separation parameters such as sample volume, and sample concentration were first optimized on analytical HSCCC, and then scaled up to preparative HSCCC. 0.65 g capsaicin and 0.28 g dihydrocapsaicin were obtained from 1.2 g crude extract and their purities were 98.5 and 97.8%, respectively. The recoveries of the two compounds were 86.3 and 85.4%, respectively. The purity of the isolated compounds was analyzed by high-performance liquid chromatography (HPLC) and their structures were identified by (1)H nuclear magnetic resonance (NMR) and (13)C NMR analysis.

Virus elimination during the recycling of chromatographic columns used during the manufacture of coagulation factors.

PubMed

Roberts, Peter L

2014-07-01

Various chromatographic procedures are used during the purification and manufacture of plasma products such as coagulation factors. These steps contribute to the overall safety of such products by removing potential virus contamination. Virus removal by two affinity chromatography procedures, i.e. monoclonal antibody chromatography and metal chelate chromatography (immobilised metal ion affinity chromatography), used during the manufacture of the high purity factor VIII (Replenate®) and factor IX (Replenine®-VF), respectively, has been investigated. In addition, as these columns are recycled after use, the effectiveness of the sanitisation procedures for preventing possible cross-contamination, has also been investigated. Both chromatographic steps proved effective for eliminating a range of model enveloped and non-enveloped viruses by 4 to >6 and 5 to >8 log for the monoclonal and metal chelate columns, respectively. The effectiveness of the relatively mild column sanitisation conditions used, i.e. ethanol for factor IX and acetic acid for factor VIII, was confirmed using non-spiked column runs. The chemicals used contributed to virus elimination by inactivation and/or by physical removal of the virus. In summary, these studies demonstrate that potential virus contamination between chromatographic runs can be prevented when an effective column recycling and sanitisation procedure is included. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

High-pressure liquid chromatography with direct injection of gas sample.

PubMed

Astanin, Anton I; Baram, Grigory I

2017-06-09

The conventional method of using liquid chromatography to determine the composition of a gaseous mixture entails dissolving vapors in a suitable solvent, then obtaining a chromatograph of the resulting solution. We studied the direct introduction of a gaseous sample into a C18 reversed-phase column, followed by separation of the components by HPLC with UV detection. Since the chromatography was performed at high pressure, vapors readily dissolved in the eluent and the substances separated in the column as effectively as in liquid samples. Samples were injected into the column in two ways: a) through the valve without a flow stop; b) after stopping the flow and relieving all pressure. We showed that an injectable gas volume could reach 70% of column dead volume. When an injected gaseous sample volume was less than 10% of the column dead volume, the resulting peaks were symmetrical and the column efficiency was high. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative determination of triterpenes from Amphiptherygium adstringens by liquid chromatography and thin-layer chromatography and morphological analysis of cuachalalate preparations.

PubMed

Navarrete, Andres; Avula, Bharathi; Joshi, Vaishali C; Ji, Xiuhong; Hersh, Paul; Khan, Ikhlas A

2006-01-01

Amphiptherygium adstringens (Anacardiaceae/Julianaceae), local name "cuachalalate," is used in folk medicine for the treatment of cholelithiasis, fevers, fresh wounds, hypercholesterolemia, gastritis, gastric ulcers, and cancer of the gastrointestinal tract. The development of column high-performance liquid chromatography-photodiode array detector (LC-PDA) and high-performance thin-layer chromatography (HPTLC)-densitometry methods for the determination of masticadienonic acid and 3-hydroxymasticadienonic acid in cuachalalate preparations is described in this paper. Good separation of the compounds could be achieved by both methods. Either might be preparable depending on the requirements. The LC separation was performed on a Phenomenex Synergi MAX-RP 80A reversed-phase column operated at 40 degrees C with detection at 215 nm. The plant materials were extracted with methanol by sonication. The triterpenes present in the plant material and commercial extracts were separated with an acetonitrile-water reagent alcohol isocratic system. The limit of detection was 0.1-0.2 microg/mL. The relative standard deviation values for the determination of triterpenes in plant extracts were less than 1.00%. This is the first report of an analytical method developed for the quantitative analysis of triterpenes from Amphiptherygium adstringens by LC-PDA and HPTLC. The stem bark showed higher amounts of triterpenes, and low amounts in root and stem root. The microscopic description of the crude drug of cuachalalate was also provided.

Simultaneous analysis of steviol and steviol glycosides by liquid chromatography with ultraviolet detection on a mixed-mode column: application to Stevia plant material and Stevia-containing dietary supplements.

PubMed

Jaworska, Karolina; Krynitsky, Alexander J; Rader, Jeanne I

2012-01-01

Simultaneous separation of steviol and steviol glycosides is challenging because of differences in their polarity and chemical structure. In this study, simultaneous analysis of steviol and steviol glycosides was achieved by LC with UV detection using a mixed-mode RP weak anion exchange chromatography column. Steviol and seven steviol glycosides were analyzed on an Acclaim Mixed-Mode Wax-1 (Dionex) column with a linear gradient of deionized water adjusted to pH 3.00 with phosphoric acid and acetonitrile. The extraction was performed by sonicating dry plant material at 40 degreesC in acetonitrile-water (30 + 70, v/v). LOQ values (mg/g dry weight of plant material) were rebaudioside B, 0.50; steviol, 0.70, dulcoside A, 1.0; steviolbioside, 1.2; stevioside and rebaudioside C, 2.0; rebaudioside D, 3.3; and rebaudioside A, 5.0. The method demonstrated suitable performance for all analytes tested with respect to accuracy (mean recoveries 95-99%), intraday and interday precision for retention times (0.070-0.28% and 0.33-1.0% RSD, respectively), and linearity. The method was used to authenticate steviol glycosides in several samples of Stevia plant material as well as to quantitate steviol glycosides in dietary supplements containing Stevia.

Determination of Poly-β-Hydroxybutyrate and Poly-β-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography

PubMed Central

Comeau, Yves; Hall, Kenneth J.; Oldham, William K.

1988-01-01

A convenient gas-liquid chromatography procedure to quantify poly-β-hydroxybutyrate and poly-β-hydroxyvalerate in activated sludge was developed by combining lyophilization of the samples, purification of the chloroform phase by water reextraction, and the use of capillary columns. With a flame ionization detector the sensitivity was estimated at 10−5 g/liter. PMID:16347745

Separation of 'Uncharged' Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH

NASA Technical Reports Server (NTRS)

Schmidt, Jurgen G.; Orgel, Leslie E.; Nielsen, Peter E.

1996-01-01

Ion-exchange chromatography is a well-established method for the analysis and purification of phosphodiester-linked oligonucleotides. If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deprotonated at pH 10. In recent work on peptide-nucleic acids (PNAs) we noted that mixtures of PNA oligomers G(sub 4), G(sub 6), G(sub 8), and G9(sub 10) are readily separated by elution at pH 12 on an RPC-5 column. Here we show that this separation method is more generally applicable.

Separation of Uncharged Oligodeoxynucleotide Analogs by Anion-Exchange Chromatography at High pH

NASA Technical Reports Server (NTRS)

Schmidt, Jurgen G.; Nielsen, Peter E.; Orgel, Leslie

1996-01-01

Ion-exchange chromatography is a well-established method for the analysis and purification of phosphodiester-linked oligonucleotides. If elution is carried out under alkaline conditions, the secondary structure of G- and C-rich oligomers is disrupted. Furthermore, elution times become more sensitive to the G and T content of the oligomer, because G and T are deprotonated at pH 10. In recent work on peptide-nucleic acids (PNAs) we noted that mixtures of PNA oligomers G(sub 4), G(Sub 6), G(sub 8), and G(sub 10) are readily separated by elution at pH 12 on an RPC-5 column. Here we show that this separation method is more generally applicable.

Comparison of separations of fatty acids from fish products using a 30-m Supelcowax-10 and a 100-m SP-2560 column.

PubMed

Santercole, Viviana; Delmonte, Pierluigi; Kramer, John K G

2012-03-01

Commercial fish oils and foods containing fish may contain trans and/or isomerized fatty acids (FA) produced during processing or as part of prepared foods. The current American Oil Chemists' Society (AOCS) official method for marine oils (method Ce 1i-07) is based on separation by use of poly(ethylene glycol) (PEG) columns, for example Supelcowax-10 or equivalent, which do not resolve most unsaturated FA geometric isomers. Highly polar 100-m cyanopropyl siloxane (CPS) columns, for example SP-2560 and CP Sil 88 are recommended for separation of geometric FA isomers. Complementary separations were achieved by use of two different elution temperature programs with the same CPS column. This study is the first direct comparison of the separations achieved by use of 30-m Supelcowax-10 and 100-m SP-2560 columns for fatty acid methyl esters (FAME) prepared from the same fish oil and fish muscle sample. To simplify the identification of the FA in these fish samples, FA were fractionated on the basis of the number and type of double bonds by silver-ion solid-phase extraction (Ag⁺-SPE) before GC analysis. The results showed that a combination of the three GC separations was necessary to resolve and identify most of the unsaturated FA, FA isomers, and other components of fish products, for example phytanic and phytenic acids. Equivalent chain length (ECL) values of most FAME in fish were calculated from the separations achieved by use of both GC columns; the values obtained were shown to be consistent with previously reported values for the Supelcowax-10 column. ECL values were also calculated for the FA separated on the SP-2560 column. The calculated ECL values were equally valid under isothermal and temperature-programmed elution GC conditions, and were valuable for confirmation of the identity of several unsaturated FAME in the fish samples. When analyzing commercially prepared fish foods, deodorized marine oils, or foods fortified with marine oils it is strongly

Trace analysis of impurities in bulk gases by gas chromatography-pulsed discharge helium ionization detection with "heart-cutting" technique.

PubMed

Weijun, Yao

2007-10-12

A method has been developed for the detection of low-nL/L-level impurities in bulk gases such as H(2), O(2), Ar, N(2), He, methane, ethylene and propylene, respectively. The solution presented here is based upon gas chromatography-pulsed discharge helium ionization detection (GC-PDHID) coupled with three two-position valves, one two-way solenoid valve and four packed columns. During the operation, the moisture and heavy compounds are first back-flushed via a pre-column. Then the trace impurities (except CO(2) which is diverted to a separate analytical column for separation and detection) together with the matrix enter onto a main column, followed by the heart-cut of the impurities onto a longer analytical column for complete separation. Finally the detection is performed by PDHID. This method has been applied to different bulk gases and the applicability of detecting impurities in H(2), Ar, and N(2) are herewith demonstrated. As an example, the resulting detection limit of 100 nL/L and a dynamic range of 100-1000 nL/L have been obtained using an Ar sample containing methane.

Separation of actinides using capillary extraction chromatography-inductively coupled plasma mass spectrometry.

PubMed

Peterson, Dominic S; Montoya, Velma M

2009-08-01

Trace levels of actinides have been separated on capillary extraction chromatography columns. Detection of the actinides was achieved using an inductively coupled plasma mass spectrometer, which was coupled with the extraction chromatography system. In this study, we compare 30-cm long, 4.6 mm i.d. columns to capillary columns (750 microm i.d.) with lengths from 30 cm up to 150 cm. The columns that were tested were packed with TRU resin. We were able to separate a mixture of five actinides ((232)Th, (238)U, (237)Np, (239)Pu, and (241)Am). This work has application to rapid bioassay as well as automated separations of actinide materials.

Determination of molindone enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry using macrocyclic antibiotic chiral stationary phases.

PubMed

Jiang, Hongliang; Li, Yinghe; Pelzer, Mary; Cannon, Michelle J; Randlett, Christopher; Junga, Heiko; Jiang, Xiangyu; Ji, Qin C

2008-05-30

A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography-tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm x 2.1 mm) over the curve range 0.100-100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD

Development and validation of high-performance liquid chromatography and high-performance thin-layer chromatography methods for the quantification of khellin in Ammi visnaga seed

PubMed Central

Kamal, Abid; Khan, Washim; Ahmad, Sayeed; Ahmad, F. J.; Saleem, Kishwar

2015-01-01

Objective: The present study was used to design simple, accurate and sensitive reversed phase-high-performance liquid chromatography RP-HPLC and high-performance thin-layer chromatography (HPTLC) methods for the development of quantification of khellin present in the seeds of Ammi visnaga. Materials and Methods: RP-HPLC analysis was performed on a C18 column with methanol: Water (75: 25, v/v) as a mobile phase. The HPTLC method involved densitometric evaluation of khellin after resolving it on silica gel plate using ethyl acetate: Toluene: Formic acid (5.5:4.0:0.5, v/v/v) as a mobile phase. Results: The developed HPLC and HPTLC methods were validated for precision (interday, intraday and intersystem), robustness and accuracy, limit of detection and limit of quantification. The relationship between the concentration of standard solutions and the peak response was linear in both HPLC and HPTLC methods with the concentration range of 10–80 μg/mL in HPLC and 25–1,000 ng/spot in HPTLC for khellin. The % relative standard deviation values for method precision was found to be 0.63–1.97%, 0.62–2.05% in HPLC and HPTLC for khellin respectively. Accuracy of the method was checked by recovery studies conducted at three different concentration levels and the average percentage recovery was found to be 100.53% in HPLC and 100.08% in HPTLC for khellin. Conclusions: The developed HPLC and HPTLC methods for the quantification of khellin were found simple, precise, specific, sensitive and accurate which can be used for routine analysis and quality control of A. visnaga and several formulations containing it as an ingredient. PMID:26681890

Development of a high-performance liquid chromatography method for the determination of florfenicol in animal feedstuffs.

PubMed

Yang, JinJing; Sun, GuiZhi; Qian, MingRong; Huang, LingLi; Ke, XianBing; Yang, Bo

2017-11-15

An effective thin layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of florfenicol (FF) in pig, chicken and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C 18 column using an isocratic procedure with acetonitrile-water (35:65, v/v) at 0.6mL/min. The ultraviolet (UV) detector was set at a wavelength of 225nm. The FF concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y=159075x-15054, r>0.9999) were achieved within the concentration range of 0.05-200μg/mL. The recoveries of FF spiked at levels of 1, 100 and 1000μg/g ranged from 80.6% to 105.3% with the intra-day and inter-day relative standard deviation (RSD) less than 9.3%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06mg/kg for pig feedstuffs, 0.02 and 0.07mg/kg for chicken feedstuffs, and 0.02 and 0.05mg/kg for fish feedstuffs, respectively. This reliable, simple and cost-effective method could be applied to the routine monitoring of FF in animal feedstuffs. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of alkyl phosphates in petroleum samples by comprehensive two-dimensional gas chromatography with nitrogen phosphorus detection and post-column Deans switching.

PubMed

Nizio, Katie D; Harynuk, James J

2012-08-24

Alkyl phosphate based gellants used as viscosity builders for fracturing fluids used in the process of hydraulic fracturing have been implicated in numerous refinery-fouling incidents in North America. In response, industry developed an inductively coupled plasma optical emission spectroscopy (ICP-OES) based method for the analysis of total volatile phosphorus in distillate fractions of crude oil; however, this method is plagued by poor precision and a high limit of detection (0.5±1μg phosphorus mL(-1)). Furthermore this method cannot provide speciation information, which is critical for developing an understanding of the challenge of alkyl phosphates at a molecular level. An approach using comprehensive two-dimensional gas chromatography with nitrogen phosphorus detection (GC×GC-NPD) and post-column Deans switching is presented. This method provides qualitative and quantitative profiles of alkyl phosphates in industrial petroleum samples with increased precision and at levels comparable to or below those achievable by ICP-OES. A recovery study in a fracturing fluid sample and a profiling study of alkyl phosphates in four recovered fracturing fluid/crude oil mixtures (flowback) are also presented. Copyright © 2012 Elsevier B.V. All rights reserved.

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,…

[Examples for using capillary gas chromatography with wide bore columns in occupational health].

PubMed

Frank, H; Senf, L; Welsch, T

1990-12-01

Wide bore capillary columns (0.4-0.75 mm ID) can be easily and inexpensively installed in packed column GCs. The analytical advantages cause an expanding market for such capillaries and interconverting hardware kits. It is illustrated with some examples that often individual exposition levels can be determined exactly only by using capillary columns: ethylbenzene may be separated from the C8-isomers also in complex mixtures, the marker PBN for rubber smoke expositions can be determined with 30 min sampling time, the detection sensitivity of the FID is sufficient also for chlorinated pesticides and the analyses of high-boiling compounds profit by the high phase ratio of wide bore capillary columns. A single capillary column substitutes a variety of different packed columns, so saving time and money and protecting the analyst from failures and frustrating compromises.

Liposome retention in size exclusion chromatography

PubMed Central

Ruysschaert, Tristan; Marque, Audrey; Duteyrat, Jean-Luc; Lesieur, Sylviane; Winterhalter, Mathias; Fournier, Didier

2005-01-01

Background Size exclusion chromatography is the method of choice for separating free from liposome-encapsulated molecules. However, if the column is not presaturated with lipids this type of chromatography causes a significant loss of lipid material. To date, the mechanism of lipid retention is poorly understood. It has been speculated that lipid binds to the column material or the entire liposome is entrapped inside the void. Results Here we show that intact liposomes and their contents are retained in the exclusion gel. Retention depends on the pore size, the smaller the pores, the higher the retention. Retained liposomes are not tightly fixed to the beads and are slowly released from the gels upon direct or inverted eluent flow, long washing steps or column repacking. Further addition of free liposomes leads to the elution of part of the gel-trapped liposomes, showing that the retention is transitory. Trapping reversibility should be related to a mechanism of partitioning of the liposomes between the stationary phase, water-swelled polymeric gel, and the mobile aqueous phase. Conclusion Retention of liposomes by size exclusion gels is a dynamic and reversible process, which should be accounted for to control lipid loss and sample contamination during chromatography. PMID:15885140

The impact of column connection on band broadening in very high pressure liquid chromatography.

PubMed

Stankovich, Joseph J; Gritti, Fabrice; Stevenson, Paul G; Guiochon, Georges

2013-09-01

A series of experiments was conducted to evaluate the degree of band broadening in very high pressure LC due to column connections. Different column manufacturers use slightly different designs for their column fittings. If the same column connections are repeatedly used to attach columns of different origins, different void volumes form between capillary tubes and column inlets. An Agilent Ultra Low Dispersion Kit (tubing id 75 μm) was installed on an Agilent Infinity 1290 ultra HPLC and used to connect successively an Agilent, a Phenomenex, and a Waters column. A series of uracil (unretained) samples were injected and eluted at a wide range of flow rates with a water/acetonitrile mixture as eluent. In order to determine the variance contribution from column connections as accurately as possible a nonretained probe compound was selected because the variance contribution from the column is the smallest for analytes, which have very low k values. Yet, this effect still has an impact on the resolution for moderately retained compounds (k > 2) for narrow-bore columns packed with fine particles, since variance contributions are additive for linear chromatographic systems. Each injection was replicated five times under the same experimental conditions. Then NanoViper column connections (tubing id 75 μm) were used and the same injections were made. This system was designed to minimize connection void volumes for any column. Band variances were calculated as the second central moment of elution peaks and used to assess the degree of band broadening due to the column connections. Band broadening may increase from 3.8 to 53.9% when conventional metal ferrules were used to join columns to connection sites. The results show that the variance contribution from improper connections can generate as much as 60.5% of the total variance observed. This demonstrates that column connections can play a larger role than the column packing with respect to band dispersion. © 2013 WILEY

WATER COLUMN DATA AND SPECTRAL IRRADIANCE MODEL

EPA Science Inventory

Water samples collected monthly, for 18 months, from six sites in the Laguna Madre were analyzed to identify and quantify phytopigments using High Performance Liquid Chromatography (HPLC). In addition, water column pigment and nutrient data were acquired at 12 stations in Upper ...

[Multi-residue method for screening of pesticides in crops by liquid chromatography with tandem mass spectrometry].

PubMed

Tanizawa, Haruna; Shima, Mikie; Ikehara, Chieko; Kobata, Masakazu; Sato, Motoaki

2005-10-01

A simple and rapid method was developed for the screening of 82 pesticides/metabolites in a wide variety of crops, using solid-phase extraction and liquid chromatography with tandem mass spectrometry (LC/MS/MS). After extraction with methanol, the filtered extracts were made up to 100 mL and a 2 mL aliquot was subjected to solid-phase extraction. Co-extractives were removed with a C18 mini-column, while pesticides were retained on 3 kinds of mini-columns (HLB, SAX, activated carbon), and then eluted with acetonitrile. Analysis was performed by LC/MS/MS, and MS acquisition parameters were established in positive and negative ESI modes. The utility of the method was demonstrated by the analysis of 6 crops (carrot, cabbage, onion, spinach, lemon, brown rice) and one mixed vegetable juice. Of 82 compounds tested, 75 in carrot and 62 in lemon were obtained with recoveries ranging from 70-120%. For all samples tested, 75 compounds could be obtained with recoveries of over 50%, and the detection limits of most compounds were lower than 0.01 microg/g. This method provides acceptable performance for analysis of these 75 compounds. Further, by using aliquots of the extracts with small-scale mini-columns, purified samples could be obtained. This proposed method with small matrix effects, is effective and suitable for screening of multiple residual pesticides by using LC/MS/MS.

Study of Separation and Identification of the Active Ingredients in Gardenia jasminoides Ellis Based on a Two-Dimensional Liquid Chromatography by Coupling Reversed Phase Liquid Chromatography and Hydrophilic Interaction Liquid Chromatography.

PubMed

Zhou, Xuan; Chen, Cen; Ye, Xiaolan; Song, Fenyun; Fan, Guorong; Wu, Fuhai

2017-01-01

In this paper, by coupling reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), a two-dimensional liquid chromatography system was developed for separation and identification of the active ingredients in Gardenia jasminoides Ellis (GJE). By applying the semi-preparative C18 column as the first dimension and the core-shell column as the second dimension, a total of 896 peaks of GJE were separated. Among the 896 peaks, 16 active ingredients including geniposide, gardenoside, gardoside, etc. were identified by mass spectrometry analysis. The results indicated that the proposed two-dimensional RPLC/HILIC system was an effective method for the analysis of GJE and might hold a high potential to become a useful tool for analysis of other complex mixtures. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Possibilities and limitations of the kinetic plot method in supercritical fluid chromatography.

PubMed

De Pauw, Ruben; Desmet, Gert; Broeckhoven, Ken

2013-08-30

Although supercritical fluid chromatography (SFC) is becoming a technique of increasing importance in the field of analytical chromatography, methods to compare the performance of SFC-columns and separations in an unbiased way are not fully developed. The present study uses mathematical models to investigate the possibilities and limitations of the kinetic plot method in SFC as this easily allows to investigate a wide range of operating pressures, retention and mobile phase conditions. The variable column length (L) kinetic plot method was further investigated in this work. Since the pressure history is identical for each measurement, this method gives the true kinetic performance limit in SFC. The deviations of the traditional way of measuring the performance as a function of flow rate (fixed back pressure and column length) and the isopycnic method with respect to this variable column length method were investigated under a wide range of operational conditions. It is found that using the variable L method, extrapolations towards other pressure drops are not valid in SFC (deviation of ∼15% for extrapolation from 50 to 200bar pressure drop). The isopycnic method provides the best prediction but its use is limited when operating closer towards critical point conditions. When an organic modifier is used, the predictions are improved for both methods with respect to the variable L method (e.g. deviations decreases from 20% to 2% when 20mol% of methanol is added). Copyright © 2013 Elsevier B.V. All rights reserved.

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

Determination of Aristolochic Acid in Botanicals and Dietary Supplements by Liquid Chromatography with Ultraviolet Detection and by Liquid Chromatography/Mass Spectrometry: Single Laboratory Validation Confirmation

PubMed Central

Trujillo, William A.; Sorenson, Wendy R.; La Luzerne, Paul; Austad, John W.; Sullivan, Darryl

2008-01-01

The presence of aristolochic acid in some dietary supplements is a concern to regulators and consumers. A method has been developed, by initially using a reference method as a guide, during single laboratory validation (SLV) for the determination of aristolochic acid I, also known as aristolochic acid A, in botanical species and dietary supplements at concentrations of approximately 2 to 32 μg/g. Higher levels were determined by dilution to fit the standard curve. Through the SLV, the method was optimized for quantification by liquid Chromatography with ultraviolet detection (LC-UV) and LC/mass Spectrometry (MS) confirmation. The test samples were extracted with organic solvent and water, then injected on a reverse phase LC column. Quantification was achieved with linear regression using a laboratory automation system. The SLV study included systematically optimizing the LC-UV method with regard to test sample size, fine grinding of solids, and solvent extraction efficiency. These parameters were varied in increments (and in separate optimization studies), in order to ensure that each parameter was individually studied; the test results include corresponding tables of parameter variations. In addition, the chromatographic conditions were optimized with respect to injection volume and detection wavelength. Precision studies produced overall relative standard deviation values from 2.44 up to 8.26% for aristolochic acid I. Mean recoveries were between 100 and 103% at the 2 μg/g level, between 102 and 103% at the 10 μg/g level, and 104% at the 30 μg/g level. PMID:16915829

Tutorial: simulating chromatography with Microsoft Excel Macros.

PubMed

Kadjo, Akinde; Dasgupta, Purnendu K

2013-04-22

Chromatography is one of the cornerstones of modern analytical chemistry; developing an instinctive feeling for how chromatography works will be invaluable to future generation of chromatographers. Specialized software programs exist that handle and manipulate chromatographic data; there are also some that simulate chromatograms. However, the algorithm details of such software are not transparent to a beginner. In contrast, how spreadsheet tools like Microsoft Excel™ work is well understood and the software is nearly universally available. We show that the simple repetition of an equilibration process at each plate (a spreadsheet row) followed by discrete movement of the mobile phase down by a row, easily automated by a subroutine (a "Macro" in Excel), readily simulates chromatography. The process is readily understood by a novice. Not only does this permit simulation of isocratic and simple single step gradient elution, linear or multistep gradients are also easily simulated. The versatility of a transparent and easily understandable computational platform further enables the simulation of complex but commonly encountered chromatographic scenarios such as the effects of nonlinear isotherms, active sites, column overloading, on-column analyte degradation, etc. These are not as easily simulated by available software. Views of the separation as it develops on the column and as it is seen by an end-column detector are both available in real time. Excel 2010™ also permits a 16-level (4-bit) color gradation of numerical values in a column/row; this permits visualization of a band migrating down the column, much as Tswett may have originally observed, but in a numerical domain. All parameters of relevance (partition constants, elution conditions, etc.) are readily changed so their effects can be examined. Illustrative Excel spreadsheets are given in the Supporting Information; these are easily modified by the user or the user can write his/her own routine. Copyright

Multiplex gas chromatography for use in space craft

NASA Technical Reports Server (NTRS)

Valentin, J. R.

1985-01-01

Gas chromatography is a powerful technique for the analysis of gaseous mixtures. Some limitations in this technique still exist which can be alleviated with multiplex gas chromatography (MGC). In MGC, rapid multiple sample injections are made into the column without having to wait for one determination to be finished before taking a new sample. The resulting data must then be reduced using computational methods such as cross correlation. In order to efficiently perform multiplexgas chromatography, experiments in the laboratory and on board future space craft, skills, equipment, and computer software were developed. Three new techniques for modulating, i.e., changing, sample concentrations were demonstrated by using desorption, decomposition, and catalytic modulators. In all of them, the need for a separate gas stream as the carrier was avoided by placing the modulator at the head of the column to directly modulate a sample stream. Finally, the analysis of an environmental sample by multiplex chromatography was accomplished by employing silver oxide to catalytically modulate methane in ambient air.

Pressure, temperature and density drops along supercritical fluid chromatography columns in different thermal environments. III. Mixtures of carbon dioxide and methanol as the mobile phase.

PubMed

Poe, Donald P; Veit, Devon; Ranger, Megan; Kaczmarski, Krzysztof; Tarafder, Abhijit; Guiochon, Georges

2014-01-03

The pressure, temperature and density drops along SFC columns eluted with a CO2/methanol mobile phase were measured and compared with theoretical values. For columns packed with 3- and 5-μm particles the pressure and temperature drops were measured using a mobile phase of 95% CO2 and 5% methanol at a flow rate of 5mL/min, at temperatures from 20 to 100°C, and outlet pressures from 80 to 300bar. 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 experimental measurements were compared to theoretical results obtained by numerical simulation. For the convective air condition at outlet pressures above 100bar the average difference between the experimental and calculated temperature drops and pressure drops were 0.1°C and 0.7% for the bare 3-μm column, respectively, and were 0.6°C and 4.1% for the insulated column. The observed temperature drops for the insulated columns are consistent with those predicted by the Joule-Thomson coefficients for isenthalpic expansion. The dependence of the temperature and the pressure drops on the Joule-Thomson coefficient and kinematic viscosity are described for carbon dioxide mobile phases containing up to 20% methanol. Copyright © 2013 Elsevier B.V. All rights reserved.

Study on the extraction and purification of glycoprotein from the yellow seahorse, Hippocampus kuda Bleeker

PubMed Central

Su, Yuting; Xu, Yongjian

2015-01-01

The optimum parameters of extraction for glycoprotein from seahorse were examined and determined by Box-Behnken combined with ultrasonic extraction technology. Column chromatography of glycoprotein was used for further purification. The optimal extraction conditions of seahorse glycoprotein were extracting time 4.3 h, salt concentration 0.08 mol/L, extracting temperature 73°C, raw material, and water ratio 1:6. At the optimal conditions, the yield of saccharide reached to 1.123%, and the yield of protein reached to 5.898%. For purifying the crude glycoprotein, the stage renounces of DEAE-52 column chromatography were done, respectively, with 0.05, 0.1, 0.5 mol/L NaHCO3 solution, and further purification was done with Sephadex G-100 column chromatography. Finally, two pieces of seahorse glycoprotein were obtained by the column chromatography, that is, HG-11 and HG-21. The saccharide content was 56.7975% and 39.479%, the protein content was 30.5475% and 51.747%, respectively. PMID:26288722

Qualitative and Quantitative Analysis of Volatile Components of Zhengtian Pills Using Gas Chromatography Mass Spectrometry and Ultra-High Performance Liquid Chromatography.

PubMed

Liu, Cui-Ting; Zhang, Min; Yan, Ping; Liu, Hai-Chan; Liu, Xing-Yun; Zhan, Ruo-Ting

2016-01-01

Zhengtian pills (ZTPs) are traditional Chinese medicine (TCM) which have been commonly used to treat headaches. Volatile components of ZTPs extracted by ethyl acetate with an ultrasonic method were analyzed by gas chromatography mass spectrometry (GC-MS). Twenty-two components were identified, accounting for 78.884% of the total components of volatile oil. The three main volatile components including protocatechuic acid, ferulic acid, and ligustilide were simultaneously determined using ultra-high performance liquid chromatography coupled with diode array detection (UHPLC-DAD). Baseline separation was achieved on an XB-C18 column with linear gradient elution of methanol-0.2% acetic acid aqueous solution. The UHPLC-DAD method provided good linearity (R (2) ≥ 0.9992), precision (RSD < 3%), accuracy (100.68-102.69%), and robustness. The UHPLC-DAD/GC-MS method was successfully utilized to analyze volatile components, protocatechuic acid, ferulic acid, and ligustilide, in 13 batches of ZTPs, which is suitable for discrimination and quality assessment of ZTPs.

Instrument platforms for nano liquid chromatography.

PubMed

Šesták, Jozef; Moravcová, Dana; Kahle, Vladislav

2015-11-20

The history of liquid chromatography started more than a century ago and miniaturization and automation are two leading trends in this field. Nanocolumn liquid chromatography (nano LC) and largely synonymous capillary liquid chromatography (capillary LC) are the most recent results of this process where miniaturization of column dimensions and sorbent particle size play crucial role. Very interesting results achieved in the research of extremely miniaturized LC columns at the end of the last century lacked distinctive raison d'être and only advances in mass spectrometry brought a real breakthrough. Configuration of nano LC-electrospray ionization mass spectrometry (LC-ESI-MS) has become a basic tool in bioanalytical chemistry, especially in proteomics. This review discusses and summarizes past and current trends in the realization of nano liquid chromatography (nano LC) platforms. Special attention is given to the mobile phase delivery under nanoflow rates (isocratic, gradient) and sample injection to the nanocolumn. Available detection techniques applied in nano LC separations are also briefly discussed. We followed up the key themes from the original scientific reports over gradual improvements up to the contemporary commercial solutions. Copyright © 2015 Elsevier B.V. All rights reserved.

Optimization of post-column reactor radius in capillary high performance liquid chromatography Effect of chromatographic column diameter and particle diameter

PubMed Central

Xu, Hongjuan; Weber, Stephen G.

2006-01-01

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 (Nobs) 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 (ar) 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 ar 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 μm radius is suitable for all columns smaller than 150 μm (radius) packed with 2–5 μm particles. For 1 μm packing particles, only columns smaller than 42.5 μm (radius) can be used and the reactor radius needs to be 5 μm. PMID:16494886

Analysis of anthocyanins in commercial fruit juices by using nano-liquid chromatography-electrospray-mass spectrometry and high-performance liquid chromatography with UV-vis detector.

PubMed

Fanali, Chiara; Dugo, Laura; D'Orazio, Giovanni; Lirangi, Melania; Dachà, Marina; Dugo, Paola; Mondello, Luigi

2011-01-01

Nano-LC and conventional HPLC techniques were applied for the analysis of anthocyanins present in commercial fruit juices using a capillary column of 100 μm id and a 2.1 mm id narrow-bore C(18) column. Analytes were detected by UV-Vis at 518 nm and ESI-ion trap MS with HPLC and nano-LC, respectively. Commercial blueberry juice (14 anthocyanins detected) was used to optimize chromatographic separation of analytes and other analysis parameters. Qualitative identification of anthocyanins was performed by comparing the recorded mass spectral data with those of published papers. The use of the same mobile phase composition in both techniques revealed that the miniaturized method exhibited shorter analysis time and higher sensitivity than narrow-bore chromatography. Good intra-day and day-to-day precision of retention time was obtained in both methods with values of RSD less than 3.4 and 0.8% for nano-LC and HPLC, respectively. Quantitative analysis was performed by external standard curve calibration of cyanidin-3-O-glucoside standard. Calibration curves were linear in the concentration ranges studied, 0.1-50 and 6-50 μg/mL for HPLC-UV/Vis and nano-LC-MS, respectively. LOD and LOQ values were good for both methods. In addition to commercial blueberry juice, qualitative and quantitative analysis of other juices (e.g. raspberry, sweet cherry and pomegranate) was performed. The optimized nano-LC-MS method allowed an easy and selective identification and quantification of anthocyanins in commercial fruit juices; it offered good results, shorter analysis time and reduced mobile phase volume with respect to narrow-bore HPLC. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The separation of flavonoids from Pongamia pinnata using combination columns in high-speed counter-current chromatography with a three-phase solvent system.

PubMed

Yin, Hao; Zhang, Si; Long, Lijuan; Yin, Hang; Tian, Xinpeng; Luo, Xiongming; Nan, Haihan; He, Sha

2013-11-08

The mangrove plant Pongamia pinnata (Leguminosae) is well known as a plant pesticide. Previous studies have indicated that the flavonoids are responsible of the biological activities of the plant. A new high-speed counter-current chromatography (HSCCC) method for the separation of three flavonoids, karanjin (1), pinnatin (2), and pongaflavone (3), from P. pinnata was developed in the present study. The lower and intermediate phase (LP and IP) of a new three-phase solvent system, n-hexane-acetonitrile-dichloromethane-water, at a volume ratio of 5:5:1:5, were used as the stationary phases, while the upper phase (UP) was used as the mobile phase, and the volume ratio between the stationary phases in the CCC column could be tuned by varying the initial pumped volume ratio of the stationary phases. The CCC columns containing all three phases of the solvent system were considered combination columns. According to the theories of combination column, it is possible to optimize the retention time of the target compounds by varying the volume ratio of the stationary phases in the HSCCC combination columns, as well as the suitable volume ratios of the stationary phases for the separation of the target compounds were predicted from the partition coefficients of the compounds in the three-phase solvent system. Then, three HSCCC separations using the combination columns with initial pumped LP:IP volume ratios of 1:0, 0.9:0.1, and 0.7:0.3 were performed separately based on the prediction. Three target compounds were prepared with high purity when the initial pumped volume ratio of the stationary phases was 0.9:0.1. The baseline separation of compounds 2 and 3 was achieved on the combination column with an initial pumped volume ratio of 0.7:0.3. Furthermore, the three experiments clearly demonstrated that the retentions and resolutions of the target compounds increased with an increasing volume ratio of IP, which is consistent with the prediction for the retention times for the

New methodology for the analysis of volatile organic compounds (VOCs) in bioethanol by gas chromatography coupled to mass spectrometry

NASA Astrophysics Data System (ADS)

Campos, M. S. G.; Sarkis, J. E. S.

2018-03-01

The present study presents a new analytical methodology for the determination of 11 compounds present in ethanol samples through the gas chromatography coupled to mass spectrometry (GC-MS) technique using a medium polarity chromatography column composed of 6% cyanopropyl-phenyl and 94% dimethyl polysiloxane. The validation parameters were determined according to NBR ISO 17025:2005. The recovery rates of the studied compounds were 100.4% to 114.7%. The limits of quantification are between 2.4 mg.kg-1 and 5.8 mg.kg-1. The uncertainty of the measurement was estimate in circa of 8%.

Calcium Isotope Analysis with "Peak Cut" Method on Column Chemistry

NASA Astrophysics Data System (ADS)

Zhu, H.; Zhang, Z.; Liu, F.; Li, X.

2017-12-01

To eliminate isobaric interferences from elemental and molecular isobars (e.g., 40K+, 48Ti+, 88Sr2+, 24Mg16O+, 27Al16O+) on Ca isotopes during mass determination, samples should be purified through ion-exchange column chemistry before analysis. However, large Ca isotopic fractionation has been observed during column chemistry (Russell and Papanastassiou, 1978; Zhu et al., 2016). Therefore, full recovery during column chemistry is greatly needed, otherwise uncertainties would be caused by poor recovery (Zhu et al., 2016). Generally, matrix effects could be enhanced by full recovery, as other elements might overlap with Ca cut during column chemistry. Matrix effects and full recovery are difficult to balance and both need to be considered for high-precision analysis of stable Ca isotopes. Here, we investigate the influence of poor recovery on δ44/40Ca using TIMS with the double spike technique. The δ44/40Ca values of IAPSO seawater, ML3B-G and BHVO-2 in different Ca subcats (e.g., 0-20, 20-40, 40-60, 60-80, 80-100%) with 20% Ca recovery on column chemistry display limited variation after correction by the 42Ca-43Ca double spike technique with the exponential law. Notably, δ44/40Ca of each Ca subcut is quite consistent with δ44/40Ca of Ca cut with full recovery within error. Our results indicate that the 42Ca-43Ca double spike technique can simultaneously correct both of the Ca isotopic fractionation that occurred during column chemistry and thermal ionization mass spectrometry (TIMS) determination properly, because both of the isotopic fractionation occurred during analysis follow the exponential law well. Therefore, we propose the "peak cut" method on Ca column chemistry for samples with complex matrix effects. Briefly, for samples with low Ca contents, we can add the double spike before column chemistry, and only collect the middle of the Ca eluate and abandon the both sides of Ca eluate that might overlap with other elements (e.g., K, Sr). This method would

"Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

PubMed

Görgens, Christian; Guddat, Sven; Orlovius, Anne-Katrin; Sigmund, Gerd; Thomas, Andreas; Thevis, Mario; Schänzer, Wilhelm

2015-07-01

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC). Despite its great potential and versatile advantages for the separation of highly polar compounds, HILIC is up to now not very common in doping analysis, although most manufacturers offer a variety of HILIC columns in their portfolio. In this study, a novel multi-target approach based on HILIC high resolution/high accuracy mass spectrometry is presented to screen for various polar stimulants, stimulant sulfo-conjugates, glycerol, AICAR, ethyl glucuronide, morphine-3-glucuronide, and myo-inositol trispyrophosphate after direct injection of diluted urine specimens. The usage of an effective online sample cleanup and a zwitterionic HILIC analytical column in combination with a new generation Hybrid Quadrupol-Orbitrap® mass spectrometer enabled the detection of highly polar analytes without any time-consuming hydrolysis or further purification steps, far below the required detection limits. The methodology was fully validated for qualitative and quantitative (AICAR, glycerol) purposes considering the parameters specificity; robustness (rRT < 2.0%); linearity (R > 0.99); intra- and inter-day precision at low, medium, and high concentration levels (CV < 20%); limit of detection (stimulants and stimulant sulfo-conjugates < 10 ng/mL; norfenefrine; octopamine < 30 ng/mL; AICAR < 10 ng/mL; glycerol 100 μg/mL; ETG < 100 ng/mL); accuracy (AICAR 103.8-105.5%, glycerol 85.1-98.3% at three concentration levels) and ion suppression/enhancement effects.

Liquid Chromatography at Critical Conditions: Balancing size exclusion and adsorption in nanopores

NASA Astrophysics Data System (ADS)

Abdulahad, Asem; Amos, Jeffrey; Ryu, Chang

2009-03-01

Liquid chromatography at critical condition (LCCC) is a measure to identify thermodynamic conditions, in which polymers elute independently of molar mass during high performance liquid chromatography. Under these critical conditions the entropic exclusions that dominate size exclusion chromatography (SEC) and the enthalpic adsorption that governs adsorption-based interaction chromatography (IC) are said to negate one another resulting in simultaneous elution of the polymer of different molecular weights. Using multiple C18-bonded silica columns with different average nanopore sizes (from 5 nm to 30 nm), we will study the LCCC conditions of PS in methylene chloride/acetonitrile solvent mixture at different temperature. In addition, we will show that the separation of polystyrene can be fine tuned using a refined temperature gradient interaction chromatography (TGIC) that employs multiple columns of varying pore size in sequence.

Size-exclusion chromatography using core-shell particles.