Controlled method of reducing electrophoretic mobility of macromolecules, particles, or cells

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1992-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and other substances is provided which comprises interacting in a conventional electrophoretic separating procedure, the substances with a polymer-linked affinity compound comprised of a hydrophilic neutral polymer such as polyethylene glycol bound to a second component such as a hydrophobic compound, an immunocompound such as an antibody or antibody active fragment, or a ligand such as a hormone, drug, antigen, or a hapten. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and such reduction can comprise up to 100 percent for particular particles and cells. The present invention is advantageous in that electrophoretic separation can now be achieved for substances whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of the specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions.

Electrophoretic separation of kidney and pituitary cells on STS-8

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Nachtwey, D. S.; Barlow, G. H.; Cleveland, C.; Lanham, J. W.; Farrington, M. A.; Hatfield, J. M.; Hymer, W. C.; Grindeland, R.; Lewis, M. L.

1984-01-01

Specific secretory cells were separated from suspensions of cultured primary human embryonic cells and rat pituitary cells in microgravity conditions, with an objective of isolating the subfractions of kidney cells that produce the largest amount of urakinase, and the subfractions of rat pituitary cells that secrete growth hormones (GH), prolactin (PRL), and other hormones. It is inferred from the experimental observations that the surface charge distributions of the GH-containing cells differ from those of the PRL-containing cells, which is explained by the presence of secretory products on the surface of pituitary cells. For kidney cells, the electrophoretic mobility distributions in flight experiments were spread more than the ground controls.

Hormone purification by isoelectric focusing in space

NASA Technical Reports Server (NTRS)

Bier, M.

1988-01-01

The objective of the program was the definition and development of optimal methods for electrophoretic separations in microgravity. The approach is based on a triad consisting of ground based experiments, mathematical modeling and experiments in microgravity. Zone electrophoresis is a rate process, where separation is achieved in uniform buffers on the basis of differences in electrophoretic mobilities. Optimization and modeling of continuous flow electrophoresis mainly concern the hydrodynamics of the flow process, including gravity dependent fluid convection due to density gradients and gravity independent electroosmosis. Optimization of focusing requires a more complex model describing the molecular transport processes involved in electrophoresis of interacting systems. Three different focusing instruments were designed, embodying novel principles of fluid stabilization. Fluid stability was achieved by: (1) flow streamlining by means of membrane elements in combination with rapid fluid recycling; (2) apparatus rotation in combination with said membrane elements; and (3) shear stress induced by rapid recycling through a narrow gap channel.

Electrophoretic interactions and aggregation of colloidal biological particles

NASA Technical Reports Server (NTRS)

Davis, Robert H.; Nichols, Scott C.; Loewenberg, Michael; Todd, Paul

1994-01-01

The separation of cells or particles from solution has traditionally been accomplished with centrifuges or by sedimentation; however, many particles have specific densities close to unity, making buoyancy-driven motion slow or negligible, but most cells and particles carry surface charges, making them ideal for electrophoretic separation. Both buoyancy-driven and electrophoretic separation may be influenced by hydrodynamic interactions and aggregation of neighboring particles. Aggregation by electrophoresis was analyzed for two non-Brownian particles with different zeta potentials and thin double layers migrating through a viscous fluid. The results indicate that the initial rate of electrophoretically-driven aggregation may exceed that of buoyancy-driven aggregation, even under conditions in which buoyancy-driven relative motion of noninteracting particles is dominant.

Electrophoretic Separation of Single Particles Using Nanoscale Thermoplastic Columns.

PubMed

Weerakoon-Ratnayake, Kumuditha M; Uba, Franklin I; Oliver-Calixte, Nyoté J; Soper, Steven A

2016-04-05

Phenomena associated with microscale electrophoresis separations cannot, in many cases, be applied to the nanoscale. Thus, understanding the electrophoretic characteristics associated with the nanoscale will help formulate relevant strategies that can optimize the performance of separations carried out on columns with at least one dimension below 150 nm. Electric double layer (EDL) overlap, diffusion, and adsorption/desorption properties and/or dielectrophoretic effects giving rise to stick/slip motion are some of the processes that can play a role in determining the efficiency of nanoscale electrophoretic separations. We investigated the performance characteristics of electrophoretic separations carried out in nanoslits fabricated in poly(methyl methacrylate), PMMA, devices. Silver nanoparticles (AgNPs) were used as the model system with tracking of their transport via dark field microscopy and localized surface plasmon resonance. AgNPs capped with citrate groups and the negatively charged PMMA walls (induced by O2 plasma modification of the nanoslit walls) enabled separations that were not apparent when these particles were electrophoresed in microscale columns. The separation of AgNPs based on their size without the need for buffer additives using PMMA nanoslit devices is demonstrated herein. Operational parameters such as the electric field strength, nanoslit dimensions, and buffer composition were evaluated as to their effects on the electrophoretic performance, both in terms of efficiency (plate numbers) and resolution. Electrophoretic separations performed at high electric field strengths (>200 V/cm) resulted in higher plate numbers compared to lower fields due to the absence of stick/slip motion at the higher electric field strengths. Indeed, 60 nm AgNPs could be separated from 100 nm particles in free solution using nanoscale electrophoresis with 100 μm long columns.

Electrophoretic separation and analysis of living cells from solid tissues by several methods - Human embryonic kidney cell cultures as a model

NASA Technical Reports Server (NTRS)

Todd, Paul; Plank, Lindsay D.; Kunze, M. Elaine; Lewis, Marian L.; Morrison, Dennis R.

1986-01-01

The use of free-fluid electrophoresis methods to separate tissue cells having a specific function is discussed. It is shown that cells suspended by trypsinization from cultures of human embryonic kidney are electrophoretically heterogeneous and tolerate a wide range of electrophoresis buffers and conditions without significant attenuation of function. Moreover, these cells do not separate electrophoretically on the basis of size or cell position alone and can be separated according to their ability to give rise to progeny that produce specific plasminogen activators.

Sample injection and electrophoretic separation on a simple laminated paper based analytical device.

PubMed

Xu, Chunxiu; Zhong, Minghua; Cai, Longfei; Zheng, Qingyu; Zhang, Xiaojun

2016-02-01

We described a strategy to perform multistep operations on a simple laminated paper-based separation device by using electrokinetic flow to manipulate the fluids. A laminated crossed-channel paper-based separation device was fabricated by cutting a filter paper sheet followed by lamination. Multiple function units including sample loading, sample injection, and electrophoretic separation were integrated on a single paper based analytical device for the first time, by applying potential at different reservoirs for sample, sample waste, buffer, and buffer waste. As a proof-of-concept demonstration, mixed sample solution containing carmine and sunset yellow were loaded in the sampling channel, and then injected into separation channel followed by electrophoretic separation, by adjusting the potentials applied at the four terminals of sampling and separation channel. The effects of buffer pH, buffer concentration, channel width, and separation time on resolution of electrophoretic separation were studied. This strategy may be used to perform multistep operations such as reagent dilution, sample injection, mixing, reaction, and separation on a single microfluidic paper based analytical device, which is very attractive for building micro total analysis systems on microfluidic paper based analytical devices. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic purification of cells in space - Evaluation of results from STS-3

NASA Technical Reports Server (NTRS)

Sarnoff, B. E.; Kunze, M. E.; Todd, P.

1983-01-01

The procedure and results of Electrophoresis Equipment Verification Test, designed to examine electrophoretic behavior of animal cells is suspension more concentrated than possible on earth and flown on the Shuttle flight STS-3, were discussed. Ground-based laboratory values of electrophoretic mobilities of a mixture of human and rabbit aldehyde-fixed red blood cells (RBC) were compared with those recorded at 11 minute intervals on the Shuttle STS-3. RBC migration and separation observed through photographic records were not as expected. However, cell mobilities and migrating band profiles were consistent with the results of laboratory simulation experiments. It was concluded that zero G electrophoresis of very high concentrations (1 x 10 to the 9th) is possible and similar to electrophoresis of normal cell concentrations on earth.

Effect of surfactant species and electrophoretic medium composition on the electrophoretic behavior of neutral and water-insoluble linear synthetic polymers in nonaqueous capillary zone electrophoresis.

PubMed

Fukai, Nao; Kitagawa, Shinya; Ohtani, Hajime

2017-07-01

We have recently demonstrated the separation of neutral and water-insoluble linear synthetic polymers in nonaqueous capillary zone electrophoresis (NACZE) using a cationic surfactant of cetyltrimethylammonium chloride (CTAC). In this study, eight ionic surfactants were investigated for the separation of four synthetic polymers (polystyrene, polymethylmethacrylates, polybutadiene, and polycarbonate); only three surfactants (CTAC, dimethyldioctadecylammonium bromide, and sodium dodecylsulfate) caused their separation. The order of the interaction between the polymers and the surfactants depended on both the surfactant species and the composition of the electrophoretic medium. Their investigation revealed that the separation is majorly affected by the hydrophobic interactions between the polymers and the ionic surfactants. In addition, the electrophoretic behavior of polycarbonate suggested that electrostatic interaction also affects the selectivity of the polymers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic separation of cells and particles from rat pituitary and rat spleen

NASA Technical Reports Server (NTRS)

Hymer, Wesley C.

1993-01-01

There are 3 parts to the IML-2 TX-101 experiment. Part 1 is a pituitary cell culture experiment. Part 2 is a pituitary cell separation experiment using the Japanese free flow electrophoresis unit (FFEU). Part 3 is a pituitary secretory granule separation experiment using the FFEU. The objectives of this three part experiment are: (1) to determine the kinetics of production of biologically active growth hormone (GH) and prolactin (PRL) in rat pituitary GH and PRL cells in microgravity (micro-g); (2) to investigate three mechanisms by which a micro-g-induced lesion in hormone production may occur; and (3) to determine the quality of separations of pituitary cells and organelles by continuous flow electrophoresis (CFE) in micro-g under conditions where buoyancy-induced convection is eliminated.

Certain problems of space biotechnology

NASA Technical Reports Server (NTRS)

Gilyarov, V. N.

1980-01-01

Experiments in the field of biotechnology conducted by the USA Apollo and Skylab space probes are described, as well as the joint Soviet-American Apollo-Soyuz Test Project (ASTP). Experiments in electrophoretic separation in space of biological compounds in a liquid medium are detailed. Space processing of vaccines and separation of human and animal cells are described. Methyl-cellulose, a coating for use in electrophoresis was developed. Erythropoietin, which stimulates the formation of red blood corpuscles in bone marrow, was obtained in pure form.

Separability of electrostatic and hydrodynamic forces in particle electrophoresis

NASA Astrophysics Data System (ADS)

Todd, Brian A.; Cohen, Joel A.

2011-09-01

By use of optical tweezers we explicitly measure the electrostatic and hydrodynamic forces that determine the electrophoretic mobility of a charged colloidal particle. We test the ansatz of O'Brien and White [J. Chem. Soc. Faraday IIJCFTBS0300-923810.1039/f29787401607 74, 1607 (1978)] that the electrostatically and hydrodynamically coupled electrophoresis problem is separable into two simpler problems: (1) a particle held fixed in an applied electric field with no flow field and (2) a particle held fixed in a flow field with no applied electric field. For a system in the Helmholtz-Smoluchowski and Debye-Hückel regimes, we find that the electrostatic and hydrodynamic forces measured independently accurately predict the electrophoretic mobility within our measurement precision of 7%; the O'Brien and White ansatz holds under the conditions of our experiment.

Advances in electrophoretic separations

NASA Technical Reports Server (NTRS)

Snyder, R. S.; Rhodes, P. H.

1984-01-01

Free fluid electrophoresis is described using laboratory and space experiments combined with extensive mathematical modeling. Buoyancy driven convective flows due to thermal and concentration gradients are absent in the reduced gravity environment of space. The elimination of convection in weightlessness offers possible improvements in electrophoresis and other separation methods which occur in fluid media. The mathematical modeling suggests new ways of doing electrophoresis in space and explains various phenomena observed during past experiments. The extent to which ground based separation techniques are limited by gravity induced convection is investigated and space experiments are designed to evaluate specific characteristics of the fluid/particle environment. A series of experiments are proposed that require weightlessness and apparatus is developed that can be used to carry out these experiments in the near future.

Differential electrophoretic separation of cells and its effect on cell viability

NASA Technical Reports Server (NTRS)

Leise, E. M.; Lesane, F.

1974-01-01

An electrophoretic separation method was applied to the separation of cells. To determine the efficiency of the separation, it was necessary to apply existing methodology and develop new methods to assess the characteristics and functions of the separated subpopulations. Through appropriate application of the widely used isoelectric focusing procedure, a reproducible separation method was developed. Cells accumulated at defined pH and 70-80% remained viable. The cells were suitable for further biologic, biochemical and immunologic studies.

Separation of lymphocytes by electrophoresis under terrestrial conditions and at zero gravity, phase 3

NASA Technical Reports Server (NTRS)

Rubin, A. L.; Stenzel, K. H.; Cheigh, J. S.; Seaman, G. V. F.; Novogrodsky, A.

1977-01-01

Electrophoretic mobilities (EPM) of peripheral lymphocytes were studied from normal subjects, chronic hemodialysis patients and kidney transplant recipients. A technique to separate B lymphocytes and null cells from non-T lymphocyte preparation was developed. The experiments were designed to determine which subpopulation of the non-T lymphocytes is primarily affected and shows a decreased EPM in chronic hemodialysis patients and kidney transplant recipients.

Multistage Electrophoretic Separators

NASA Technical Reports Server (NTRS)

Thomas, Nathan; Doyle, John F.; Kurk, Andy; Vellinger, John C.; Todd, Paul

2006-01-01

A multistage electrophoresis apparatus has been invented for use in the separation of cells, protein molecules, and other particles and solutes in concentrated aqueous solutions and suspensions. The design exploits free electrophoresis but overcomes the deficiencies of prior free-electrophoretic separators by incorporating a combination of published advances in mathematical modeling of convection, sedimentation, electro-osmotic flow, and the sedimentation and aggregation of droplets. In comparison with other electrophoretic separators, these apparatuses are easier to use and are better suited to separation in relatively large quantities characterized in the art as preparative (in contradistinction to smaller quantities characterized in the art as analytical). In a multistage electrophoretic separator according to the invention, an applied vertical steady electric field draws the electrically charged particles of interest from within a cuvette to within a collection cavity that has been moved into position of the cuvette. There are multiple collection cavities arranged in a circle; each is aligned with the cuvette for a prescribed short time. The multistage, short-migration-path character of the invention solves, possibly for the first time, the fluid-instability problems associated with free electrophoresis. The figure shows a prototype multistage electrophoretic separator that includes four sample stations and five collection stages per sample. At each sample station, an aqueous solution or suspension containing charged species to be separated is loaded into a cuvette, which is machined into a top plate. The apparatus includes a lower plate, into which 20 collection cavities have been milled. Each cavity is filled with an electrophoresis buffer solution. For the collection of an electrophoretic fraction, the lower plate is rotated to move a designated collection cavity into alignment with the opening of the cuvette. An electric field is then applied between a non-gassing electrode in the collection cavity and an electrolyte compartment, which is separated from the cuvette by a semipermeable membrane. The electrolyte is refreshed by circulation by use of a peristaltic pump. In subsequent steps, the lower plate is rotated to collect other electrophoretic fractions. Later, the collected fractions are removed from the collection cavities through ports that have threaded plugs. The base of the apparatus contains power supplies and a computer interface. The design includes provisions for monitoring and feedback control of cavity position, electric field, and temperature. The operation of the apparatus can easily be automated, as demonstrated by use of software that has already been written for this purpose.

Density gradient electrophoresis of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Giranda, V.; Todd, P. W.

1985-01-01

Ground based confirmation of the electrophoretic heterogeneity of human embryonic kidney cell cultures, the general characterization of their electrophoretic migration, and observations on the general properties of cultures derived from electrophoretic subpopulations were studied. Cell migration in a density gradient electrophoresis column and cell electrophoretic mobility was determined. The mobility and heterogeneity of cultured human embryonic kidney cells with those of fixed rat erythrocytes as model test particle was compared. Electrophoretically separated cell subpopulations with respect to size, viability, and culture characteristics were examined.

Controlled method of reducing electrophoretic mobility of various substances

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1989-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and the like is provided. The method comprises interacting the particles or cells with a polymer-linked affinity compound composed of: a hydrophilic neutral polymer such as polyethylene glycol, and an affinity component consisting of a hydrophobic compound such as a fatty acid ester, an immunocompound such as an antibody or active fragment thereof or simular macromolecule, or other ligands. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and the mobility reduction obtainable is up to 100 percent for particular particles and cells. The present invention is advantageous in that analytical electrophoretic separation can not be achieved for macromolecules, particles, and cells whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions. The present method is also advantageous in that it can be used in a variety of standard laboratory electrophoresis equipment.

Method and apparatus for continuous electrophoresis

DOEpatents

Watson, Jack S.

1992-01-01

A method and apparatus for conducting continuous separation of substances by electrophoresis are disclosed. The process involves electrophoretic separation combined with couette flow in a thin volume defined by opposing surfaces. By alternating the polarity of the applied potential and producing reciprocating short rotations of at least one of the surfaces relative to the other, small increments of separation accumulate to cause substantial, useful segregation of electrophoretically separable components in a continuous flow system.

Electrophoretic separations on paper: Past, present, and future-A review.

PubMed

Nanthasurasak, Pavisara; Cabot, Joan Marc; See, Hong Heng; Guijt, Rosanne M; Breadmore, Michael C

2017-09-08

Point-of-collection (POC) devices aim for a fast, on-site detection for medical and environmental purposes. In this area, microfluidic Paper-based Analytical Devices (μPADs) have recently gained popularity because these are potentially cheap and environmentally friendly to produce, and easy to use. From an analytical perspective, paper is well known for its use as a substrate for chromatography, but less known for its use in electrophoretic separations. With the recent interest in μPADs, most applications are based on rather simple assays with relatively few applications incorporating an analytical separation. The focus of this review is on paper-based electrophoresis, originating with the key developments in the 1940s and 1950s as well as the recent developments of electrophoretic μPADs, and concluding with a critical discussion of the opportunities and challenges for electrophoretic μPADS in the future. Copyright © 2017. Published by Elsevier B.V.

Automated space processing payloads study. Volume 2, book 2: Technical report, appendices A through E. [instrument packages and space shuttles

NASA Technical Reports Server (NTRS)

1975-01-01

Experiment hardware and operational requirements for space shuttle experiments are discussed along with payload and system concepts. Appendixes are included in which experiment data sheets, chamber environmental control and monitoring, method for collection and storage of electrophoretically-separated samples, preliminary thermal evaluation of electromagnetic levitation facilities L1, L2, and L3, and applicable industrial automation equipment are discussed.

Physicochemical characteristics of LR3-IGF1 protein inclusion bodies: electrophoretic mobility studies.

PubMed

Wangsa-Wirawan, N D; O'Neill, B K; Middelberg, A P

2001-01-01

A knowledge of the physicochemical properties of inclusion bodies is important for the rational design of potential recovery processes such as flotation and precipitation. In this study, measurement of the size and electrophoretic mobility of protein inclusion bodies and cell debris was undertaken. SDS-PAGE analysis of protein inclusion bodies subjected to different cleaning regimes suggested that electrophoretic mobility provides a qualitative measure of protein inclusion body purity. Electrophoretic mobility as a function of electrolyte type and ionic strength was investigated. The presence of divalent ions produced a stronger effect on electrophoretic mobility compared with monovalent ions. The isoelectric point of cell debris was significantly lower than that for the inclusion bodies. Hence, the contaminating cell debris may be separated from inclusion bodies using flotation by exploiting this difference in isoelectric points. Separation by this method is simple, convenient, and a possible alternative to the conventional route of centrifugation.

Protein Separation by Electrophoretic-Electroosmotic Focusing on Supported Lipid Bilayers

PubMed Central

Liu, Chunming; Monson, Christopher F.; Yang, Tinglu; Pace, Hudson; Cremer, Paul S.

2011-01-01

An electrophoretic-electroosmotic focusing (EEF) method was developed and used to separate membrane-bound proteins and charged lipids based on their charge-to-size ratio from an initially homogeneous mixture. EEF uses opposing electrophoretic and electroosmotic forces to focus and separate proteins and lipids into narrow bands on supported lipid bilayers (SLBs). Membrane-associated species were focused into specific positions within the SLB in a highly repeatable fashion. The steady-state focusing positions of the proteins could be predicted and controlled by tuning experimental conditions, such as buffer pH, ionic strength, electric field and temperature. Careful tuning of the variables should enable one to separate mixtures of membrane proteins with only subtle differences. The EEF technique was found to be an effective way to separate protein mixtures with low initial concentrations, and it overcame diffusive peak broadening to allow four bands to be separated simultaneously within a 380 μm wide isolated supported membrane patch. PMID:21958061

Separation of very hydrophobic analytes by micellar electrokinetic chromatography IV. Modeling of the effective electrophoretic mobility from carbon number equivalents and octanol-water partition coefficients.

PubMed

Huhn, Carolin; Pyell, Ute

2008-07-11

It is investigated whether those relationships derived within an optimization scheme developed previously to optimize separations in micellar electrokinetic chromatography can be used to model effective electrophoretic mobilities of analytes strongly differing in their properties (polarity and type of interaction with the pseudostationary phase). The modeling is based on two parameter sets: (i) carbon number equivalents or octanol-water partition coefficients as analyte descriptors and (ii) four coefficients describing properties of the separation electrolyte (based on retention data for a homologous series of alkyl phenyl ketones used as reference analytes). The applicability of the proposed model is validated comparing experimental and calculated effective electrophoretic mobilities. The results demonstrate that the model can effectively be used to predict effective electrophoretic mobilities of neutral analytes from the determined carbon number equivalents or from octanol-water partition coefficients provided that the solvation parameters of the analytes of interest are similar to those of the reference analytes.

Investigation of electrophoretic exclusion method for the concentration and differentiation of proteins.

PubMed

Meighan, Michelle M; Vasquez, Jared; Dziubcynski, Luke; Hews, Sarah; Hayes, Mark A

2011-01-01

This work presents a technique termed as "electrophoretic exclusion" that is capable of differentiation and concentration of proteins in bulk solution. In this method, a hydrodynamic flow is countered by the electrophoretic velocity to prevent a species from entering into a channel. The separation can be controlled by changing the flow rate or applied electric potential in order to exclude a certain species selectively while allowing others to pass through the capillary. The exclusion of various proteins is investigated using a flow-injection regime of the method. Concentration of myoglobin of up to 1200 times the background concentration in 60 s was demonstrated. Additionally, negatively charged myoglobin was separated from a solution containing negatively charged allophycocyanin. Cationic cytochrome c was also differentiated from a solution with allophycocyanin. The ability to differentially transport species in bulk solution enables parallel and serial separation modes not available with other separations schemes.

Free zone electrophoresis simulation of static column electrophoresis in microgravity on shuttle flight STS-3

NASA Technical Reports Server (NTRS)

Todd, P. W.; Hjerten, S.

1985-01-01

Experiments were designed to replicate, as closely as possible in 1-G, the conditions of the STS-3 red blood cell (RBC) experiments. Free zone electrophoresis was the method of choice, since it minimizes the role of gravity in cell migration. The physical conditions of the STS-3 experiments were used, and human and rabbit RBC's fixed by the same method were the test particles. The effects of cell concentration, electroosmotic mobility, and sample composition were tested in order to seek explanations for the STS-3 results and to provide data on cell concentration effects for future zero-G separation on the continuous-flow zero-G electrophoretics separator.

Integrated microreactor for enzymatic reaction automation: An easy step toward the quality control of monoclonal antibodies.

PubMed

Ladner, Yoann; Mas, Silvia; Coussot, Gaelle; Bartley, Killian; Montels, Jérôme; Morel, Jacques; Perrin, Catherine

2017-12-15

The main purpose of the present work is to provide a fully integrated miniaturized electrophoretic methodology in order to facilitate the quality control of monoclonal antibodies (mAbs). This methodology called D-PES, which stands for Diffusion-mediated Proteolysis combined with an Electrophoretic Separation, permits to perform subsequently mAb tryptic digestion and electrophoresis separation of proteolysis products in an automated manner. Tryptic digestion conditions were optimized regarding the influence of enzyme concentration and incubation time in order to achieve similar enzymatic digestion efficiency to that obtained with the classical methodology (off-line). Then, the optimization of electrophoretic separation conditions concerning the nature of background electrolyte (BGE), ionic strength and pH was realized. Successful and repeatable electrophoretic profiles of three mAbs digests (Trastuzumab, Infliximab and Tocilizumab), comparable to the off-line digestion profiles, were obtained demonstrating the feasibility and robustness of the proposed methodology. In summary, the use of the proposed and optimized in-line approach opens a new, fast and easy way for the quality control of mAbs. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of polymeric coatings for control of electro-osmotic flow in ASTP MA-011 electrophoresis technology experiment

NASA Technical Reports Server (NTRS)

Patterson, W. J.

1976-01-01

The development of a methyl cellulose based coating system for control of electro-osmotic flow at the walls of electrophoresis cells is described. Flight electrophoresis columns were coated with this system, resulting in a flight set of six columns. In flight photography of MA-011 electrophoretic separations verified control of electro-osmotic flow.

Pre-flight report on cultured human embryonic kidney cell handling and cell electrophoresis. Prepared prior to continuous-flow electrophoretic separation experiments aboard space shuttle flight STS-8

NASA Technical Reports Server (NTRS)

Todd, P. W.; Sarnoff, B. E.; Li, Z. K.

1985-01-01

Studies of the physical properties of continuous-flow zero-G electrophoretic separator (CFES) buffer, the electrokinetic properties of human erythrocytes in the CFES buffer, the electrokinetic properties of human embryonic kidney cells in the CFES buffer, and the viability and yield of human embryonc kidney cells subjected to flight handling procedures are discussed. In general, the procedure for cell handling and electrophoresis of HEK-8514 cells in 1st or 2nd passage on STS-8 is acceptable if executed properly. The CFES buffer has ionic strength that is barely compatible with cell viability and membrane stability, as seen in experiments with human erythrocytes and trypan-blue staining of human kidney cells. Cells suspended in 10% dialysed horse serum for 3 days in the cold appear to be more stable than freshly trypsinized cells. 10% horse serum appears to be superior to 5% horse serum for this purpose. The mean absolute raw mobility of HEK-8514 cells in CFES buffer at 6 degrees, conductivity 0.055 mmho/cm, is 1.1 to 1.4 um-cm/V-sec, with a range of nearly a whole mobility unit.

Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

NASA Technical Reports Server (NTRS)

Williams, K. B.; Kunze, M. E.; Todd, P. W.

1985-01-01

Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

Kidney cell electrophoresis, continuing task

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

Materials and procedures for microgravity electrophoresis of living human embryonic kidney cells were evaluated to provide ground support in the form of analytical cell electrophoresis and flow cytometry. Preflight culture media, electrophoresis buffer, fraction collection media, temperature profiles, and urokinase assay procedures were tested prior to flight. Electrophoretic mobility distributions of aliquots of the cell population to be fractionated in flight were obtained. Cells were prepared in suspension prior to flight in electrophoresis buffer and 10% calf serum. Electrophoretic separation proceeded in electrophoresis buffer without serum in the Continuous Flow Electrophoretic Separator, and fractions were collected into sample bags containing culture medium and concentrated serum. Fractions that yielded enough progeny cells were analyzed for morphology and electrophoretic mobility distributions. It is noted that the lowest mobility fraction studied produced higher mobility progeny while the other fractions produced progeny cells with mobilities related to the fractions from which they were collected.

Ultrafast electrokinetics.

PubMed

Rouhi Youssefi, Mehrnaz; Diez, Francisco Javier

2016-03-01

The influence of a high electric field applied on both fluid flow and particle velocities is quantified at large Peclet numbers. The experiments involved simultaneous particle image velocimetry and flow rate measurements. These are conducted in polydimethylsiloxane channels with spherical nonconducting polystyrene particles and DI water as the background flow. The high electric field tests produced up to three orders of magnitude higher electrokinetic velocities than any previous reports. The maximum electroosmotic velocity and electrophoretic velocity measured were 3.55 and 2.3 m/s. Electrophoretic velocities are measured over the range of 100 V/cm < E < 250 000 V/cm. The results are separated according to the different nonlinear theoretical models, including low and high Peclet numbers, and weak and strong concentration polarization. They show good agreement with the models. Such fast velocities could be used for flow separation, mixing, transport, control, and manipulation of suspended particles as well as microthrust generation among other applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detailed results of ASTP experiment MA-011. [biological processing facility in space

NASA Technical Reports Server (NTRS)

Seaman, G. V. F.; Allen, R. E.; Barlow, G. H.; Bier, M.

1976-01-01

This experiment was developed in order to conduct engineering and operational tests of electrokinetic equipment in a micro-gravity environment. The experimental hardware in general functioned as planned and electrophoretic separations were obtained in space. The results indicated the development of satisfactory sample collection, return, and preservation techniques. The application of a near-zero zeta potential interior wall coating to the experimental columns, confirmation of biocompatibility of all appropriate hardware components, and use of a sterile operating environment provided a significant step forward in the development of a biological processing facility in space. A separation of a test of aldehyde-fixed rabbit, human, and horse red blood cells was obtained. Human kidney cells were separated into several components and viable cells returned to earth. The isotachophoretic separation of red cells was also demonstrated. Problems associated with the hardware led to a lack of success in the attempt to separate subpopulations of human lymphocytes.

Chiral ionic liquids in chromatographic and electrophoretic separations.

PubMed

Kapnissi-Christodoulou, Constantina P; Stavrou, Ioannis J; Mavroudi, Maria C

2014-10-10

This report provides an overview of the application of chiral ionic liquids (CILs) in separation technology, and particularly in capillary electrophoresis and both gas and liquid chromatography. There is a large number of CILs that have been synthesized and designed as chiral agents. However, only a few have successfully been applied in separation technology. Even though this application of CILs is still in its early stages, the scientific interest is increasing dramatically. This article is focused on the use of CILs as chiral selectors, background electrolyte additives, chiral ligands and chiral stationary phases in electrophoretic and chromatographic techniques. Different examples of CILs, which contain either a chiral cation, a chiral anion or both, are presented in this review article, and their major advantages along with their potential applications in chiral electrophoretic and chromatographic recognition are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Cell and Particle Interactions and Aggregation During Electrophoretic Motion

NASA Technical Reports Server (NTRS)

Wang, Hua; Zeng, Shulin; Loewenberg, Michael; Todd, Paul; Davis, Robert H.

1996-01-01

The stability and pairwise aggregation rates of small spherical particles under the collective effects of buoyancy-driven motion and electrophoretic migration are analyzed. The particles are assumed to be non-Brownian, with thin double-layers and different zeta potentials. The particle aggregation rates may be enhanced or reduced, respectively, by parallel and antiparallel alignments of the buoyancy-driven and electrophoretic velocities. For antiparallel alignments, with the buoyancy-driven relative velocity exceeding the electrophoretic relative velocity between two widely-separated particles, there is a 'collision-forbidden region' in parameter space due to hydrodynamic interactions; thus, the suspension becomes stable against aggregation.

Electrophoretic separation of gold nanoparticles according to bifunctional molecules-induced charge and size.

PubMed

Kim, Jong-Yeob; Kim, Hyung-Bae; Jang, Du-Jeon

2013-03-01

Gold nanospheres modified with bifunctional molecules have been separated and characterized by using agarose gel electrophoresis as well as optical spectroscopy and electron microscopy. The electrophoretic mobility of a gold nanosphere capped with 11-mercaptoundecanoic acid (MUA) has been found to depend on the number of MUA molecules per gold nanosphere, indicating that it increases with the surface charge of the nanoparticle. The extinction spectrum of gold nanospheres capped with MUA at an MUA molecules per gold nanosphere value of 1000 and connected via 1,6-hexanedithiol (HDT) decreases by 33% in magnitude and shifts to the red as largely as 22 nm with the increase of the molar ratio of HDT to MUA (R(HM)). Gold nanospheres capped with MUA and connected via HDT have been separated successfully using gel electrophoresis and characterized by measuring reflectance spectra of discrete electrophoretic bands directly in the gel and by monitoring transmission electron microscope images of gold nanoparticles collected from the discrete bands. Electrophoretic mobility has been found to decrease substantially with the increment of HDT to MUA, indicating that the size of aggregated gold nanoparticles increases with the concentration of HDT. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Urokinase production by electrophoretically separated cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Plank, L. D.; Giranda, V.; Sedor, K.; Todd, P. W.

1985-01-01

Urokinase is a plasminogen activator found in urine. Relatively pure preparations have been tested in Europe, Japan and the United States for the treatment of deep vein thrombosis and other dangerous blood clots. Human embryonic kidney cell cultures have been found to produce urokinase at much higher concentrations, but less than 5% of the cells in typical cultures are producers. Since human diploid cells become senescent in culture the selection of clones derived from single cells will not provide enough material to be useful, so a bulk purification method is needed for the isolation of urokinase producing cell populations. Preparative cell electrophoresis was chosen as the method, since evidence exists that human embryonic cell cultures are richly heterogeneous with respect to electrophoretic mobility, and preliminary electrophoretic separations on the Apollo-Soyuz space flight produced cell populations that were rich in urokinase production. Similarly, erythropoietin is useful in the treatment of certain anemias and is a kidney cell duct, and electrophoretically enriched cell populations producing this product have been reported. Thus, there is a clear need for diploid human cells that produce these products, and there is evidence that such cells should be separable by free-flow cell electrophoresis.

Electrophoretic separation techniques and their hyphenation to mass spectrometry in biological inorganic chemistry.

PubMed

Holtkamp, Hannah; Grabmann, Gerlinde; Hartinger, Christian G

2016-04-01

Electrophoretic methods have been widely applied in research on the roles of metal complexes in biological systems. In particular, CE, often hyphenated to a sensitive MS detector, has provided valuable information on the modes of action of metal-based pharmaceuticals, and more recently new methods have been added to the electrophoretic toolbox. The range of applications continues to expand as a result of enhanced CE-to-MS interfacing, with sensitivity often at picomolar level, and evolved separation modes allowing for innovative sample analysis. This article is a followup to previous reviews about CE methods in metallodrug research (Electrophoresis, 2003, 24, 2023-2037; Electrophoresis, 2007, 28, 3436-3446; Electrophoresis, 2012, 33, 622-634), also providing a comprehensive overview of metal species studied by electrophoretic methods hyphenated to MS. It highlights the latest CE developments, takes a sneak peek into gel electrophoresis, traces biomolecule labeling, and focuses on the importance of early-stage drug development. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

INFLUENCE OF BORATE BUFFERS ON THE ELECTROPHORETIC BEHAVIOR OF HUMIC SUBSTANCES IN CAPILLARY ZONE ELECTROPHORESIS

EPA Science Inventory

The influence of tetrahydroxyborate ions on the electrophoretic mobility of humic acids was evaluated by capillary electrophoresis (CE). Depending on the molarity of borate ions in the separation buffer, the humic acids exhibit electropherograms with sharp peaks consistently exte...

Continuous particle separation using pressure-driven flow-induced miniaturizing free-flow electrophoresis (PDF-induced μ-FFE).

PubMed

Jeon, Hyungkook; Kim, Youngkyu; Lim, Geunbae

2016-01-28

In this paper, we introduce pressure-driven flow-induced miniaturizing free-flow electrophoresis (PDF-induced μ-FFE), a novel continuous separation method. In our separation system, the external flow and electric field are applied to particles, such that particle movement is affected by pressure-driven flow, electroosmosis, and electrophoresis. We then analyzed the hydrodynamic drag force and electrophoretic force applied to the particles in opposite directions. Based on this analysis, micro- and nano-sized particles were separated according to their electrophoretic mobilities with high separation efficiency. Because the separation can be achieved in a simple T-shaped microchannel, without the use of internal electrodes, it offers the advantages of low-cost, simple device fabrication and bubble-free operation, compared with conventional μ-FFE methods. Therefore, we expect the proposed separation method to have a wide range of filtering/separation applications in biochemical analysis.

Continuous particle separation using pressure-driven flow-induced miniaturizing free-flow electrophoresis (PDF-induced μ-FFE)

PubMed Central

Jeon, Hyungkook; Kim, Youngkyu; Lim, Geunbae

2016-01-01

In this paper, we introduce pressure-driven flow-induced miniaturizing free-flow electrophoresis (PDF-induced μ-FFE), a novel continuous separation method. In our separation system, the external flow and electric field are applied to particles, such that particle movement is affected by pressure-driven flow, electroosmosis, and electrophoresis. We then analyzed the hydrodynamic drag force and electrophoretic force applied to the particles in opposite directions. Based on this analysis, micro- and nano-sized particles were separated according to their electrophoretic mobilities with high separation efficiency. Because the separation can be achieved in a simple T-shaped microchannel, without the use of internal electrodes, it offers the advantages of low-cost, simple device fabrication and bubble-free operation, compared with conventional μ-FFE methods. Therefore, we expect the proposed separation method to have a wide range of filtering/separation applications in biochemical analysis. PMID:26819221

Hydrodynamic sample injection into short electrophoretic capillary in systems with a flow-gating interface.

PubMed

Opekar, František; Tůma, Petr

2017-01-13

An electrophoretic apparatus with a flow-gating interface has been developed, enabling hydrodynamic sequence injection of the sample into the separation capillary from the liquid flow by underpressure generated in the outlet electrophoretic vessel. The properties of the apparatus were tested on an artificial sample of an equimolar mixture of 100μM potassium and sodium ions and arginine. The repeatability of the injection of the tested ions expressed as RSD (in%) for the peak area, peak height and migration time was in the range 0.76-2.08, 0.18-0.68 and 0.28-0.48, respectively. Under optimum conditions, the apparatus was used for sequence monitoring of the reaction between the antidiabetic drug phenyl biguanide and the glycation agent methyl glyoxal. The reaction solution was continuously sampled by a microdialysis probe from a thermostated external vessel using a syringe pump at a flow rate of 3μLmin -1 and was injected into a separation capillary at certain time intervals. The electrophoretic separation progressed in a capillary with an internal diameter of 50μm with a length of 11.5cm and was monitored using a contactless conductivity detector. Copyright © 2016 Elsevier B.V. All rights reserved.

GESA--a two-dimensional processing system using knowledge base techniques.

PubMed

Rowlands, D G; Flook, A; Payne, P I; van Hoff, A; Niblett, T; McKee, S

1988-12-01

The successful analysis of two-dimensional (2-D) polyacrylamide electrophoresis gels demands considerable experience and understanding of the protein system under investigation as well as knowledge of the separation technique itself. The present work concerns the development of a computer system for analysing 2-D electrophoretic separations which incorporates concepts derived from artificial intelligence research such that non-experts can use the technique as a diagnostic or identification tool. Automatic analysis of 2-D gel separations has proved to be extremely difficult using statistical methods. Non-reproducibility of gel separations is also difficult to overcome using automatic systems. However, the human eye is extremely good at recognising patterns in images, and human intervention in semi-automatic computer systems can reduce the computational complexities of fully automatic systems. Moreover, the expertise and understanding of an "expert" is invaluable in reducing system complexity if it can be encapsulated satisfactorily in an expert system. The combination of user-intervention in the computer system together with the encapsulation of expert knowledge characterises the present system. The domain within which the system has been developed is that of wheat grain storage proteins (gliadins) which exhibit polymorphism to such an extent that cultivars can be uniquely identified by their gliadin patterns. The system can be adapted to other domains where a range of polymorpic protein sub-units exist. In its generalised form, the system can also be used for comparing more complex 2-D gel electrophoretic separations.

Native red electrophoresis--a new method suitable for separation of native proteins.

PubMed

Dráb, Tomáš; Kračmerová, Jana; Tichá, Ivana; Hanzlíková, Eva; Tichá, Marie; Ryšlavá, Helena; Doubnerová, Veronika; Maňásková-Postlerová, Pavla; Liberda, Jiří

2011-12-01

A new type of native electrophoresis was developed to separate and characterize proteins. In this modification of the native blue electrophoresis, the dye Ponceau Red S is used instead of Coomassie Brilliant Blue to impose uniform negative charge on proteins to enable their electrophoretic separation according to their relative molecular masses. As Ponceau Red S binds less tightly to proteins, in comparison with Coomassie Blue, it can be easily removed after the electrophoretic separation and a further investigation of protein properties is made possible (e.g. an enzyme detection or electroblotting). The tested proteins also kept their native properties (enzyme activity or aggregation state). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic-like gating used to control metal-insulator transitions in electronically phase separated manganite wires.

PubMed

Guo, Hangwen; Noh, Joo H; Dong, Shuai; Rack, Philip D; Gai, Zheng; Xu, Xiaoshan; Dagotto, Elbio; Shen, Jian; Ward, T Zac

2013-08-14

Electronically phase separated manganite wires are found to exhibit controllable metal-insulator transitions under local electric fields. The switching characteristics are shown to be fully reversible, polarity independent, and highly resistant to thermal breakdown caused by repeated cycling. It is further demonstrated that multiple discrete resistive states can be accessed in a single wire. The results conform to a phenomenological model in which the inherent nanoscale insulating and metallic domains are rearranged through electrophoretic-like processes to open and close percolation channels.

Electrophoresis tests on STS-3 and ground control experiments - A basis for future biological sample selections

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Lewis, M. L.

1982-01-01

Static zone electrophoresis is an electrokinetic method of separating macromolecules and small particles. However, its application for the isolation of biological cells and concentrated protein solutions is limited by sedimentation and convection. Microgravity eliminates or reduces sedimentation, floatation, and density-driven convection arising from either Joule heating or concentration differences. The advantages of such an environment were first demonstrated in space during the Apollo 14 and 16 missions. In 1975 the Electrophoresis Technology Experiment (MA-011) was conducted during the Apollo-Soyuz Test Project flight. In 1979 a project was initiated to repeat the separations of human kidney cells. One of the major objectives of the Electrophoresis Equipment Verification Tests (EEVT) on STS-3 was to repeat and thereby validate the first successful electrophoretic separation of human kidney cells. Attention is given to the EEVT apparatus, the preflight electrophoresis, and inflight operational results.

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection.

PubMed

Duffy, Ciarán F; MacCraith, Brian; Diamond, Dermot; O'Kennedy, Richard; Arriaga, Edgar A

2006-08-01

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.

Vertical ascending electrophoresis of cells with a minimal stabilizing medium

NASA Technical Reports Server (NTRS)

Omenyi, S. N.; Snyder, R. S.

1983-01-01

Vertical fractionation of a mixture of fixed horse and human red blood cells layered over a stabilizing support medium was done to give a valid comparison with proposed space experiments. In particular, the effects of sample thickness and concentration on zone migration rate were investigated. Electrophoretic mobilities of horse and human cells calculated from zone migration rates were compatible with those obtained by microelectrophoresis. Complete cell separation was observed when low power and effective cooling were employed.

Improved separation and size characterization of gold nanoparticles through a novel capillary zone electrophoresis method using poly(sodium4-styrenesulfonate) as stabiliser and a stepwise field strength gradient.

PubMed

Ciriello, Rosanna; Iallorenzi, Pina Teresa; Laurita, Alessandro; Guerrieri, Antonio

2017-03-01

A novel capillary zone electrophoresis (CZE) method was developed for an improved separation and size characterization of pristine gold nanoparticles (AuNP) using uncoated fused-silica capillaries with UV-Vis detection at 520 nm. To avoid colloid aggregation and/or adsorption during runs, poly(sodium 4-styrenesulfonate) (PSS) was added (1%, w/v) in the running buffer (CAPS 10 mM, pH 11). This polyelectrolyte conferred an enhanced stabilization to AuNP, both steric and electrostatic, exalting at the same time their differences in electrophoretic mobility. Resolution was further and successfully improved through a stepwise field strength gradient by the application of 25 kV for the first 5 min and then 10 kV. Migration times varied linearly with particles diameters showing relative standard deviations better than 1% for daily experiments and 3% for interday experiments. A comparison with the size distribution obtained by transmission electron microscopy (TEM) allowed assessing that the electrophoretic profile can reasonably be considered as representative of the effective size heterogeneity of each colloid. Finally, the practical utility of the proposed method was demonstrated by measuring the core diameter of a gold colloid sample produced by chemical synthesis which was in good agreement with the value obtained by TEM measurements. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combined electrophoretic-separation and electrospray method and system

DOEpatents

Smith, Richard D.; Olivares, Jose A.

1989-01-01

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary zone electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5-100 KVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., .+-.2-8 KVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit.

Use of hydrophilic polymer coatings for control of electroosmosis and protein adsorption

NASA Technical Reports Server (NTRS)

Harris, J. Milton

1987-01-01

The purpose of this project was to examine the utility of polyethylene glycol (PEG) and dextran coatings for control of electroosmosis and protein adsorption; electroosmosis is an important, deleterious process affecting electrophoretic separations, and protein adsorption is a factor which needs to be controlled during protein crystal growth to avoid multiple nucleation sites. Performance of the project required use of X-ray photoelectron spectroscopy to refine previously developed synthetic methods. The results of this spectroscopic examination are reported. Measurements of electroosmotic mobility of charged particles in appropriately coated capillaries reveals that a new, one-step route to coating capillaries gives a surface in which electroosmosis is dramatically reduced. Similarly, both PEG and dextran coatings were shown by protein adsorption measurements to be highly effective at reducing protein adsorption on solid surfaces. These results should have impact on future low-g electrophoretic and protein crystal growth experiments.

Dynamic computer simulations of electrophoresis: three decades of active research.

PubMed

Thormann, Wolfgang; Caslavska, Jitka; Breadmore, Michael C; Mosher, Richard A

2009-06-01

Dynamic models for electrophoresis are based upon model equations derived from the transport concepts in solution together with user-inputted conditions. They are able to predict theoretically the movement of ions and are as such the most versatile tool to explore the fundamentals of electrokinetic separations. Since its inception three decades ago, the state of dynamic computer simulation software and its use has progressed significantly and Electrophoresis played a pivotal role in that endeavor as a large proportion of the fundamental and application papers were published in this periodical. Software is available that simulates all basic electrophoretic systems, including moving boundary electrophoresis, zone electrophoresis, ITP, IEF and EKC, and their combinations under almost exactly the same conditions used in the laboratory. This has been employed to show the detailed mechanisms of many of the fundamental phenomena that occur in electrophoretic separations. Dynamic electrophoretic simulations are relevant for separations on any scale and instrumental format, including free-fluid preparative, gel, capillary and chip electrophoresis. This review includes a historical overview, a survey of current simulators, simulation examples and a discussion of the applications and achievements of dynamic simulation.

Scanning and storage of electrophoretic records

DOEpatents

McKean, Ronald A.; Stiegman, Jeff

1990-01-01

An electrophoretic record that includes at least one gel separation is mounted for motion laterally of the separation record. A light source is positioned to illuminate at least a portion of the record, and a linear array camera is positioned to have a field of view of the illuminated portion of the record and orthogonal to the direction of record motion. The elements of the linear array are scanned at increments of motion of the record across the field of view to develop a series of signals corresponding to intensity of light at each element at each scan increment.

Investigation of the free flow electrophoretic process. Volume 2: Technical analysis

NASA Technical Reports Server (NTRS)

Weiss, R. A.; Lanham, J. W.; Richman, D. W.; Walker, C. D.

1979-01-01

The effect of gravity on the free flow electrophoretic process was investigated. The demonstrated effects were then compared with predictions made by mathematical models. Results show that the carrier buffer flow was affected by gravity induced thermal convection and that the movement of the separating particle streams was affected by gravity induced buoyant forces. It was determined that if gravity induced buoyant forces were included in the mathematical models, then effective predictions of electrophoresis chamber separation performance were possible. The results of tests performed using various methods of electrophoresis using supportive media show that the mobility and the ability to separate were essentially independent of concentration, providing promise of being able to perform electrophoresis with higher inlet concentrations in space.

Simplification and improvement of protein detection in two-dimensional electrophoresis gels with SERVA HPE™ lightning red.

PubMed

Griebel, Anja; Obermaier, Christian; Westermeier, Reiner; Moche, Martin; Büttner, Knut

2013-07-01

A new fluorescent amino-reactive dye has been tested for both labelling proteins prior to electrophoretic separations and between the two steps of two-dimensional electrophoresis. A series of experiments showed, that the labelling of lysines with this dye is compatible with all standard additives used for sample preparation, including reducing substances and carrier ampholytes. Using this dye for pre-labelling considerably simplifies the electrophoresis and detection workflow and provides highly sensitive and quantitative visualisation of proteins.

Combined electrophoretic-separation and electrospray method and system

DOEpatents

Smith, R.D.; Olivares, J.A.

1989-06-27

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary zone electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5--100 kVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., [+-]2--8 kVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit. 10 figs.

Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

PubMed

Maccari, Francesca; Volpi, Nicola

2002-09-01

We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

Nonaqueous capillary electrophoresis of dextromethorphan and its metabolites.

PubMed

Pelcová, Marta; Langmajerová, Monika; Cvingráfová, Eliška; Juřica, Jan; Glatz, Zdeněk

2014-10-01

This study deals with the nonaqueous capillary electrophoretic separation of dextromethorphan and its metabolites using a methanolic background electrolyte. The optimization of separation conditions was performed in terms of the resolution of dextromethorphan and dextrorphan and the effect of separation temperature, voltage, and the characteristics of the background electrolyte were studied. Complete separation of all analytes was achieved in 40 mM ammonium acetate dissolved in methanol. Hydrodynamic injection was performed at 3 kPa for 4 s. The separation voltage was 20 kV accompanied by a low electric current. The ultraviolet detection was performed at 214 nm, the temperature of the capillary was 25°C. These conditions enabled the separation of four analytes plus the internal standard within 9 min. Further, the developed method was validated in terms of linearity, sensitivity, and repeatability. Rat liver perfusate samples were subjected to the nonaqueous capillary electrophoretic method to illustrate its applicability. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Polystyrene latex separations by continuous flow electrophoresis on the Space Shuttle

NASA Technical Reports Server (NTRS)

Snyder, R. S.; Rhodes, P. H.; Miller, T. Y.; Micale, F. J.; Mann, R. V.

1986-01-01

The seventh mission of the Space Shuttle carried two NASA experiments in the McDonnell Douglas Astronautics Corporation continuous flow electrophoresis system. The objectives were to test the operation of continuous flow electrophoresis in a reduced gravity environment using stable particles with established electrokinetic properties and specifically to evaluate the influence of the electrical properties of the sample constituents on the resolution of the continuous flow electrophoretic device. Polystrene latex microspheres dispersed in a solution with three times the electrical conductivity of the curtain buffer separated with a significantly larger band spread compared to the second experiment under matched conductivity conditions. It is proposed that the sample of higher electrical conductivity distorted the electric field near the sample stream so that the polystyrene latex particles migrated toward the chamber walls where electroosmosis retarded and spread the sample.

Electrophoretic cell separation using microspheres. [purification of lymphocytes

NASA Technical Reports Server (NTRS)

Smolka, A.; Sachs, G.

1980-01-01

Methods of cell separation based on the electrokinetic properties of the cell membrane offer a degree of discrimination among cell populations which is not available with methods based on cell size or density alone. Studies aimed at extending red cell separations using microspheres to purification of lymphocytes.

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

Thornton, Michelle

Capillary electrophoresis (CE) is an effective method for separating ionic species according to differences in their electrophoretic mobilities. CE separations of amino acids by direct detection are difficult due to their similar electrophoretic mobilities and low absorbances. However, native amino acids can be separated by CE as cations at a low pH by adding an alkanesulfonic acid to the electrolyte carrier which imparts selectivity to the system. Derivatization is unnecessary when direct UV detection is used at 185 nm. Simultaneous speciation of metal cations such as vanadium (IV) and vanadium (V) can easily be performed without complexation prior to analysis.more » An indirect UV detection scheme for acidic conditions was also developed using guanidine as the background carrier electrolyte (BCE) for the indirect detection of metal cations. Three chapters have been removed for separate processing. This report contains introductory material, references, and general conclusions. 80 refs.« less

Separation of saccharides derivatized with 2-aminobenzoic acid by capillary electrophoresis and their structural consideration by nuclear magnetic resonance.

PubMed

He, Liping; Sato, Kae; Abo, Mitsuru; Okubo, Akira; Yamazaki, Sunao

2003-03-01

Saccharides including mono- and disaccharides were quantitatively derivatized with 2-aminobenzoic acid (2-AA). These derivatives were then separated by capillary zone electrophoresis with UV detection using 50mM sodium phosphate buffer as the running electrolyte solution. In particular, the saccharide derivatives with the same molecular weight as 2-AA aldohexoses (mannose and glucose) and 2-AA aldopentoses (ribose and xylose) were well separated. The underlying reasons for separation were explored by studying their structural data using 1H and 13C NMR. It was found that the configurational difference between their hydroxyl group at C2 or C3 could cause the difference in Stokes' radii between their molecules and thus lead to different electrophoretic mobilities. The correlation between the electrophoretic behavior of these carbohydrate derivatives and their structures was studied utilizing the calculated molecular models of the 2-AA-labeled mannose, glucose, ribose, and xylose.

Kidney cell electrophoresis in space flight: Rationale, methods, results and flow cytometry applications

NASA Technical Reports Server (NTRS)

Todd, P.; Morrison, Dennis R.; Barlow, Grant H.; Lewis, Marian L.; Lanham, J. W.; Cleveland, C.; Williams, K.; Kunze, M. E.; Goolsby, C. L.

1988-01-01

Cultures of human embryonic kidney cells consistently contain an electrophoretically separable subpopulation of cells that produce high levels of urokinase and have an electrophoretic mobility about 85 percent as high as that of the most mobile human embryonic kidney cells. This subpopulation is rich in large epithelioid cells that have relatively little internal structure. When resolution and throughput are adequate, free fluid electrophoresis can be used to isolate a broad band of low mobility cells which also produces high levels of plasminogen activators (PAs). In the course of performing this, it was discovered that all electrophoretic subpopulations of cultured human embryonic kidney cells produce some PAs and that separate subpopulations produce high quantities of different types of PA's. This information and the development of sensitive assays for this project have provided new insights into cell secretion mechanisms related to fibrinolysis. These advances would probably not have been made without the NASA program to explore fundamental questions of free fluid electrophoresis in space.

Comprehensive and Critical Literature Review on Insitu Micro-Sensors for Application in Tribology

DTIC Science & Technology

1994-04-01

Electroosmotic flow provides a pumping method that is convenient for small capillaries. Electrophoretic separation is shown to be useful. On the left hand...analysis systems on glass chips (1 centimeter by 2 centimeters or larger) that utilize electroosmotic pumping to drive fluid flow and electrophoretic...elucidate the interaction mechanism. Additionally, using two types of sensors in a mixed array increases selectivity by providing different information

Free-Flow Open-Chamber Electrophoresis

NASA Technical Reports Server (NTRS)

Sharnez, Rizwan; Sammons, David W.

1994-01-01

Free-flow open-chamber electrophoresis variant of free-flow electrophoresis performed in chamber with open ends and in which velocity of electro-osmotic flow adjusted equal to and opposite mean electrophoretic velocity of sample. Particles having electrophoretic mobilities greater than mean mobility of sample particles move toward cathode, those with mobilities less move toward anode. Technique applied to separation of components of mixtures of biologically important substances. Sensitivity enhanced by use of tapered chamber.

[Electrophoretic patterns of cell wall protein as a criterion for the identification and classification of Corynebacteria].

PubMed

Mykhal's'kyĭ, L O; Furtat, I M; Dem'ianenko, F P; Kostiuchyk, A A

2001-01-01

Electrophoretic patterns of cell wall protein of three industrial strains, that were used for production of lysin, and eight collection strains from the genus Corynevacterium were studied to analyze their similarity as well as to estimate an opportunity of using this parameter as an additional criterion for identification and classification of corynebacteria. Similarity coefficient of cell wall overall and main protein electrophoretic patterns were determined by a specially created computer program. Electrophoretic analysis showed that every specie had an individual protein profile. There were determined biopolymers common for the specie, genus and individual among the overall majors and minors. The obtained results showed, that the patterns of main proteins were more conservative and informative in comparison with those ones of overall proteins. The definition of similarity coefficient by the main protein patterns has correlated with the protein profile characteristics of every analyzed strain, and it managed to distribute them into the separate groups. The similarity coefficient of preparations by the main protein patterns allows to separate one specie or a strain from another, and that gives us a chance to claim that this parameter could be used as an additional criterion for differentiation and referring the corynebacteria to a certain taxonomic group.

Purification and Cultivation of Human Pituitary Growth Hormones Secreting Cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Todd, P.; Grindeland, R.; Lanham, W.; Morrison, D.

1985-01-01

The rat and human pituitary gland contains a mixture of hormone producing cell types. The separation of cells which make growth hormone (GH) is attempted for the purpose of understanding how the hormone molecule is made within the pituitary cell; what form(s) it takes within the cell; and what form(s) GH assumes as it leaves the cell. Since GH has a number of biological targets (e.g., muscle, liver, bone), the assessment of the activities of the intracellular/extracellular GH by new and sensitive bioassays. GH cells contained in the mixture was separated by free flow electrophoresis. These experiments show that GH cells have different electrophoretic mobilities. This is relevant to NASA since a lack of GH could be a prime causative factor in muscle atrophy. Further, GH has recently been implicated in the etiology of motion sickness in space. Continous flow electrophoresis experiment on STS-8 showed that GH cells could be partially separated in microgravity. However, definitive cell culture studies could not be done due to insufficient cell recoveries.

Reproductive cell separation: A concept

NASA Technical Reports Server (NTRS)

Cutaia, A. J.

1973-01-01

Attempt has been made to separate mammalian male (Y) bearing sperm from female (X) bearing sperm. Both types of sperm are very dependent on gravity for their direction of movement. Proposed concept suggests electrophoretic force of suitable magnitude and direction may be effective means of separating X and Y sperm under zero gravity.

Monolithic integration of fine cylindrical glass microcapillaries on silicon for electrophoretic separation of biomolecules

PubMed Central

Cao, Zhen; Ren, Kangning; Wu, Hongkai; Yobas, Levent

2012-01-01

We demonstrate monolithic integration of fine cylindrical glass microcapillaries (diameter ∼1 μm) on silicon and evaluate their performance for electrophoretic separation of biomolecules. Such microcapillaries are achieved through thermal reflow of a glass layer on microstructured silicon whereby slender voids are moulded into cylindrical tubes. The process allows self-enclosed microcapillaries with a uniform profile. A simplified method is also described to integrate the microcapillaries with a sample-injection cross without the requirement of glass etching. The 10-mm-long microcapillaries sustain field intensities up to 90 kV/m and limit the temperature excursions due to Joule heating to a few degrees Celsius only. PMID:23874369

Electrophoretic separation of cells and particles from rat pituitary. [analysis of pituitary cell electrophoresis experiment done on IML-2 (7/94)

NASA Technical Reports Server (NTRS)

Hymer, W. C.

1995-01-01

In spite of the fact that a vast majority of the electrophoresis effort (approximately 90%) could not be done on this mission (IML-2) due to failure of FFEU hardware, we find some interesting differences in flight samples obtained from other parts of the experiment. These differences are entirely novel and sometimes unexpected. This report is organized into 4 parts. Each part describes the data collected thus far from each of the 4 cell culture kits (CCK) which flew in space. Each CCK was loaded with 40x10(exp 6) fresh pituitary cells; all CCK's were identical at the start of the experiment because we prepared one pool of cells.

Biologically driven neural platform invoking parallel electrophoretic separation and urinary metabolite screening.

PubMed

Page, Tessa; Nguyen, Huong Thi Huynh; Hilts, Lindsey; Ramos, Lorena; Hanrahan, Grady

2012-06-01

This work reveals a computational framework for parallel electrophoretic separation of complex biological macromolecules and model urinary metabolites. More specifically, the implementation of a particle swarm optimization (PSO) algorithm on a neural network platform for multiparameter optimization of multiplexed 24-capillary electrophoresis technology with UV detection is highlighted. Two experimental systems were examined: (1) separation of purified rabbit metallothioneins and (2) separation of model toluene urinary metabolites and selected organic acids. Results proved superior to the use of neural networks employing standard back propagation when examining training error, fitting response, and predictive abilities. Simulation runs were obtained as a result of metaheuristic examination of the global search space with experimental responses in good agreement with predicted values. Full separation of selected analytes was realized after employing optimal model conditions. This framework provides guidance for the application of metaheuristic computational tools to aid in future studies involving parallel chemical separation and screening. Adaptable pseudo-code is provided to enable users of varied software packages and modeling framework to implement the PSO algorithm for their desired use.

High-resolution slab gel isoelectric focusing: methods for quantitative electrophoretic transfer and immunodetection of proteins as applied to the study of the multiple isoelectric forms of ornithine decarboxylase.

PubMed

Reddy, S G; Cochran, B J; Worth, L L; Knutson, V P; Haddox, M K

1994-04-01

A high-resolution isoelectric focusing vertical slab gel method which can resolve proteins which differ by a single charge was developed and this method was applied to the study of the multiple isoelectric forms of ornithine decarboxylase. Separation of proteins at this high level of resolution was achieved by increasing the ampholyte concentration in the gels to 6%. Various lots of ampholytes, from the same or different commercial sources, differed significantly in their protein binding capacity. Ampholytes bound to proteins interfered both with the electrophoretic transfer of proteins from the gel to immunoblotting membranes and with the ability of antibodies to interact with proteins on the immunoblotting membranes. Increasing the amount of protein loaded into a gel lane also decreased the efficiency of the electrophoretic transfer and immunodetection. To overcome these problems, both gel washing and gel electrophoretic transfer protocols for disrupting the ampholyte-protein binding and enabling a quantitative electrophoretic transfer of proteins were developed. Two gel washing procedures, with either thiocyanate or borate buffers, and a two-step electrophoretic transfer method are described. The choice of which method to use to optimally disrupt the ampholyte-protein binding was found to vary with each lot of ampholytes employed.

The effect of small temperature gradients on flow in a continuous flow electrophoresis chamber

NASA Technical Reports Server (NTRS)

Rhodes, P. H.; Snyder, R. S.

1982-01-01

Continuous flow electrophoresis employs an electric field to separate biological cells suspended in a flowing liquid buffer solution. Good separations based on differences in electrophoretic mobility are obtained only when a unidirectional flow is maintained. The desired flow has a parabolic structure in the narrow dimension of the chamber and is uniform acros the width, except near the edges where the no-slip condition prevails. However, because of buoyancy, very small laterall or axial temperature gradients deform the flow significantly. The results of experiments conducted with a specially instrumented chamber show the origin and structure of the buoyancy-driven perturbations. It is found that very small temperature gradients can disturb the flow significantly, as was predicted by earlier theoretical work.

Peak capacity and peak capacity per unit time in capillary and microchip zone electrophoresis.

PubMed

Foley, Joe P; Blackney, Donna M; Ennis, Erin J

2017-11-10

The origins of the peak capacity concept are described and the important contributions to the development of that concept in chromatography and electrophoresis are reviewed. Whereas numerous quantitative expressions have been reported for one- and two-dimensional separations, most are focused on chromatographic separations and few, if any, quantitative unbiased expressions have been developed for capillary or microchip zone electrophoresis. Making the common assumption that longitudinal diffusion is the predominant source of zone broadening in capillary electrophoresis, analytical expressions for the peak capacity are derived, first in terms of migration time, diffusion coefficient, migration distance, and desired resolution, and then in terms of the remaining underlying fundamental parameters (electric field, electroosmotic and electrophoretic mobilities) that determine the migration time. The latter expressions clearly illustrate the direct square root dependence of peak capacity on electric field and migration distance and the inverse square root dependence on solute diffusion coefficient. Conditions that result in a high peak capacity will result in a low peak capacity per unit time and vice-versa. For a given symmetrical range of relative electrophoretic mobilities for co- and counter-electroosmotic species (cations and anions), the peak capacity increases with the square root of the electric field even as the temporal window narrows considerably, resulting in a significant reduction in analysis time. Over a broad relative electrophoretic mobility interval [-0.9, 0.9], an approximately two-fold greater amount of peak capacity can be generated for counter-electroosmotic species although it takes about five-fold longer to do so, consistent with the well-known bias in migration time and resolving power for co- and counter-electroosmotic species. The optimum lower bound of the relative electrophoretic mobility interval [μ r,Z , μ r,A ] that provides the maximum peak capacity per unit time is a simple function of the upper bound, but its direct application is limited to samples with analytes whose electrophoretic mobilities can be varied independently of electroosmotic flow. For samples containing both co- and counter-electroosmotic ions whose electrophoretic mobilities cannot be easily manipulated, comparable levels of peak capacity and peak capacity per unit time for all ions can be obtained by adjusting the EOF to devote the same amount of time to the separation of each class of ions; this corresponds to μ r,Z =-0.5. Copyright © 2017 Elsevier B.V. All rights reserved.

Affinity monolith-integrated poly(methyl methacrylate) microchips for on-line protein extraction and capillary electrophoresis.

PubMed

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T

2008-07-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with eight reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in a porogenic solvent consisting of 70% 1-dodecanol and 30% cyclohexanol. Antifluorescein isothiocyanate was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of fluorescein isothiocyanate (FITC)-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis.

Affinity Monolith-Integrated Poly(methyl Methacrylate) Microchips for On-Line Protein Extraction and Capillary Electrophoresis

PubMed Central

Sun, Xiuhua; Yang, Weichun; Pan, Tao; Woolley, Adam T.

2008-01-01

Immunoaffinity monolith pretreatment columns have been coupled with capillary electrophoresis separation in poly(methyl methacrylate) (PMMA) microchips. Microdevices were designed with 8 reservoirs to enable the electrically controlled transport of selected analytes and solutions to carry out integrated immunoaffinity extraction and electrophoretic separation. The PMMA microdevices were fabricated reproducibly and with high fidelity by solvent imprinting and thermal bonding methods. Monoliths with epoxy groups for antibody immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene dimethacrylate in a porogenic solvent consisting of 70% dodecanol and 30% hexanol. Anti-fluorescein isothiocyanate (FITC) was utilized as a model affinity group in the monoliths, and the immobilization process was optimized. A mean elution efficiency of 92% was achieved for the monolith-based extraction of FITC-tagged human serum albumin. FITC-tagged proteins were purified from a contaminant protein and then separated electrophoretically using these devices. The developed immunoaffinity column/capillary electrophoresis microdevices show great promise for combining sample pretreatment and separation in biomolecular analysis. PMID:18479142

Electrokinetic Sample Preconcentration and Hydrodynamic Sample Injection for Microchip Electrophoresis Using a Pneumatic Microvalve

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

Cong, Yongzheng; Katipamula, Shanta; Geng, Tao

2016-02-01

A microfluidic platform was developed to perform online electrokinetic sample preconcentration and rapid hydrodynamic sample injection for electrophoresis using a single microvalve. The PDMS microchip consists of a separation channel, a side channel for sample introduction, and a control channel which is used as a pneumatic microvalve aligned at the intersection of the two flow channels. The closed microvalve, created by multilayer soft lithography, can serve as a preconcentrator under an applied electric potential, enabling current to pass through while blocking bulk flow. Once analytes are concentrated, the valve is briefly opened and the stacked sample is pressure injected intomore » the separation channel for electrophoretic separation. Fluorescently labeled peptides were enriched by a factor of ~450 in 230 s. The performance of the platform was validated by the online preconcentration, injection and electrophoretic separation of fluorescently labeled peptides. This method enables both rapid analyte concentration and controlled injection volume for high sensitivity, high resolution capillary electrophoresis.« less

Summary electrophoretic data base on human embryonic kidney cell strain 8514

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Arquiza, M. V.; Morrison, D. R.; Todd, P. W.

1985-01-01

To properly plan the electrophoresis equipment verification test (EEVT) and continuous flow electrophoresis system (CFES) experiments with human embryonic kidney cells, first a candidate cell lot had to be chosen on the basis of electrophoretic heterogeneity, growth potential, cytogenetics, and urokinase production. Cell lot 8514 from MA Bioproducts, Inc. was chosen for this purpose, and several essential analytical electrophoresis experiments were performed to test its final suitability for these experiments.

Siderosomal ferritin. The missing link between ferritin and haemosiderin?

PubMed Central

Andrews, S C; Treffry, A; Harrison, P M

1987-01-01

A minor electrophoretically fast component was found in ferritin from iron-loaded rat liver in addition to a major electrophoretically slow ferritin similar to that observed in control rats. The electrophoretically fast ferritin showed immunological identity with the slow component, but on electrophoresis in SDS it gave a peptide of 17.3 kDa, in contrast with the electrophoretically slow ferritin, which gave a major band corresponding to the L-subunit (20.7 kDa). Thus the electrophoretically fast ferritin resembles that reported by Massover [(1985) Biochim. Biophys. Acta 829, 377-386] in livers of mice with short-term parenteral iron overload. The electrophoretically fast ferritin had a lower iron content (2000 Fe atoms/molecule) than the electrophoretically slow ferritin (3000 Fe atoms/molecule). Removal and re-incorporation of iron was possible without effect on the electrophoretic mobility of either ferritin species. On subcellular fractionation the electrophoretically fast ferritin was enriched in pellet fractions and was the sole soluble ferritin isolated from iron-laden secondary lysosomes (siderosomes). The amount and relative proportion of the electrophoretically fast species increased with iron loading. Haemosiderin isolated from siderosomes was found to contain a peptide reactive to anti-ferritin serum and corresponding to the 17.3 kDa peptide of the electrophoretically fast ferritin species. Unlike the electrophoretically slow ferritin, the electrophoretically fast ferritin did not become significantly radioactive in a 1 h biosynthetic labelling experiment. We conclude that the minor ferritin is not, as has been suggested for mouse liver ferritin, 'a completely new species of smaller holoferritin that represents a shift in the ferritin phenotype' in response to siderosis, but a precursor of haemosiderin, in agreement with the proposal by Richter [(1984) Lab. Invest. 50, 26-35] concerning siderosomal ferritin. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:3663170

Method for in-situ calibration of electrophoretic analysis systems

DOEpatents

Liu, Changsheng; Zhao, Hequan

2005-05-08

An electrophoretic system having a plurality of separation lanes is provided with an automatic calibration feature in which each lane is separately calibrated. For each lane, the calibration coefficients map a spectrum of received channel intensities onto values reflective of the relative likelihood of each of a plurality of dyes being present. Individual peaks, reflective of the influence of a single dye, are isolated from among the various sets of detected light intensity spectra, and these can be used to both detect the number of dye components present, and also to establish exemplary vectors for the calibration coefficients which may then be clustered and further processed to arrive at a calibration matrix for the system. The system of the present invention thus permits one to use different dye sets to tag DNA nucleotides in samples which migrate in separate lanes, and also allows for in-situ calibration with new, previously unused dye sets.

Chromatographic and electrophoretic approaches in ink analysis.

PubMed

Zlotnick, J A; Smith, F P

1999-10-15

Inks are manufactured from a wide variety of substances that exhibit very different chemical behaviors. Inks designed for use in different writing instruments or printing methods have quite dissimilar components. Since the 1950s chromatographic and electrophoretic methods have played important roles in the analysis of inks, where compositional information may have bearing on the investigation of counterfeiting, fraud, forgery, and other crimes. Techniques such as paper chromatography and electrophoresis, thin-layer chromatography, high-performance liquid chromatography, gas chromatography, gel electrophoresis, and the relatively new technique of capillary electrophoresis have all been explored as possible avenues for the separation of components of inks. This paper reviews the components of different types of inks and applications of the above separation methods are reviewed.

Modeling the electrophoretic separation of short biological molecules in nanofluidic devices

NASA Astrophysics Data System (ADS)

Fayad, Ghassan; Hadjiconstantinou, Nicolas

2010-11-01

Via comparisons with Brownian Dynamics simulations of the worm-like-chain and rigid-rod models, and the experimental results of Fu et al. [Phys. Rev. Lett., 97, 018103 (2006)], we demonstrate that, for the purposes of low-to-medium field electrophoretic separation in periodic nanofilter arrays, sufficiently short biomolecules can be modeled as point particles, with their orientational degrees of freedom accounted for using partition coefficients. This observation is used in the present work to build a particularly simple and efficient Brownian Dynamics simulation method. Particular attention is paid to the model's ability to quantitatively capture experimental results using realistic values of all physical parameters. A variance-reduction method is developed for efficiently simulating arbitrarily small forcing electric fields.

Fabrication of superhydrophobic nano-aluminum films on stainless steel meshes by electrophoretic deposition for oil-water separation

NASA Astrophysics Data System (ADS)

Xu, Zhe; Jiang, Deyi; Wei, Zhibo; Chen, Jie; Jing, Jianfeng

2018-01-01

Stainless steel meshes with superhydrophobic surfaces were successfully fabricated via a facile electrophoretic deposition process. The surface morphology and chemical compositions were characterized by a field emission scanning electron microscope (FE-SEM), energy-dispersive X-ray spectroscope (EDS), X-ray diffraction (XRD) and fourier-transform infrared spectrophotometer (FTIR). After stearic acid modification, the obtained nano-aluminum films on stainless steel meshes showed an excellent superhydrophobic properties with a water contact angle of 160° ± 1.2° and a water sliding angle of less than 5°. In addition, on the basis of the superhydrophobic meshes, a simple, continuous oil-water separation apparatus was designed, and the oil-water separation efficiency was up to 95.8% ± 0.9%. Meanwhile, after 20 oil-water separation cycles, the separation efficiency without significant reduction suggested the stable performance of superhydrophobic stainless steel meshes on the oil-water separation. Moreover, the flow rate of oil-water mixture and effective separation length were investigated to determine their effects on the oil-water separation efficiency, respectively. Our work provides a cost-efficient method to prepare stable superhydrophobic nano-Al films on stainless steel meshes, and it has promising practical applications on oil-water separation.

Coil planet centrifugation as a means for small particle separation

NASA Technical Reports Server (NTRS)

Herrmann, F. T.

1983-01-01

The coil planet centrifuge uses a centrifugal force field to provide separation of particles based on differences in sedimentation rates by flow through a rotating coiled tube. Three main separations are considered: (1) single phase fresh sheep and human erythrocytes, (2) single phase fixed heep and human erythrocytes, and (3) electrophoretically enhanced single phase fresh sheep and human erythrocytes.

Biased Cyclical Electrical Field-Flow Fractionation for Separation of Submicron Particles

PubMed Central

Ornthai, Mathuros; Siripinyanond, Atitaya; Gale, Bruce K.

2015-01-01

The potential of biased cyclical electrical field flow fractionation (BCyElFFF), which applies the positive cycle voltage longer than the negative cycle voltage, for characterization of submicron particles, was investigated. Parameters affecting separation and retention such as voltage, frequency, and duty cycle were examined. The results suggest that the separation mechanism in BCyElFFF in many cases is more related to the size of particles, as is the case with normal ElFFF, in the studied conditions, than the electrophoretic mobility, which is what the theory predicts for CyElFFF. However, better resolution was obtained when separating using BCyElFFF mode than when using normal CyElFFF. BCyElFFF was able to demonstrate simultaneous baseline separations of a mixture of 0.04, 0.1, and 0.2 μm particles and near separation of 0.5 μm particles. This study has shown the applicability of the BCyElFFF for separation and characterization of submicron particles greater than 0.1 μm in size, which had not been demonstrated previously. The separation and retention results suggest that for particles of this size, retention is based more on particle size than on electrophoretic mobility, which is contrary to existing theory for CyElFFF. PMID:26612733

Biased cyclical electrical field-flow fractionation for separation of submicron particles.

PubMed

Ornthai, Mathuros; Siripinyanond, Atitaya; Gale, Bruce K

2016-01-01

The potential of biased cyclical electrical field-flow fractionation (BCyElFFF), which applies the positive cycle voltage longer than the negative cycle voltage, for characterization of submicron particles, was investigated. Parameters affecting separation and retention such as voltage, frequency, and duty cycle were examined. The results suggest that the separation mechanism in BCyElFFF in many cases is more related to the size of particles, as is the case with normal ElFFF, in the studied conditions, than the electrophoretic mobility, which is what the theory predicts for CyElFFF. However, better resolution was obtained when separating using BCyElFFF mode than when using normal CyElFFF. BCyElFFF was able to demonstrate simultaneous baseline separations of a mixture of 0.04-, 0.1-, and 0.2-μm particles and near separation of 0.5-μm particles. This study has shown the applicability of BCyElFFF for separation and characterization of submicron particles greater than 0.1-μm in size, which had not been demonstrated previously. The separation and retention results suggest that for particles of this size, retention is based more on particle size than on electrophoretic mobility, which is contrary to existing theory for CyElFFF.

A new electrophoretic focusing principle: focusing of nonamphoteric weak ionogenic analytes using inverse electromigration dispersion profiles.

PubMed

Gebauer, Petr; Malá, Zdena; Bocek, Petr

2010-03-01

This contribution introduces a new separation principle in CE which offers focusing of weak nonamphoteric ionogenic species and their inherent transport to the detector. The prerequisite condition for application of this principle is the existence of an inverse electromigration dispersion profile, i.e. a profile where pH is decreasing toward the anode or cathode for focusing of anionic or cationic weak analytes, respectively. The theory presented defines the principal conditions under which an analyte is focused on a profile of this type. Since electromigration dispersion profiles are migrating ones, the new principle offers inherent transport of focused analytes into the detection cell. The focusing principle described utilizes a mechanism different from both CZE (where separation is based on the difference in mobilities) and IEF (where separation is based on difference in pI), and hence, offers another separation dimension in CE. The new principle and its theory presented here are supplemented by convincing experiments as their proof.

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis.

PubMed

Scherer, James R; Liu, Peng; Mathies, Richard A

2010-11-01

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ~20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex(®) 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.

Design and operation of a portable scanner for high performance microchip capillary array electrophoresis

NASA Astrophysics Data System (ADS)

Scherer, James R.; Liu, Peng; Mathies, Richard A.

2010-11-01

We have developed a compact, laser-induced fluorescence detection scanner, the multichannel capillary array electrophoresis portable scanner (McCAEPs) as a platform for electrophoretic detection and control of high-throughput, integrated microfluidic devices for genetic and other analyses. The instrument contains a confocal optical system with a rotary objective for detecting four different fluorescence signals, a pneumatic system consisting of two pressure/vacuum pumps and 28 individual addressable solenoid valves for control of on-chip microvalves and micropumps, four Polymerase Chain Reaction (PCR) temperature control systems, and four high voltage power supplies for electrophoresis. The detection limit of the instrument is ˜20 pM for on-chip capillary electrophoresis of fluorescein dyes. To demonstrate the system performance for forensic short tandem repeat (STR) analysis, two experiments were conducted: (i) electrophoretic separation and detection of STR samples on a 96-lane microfabricated capillary array electrophoresis microchip. Fully resolved PowerPlex® 16 STR profiles amplified from 1 ng of 9947A female standard DNA were successfully obtained; (ii) nine-plex STR amplification, sample injection, separation, and fluorescence detection of 100-copy 9948 male standard DNA in a single integrated PCR- capillary electrophoresis microchip. These results demonstrate that the McCAEPs can be used as a versatile control and detection instrument that operates integrated microfluidic devices for high-performance forensic human identification.

Atomic-force-controlled capillary electrophoretic nanoprinting of proteins.

PubMed

Lovsky, Yulia; Lewis, Aaron; Sukenik, Chaim; Grushka, Eli

2010-01-01

The general nanoprinting and nanoinjection of proteins on non-conducting or conducting substrates with a high degree of control both in terms of positional and timing accuracy is an important goal that could impact diverse fields from biotechnology (protein chips) to molecular electronics and from fundamental studies in cell biology to nanophotonics. In this paper, we combine capillary electrophoresis (CE), a separation method with considerable control of protein movement, with the unparalleled positional accuracy of an atomic force microscope (AFM). This combination provides the ability to electrophoretically or electroosmotically correlate the timing of protein migration with AFM control of the protein deposition at a high concentration in defined locations and highly confined volumes estimated to be 2 al. Electrical control of bovine serum albumin printing on standard protein-spotting glass substrates is demonstrated. For this advance, fountain pen nanolithography (FPN) that uses cantilevered glass-tapered capillaries is amended with the placement of electrodes on the nanopipette itself. This results in imposed voltages that are three orders of magnitude less than what is normally used in capillary electrophoresis. The development of atomic-force-controlled capillary electrophoretic printing (ACCEP) has the potential for electrophoretic separation, with high resolution, both in time and in space. The large voltage drop at the tip of the tapered nanopipettes allows for significant increases in concentration of protein in the small printed volumes. All of these attributes combine to suggest that this methodology should have a significant impact in science and technology.

An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution.

PubMed

Lee, H G; Cowman, M K

1994-06-01

An electrophoretic method is described for determining the molecular weight distribution of hyaluronan (HA). The method involves separation of HA by electrophoresis on a 0.5% agarose gel, followed by detection of HA using the cationic dye Stains-All (3,3'-dimethyl-9-methyl-4,5,4'5'-dibenzothiacarbocyanine). The recommended sample load is 7 micrograms. Calibration of the method with HA standards of known molecular weight has established a linear relationship between electrophoretic mobility and the logarithm of the weight-average molecular weight over the range of approximately 0.2-6 x 10(6). The separated HA pattern may also be visualized after electrotransfer of HA from the agarose gel to a nylon membrane. The membrane may be stained with the dye alcian blue. Alternatively, specific detection of HA from impure samples can be achieved by probing the nylon membrane with biotin-labeled HA-binding protein and subsequent interaction with a streptavidin-linked gold reagent and silver staining for amplification. The electrophoretic method was used to analyze HA in two different liquid connective tissues. Normal human knee joint synovial fluid showed a narrow HA molecular weight distribution, with a peak at 6-7 x 10(6). Owl monkey vitreous HA also showed a narrow molecular weight distribution, with a peak at 5-6 x 10(6). These results agree well with available published data and indicate the applicability of the method to the analysis of impure HA samples which may be available in limited amounts.

Mathematical modeling of sample stacking methods in microfluidic systems

NASA Astrophysics Data System (ADS)

Horek, Jon

Gradient focusing methods are a general class of experimental techniques used to simultaneously separate and increase the cross-sectionally averaged concentration of charged particle mixtures. In comparison, Field Amplified Sample Stacking (FASS) techniques first concentrate the collection of molecules before separating them. Together, we denote gradient focusing and FASS methods "sample stacking" and study the dynamics of a specific method, Temperature Gradient Focusing (TGF), in which an axial temperature gradient is applied along a channel filled with weak buffer. Gradients in electroosmotic fluid flow and electrophoretic species velocity create the simultaneous separating and concentrating mechanism mentioned above. In this thesis, we begin with the observation that very little has been done to model the dynamics of gradient focusing, and proceed to solve the fundamental equations of fluid mechanics and scalar transport, assuming the existence of slow axial variations and the Taylor-Aris dispersion coefficient. In doing so, asymptotic methods reduce the equations from 3D to 1D, and we arrive at a simple 1D model which can be used to predict the transient evolution of the cross-sectionally averaged analyte concentration. In the second half of this thesis, we run several numerical focusing experiments with a 3D finite volume code. Comparison of the 1D theory and 3D simulations illustrates not only that the asymptotic theory converges as a certain parameter tends to zero, but also that fairly large axial slip velocity gradients lead to quite small errors in predicted steady variance. Additionally, we observe that the axial asymmetry of the electrophoretic velocity model leads to asymmetric peak shapes, a violation of the symmetric Gaussians predicted by the 1D theory. We conclude with some observations on the effect of Peclet number and gradient strength on the performance of focusing experiments, and describe a method for experimental optimization. Such knowledge is useful for design of lab-on-a-chip devices.

DIGE Analysis of Human Tissues.

PubMed

Gelfi, Cecilia; Capitanio, Daniele

2018-01-01

Two-dimensional difference gel electrophoresis (2-D DIGE) is an advanced and elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2-DE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The two-dye or three-dye systems can be adopted and their choice depends on specific applications. Furthermore, the use of an internal pooled standard makes 2-D DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. The image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful.

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components.

PubMed

Xiang, Rong; Horváth, Csaba

2002-02-15

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.

The role of cell size in density gradient electrophoretic separation of mouse leukemia cells according to position in the cell cycle

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

Cultured mouse leukemia cells line L5178Y were subjected to upward electrophoresis in a density gradient and the slower migrating cell populations were enriched in G2 cells. It is indicated that this cell line does not change electrophoretic mobility through the cell cycle. The possibility that increased sedimentation downward on the part of the larger G2 cells caused this separation was explored. Two different cell populations were investigated. The log phase population was found to migrate upward faster than the G2 population, and a similar difference between their velocities and calculated on the basis of a 1 um diameter difference between the two cell populations. The G2 and G1 enriched populations were isolated by Ficoll density gradient sedimentation. The bottom fraction was enriched in G2 cells and the top fraction was enriched with G1 cells, especially when compared with starting materials. The electrophoretic mobilities of these two cell populations did not differ significantly from one another. Cell diameter dependent migration curves were calculated and were found to be different. Families of migration curves that differ when cell size is considered as a parameter are predicted.

A Student-Made Microfluidic Device for Electrophoretic Separation of Food Dyes

ERIC Educational Resources Information Center

Teerasong, Saowapak; McClain, Robert L.

2011-01-01

We have developed an undergraduate laboratory activity to introduce students to microfluidics. In the activity, each student constructs their own microfluidic device using simple photolithographic techniques and then uses the device to separate a food dye mixture by electrophoresis. Dyes are used so that students are able to visually observe the…

Sample detection and analysis techniques for electrophoretic separation

NASA Technical Reports Server (NTRS)

Falb, R. D.; Hughes, K. E.; Powell, T. R.

1975-01-01

Methods for detecting and analyzing biological agents suitable for space flight operations were studied primarily by literature searches which were conducted of cell separation techniques. Detection methods discussed include: photometrometric, electric, radiometric, micrometry, ultrasonic, microscopic, and photographic. A bibliography, and a directory of vendors are included along with an index of commercial hardware.

A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

ERIC Educational Resources Information Center

Chang, Ming-Mei; Lovett, Janice

2011-01-01

Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

Analysis of oligonucleotide photoproducts produced by UV-A light and a riboflavin photosensitizer

NASA Astrophysics Data System (ADS)

Gelhaus, Stacy L.; LaCourse, William R.

2004-12-01

DNA damage is caused by a variety of foreign and endogenous compounds. There are endogenous photosensitizers in cells, such as porphyrins and flavins, which may create damage in the presence of UV-A light. Typically, samples are analyzed by 32P-postlabelling and electrophoretic separation or by LC-MS separation and detection. Separation by HPLC is common; however, in all instances, the DNA sample is hydrolyzed down to nucleosides prior to analysis. It will be shown here that ion-pairing reversed phase high performance liquid chromatography (IP-RPLC) has the ability to provide biophysical information concerning the sites of UV-A induced photosensitizer damage on an intact oligonucleotide concurrent with the separation. IP-RPLC is less labor intensive and faster than electrophoretic methods and it is less costly than LC-MS. IP-RPLC can also be used to purify modified oligonucleotides for further use and analysis. This technique is sensitive to the charge, conformation, and sequence characteristics of the nucleic acid sample and may be used to determine the damage or modifications made to DNA by a variety of compounds.

Characterization of polymerized liposomes using a combination of dc and cyclical electrical field-flow fractionation.

PubMed

Sant, Himanshu J; Chakravarty, Siddharth; Merugu, Srinivas; Ferguson, Colin G; Gale, Bruce K

2012-10-02

Characterization of polymerized liposomes (PolyPIPosomes) was carried out using a combination of normal dc electrical field-flow fractionation and cyclical electrical field-flow fractionation (CyElFFF) as an analytical technique. The constant nature of the carrier fluid and channel configuration for this technique eliminates many variables associated with multidimensional analysis. CyElFFF uses an oscillating field to induce separation and is performed in the same channel as standard dc electrical field-flow fractionation separation. Theory and experimental methods to characterize nanoparticles in terms of their sizes and electrophoretic mobilities are discussed in this paper. Polystyrene nanoparticles are used for system calibration and characterization of the separation performance, whereas polymerized liposomes are used to demonstrate the applicability of the system to biomedical samples. This paper is also the first to report separation and a higher effective field when CyElFFF is operated at very low applied voltages. The technique is shown to have the ability to quantify both particle size and electrophoretic mobility distributions for colloidal polystyrene nanoparticles and PolyPIPosomes.

Fast and simultaneous detection of prominent natural antioxidants using analytical microsystems for capillary electrophoresis with a glassy carbon electrode: a new gateway to food environments.

PubMed

Blasco, Antonio Javier; Barrigas, Inés; González, María Cristina; Escarpa, Alberto

2005-12-01

This paper examines for the first time the analytical possibilities of fast and simultaneous detection of prominent natural antioxidants including examples of flavonoids and vitamins using a CE microchip with electrochemical detection (ED). Unpinched injection conditions, zone electrophoretic separation and amperometric detection were carefully assayed and optimised. Analysis involved the zone electrophoretic separation of arbutin, (+)-catechin and ascorbic acid in less than 4 min using a borate buffer (pH 9.0, 50 mM), employing 2 kV as the separation voltage and +1.0 V as the detection potential. In addition, the separation of different 'couples' of natural antioxidants of food significance including (+)-catechin and ascorbic acid, (+)-catechin and rutin, as well as arbutin and phlorizdin is proposed. To demonstrate the potential and future role of CE microsystems, analytical possibilities and a new route in the raw sample analysis are presented. The preliminary results obtained allow the proposal of CE-ED microchips as a real gateway to microanalysis in foods.

In-gel staining of proteins in native polyacrylamide gel electrophoresis using meso-tetrakis(4-sulfonatophenyl) porphyrin.

PubMed

Divakar, K; Devi, G Nandhini; Gautam, Pennathur

2012-01-01

Protein identification in polyacrylamide gel electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining, and destaining of the gel, which are time-consuming and cumbersome. A new method for direct visualization of protein bands in PAGE has been developed using meso-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) as a dye without the need for any post-electrophoretic steps; thus, separation and recovery of enzymes become much easier for further analysis. Activity staining was carried out to show that the biochemical activity of the enzymes was preserved after electrophoresis.

CZE separation of strawberry anthocyanins with acidic buffer and comparison with HPLC.

PubMed

Comandini, Patrizia; Blanda, Giampaolo; Cardinali, Andrea; Cerretani, Lorenzo; Bendini, Alessandra; Caboni, Maria Fiorenza

2008-10-01

Anthocyanins, the major colourants of strawberries, are polar pigments that are positively charged at low pH. Herein, we have assessed a new analytical method for the separation of anthocyanins using CZE. Acidic buffer solutions (pH <2) were employed in order to maintain pigments in the cation flavylium form and achieve high molar absorptivity at 510 nm. These spectral properties enabled us to identify strawberry anthocyanins in a preliminary stage by detection in the visible range, although the method was optimised at 280 nm to obtain the best S/N. The effects of buffer composition highlighted the necessity of adding an organic modifier to the running buffer to obtain a suitable separation. The electrophoretic method permitted the separation of the three main anthocyanins of strawberry extracts, namely pelargonidin 3-glucoside (Pg-glu), pelargonidin 3-rutinoside and cyanidin 3-glucoside. The electrophoretic results, expressed as retention time and separation efficiency of the major anthocyanin (Pg-glu), were compared to those achieved in HPLC, the analytical technique traditionally used for the investigation of anthocyanins in vegetable matrix. The content of Pg-glu in strawberries (cv. Camarosa), calculated with HPCE and HPLC methods, resulted respectively in 11.41 mg/L and 11.37 mg/L.

Assessing the scalability of dynamic field gradient focusing by linear modeling

PubMed Central

Tracy, Noah I.; Ivory, Cornelius F.

2010-01-01

Dynamic field gradient focusing (DFGF) separates and concentrates proteins in native buffers, where proteins are most soluble, using a computer-controlled electric field gradient which lets the operator adjust the pace and resolution of the separation in real-time. The work in this paper assessed whether DFGF could be scaled up from microgram analytical-scale protein loads to milligram preparative-scale loads. Linear modeling of the electric potential, protein transport, and heat transfer simulated the performance of a preparative-scale DFGF instrument. The electric potential model showed where the electrodes should be placed to optimize the shape and strength of the electric field gradient. Results from the protein transport model suggested that in 10 min the device should separate 10 mg each of two proteins whose electrophoretic mobilities differ by 5 ×. Proteins with electrophoretic mobilities differing by only 5% should separate in 3 h. The heat transfer model showed that the preparative DFGF design could dissipate 1 kW of Joule heat while keeping the separation chamber at 25°C. Model results pointed to DFGF successfully scaling up by 1000 × using the proposed instrument design. PMID:18196522

Chemical vapor deposition of aminopropyl silanes in microfluidic channels for highly efficient microchip capillary electrophoresis-electrospray ionization-mass spectrometry.

PubMed

Batz, Nicholas G; Mellors, J Scott; Alarie, Jean Pierre; Ramsey, J Michael

2014-04-01

We describe a chemical vapor deposition (CVD) method for the surface modification of glass microfluidic devices designed to perform electrophoretic separations of cationic species. The microfluidic channel surfaces were modified using aminopropyl silane reagents. Coating homogeneity was inferred by precise measurement of the separation efficiency and electroosmotic mobility for multiple microfluidic devices. Devices coated with (3-aminopropyl)di-isopropylethoxysilane (APDIPES) yielded near diffusion-limited separations and exhibited little change in electroosmotic mobility between pH 2.8 and pH 7.5. We further evaluated the temporal stability of both APDIPES and (3-aminopropyl)triethoxysilane (APTES) coatings when stored for a total of 1 week under vacuum at 4 °C or filled with pH 2.8 background electrolyte at room temperature. Measurements of electroosmotic flow (EOF) and separation efficiency during this time confirmed that both coatings were stable under both conditions. Microfluidic devices with a 23 cm long, serpentine electrophoretic separation channel and integrated nanoelectrospray ionization emitter were CVD coated with APDIPES and used for capillary electrophoresis (CE)-electrospray ionization (ESI)-mass spectrometry (MS) of peptides and proteins. Peptide separations were fast and highly efficient, yielding theoretical plate counts over 600,000 and a peak capacity of 64 in less than 90 s. Intact protein separations using these devices yielded Gaussian peak profiles with separation efficiencies between 100,000 and 400,000 theoretical plates.

Model of separation performance of bilinear gradients in scanning format counter-flow gradient electrofocusing techniques.

PubMed

Shameli, Seyed Mostafa; Glawdel, Tomasz; Ren, Carolyn L

2015-03-01

Counter-flow gradient electrofocusing allows the simultaneous concentration and separation of analytes by generating a gradient in the total velocity of each analyte that is the sum of its electrophoretic velocity and the bulk counter-flow velocity. In the scanning format, the bulk counter-flow velocity is varying with time so that a number of analytes with large differences in electrophoretic mobility can be sequentially focused and passed by a single detection point. Studies have shown that nonlinear (such as a bilinear) velocity gradients along the separation channel can improve both peak capacity and separation resolution simultaneously, which cannot be realized by using a single linear gradient. Developing an effective separation system based on the scanning counter-flow nonlinear gradient electrofocusing technique usually requires extensive experimental and numerical efforts, which can be reduced significantly with the help of analytical models for design optimization and guiding experimental studies. Therefore, this study focuses on developing an analytical model to evaluate the separation performance of scanning counter-flow bilinear gradient electrofocusing methods. In particular, this model allows a bilinear gradient and a scanning rate to be optimized for the desired separation performance. The results based on this model indicate that any bilinear gradient provides a higher separation resolution (up to 100%) compared to the linear case. This model is validated by numerical studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic studies of polygalacturonate oligomers and their interactions with metal ions.

PubMed

Wiedmer, S K; Cassely, A; Hong, M; Novotny, M V; Riekkola, M L

2000-09-01

Polygalacturonic acid, a linear homopolysaccharide, was investigated by capillary electrophoresis (CE) using linear polyacrylamide-coated capillaries and laser-induced fluorescence (LIF) detection. A successful separation of its fluorescently labeled oligomers was achieved through sieving in polyacrylamide entangled matrices. The reaction conditions for the derivatization of polygalacturonic acid were optimized. In studying the interactions between polygalacturonic acid and various metal ions, the end-label, free-solution electrophoretic (ELFSE) technique, developed earlier in our laboratory (Sudor, J., Novotny, M. V., Anal. Chem. 1995, 67, 4205-4209) was found preferable to the sieving method. ELFSE is fast and convenient in that no polymer solutions are needed for the separation. The investigation showed that for the moderately large oligomers, the strongest binding occurred with calcium and cadmium ions, while the smallest interaction was observed with magnesium ions.

Candidate space processing techniques for biomaterials other than preparative electrophoresis

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1976-01-01

The advantages of performing the partition and countercurrent distribution (CCD) of cells in phase separated aqueous polymer systems under reduced gravity were assessed. Other possible applications considered for the space processing program include the freezing front separation of cells, adsorption of cells at the air-water interface, and the macrophage electrophoretic mobility test for cancer.

Enhanced Microchip Electrophoresis Separations Combined with Electrochemical Detection Utilizing a Capillary Embedded in Polystyrene.

PubMed

Mehl, Benjamin T; Martin, R Scott

2018-01-07

The ability to use microchip-based electrophoresis for fast, high-throughput separations provides researchers with a tool for close-to real time analysis of biological systems. While PDMS-based electrophoresis devices are popular, the separation efficiency is often an issue due to the hydrophobic nature of PDMS. In this study, a hybrid microfluidic capillary device was fabricated to utilize the positive features of PDMS along with the electrophoretic performance of fused silica. A capillary loop was embedded in a polystyrene base that can be coupled with PDMS microchannels at minimal dead volume interconnects. A method for cleaning out the capillaries after a wet-polishing step was devised through the use of 3D printed syringe attachment. By comparing the separation efficiency of fluorescein and CBI-glycine with both a PDMS-based serpentine device and the embedded capillary loop device, it was shown that the embedded capillary loop device maintained higher theoretical plates for both analytes. A Pd decoupler with a carbon or Pt detection electrode were embedded along with the loop allowing integration of the electrophoretic separation with electrochemical detection. A series of catecholamines were separated to show the ability to resolve similar analytes and detect redox active species. The release of dopamine and norepinephrine from PC 12 cells was also analyzed showing the compatibility of these improved microchip separations with high ionic cell buffers associated with cell culture.

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P.

1980-01-01

The following aspects of kidney cell electrophoresis are discussed: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characterization of kidney cells.

Kidney cell electrophoresis

NASA Technical Reports Server (NTRS)

Todd, P.

1979-01-01

A kidney cell electrophoresis technique is described in four parts: (1) the development and testing of electrophoresis solutions; (2) optimization of freezing and thawing; (3) procedures for evaluation of separated kidney cells; and (4) electrophoretic mobility characteristics of kidney cells.

Strategies for the capillary electrophoretic separation of indole alkaloids in Psilocybe semilanceata.

PubMed

Pedersen-Bjergaard, S; Rasmussen, K E; Sannes, E

1998-01-01

While the hallucinogenic mushrooms Psilocybe semilanceata have previously been analyzed for the indole alkaloids psilocybin and baeocystin by capillary zone electrophoresis (CZE) at pH 11.5, the present work focused on the development of an alternative and complementary capillary electrophoretic method for their identification. Owing to their structural similarity and zwitterionic nature, the compounds were difficult to resolve based on different interactions with cationic or anionic micelles. However, while the attempts with micellar electrokinetic chromatography (MEKC) were unsuccessful, rapid derivatization with propyl chloroformate and reanalysis by CZE at pH 11.5 was effective to support identification of the two indole alkaloids. Psilocin was difficult to analyze by CZE at pH 11.5 owing to comigration with the electroosmotic flow. For this compound, the pH of the running buffer was reduced to 7.2 to effectively enhance the electrophoretic mobility.

Methods for separating particles and/or nucleic acids using isotachophoresis

DOEpatents

Jung, Byoungsok; Ness, Kevin; Rose, Klint A.

2016-03-15

According to one embodiment, a method includes co-feeding fluids comprising a leading electrolyte, a trailing electrolyte, and at least one of DNA and RNA to a channel, and applying an electric field to the fluids in a direction perpendicular to an axis of the channel for inducing transverse isotachophoresis. In another embodiment, a method includes co-feeding fluids to a channel. The fluids include a leading electrolyte, a trailing electrolyte, biological objects, at least one of DNA and RNA, and a spacer electrolyte having an electrophoretic mobility that is between an electrophoretic mobility of at least some of the biological objects and an electrophoretic mobility of the at least one of the DNA and the RNA. The method also includes applying an electric field to the fluids in a direction perpendicular to an axis of the channel for inducing transverse isotachophoresis. Other methods of isotachophoresis are disclosed in addition to these.

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles

PubMed Central

Gondikas, Andreas; von der Kammer, Frank; Hofmann, Thilo; Marchetti-Deschmann, Martina; Allmaier, Günter; Marko-Varga, György; Andersson, Roland

2017-01-01

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability. PMID:27639623

Free-zone electrophoresis of animal cells. 1: Experiments on cell-cell interactions

NASA Technical Reports Server (NTRS)

Todd, P. W.; Hjerten, S.

1985-01-01

The electrophoretically migrating zones wasa monitored. The absence of fluid flows in the direction of migration permits direct measurement of electrophoretic velocities of any material. Sedimentation is orthogonal to electrokinetic motion and the effects of particle-particle interaction on electrophoretic mobility is studied by free zone electrophoresis. Fixed erythrocytes at high concentrations, mixtures of fixed erythrocytes from different animal species, and mixtures of cultured human cells were studied in low ionic strength buffers. The electrophoretic velocity of fixed erythrocytes was not altered by increasing cell concentration or by the mixing of erythrocytes from different species. When zones containing cultured human glial cells and neuroblastoma cells are permitted to interact during electrophoresis, altered migration patterns occur. It is found that cell-cell interactions depends upon cell type.

Characterizing the interaction between enantiomers of eight psychoactive drugs and highly sulfated-β-cyclodextrin by counter-current capillary electrophoresis.

PubMed

Asensi-Bernardi, Lucía; Escuder-Gilabert, Laura; Martín-Biosca, Yolanda; Sagrado, Salvador; Medina-Hernández, María José

2014-01-01

The estimation of apparent binding constants and limit mobilities of the complexes of the enantiomers that characterize the interaction of enantiomers with chiral selectors, in this case highly sulfated β-cyclodextrin, was approached using a simple and economic electrophoretic modality, the complete filling technique (CFT) in counter-current mode. The enantiomers of eight psychoactive drugs, four antihistamines (dimethindene, promethazine, orphenadrine and terfenadine) and four antidepressants (bupropion, fluoxetine, nomifensine and viloxazine) were separated for the first time for this cyclodextrin (CD). Estimations of thermodynamic and electrophoretic enantioselectivies were also performed. Results indicate that, in general, thermodynamic enantioselectivity is the main component explaining the high resolution found, but also one case suggests that electrophoretic enantioselectivity itself is enough to obtain a satisfactory resolution. CFT results advantageous compared with conventional capillary electrophoresis (CE) and partial filling technique (PFT) for the study of the interaction between drugs and chiral selectors. It combines the use of a simple fitting model (as in CE), when the enantiomers do not exit the chiral selector plug during the separation (i.e. mobility of electroosmotic flow larger than mobility of CD), and drastic reduction of the consumption (and cost; ~99.7%) of the CD reagent (as in PFT) compared with the conventional CE. Copyright © 2013 John Wiley & Sons, Ltd.

DOE-FG02-00ER62797 Final Report

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

Sweedler, J.V.

2004-12-01

Specific Aims The overall goal of this proposal has been to develop and interface a new technology, molecular gates, with microfabricated systems to add an important capability to microfabricated DNA measurement systems. This project specifically focused on demonstrating how molecular gates could be used to capture a single analyte band, among a stream of bands from a separation or a flow injection analysis experiment, and release it for later measurement, thus allowing further manipulations on the selected analyte. Since the original proposal, the molecular gate concept has been greatly expanded to allow the gates to be used as externally controllablemore » intelligent interconnects in multilayer microfluidic networks. We have demonstrated: (1) the ability of the molecular gates to work with a much wider range of biological molecules including DNA, proteins and small metabolites; and (2) the capability of performing an electrophoretic separation and sequestering individual picoliter volume components (or even classes of components) into separate channels for further analysis. Both capabilities will enable characterization of small mass amounts of complex mixtures of DNA, proteins and even small molecules--allowing them to be further separated and chemically characterized.« less

Electrophoretic mobilities of erythrocytes in various buffers

NASA Technical Reports Server (NTRS)

Plank, L. D.; Kunze, M. E.; Todd, P. W.

1985-01-01

The calibration of space flight equipment depends on a source of standard test particles, this test particle of choice is the fixed erythrocyte. Erythrocytes from different species have different electrophoretic mobilities. Electrophoretic mobility depends upon zeta potential, which, in turn depends upon ionic strength. Zeta potential decreases with increasing ionic strength, so cells have high electrophoretic mobility in space electrophoresis buffers than in typical physiological buffers. The electrophoretic mobilities of fixed human, rat, and rabbit erythrocytes in 0.145 M salt and buffers of varying ionic strength, temperature, and composition, to assess the effects of some of the unique combinations used in space buffers were characterized. Several effects were assessed: glycerol or DMSO (dimethylsulfoxide) were considered for use as cryoprotectants. The effect of these substances on erythrocyte electrophoretic mobility was examined. The choice of buffer depended upon cell mobility. Primary experiments with kidney cells established the choice of buffer and cryoprotectant. A nonstandard temperature of EPM in the suitable buffer was determined. A loss of ionic strength control occurs in the course of preparing columns for flight, the effects of small increases in ionic strength over the expected low values need to be evaluated.

Problems with multiple use of transfer buffer in protein electrophoretic transfer.

PubMed

Dorri, Yaser; Kurien, Biji T; Scofield, R Hal

2010-04-01

Two-dimensional gel electrophoresis (2DE) and SDS-PAGE are the two most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are usually transferred to membranes using a variety of methods, such as electrophoretic transfer, heat-mediated transfer, or nonelectrophoretic transfer, for specific protein detection and/or analysis. In a recent study, Pettegrew et al. claim to reuse transfer buffer containing methanol for at least five times for transferring proteins from SDS-PAGE to polyvinylidene difluoride. They add 150-200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is not accurate and useful for protein analysis, and it can cause background binding as well as inaccurate protein analysis.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy E; Singh, Anup K; Throckmorton, Daniel J

2015-02-24

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Fingerprinting postblast explosive residues by portable capillary electrophoresis with contactless conductivity detection.

PubMed

Kobrin, Eeva-Gerda; Lees, Heidi; Fomitšenko, Maria; Kubáň, Petr; Kaljurand, Mihkel

2014-04-01

A portable capillary electrophoretic system with contactless conductivity detection was used for fingerprint analysis of postblast explosive residues from commercial organic and improvised inorganic explosives on various surfaces (sand, concrete, metal witness plates). Simple extraction methods were developed for each of the surfaces for subsequent simultaneous capillary electrophoretic analysis of anions and cations. Dual-opposite end injection principle was used for fast (<4 min) separation of 10 common anions and cations from postblast residues using an optimized separation electrolyte composed of 20 mM MES, 20 mM l-histidine, 30 μM CTAB and 2 mM 18-crown-6. The concentrations of all ions obtained from the electropherograms were subjected to principal component analysis to classify the tested explosives on all tested surfaces, resulting in distinct cluster formations that could be used to verify (each) type of the explosive. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microchannel gel electrophoretic separation systems and methods for preparing and using

DOEpatents

Herr, Amy; Singh, Anup K; Throckmorton, Daniel J

2013-09-03

A micro-analytical platform for performing electrophoresis-based immunoassays was developed by integrating photopolymerized cross-linked polyacrylamide gels within a microfluidic device. The microfluidic immunoassays are performed by gel electrophoretic separation and quantifying analyte concentration based upon conventional polyacrylamide gel electrophoresis (PAGE). To retain biological activity of proteins and maintain intact immune complexes, native PAGE conditions were employed. Both direct (non-competitive) and competitive immunoassay formats are demonstrated in microchips for detecting toxins and biomarkers (cytokines, c-reactive protein) in bodily fluids (serum, saliva, oral fluids). Further, a description of gradient gels fabrication is included, in an effort to describe methods we have developed for further optimization of on-chip PAGE immunoassays. The described chip-based PAGE immunoassay method enables immunoassays that are fast (minutes) and require very small amounts of sample (less than a few microliters). Use of microfabricated chips as a platform enables integration, parallel assays, automation and development of portable devices.

Polyacrylamide medium for the electrophoretic separation of biomolecules

DOEpatents

Madabhushi, Ramakrishna S.; Gammon, Stuart A.

2003-11-11

A polyacryalmide medium for the electrophoretic separation of biomolecules. The polyacryalmide medium comprises high molecular weight polyacrylamides (PAAm) having a viscosity average molecular weight (M.sub.v) of about 675-725 kDa were synthesized by conventional red-ox polymerization technique. Using this separation medium, capillary electrophoresis of BigDye DNA sequencing standard was performed. A single base resolution of .about.725 bases was achieved in .about.60 minute in a non-covalently coated capillary of 50 .mu.m i.d., 40 cm effective length, and a filed of 160 V/cm at 40.degree. C. The resolution achieved with this formulation to separate DNA under identical conditions is much superior (725 bases vs. 625 bases) and faster (60 min. vs. 75 min.) to the commercially available PAAm, such as supplied by Amersham. The formulation method employed here to synthesize PAAm is straight-forward, simple and does not require cumbersome methods such as emulsion polymerizaiton in order to achieve very high molecular weights. Also, the formulation here does not require separation of PAAm from the reaction mixture prior to reconstituting the polymer to a final concentration. Furthermore, the formulation here is prepared from a single average mol. wt. PAAm as opposed to the mixture of two different average mo. wt. PAAm previously required to achieve high resolution.

Lab-on-fiber electrophoretic trace mixture separating and detecting an optofluidic device based on a microstructured optical fiber.

PubMed

Yang, Xinghua; Guo, Xiaohui; Li, Song; Kong, Depeng; Liu, Zhihai; Yang, Jun; Yuan, Libo

2016-04-15

We report an in-fiber integrated electrophoretic trace mixture separating and detecting an optofluidic optical fiber sensor based on a specially designed optical fiber. In this design, rapid in situ separation and simultaneous detection of mixed analytes can be realized under electro-osmotic flow in the microstructured optical fiber. To visually display the in-fiber separating and detecting process, two common fluorescent indicators are adopted as the optofluidic analytes in the optical fiber. Results show that a trace amount of the mixture (0.15 μL) can be completely separated within 3.5 min under a high voltage of 5 kV. Simultaneously, the distributed information of the separated analytes in the optical fiber can be clearly obtained by scanning along the optical fiber using a 355 nm laser. The emission from the analytes can be efficiently coupled into the inner core and guides to the remote end of the optical fiber. In addition, the thin cladding around the inner core in the optical fiber can prevent the fluorescent cross talk between the analytes in this design. Compared to previous optical fiber optofluidic devices, this device first realizes simultaneously separating treatment and the detection of the mixed samples in an optical fiber. Significantly, such an in-fiber integrated separating and detecting optofluidic device can find wide applications in various analysis fields involves mixed samples, such as biology, chemistry, and environment.

Capillary electrophoresis for aluminum ion speciation: Optimized separation conditions for complex polycation mixtures.

PubMed

Ouadah, Nesrine; Moire, Claudine; Brothier, Fabien; Kuntz, Jean-François; Deschaume, Olivier; Bartic, Carmen; Cottet, Hervé

2018-06-01

Aluminum chlorohydrates (ACH) are used in numerous applications and commercial products on a global scale including water treatment, catalysis or antiperspirants. They are complex mixtures of water soluble aluminum polycations of different degrees of polymerization, that are difficult to separate and quantify due to their susceptibility to depolymerize in solution when placed out of equilibrium, which is inherent to any separation process. We recently achieved the first capillary electrophoresis separation and characterization of ACH oligomers using 4-morpholineethanesulfonic acid (MES) as background electrolyte counter-ion. MES stabilizes the separated ACH oligomers during the electrophoretic process leading to highly repeatable and fast separations. In this work, the separation of ACH oligomers was further studied and perfected by varying the ionic strength, MES concentration and pH of the background electrolyte. Complex electrophoretic behavior is reported for the separation of Al 13 , Al 30 and Na + ions according to these experimental parameters. The transformation of the electropherograms in effective mobility scale and the use of the slope-plot approach are used to better understand the observed changes in selectivity/resolution. Optimal conditions (700 mM MES at 25 mM ionic strength containing 0.1 mM didodecyldimethylammonium bromide for dynamic capillary coating, pH 4.8) obtained for the separation of ACH oligomers are used for the baseline separation of samples difficult to analyze with other methods, including different molecular, aggregated and colloidal forms of aluminum from the Al 13 , Al 30 and Na + mixture, validating the rationale of the approach. Copyright © 2018. Published by Elsevier B.V.

Electrophoretic separator for purifying biologicals

NASA Technical Reports Server (NTRS)

1976-01-01

This technique separates a single narrow zone of sample mixture in an electrolyte medium into many zones containing a single component of the mixture and electrolyte between them. Since the densities of the separated zones generally differ from that of the intervening medium, such systems are gravitationally unstable and stabilization is required. The various techniques for stabilization include using the capillary space provided by thin films, the interstices of solid material such as filter paper and a variety of gel-forming substances.

Simultaneous detection of 19 K-ras mutations by free-solution conjugate electrophoresis of ligase detection reaction products on glass microchips

PubMed Central

Albrecht, Jennifer Coyne; Kotani, Akira; Lin, Jennifer S.; Soper, Steven A.; Barron, Annelise E.

2015-01-01

We demonstrate here the power and flexibility of free-solution conjugate electrophoresis (FSCE) as a method of separating DNA fragments by electrophoresis with no sieving polymer network. Previous work introduced the coupling of FSCE with ligase detection reaction (LDR) to detect point mutations, even at low abundance compared to the wild-type DNA. Here, four large drag-tags are used to achieve free-solution electrophoretic separation of 19 LDR products ranging in size from 42–66 nt that correspond to mutations in the K-ras oncogene. LDR-FSCE enabled electrophoretic resolution of these 19 LDR-FSCE products by CE in 13.5 minutes (E = 310 V/cm) and by microchip electrophoresis in 140 seconds (E = 350 V/cm). The power of FSCE is demonstrated in the unique characteristic of free-solution separations where the separation resolution is constant no matter the electric field strength. By microchip electrophoresis, the electric field was increased to the maximum of the power supply (E = 700 V/cm), and the 19 LDR-FSCE products were separated in < 70 seconds with almost identical resolution to the separation at E = 350 V/cm. These results will aid the goal of screening K-ras mutations on integrated “sample-in/answer-out” devices with amplification, LDR, and detection all on one platform. PMID:23192597

Solvent-mediated nonelectrostatic ion-ion interactions predicting anomalies in electrophoresis.

PubMed

Goswami, Prakash; Dhar, Jayabrata; Ghosh, Uddipta; Chakraborty, Suman

2017-03-01

We study the effects of solvent-mediated nonelectrostatic ion-ion interactions on electrophoretic mobility of a charged spherical particle. To this end, we consider the case of low surface electrostatic potential resulting in the linearization of the governing equations, which enables us to deduce a closed-form analytical solution to the electrophoretic mobility. We subsequently compare our results to the standard model using Henry's approach and report the changes brought about by the nonelectrostatic potential. The classical approach to determine the electrophoretic mobility underpredicts the particle velocity when compared with experiments. We show that this issue can be resolved by taking into account nonelectrostatic interactions. Our analysis further reveals the phenomenon of electrophoretic mobility reversal that has been experimentally observed in numerous previous studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In-channel electrochemical detection in the middle of microchannel under high electric field.

PubMed

Kang, Chung Mu; Joo, Segyeong; Bae, Je Hyun; Kim, Yang-Rae; Kim, Yongseong; Chung, Taek Dong

2012-01-17

We propose a new method for performing in-channel electrochemical detection under a high electric field using a polyelectrolytic gel salt bridge (PGSB) integrated in the middle of the electrophoretic separation channel. The finely tuned placement of a gold working electrode and the PGSB on an equipotential surface in the microchannel provided highly sensitive electrochemical detection without any deterioration in the separation efficiency or interference of the applied electric field. To assess the working principle, the open circuit potentials between gold working electrodes and the reference electrode at varying distances were measured in the microchannel under electrophoretic fields using an electrically isolated potentiostat. In addition, "in-channel" cyclic voltammetry confirmed the feasibility of electrochemical detection under various strengths of electric fields (∼400 V/cm). Effective separation on a microchip equipped with a PGSB under high electric fields was demonstrated for the electrochemical detection of biological compounds such as dopamine and catechol. The proposed "in-channel" electrochemical detection under a high electric field enables wider electrochemical detection applications in microchip electrophoresis.

A new open tubular capillary microextraction and sweeping for the analysis of super low concentration of hydrophobic compounds.

PubMed

Xia, Zhining; Gan, Tingting; Chen, Hua; Lv, Rui; Wei, Weili; Yang, Fengqing

2010-10-01

A sample pre-concentration method based on the in-line coupling of in-tube solid-phase microextraction and electrophoretic sweeping was developed for the analysis of hydrophobic compounds. The sample pre-concentration and electrophoretic separation processes were simply and sequentially carried out with a (35%-phenyl)-methylpolysiloxane-coated capillary. The developed method was validated and applied to enrich and separate several pharmaceuticals including loratadine, indomethacin, ibuprofen and doxazosin. Several parameters of microextration were investigated such as temperature, pH and eluant. And the concentration of microemulsion that influences separation efficiency and microextraction efficiency were also studied. Central composite design was applied for the optimization of sampling flow rate and sampling time that interact in a very complex way with each other. The precision, sensitivity and recovery of the method were investigated. Under the optimal conditions, the maximum enrichment factors for loratadine, indomethacin, ibuprofen and doxazosin in aqueous solutions are 1355, 571, 523 and 318, respectively. In addition, the developed method was applied to determine loratadine in rabbit blood sample.

MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)

EPA Science Inventory

An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

ERIC Educational Resources Information Center

Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

2012-01-01

An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

Electrophoretic sample insertion. [device for uniformly distributing samples in flow path

NASA Technical Reports Server (NTRS)

Mccreight, L. R. (Inventor)

1974-01-01

Two conductive screens located in the flow path of an electrophoresis sample separation apparatus are charged electrically. The sample is introduced between the screens, and the charge is sufficient to disperse and hold the samples across the screens. When the charge is terminated, the samples are uniformly distributed in the flow path. Additionally, a first separation by charged properties has been accomplished.

Continuous cell introduction and rapid dynamic lysis for high-throughput single-cell analysis on microfludic chips with hydrodynamic focusing.

PubMed

Xu, Chun-Xiu; Yin, Xue-Feng

2011-02-04

A chip-based microfluidic system for high-throughput single-cell analysis is described. The system was integrated with continuous introduction of individual cells, rapid dynamic lysis, capillary electrophoretic (CE) separation and laser induced fluorescence (LIF) detection. A cross microfluidic chip with one sheath-flow channel located on each side of the sampling channel was designed. The labeled cells were hydrodynamically focused by sheath-flow streams and sequentially introduced into the cross section of the microchip under hydrostatic pressure generated by adjusting liquid levels in the reservoirs. Combined with the electric field applied on the separation channel, the aligned cells were driven into the separation channel and rapidly lysed within 33ms at the entry of the separation channel by Triton X-100 added in the sheath-flow solution. The maximum rate for introducing individual cells into the separation channel was about 150cells/min. The introduction of sheath-flow streams also significantly reduced the concentration of phosphate-buffered saline (PBS) injected into the separation channel along with single cells, thus reducing Joule heating during electrophoretic separation. The performance of this microfluidic system was evaluated by analysis of reduced glutathione (GSH) and reactive oxygen species (ROS) in single erythrocytes. A throughput of 38cells/min was obtained. The proposed method is simple and robust for high-throughput single-cell analysis, allowing for analysis of cell population with considerable size to generate results with statistical significance. Copyright © 2010 Elsevier B.V. All rights reserved.

Seed protein variations of Salicornia L. and allied taxa in Turkey.

PubMed

Yaprak, A E; Yurdakulol, E

2007-06-01

Electrophoretic seed protein patterns of a number of accessions of Salicornia europaea L. sl., S. prostrata Palas, S. fragilis P.W. Ball and Tutin, Sarcocornia fruticosa (L.) A. J. Scott, Sarcocornia perennis (Miller.) A. J. Scott, Arthrocnemum glaucum (Del.) Ung.-Sternb., Microcnemum coralloides (Loscos and Pardo) subsp. anatolicum Wagenitz and Halocnemum strobilaceum (Pall.) Bieb. were electrophoretically analysed on SDS-PAGE. In total 48 different bands were identified. The obtained data have been treated numerically using the cluster analysis method of unweighted pair group (UPGMA). Finally it was determined that all species separated according to seed protein profiles. And the cladogram obtained studied taxa have been given.

Activation energy associated with the electromigration of oligosaccharides through viscosity modifier and polymeric additive containing background electrolytes.

PubMed

Kerékgyártó, Márta; Járvás, Gábor; Novák, Levente; Guttman, András

2016-02-01

The activation energy related to the electromigration of oligosaccharides can be determined from their measured electrophoretic mobilities at different temperatures. The effects of a viscosity modifier (ethylene glycol) and a polymeric additive (linear polyacrylamide) on the electrophoretic mobility of linear sugar oligomers with α1-4 linked glucose units (maltooligosaccharides) were studied in CE using the activation energy concept. The electrophoretic separations of 8-aminopyrene-1,3,6-trisulfonate-labeled maltooligosaccharides were monitored by LIF detection in the temperature range of 20-50°C, using either 0-60% ethylene glycol (viscosity modifier) or 0-3% linear polyacrylamide (polymeric additive) containing BGEs. Activation energy curves were constructed based on the slopes of the Arrhenius plots. With the use of linear polyacrylamide additive, solute size-dependent activation energy variations were found for the maltooligosaccharides with polymerization degrees below and above maltoheptaose (DP 7), probably due to molecular conformation changes and possible matrix interaction effects. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combining gas-phase electrophoretic mobility molecular analysis (GEMMA), light scattering, field flow fractionation and cryo electron microscopy in a multidimensional approach to characterize liposomal carrier vesicles.

PubMed

Urey, Carlos; Weiss, Victor U; Gondikas, Andreas; von der Kammer, Frank; Hofmann, Thilo; Marchetti-Deschmann, Martina; Allmaier, Günter; Marko-Varga, György; Andersson, Roland

2016-11-20

For drug delivery, characterization of liposomes regarding size, particle number concentrations, occurrence of low-sized liposome artefacts and drug encapsulation are of importance to understand their pharmacodynamic properties. In our study, we aimed to demonstrate the applicability of nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyser (nES GEMMA) as a suitable technique for analyzing these parameters. We measured number-based particle concentrations, identified differences in size between nominally identical liposomal samples, and detected the presence of low-diameter material which yielded bimodal particle size distributions. Subsequently, we compared these findings to dynamic light scattering (DLS) data and results from light scattering experiments coupled to Asymmetric Flow-Field Flow Fractionation (AF4), the latter improving the detectability of smaller particles in polydisperse samples due to a size separation step prior detection. However, the bimodal size distribution could not be detected due to method inherent limitations. In contrast, cryo transmission electron microscopy corroborated nES GEMMA results. Hence, gas-phase electrophoresis proved to be a versatile tool for liposome characterization as it could analyze both vesicle size and size distribution. Finally, a correlation of nES GEMMA results with cell viability experiments was carried out to demonstrate the importance of liposome batch-to-batch control as low-sized sample components possibly impact cell viability. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

High-throughput DNA separation in nanofilter arrays.

PubMed

Choi, Sungup; Kim, Ju Min; Ahn, Kyung Hyun; Lee, Seung Jong

2014-08-01

We numerically investigated the dynamics of short double-stranded DNA molecules moving through a deep-shallow alternating nanofilter, by utilizing Brownian dynamics simulation. We propose a novel mechanism for high-throughput DNA separation with a high electric field, which was originally predicted by Laachi et al. [Phys. Rev. Lett. 2007, 98, 098106]. In this work, we show that DNA molecules deterministically move along different electrophoretic streamlines according to their length, owing to geometric constraint at the exit of the shallow region. Consequently, it is more probable that long DNA molecules pass over a deep well region without significant lateral migration toward the bottom of the deep well, which is in contrast to the long dwelling time for short DNA molecules. We investigated the dynamics of DNA passage through a nanofilter facilitating electrophoretic field kinematics. The statistical distribution of the DNA molecules according to their size clearly corroborates our assumption. On the other hand, it was also found that the tapering angle between the shallow and deep regions significantly affects the DNA separation performance. The current results show that the nonuniform field effect combined with geometric constraint plays a key role in nanofilter-based DNA separation. We expect that our results will be helpful in designing and operating nanofluidics-based DNA separation devices and in understanding the polymer dynamics in confined geometries. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biochemical analysis with microfluidic systems.

PubMed

Bilitewski, Ursula; Genrich, Meike; Kadow, Sabine; Mersal, Gaber

2003-10-01

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Assessment of Carbon- and Metal-Based Nanoparticle DNA Damage with Microfluidic Electrophoretic Separation Technology.

PubMed

Schrand, Amanda M; Powell, Thomas; Robertson, Tiffany; Hussain, Saber M

2015-02-01

In this study, we examined the feasibility of extracting DNA from whole cell lysates exposed to nanoparticles using two different methodologies for evaluation of fragmentation with microfluidic electrophoretic separation. Human lung macrophages were exposed to five different carbon- and metal-based nanoparticles at two different time points (2 h, 24 h) and two different doses (5 µg/ml, 100 µg/ml). The primary difference in the banding patterns after 2 h of nanoparticle exposure is more DNA fragmentation at the higher NP concentration when examining cells exposed to nanoparticles of the same composition. However, higher doses of carbon and silver nanoparticles at both short and long dosing periods can contribute to erroneous or incomplete data with this technique. Also comparing DNA isolation methodologies, we recommend the centrifugation extraction technique, which provides more consistent banding patterns in the control samples compared to the spooling technique. Here we demonstrate that multi-walled carbon nanotubes, 15 nm silver nanoparticles and the positive control cadmium oxide cause similar DNA fragmentation at the short time point of 2 h with the centrifugation extraction technique. Therefore, the results of these studies contribute to elucidating the relationship between nanoparticle physicochemical properties and DNA fragmentation results while providing the pros and cons of altering the DNA isolation methodology. Overall, this technique provides a high throughput way to analyze subcellular alterations in DNA profiles of cells exposed to nanomaterials to aid in understanding the consequences of exposure and mechanistic effects. Future studies in microfluidic electrophoretic separation technologies should be investigated to determine the utility of protein or other assays applicable to cellular systems exposed to nanoparticles.

Free-solution electrophoretic separations of DNA–drag-tag conjugates on glass microchips with no polymer network and no loss of resolution at increased electric field strength

PubMed Central

Albrecht, Jennifer Coyne; Kerby, Matthew B.; Niedringhaus, Thomas P.; Lin, Jennifer S.; Wang, Xiaoxiao; Barron, Annelise E.

2012-01-01

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of Free-Solution Conjugate Electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. Single-stranded DNA separations in “gels” have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE’s ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with pHEA-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags. PMID:21500207

CAPILLAR ELECTROPHORETIC SEPARATION OF TNT AND ITS TRANSFORMATION PRODUCTS. (R825513C006)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

75 FR 22118 - Notice of Invention Available for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-27

... represented by the Department of Commerce. The Department of Commerce's interest in the invention is available... microfluidic apparatus and method for performing electrophoretic separation of compounds. The apparatus... mm. By the foregoing, compounds can be sequentially detected and quantified. Dated: April 21, 2010...

Canine serum protein patterns using high-resolution electrophoresis (HRE).

PubMed

Abate, O; Zanatta, R; Malisano, T; Dotta, U

2000-03-01

Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively. In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information. Copyright 2000 Harcourt Publishers Ltd.

Nitrate and Nitrite Determination in Gunshot Residue Samples by Capillary Electrophoresis in Acidic Run Buffer.

PubMed

Erol, Özge Ö; Erdoğan, Behice Y; Onar, Atiye N

2017-03-01

Simultaneous determination of nitrate and nitrite in gunshot residue has been conducted by capillary electrophoresis using an acidic run buffer (pH 3.5). In previously developed capillary electrophoretic methods, alkaline pH separation buffers were used where nitrite and nitrate possess similar electrophoretic mobility. In this study, the electroosmotic flow has been reversed by using low pH running buffer without any additives. As a result of reversing the electroosmotic flow, very fast analysis has been actualized, well-defined and separated ion peaks emerge in less than 4 min. Besides, the limit of detection was improved by employing large volume sample stacking. Limit of detection values were 6.7 and 4.3 μM for nitrate and nitrite, respectively. In traditional procedure, mechanical agitation is employed for extraction, while in this work the extraction efficiency of ultrasound mixing for 30 min was found sufficient. The proposed method was successfully applied to authentic gunshot residue samples. © 2016 American Academy of Forensic Sciences.

Binary Oscillatory Crossflow Electrophoresis

NASA Technical Reports Server (NTRS)

Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

1996-01-01

We present preliminary results of our implementation of a novel electrophoresis separation technique: Binary Oscillatory Cross flow Electrophoresis (BOCE). The technique utilizes the interaction of two driving forces, an oscillatory electric field and an oscillatory shear flow, to create an active binary filter for the separation of charged species. Analytical and numerical studies have indicated that this technique is capable of separating proteins with electrophoretic mobilities differing by less than 10%. With an experimental device containing a separation chamber 20 cm long, 5 cm wide, and 1 mm thick, an order of magnitude increase in throughput over commercially available electrophoresis devices is theoretically possible.

Genetic differentiation among populations of marine algae

NASA Astrophysics Data System (ADS)

Innes, D. J.

1984-09-01

Most of the information for genetic differentiation among populations of marine algae is from studies on ecotypic variation. Physiological ecotypes have been described for individuals showing different responses to temperature and salinity conditions. Morphological ecotypes have also been found associated with areas differing in wave exposure or different intertidal positions. Little is known on how genetic variation is organized within and between populations of marine algae. The occurrence of ecotypic variation in some species is evidence for genetic differentiation among populations resulting from selection by the local environment. The rate of dispersal and subsequent gene flow will also affect the level of differentiation among populations. In species with low dispersal, differentiation can arise through chance founder events or random genetic drift. The few studies available have shown that species of algae exhibit a range of dispersal capabilities. This information can be useful for predicting the potential level of genetic differentiation among populations of these species. Crossing experiments with several species of algae have shown that populations separated by a considerable distance can be interfertile. In some cases individuals from these populations have been found to be morphologically distinct. Crosses have been used to study the genetic basis of this variation and are evidence for genetic differentiation among the populations sampled. Genetic variation of enzyme proteins detected by electrophoresis provides an additional method for measuring genetic variation within and between populations of marine algae. Electrophoretic methods have previously been used to study systematic problems in algae. However, there have been few attempts to use electrophoretic variation to study the genetic structure of populations of marine algae. This approach is outlined and includes some of the potential problems associated with interpreting electrophoretic data. Studies of electrophoretic variation in natural populations of Enteromorpha linza from Long island Sound are used as an example. This species was found to reproduce only asexually. Despite a dispersing spore stage, genetic differentiation was found on a microgeographic scale and was correlated with differences in the local environment of some of the populations. Similar studies on other species, and especially sexually reproducing species, will add to a growing understanding of the evolutionary genetics of marine algae.

Method and apparatus for reducing sample dispersion in turns and junctions of microchannel systems

DOEpatents

Griffiths, Stewart K.; Nilson, Robert H.

2001-01-01

The performance of microchannel devices is improved by providing turns, wyes, tees, and other junctions that produce little dispersions of a sample as it traverses the turn or junction. The reduced dispersion results from contraction and expansion regions that reduce the cross-sectional area over some portion of the turn or junction. By carefully designing the geometries of these regions, sample dispersion in turns and junctions is reduced to levels comparable to the effects of ordinary diffusion. A numerical algorithm was employed to evolve low-dispersion geometries by computing the electric or pressure field within candidate configurations, sample transport through the turn or junction, and the overall effective dispersion. These devices should greatly increase flexibility in the design of microchannel devices by permitting the use of turns and junctions that do not induce large sample dispersion. In particular, the ability to fold electrophoretic and electrochrornatographic separation columns will allow dramatic improvements in the miniaturization of these devices. The low-lispersion devices are particularly suited to electrochromatographic and electrophoretic separations, as well as pressure-driven chromatographic separation. They are further applicable to microfluidic systems employing either electroosrnotic or pressure-driven flows for sample transport, reaction, mixing, dilution or synthesis.

Electrophoretic analysis of biomarkers using capillary modification with gold nanoparticles embedded in a polycation and boron doped diamond electrode.

PubMed

Zhou, Lin; Glennon, Jeremy D; Luong, John H T

2010-08-15

Field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles (AuNPs) embedded in poly(diallyl dimethylammonium) chloride (PDDA) has been investigated for the electrophoretic separation of indoxyl sulfate, homovanillic acid (HVA), and vanillylmandelic acid (VMA). AuNPs (27 nm) exhibit ionic and hydrophobic interactions, as well as hydrogen bonding with the PDDA network to form a stable layer on the internal wall of the capillary. This approach reverses electro-osmotic flow allowing for fast migration of the analytes while retarding other endogenous compounds including ascorbic acid, uric acid, catecholamines, and indoleamines. Notably, the two closely related biomarkers of clinical significance, HVA and VMA, displayed differential interaction with PDDA-AuNPs which enabled the separation of this pair. The detection limit of the three analytes obtained by using a boron doped diamond electrode was approximately 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfering chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration.

Pencil graphite leads as simple amperometric sensors for microchip electrophoresis.

PubMed

Natiele Tiago da Silva, Eiva; Marques Petroni, Jacqueline; Gabriel Lucca, Bruno; Souza Ferreira, Valdir

2017-11-01

In this work we demonstrate, for the first time, the use of inexpensive commercial pencil graphite leads as simple amperometric sensors for microchip electrophoresis. A PDMS support containing one channel was fabricated through soft lithography and sanded pencil graphite leads were inserted into this channel to be used as working electrodes. The electrochemical and morphological characterization of the sensor was carried out. The graphite electrode was coupled to PDMS microchips in end-channel configuration and electrophoretic experiments were performed using nitrite and ascorbate as probe analytes. The analytes were successfully separated and detected in well-defined peaks with satisfactory resolution using the microfluidic platform proposed. The repeatability of the pencil graphite electrode was satisfactory (RSD values of 1.6% for nitrite and 12.3% for ascorbate, regarding the peak currents) and its lifetime was estimated to be ca. 700 electrophoretic runs over a cost of ca. $ 0.05 per electrode. The limits of detection achieved with this system were 2.8 μM for nitrite and 5.7 μM for ascorbate. For proof of principle, the pencil graphite electrode was employed for the real analysis of well water samples and nitrite was successfully quantified at levels below its maximum contaminant level established in Brazil and US. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

DOEpatents

Karger, Barry L.; Thilly, William G.; Foret, Frantisek; Khrapko, Konstaintin; Koehavong, Phouthone; Cohen, Aharon S.; Giese, Roger W.

1997-01-01

The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability.

Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels

DOEpatents

Mathies, Richard A.; Singhal, Pankaj; Xie, Jin; Glazer, Alexander N.

2002-01-01

This invention relates to a microfabricated capillary electrophoresis chip for detecting multiple redox-active labels simultaneously using a matrix coding scheme and to a method of selectively labeling analytes for simultaneous electrochemical detection of multiple label-analyte conjugates after electrophoretic or chromatographic separation.

Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

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

Vogt, R.G.; Prestwich, G.D.; Riddiford, L.M.

1988-03-15

A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-(/sup 3/H)hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specificmore » fashion with a biologically relevant odorant.« less

Binary Oscillatory Crossflow Electrophoresis

NASA Technical Reports Server (NTRS)

Molloy, Richard F.; Gallagher, Christopher T.; Leighton, David T., Jr.

1997-01-01

Electrophoresis has long been recognized as an effective analytic technique for the separation of proteins and other charged species, however attempts at scaling up to accommodate commercial volumes have met with limited success. In this report we describe a novel electrophoretic separation technique - Binary Oscillatory Crossflow Electrophoresis (BOCE). Numerical simulations indicate that the technique has the potential for preparative scale throughputs with high resolution, while simultaneously avoiding many problems common to conventional electrophoresis. The technique utilizes the interaction of an oscillatory electric field and a transverse oscillatory shear flow to create an active binary filter for the separation of charged protein species. An oscillatory electric field is applied across the narrow gap of a rectangular channel inducing a periodic motion of charged protein species. The amplitude of this motion depends on the dimensionless electrophoretic mobility, alpha = E(sub o)mu/(omega)d, where E(sub o) is the amplitude of the electric field oscillations, mu is the dimensional mobility, omega is the angular frequency of oscillation and d is the channel gap width. An oscillatory shear flow is induced along the length of the channel resulting in the separation of species with different mobilities. We present a model that predicts the oscillatory behavior of charged species and allows estimation of both the magnitude of the induced convective velocity and the effective diffusivity as a function of a in infinitely long channels. Numerical results indicate that in addition to the mobility dependence, the steady state behavior of solute species may be strongly affected by oscillating fluid into and out of the active electric field region at the ends of the cell. The effect is most pronounced using time dependent shear flows of the same frequency (cos((omega)t)) flow mode) as the electric field oscillations. Under such conditions, experiments indicate that solute is drawn into the cell from reservoirs at both ends of the cell leading to a large mass build up. As a consequence, any initially induced mass flux will vanish after short times. This effect was not captured by the infinite channel model and hence numerical and experimental results deviated significantly. The revised model including finite cell lengths and reservoir volumes allowed quantitative predictions of the time history of the concentration profile throughout the system. This latter model accurately describes the fluxes observed for both oscillatory flow modes in experiments using single protein species. Based on the results obtained from research funded under NASA grant NAG-8-1080.S, we conclude that binary separations are not possible using purely oscillatory flow modes because of end effects associated with the cos((omega)t) mode. Our research shows, however, that a combination of cos(2(omega)t) and steady flow should lead to efficient separation free of end effects. This possibility is currently under investigation.

THE EMERGENCE OF ANTIBODIES WITH EITHER IDENTICAL OR UNRELATED INDIVIDUAL ANTIGENIC SPECIFICITY DURING REPEATED IMMUNIZATIONS WITH STREPTOCOCCAL VACCINES

PubMed Central

Eichmann, Klaus; Braun, Dietmar G.; Feizi, Ten; Krause, Richard M.

1970-01-01

Electrophoretically monodisperse antibody components in rabbit antisera to the carbohydrates of the Groups A and C streptococci have been examined for their individual antigenic specificity. In these antibody components which were isolated by preparative electrophoresis, individual antigenic specificity was confined to the specific antibody and was absent in the nonantibody γ-globulin. Radioprecipitation experiments and the use of immune absorbent columns constructed from goat anti-antisera, which had been absorbed with fraction II, revealed that all the specific antibody in an electrophoretically monodisperse component was reactive with the homologous anti-antibody. Antibodies with either identical or distinct individual antigenic specificities may occur in the same rabbit with repeated immunizations. Antibodies with identical antigenic specificity had identical electrophoretic mobility, whereas antibodies with unrelated antigenic specificities had distinct electrophoretic mobilities. In the interval between immunizations, if antibody to the carbohydrate antigen was absent, there was no detectable antibody with individual antigenic specificity. PMID:4192569

Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

DOEpatents

Karger, B.L.; Thilly, W.G.; Foret, F.; Khrapko, K.; Koehavong, P.; Cohen, A.S.; Giese, R.W.

1997-05-27

The disclosure relates to a method for resolving double-stranded DNA species differing by at least one base pair. Each of the species is characterized by an iso-melting domain with a unique melting temperature contiguous with a melting domain of higher thermal stability. 18 figs.

Isoelectric focusing of red blood cells in a density gradient stabilized column

NASA Technical Reports Server (NTRS)

Smolka, A. J. K.; Miller, T. Y.

1980-01-01

The effects of Ficoll and cell application pH on red blood cell electrophoretic mobility and focusing pH were investigated by focusing cells in a density gradient stabilized column. Sample loading, cell dispersion, column conductivity, resolution of separation, and the effect of Ampholines were examined.

An analysis of genetic architecture in populations of Ponderosa Pine

Treesearch

Yan B. Linhart; Jeffry B. Mitton; Kareen B. Sturgeon; Martha L. Davis

1981-01-01

Patterns of genetic variation were studied in three populations of ponderosa pine in Colorado by using electrophoretically variable protein loci. Significant genetic differences were found between separate clusters of trees and between age classes within populations. In addition, data indicate that differential cone production and differential animal damage have...

Ionic liquids in chromatographic and electrophoretic techniques: toward additional improvements in the separation of natural compounds

PubMed Central

Freire, Carmen S. R.; Coutinho, João A. P.; Silvestre, Armando J. D.; Freire, Mara G.

2016-01-01

Due to their unique properties, in recent years, ionic liquids (ILs) have been largely investigated in the field of analytical chemistry. Particularly during the last sixteen years, they have been successfully applied in the chromatographic and electrophoretic analysis of value-added compounds extracted from biomass. Considering the growing interest in the use of ILs in this field, this critical review provides a comprehensive overview on the improvements achieved using ILs as constituents of mobile or stationary phases in analytical techniques, namely in capillary electrophoresis and its different modes, in high performance liquid chromatography, and in gas chromatography, for the separation and analysis of natural compounds. The impact of the IL chemical structure and the influence of secondary parameters, such as the IL concentration, temperature, pH, voltage and analysis time (when applied), are also critically addressed regarding the achieved separation improvements. Major conclusions on the role of ILs in the separation mechanisms and the performance of these techniques in terms of efficiency, resolution and selectivity are provided. Based on a critical analysis of all published results, some target-oriented ILs are suggested. Finally, current drawbacks and future challenges in the field are highlighted. In particular, the design and use of more benign and effective ILs as well as the development of integrated (and thus more sustainable) extraction–separation processes using IL aqueous solutions are suggested within a green chemistry perspective. PMID:27667965

Electrokinetic transport phenomena: Mobility measurement and electrokinetic instability

NASA Astrophysics Data System (ADS)

Oddy, Michael Huson

Miniaturization and integration of traditional bioassay procedures into microfabricated on-chip assay systems, commonly referred to as "Micro Total Analysis" (muTAS) systems, may have a significant impact on the fields of genomics, proteomics, and clinical analysis. These bioanalytical microsystems leverage electroosmosis and electrophoresis for sample transport, mixing, manipulation, and separation. This dissertation addresses the following three topics relevant to such systems: a new diagnostic for measuring the electrophoretic mobility of sub-micron, fluorescently-labeled particles and the electroosmotic mobility of a microchannel; a novel method and device for rapidly stirring micro- and nanoliter volume solutions for microfluidic bioanalytical applications; and a multiple-species electrokinetic instability model. Accurate measurement of the electrophoretic particle mobility and the electroosmotic mobility of microchannel surfaces is crucial to understanding the stability of colloidal suspensions, obtaining particle tracking-based velocimetry measurements of electroosmotic flow fields, and the quantification of electrokinetic bioanalytical device performance. A method for determining these mobilities from alternating and direct current electrokinetic particle tracking measurements is presented. The ability to rapidly mix fluids at low Reynolds numbers is important to the functionality of many bioanalytical, microfluidic devices. We present an electrokinetic process for rapidly stirring microflow streams by initiating an electrokinetic flow instability. The design, fabrication and performance analysis of two micromixing devices capable of rapidly stirring two low Reynolds number fluid streams are presented. Electroosmotic and electrophoretic transport in the presence of conductivity mismatches between reagent streams and the background electrolytes, can lead to an unstable flow field generating significant sample dispersion. In the multiple-species electrokinetic instability model, we consider a high aspect ratio microchannel geometry, a conductivity gradient orthogonal to the applied electric field, and a four-species chemistry model. A linear stability analysis of the depth-averaged governing equations shows unstable eigenmodes for conductivity ratios as close to unity as 1.01. Experiments and full nonlinear simulations of the governing equations were conducted for a conductivity ratio of 1.05. Images of the disturbance dye field from the nonlinear simulations show good qualitative and quantitative agreement with experiment. Species electromigration is shown to a have significant influence on the development of the conductivity field and instability dynamics in multi-ion configurations.

Capillary zone electrophoresis for analysis of phytochelatins and other thiol peptides in complex biological samples derivatized with monobromobimane.

PubMed

Perez-Rama, Mónica; Torres Vaamonde, Enrique; Abalde Alonso, Julio

2005-02-01

A new method to improve the analysis of phytochelatins and their precursors (cysteine, gamma-Glu-Cys, and glutathione) derivatized with monobromobimane (mBrB) in complex biological samples by capillary zone electrophoresis is described. The effects of the background electrolyte pH, concentration, and different organic additives (acetonitrile, methanol, and trifluoroethanol) on the separation were studied to achieve optimum resolution and number of theoretical plates of the analyzed compounds in the electropherograms. Optimum separation of the thiol peptides was obtained with 150 mM phosphate buffer at pH 1.60. Separation efficiency was improved when 2.5% v/v methanol was added to the background electrolyte. The electrophoretic conditions were 13 kV and capillary dimensions with 30 cm length from the inlet to the detector (38 cm total length) and 50 microm inner diameter. The injection was by pressure at 50 mbar for 17 s. Under these conditions, the separation between desglycyl-peptides and phytochelatins was also achieved. We also describe the optimum conditions for the derivatization of biological samples with mBrB to increase electrophoretic sensitivity and number of theoretical plates. The improved method was shown to be simple, reproducible, selective, and accurate in measuring thiol peptides in complex biological samples, the detection limit being 2.5 microM glutathione at a wavelength of 390 nm.

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

Silica-coated titania and zirconia colloids for subsurface transport field experiments

USGS Publications Warehouse

Ryan, Joseph N.; Elimelech, Menachem; Baeseman, Jenny L.; Magelky, Robin D.

2000-01-01

Silica-coated titania (TiO2) and zirconia (ZrO2) colloids were synthesized in two sizes to provide easily traced mineral colloids for subsurface transport experiments. Electrophoretic mobility measurements showed that coating with silica imparted surface properties similar to pure silica to the titania and zirconia colloids. Measurements of steady electrophoretic mobility and size (by dynamic light scattering) over a 90-day period showed that the silica-coated colloids were stable to aggregation and loss of coating. A natural gradient field experiment conducted in an iron oxide-coated sand and gravel aquifer also showed that the surface properties of the silica-coated colloids were similar. Colloid transport was traced at μg L-1 concentrations by inductively coupled plasma-atomic emission spectroscopy measurement of Ti and Zr in acidified samples.

Geographical markers for Saccharomyces cerevisiae strains with similar technological origins domesticated for rice-based ethnic fermented beverages production in North East India.

PubMed

Jeyaram, Kumaraswamy; Tamang, Jyoti Prakash; Capece, Angela; Romano, Patrizia

2011-11-01

Autochthonous strains of Saccharomyces cerevisiae from traditional starters used for the production of rice-based ethnic fermented beverage in North East India were examined for their genetic polymorphism using mitochondrial DNA-RFLP and electrophoretic karyotyping. Mitochondrial DNA-RFLP analysis of S. cerevisiae strains with similar technological origins from hamei starter of Manipur and marcha starter of Sikkim revealed widely separated clusters based on their geographical origin. Electrophoretic karyotyping showed high polymorphism amongst the hamei strains within similar mitochondrial DNA-RFLP cluster and one unique karyotype of marcha strain was widely distributed in the Sikkim-Himalayan region. We conceptualized the possibility of separate domestication events for hamei strains in Manipur (located in the Indo-Burma biodiversity hotspot) and marcha strains in Sikkim (located in Himalayan biodiversity hotspot), as a consequence of less homogeneity in the genomic structure between these two groups, their clear separation being based on geographical origin, but not on technological origin and low strain level diversity within each group. The molecular markers developed based on HinfI-mtDNA-RFLP profile and the chromosomal doublets in chromosome VIII position of Sikkim-Himalayan strains could be effectively used as geographical markers for authenticating the above starter strains and differentiating them from other commercial strains.

New findings on the purification and characterization of an anti-bothropic factor from Didelphis marsupialis (opossum) serum.

PubMed

Perales, J; Muños, R; Graterol, S; Oviedo, O; Moussatché, H

1989-01-01

We have used DEAE-Sephacel and Sephacryl S-200 to separate protein fractions from Didelphis marsupialis serum capable of protecting mice from the lethal effect of Bothrops jararaca venom. The fractions separated were homogeneous by conventional electrophoresis using cellulose acetate and polyacrylamide; however, they were heterogeneous on PAGE-SDS, showing similar electrophoretic patterns with or without mercaptoethanol. The protein bands obtained were glycoproteins with a molecular weight of 42,000 to 58,000 Daltons.

Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

ERIC Educational Resources Information Center

Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

2002-01-01

Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

Chromatographic and electrophoretic methods for Lingzhi pharmacologically active components.

PubMed

Huie, Carmen W; Di, Xin

2004-12-05

Lingzhi is the Chinese name given to the Ganoderma family of mushrooms, which was considered the most valuable medicine in ancient China and was believed to bring longevity, due to its mysterious power of healing the body and calming the mind. Today, Lingzhi is still widely revered as a valuable health supplement and herbal medicine worldwide, as studies (mostly conducted in China, Korea, Japan and the United States) into the medicinal and nutritional values of Lingzhi revealed that it does indeed contain certain bioactive ingredients (such as triterpenes and polysaccharides) that might be beneficial for the prevention and treatment of a variety of ailments, including important diseases such as hypertension, diabetes, hepatitis, cancers, and AIDS. As research into the biological activities of Lingzhi, as well as the quality assurance and quality control of Lingzhi products, require the isolation/purification of active ingredients from Lingzhi, followed by subsequent analytical and/or preparative separations, the present review summarizes the various chromatographic and electrophoretic methods (as well as sample pretreatment methods) typically employed to achieve such extraction/separation procedures.

Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

PubMed

Hall, Gordon H; Glerum, D Moira; Backhouse, Christopher J

2016-02-01

Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analytical study of electrophoretic characterization of kidney cells. [conducted during the Apollo Soyuz Test Project

NASA Technical Reports Server (NTRS)

Knox, R. J.

1978-01-01

Embryonic kidney cells were studied as a follow-up to the MA-011 Electrophoresis Technology Experiment which was conducted during the Apollo Soyuz Test Project (ASTP). The postflight analysis of the performance of the ASTP zone electrophoresis experiment involving embryonic kidney cells is reported. The feasibility of producing standard particles for electrophoresis was also studied. This work was undertaken in response to a need for standardization of methods for producing, calibrating, and storing electrophoretic particle standards which could be employed in performance tests of various types of electrophoresis equipment. Promising procedures were tested for their suitability in the production of standard test particles from red blood cells.

The laboratory technology of discrete molecular separation: the historical development of gel electrophoresis and the material epistemology of biomolecular science, 1945-1970.

PubMed

Chiang, Howard Hsueh-hao

2009-01-01

Preparative and analytical methods developed by separation scientists have played an important role in the history of molecular biology. One such early method is gel electrophoresis, a technique that uses various types of gel as its supporting medium to separate charged molecules based on size and other properties. Historians of science, however, have only recently begun to pay closer attention to this material epistemological dimension of biomolecular science. This paper substantiates the historiographical thread that explores the relationship between modern laboratory practice and the production of scientific knowledge. It traces the historical development of gel electrophoresis from the mid-1940s to the mid-1960s, with careful attention to the interplay between technical developments and disciplinary shifts, especially the rise of molecular biology in this time-frame. Claiming that the early 1950s marked a decisive shift in the evolution of electrophoretic methods from moving boundary to zone electrophoresis, I reconstruct various trajectories in which scientists such as Oliver Smithies sought out the most desirable solid supporting medium for electrophoretic instrumentation. Biomolecular knowledge, I argue, emerged in part from this process of seeking the most appropriate supporting medium that allowed for discrete molecular separation and visualization. The early 1950s, therefore, marked not only an important turning point in the history of separation science, but also a transformative moment in the history of the life sciences as the growth of molecular biology depended in part on the epistemological access to the molecular realm available through these evolving technologies.

Single molecule fluorescence burst detection of DNA fragments separated by capillary electrophoresis

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

Haab, B.B.; Mathies, R.A.

A method has been developed for detecting DNA separated by capillary gel electrophoresis (CGE) using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with the thiazole orange derivative TO6 as they passed through the nearly 2-{mu}m diameter focused laser beam. Amplified photo-electron pulses from the photomultiplier are grouped into bins of 360-450 {mu}s in duration, and the resulting histogram is stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were usedmore » to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. The optimized single molecule counting method was then applied to an electrophoretic separation of M13 DNA and to a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discreet fluorescence bursts were observed at the expected appearance time of each DNA band. The auto-correlation function of these data indicated transit times that were consistent with the observed electrophoretic velocity. These separations were easily detected when only 50-100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of nearly 100 DNA molecules/band or better. 45 refs., 10 figs.« less

Development and validation of an analytical method for the separation and determination of major bioactive curcuminoids in Curcuma longa rhizomes and herbal products using non-aqueous capillary electrophoresis.

PubMed

Anubala, S; Sekar, R; Nagaiah, K

2014-06-01

A simple, fast and efficient non-aqueous capillary electrophoresis method (NACE) was developed for the simultaneous determination of three major bioactive curcuminoids (CMNs) in Curcuma longa rhizomes and its herbal products. Good separation, resolution and reproducibility were achieved with the background electrolyte (BGE) consisting a mixture of 15.0 mM sodium tetraborate and 7.4 mM sodium hydroxide (NaOH) in 2:10:15 (v/v/v) of water, 1-propanol, and methanol. The influences of background electrolyte, sodium hydroxide, water, sodium dodecyl sulfate and hydroxylpropyl-β-cyclodextrin on separations were investigated. The separation was carried out in a fused-silica capillary tube with reverse polarity. Hydrodynamic injection of 25mbar for 12s was used for injecting samples and a voltage of 28 kV was applied for separation. The ultrasonication method was used for the extraction of CMNs from the turmeric herbal products and the extract was filtered and directly injected without any further treatments. The limits of detection and quantification were less than 5.0 and 14.6 µg/ml respectively for all CMNs. The percentage recoveries for CMNs were >97.2% (%RSD, <2.62). The results obtained by the method were compared with existing spectrophotometric and HPLC methods. The related compounds in the extract did not interfere in the determination of CMNs. The proposed NACE method is better than existing chromatographic and electrophoretic methods in terms of simple electrophoretic medium, fast analysis and good resolution. Copyright © 2014 Elsevier B.V. All rights reserved.

A method for UV-bonding in the fabrication of glass electrophoretic microchips.

PubMed

Huang, Z; Sanders, J C; Dunsmor, C; Ahmadzadeh, H; Landers, J P

2001-10-01

This paper presents an approach for the development of methodologies amenable to simple and inexpensive microchip fabrication, potentially applicable to dissimilar materials bonding and chip integration. The method involves a UV-curable glue that can be used for glass microchip fabrication bonding at room temperature. This involves nothing more than fabrication of glue "guide channels" into the microchip architecture that upon exposure to the appropriate UV light source, bonds the etched plate and cover plate together. The microchip performance was verified by capillary zone electrophoresis (CZE) of small fluorescent molecules with no microchannel surface modification carried out, as well as with a DNA fragment separation following surface modification. The performance of these UV-bonded electrophoretic microchips indicates that this method may provide an alternative to high temperature bonding.

Preparation of convection interaction media isobutyl disc monolithic column and its application to purification of secondary alcohol dehydrogenase and alcohol oxidase.

PubMed

Isobe, Kimiyasu; Kawakami, Yoshimitsu

2007-03-09

A convection interaction media (trade name CIM, BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.

Capillary electrophoretic determination of main components of natural dyes with MS detection.

PubMed

Surowiec, Izabella; Pawelec, Katarzyna; Rezeli, Melinda; Kilar, Ferenc; Trojanowicz, Marek

2008-07-01

CE with UV-Vis and MS detections was investigated as a technique for detection of main components of selected natural dyes of plant and insect origin. The BGE giving the best separation of the investigated flavonoids and anthraquinoids, suitable for MS detection consisted of 40 mM ammonium acetate solution of pH 9.5 with 40% ACN. LODs obtained with MS detection were even one order of magnitude lower than the ones obtained with UV-Vis detection. Application of MS detection enabled determination of eleven dye compounds from three different chemical groups in 15 min. and proved to be more satisfactory than diode-array detection in the electrophoretic analysis of main classes of natural dyes both in terms of selectivity and sensitivity of analysis.

Preparation of guinea pig macrophage for electrophoretic experiments in space

NASA Technical Reports Server (NTRS)

1979-01-01

Methods of storage and cultivation of macrophage cells in preparation for space experiments were investigated. Results show that freezing and thawing immediately after extraction did not cause any change in viability or electrophoretic mobility of the cells. A prolonged storage at -80 C did cause cell damage as indicated by a 95% reduction in variable cells. Cell damage was decreased when Glycerol or Dimethyl Sulfoxide (DMSO) was added as a cryogenic protective agent. A 100% viability was observed in cultivation experiments after two weeks due to the additional serum. Results from gamma-glutamyl transpeptidase study showed a zero activity rate. It is suggested that a flat stationary field be used for the collection and use of macrophage. It was found that a 24-hour delay in obtaining macrophage cells helps to maintain a pure culture.

Electrophoretic separation of fish brain esterases

USGS Publications Warehouse

Knowles, Charles O.; Arurkar, Suresh K.; Hogan, James W.

1968-01-01

Fish brains were homogenized in an all-glass Potter-Elvehjem-type tissue grinder in 40% sucrose solution. The homogenate concentration was 10 brains/ml for both the bluegill and channel catfish. The brei was centrifuged at 34,700 g for 30 min at 5 C, and 30 J.lliters of the supernatant were used per column for electrophoresis.

A strategy to modulate the electrophoretic behavior in plastic microchips using sodium polystyrene sulfonate.

PubMed

Guo, Jinxiu; Chen, Yu; Zhao, Lizhi; Sun, Ping; Li, Hongli; Zhou, Lei; Wang, Xiayan; Pu, Qiaosheng

2016-12-16

Plastic microchips have been broadly used as disposable microfluidic devices, but the poorly defined surface properties limit their application. Herein, we proved that an anionic polymer could be used as the background electrolyte (BGE) to provide a strong and stable cathodic electroosmotic flow (EOF) and modulate the electrophoretic behavior for efficient separation in relative thicker microchannels (∼75μm id). A cathodic EOF of ∼3.3×10 -4 cm 2 V -1 s -1 was maintained using sodium polystyrene sulfonate (PSSNa) with a molecular weight of 5×10 5 as the BGE, which ensured fluorescein isothiocyanate labeled biogenic amines (BAs) appeared ahead of other components in the electropherograms obtained with microchips of cyclic olefin copolymer. Four selected BAs appeared within 50s and theoretical plate numbers of 8.0×10 5 /m were achieved. The role of PSSNa was evaluated with streaming potential, dynamic light scattering, contact angle and atomic force microscopy. Its functionalities as surface modifier, viscosity regulator and pseudostationary phase were also confirmed. The proposed electrophoretic method was applied in the fast determination of BAs in fish meat samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Separation of metalloporphyrins from metallation reactions by liquid chromatography and electrophoresis.

PubMed

Duff, G A; Yeager, S A; Singhal, A K; Pestel, B C; Ressner, J M; Foster, N

1987-04-24

The analytical separation of the indium and manganese complexes of three synthetic, meso-substituted, water-soluble porphyrins from their respective free bases in metallation reaction mixtures is described. The ligands tetra-3N-methylpyridyl porphyrin, tetra-4N-methylpyridyl porphyrin and tetra-N,N,N-trimethylanilinium porphyrin are complexed with In (III) and Mn (III) and are separated from residual free base by high-performance liquid chromatography (HPLC) in acidic conditions with gradient elution on ODS bonded stationary phase. Electrophoretic separation is achieved on both cellulose polyacetate strips and polyacrylamide tube gels under basic conditions. Although analytical separations can be achieved by both HPLC and electrophoresis, only HPLC is suitable for the development of preparative scale separations. Column chromatography, ion-pairing and ion-suppression HPLC techniques fail to separate such highly charged and closely related aromatic compounds.

Capillary electrophoresis in two-dimensional separation systems: Techniques and applications.

PubMed

Kohl, Felix J; Sánchez-Hernández, Laura; Neusüß, Christian

2015-01-01

The analysis of complex samples requires powerful separation techniques. Here, 2D chromatographic separation techniques (e.g. LC-LC, GC-GC) are increasingly applied in many fields. Electrophoretic separation techniques show a different selectivity in comparison to LC and GC and very high separation efficiency. Thus, 2D separation systems containing at least one CE-based separation technique are an interesting alternative featuring potentially a high degree of orthogonality. However, the generally small volumes and strong electrical fields in CE require special coupling techniques. These technical developments are reviewed in this work, discussing benefits and drawbacks of offline and online systems. Emphasis is placed on the design of the systems, their coupling, and the detector used. Moreover, the employment of strategies to improve peak capacity, resolution, or sensitivity is highlighted. Various applications of 2D separations with CE are summarized. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fundamentals of electrochemical detection techniques for CE and MCE.

PubMed

Kubán, Pavel; Hauser, Peter C

2009-10-01

The electroanalytical techniques of amperometry, conductometry and potentiometry match well with the instrumental simplicity of CE. Indeed, all three detection approaches have been reported for electrophoretic separations. However, the characteristics of the three methods are quite distinct and these are not related to the optical methods more commonly employed. A detailed discussion of the underlying principles of each is given. The issue of possible effects of the separation voltage on the electrochemical detection techniques is considered in depth, and approaches to the elimination of such interferences are also discussed for each case.

Study for identification of beneficial uses of Space (BUS). Volume 2: Technical report. Book 1: Development and business analysis of space processed isoenzymes

NASA Technical Reports Server (NTRS)

1975-01-01

A separation method to provide reasonable yields of high specificity isoenzymes for the purpose of large scale, early clinical diagnosis of diseases and organic damage such as, myocardial infarction, hepatoma, muscular dystrophy, and infectous disorders is presented. Preliminary development plans are summarized. An analysis of required research and development and production resources is included. The costs of such resources and the potential profitability of a commercial space processing opportunity for electrophoretic separation of high specificity isoenzymes are reviewed.

Heterocoagulation of chalcopyrite and pyrite minerals in flotation separation.

PubMed

Mitchell, Timothy K; Nguyen, Anh V; Evans, Geoffrey M

2005-06-30

Heterocoagulation between various fine mineral particles contained within a mineral suspension with different structural and surface chemistry can interfere with the ability of the flotation processes to selectively separate the minerals involved. This paper examines the interactions between chalcopyrite (a copper mineral) and pyrite (an iron mineral often bearing gold) as they approach each other in suspensions with added chemicals, and relates the results to the experimental data for the flotation recovery and selectivity. The heterocoagulation was experimentally studied using the electrophoretic light scattering (ELS) technique and was modelled by incorporating colloidal forces, including the van der Waals, electrostatic double layer and hydrophobic forces. The ELS results indicated that pyrite has a positive zeta potential (zeta) up to its isoelectric point (IEP) at approximately pH 2.2, while chalcopyrite has a positive zeta up to its IEP at approximately pH 5.5. This produces heterocoagulation of chalcopyrite with pyrite between pH 2.2 and pH 5.5. The heterocoagulation was confirmed by the ELS spectra measured with a ZetaPlus instrument from Brookhaven and by small-scale flotation experiments.

New methodology for capillary electrophoresis with ESI-MS detection: Electrophoretic focusing on inverse electromigration dispersion gradient. High-sensitivity analysis of sulfonamides in waters.

PubMed

Malá, Zdena; Gebauer, Petr; Boček, Petr

2016-09-07

This article describes for the first time the combination of electrophoretic focusing on inverse electromigration dispersion (EMD) gradient, a new separation principle described in 2010, with electrospray-ionization (ESI) mass spectrometric detection. The separation of analytes along the electromigrating EMD profile proceeds so that each analyte is focused and concentrated within the profile at a particular position given by its pKa and ionic mobility. The proposed methodology combines this principle with the transport of the focused zones to the capillary end by superimposed electromigration, electroosmotic flow and ESI suction, and their detection by the MS detector. The designed electrolyte system based on maleic acid and 2,6-lutidine is suitable to create an inverse EMD gradient of required properties and its components are volatile enough to be compatible with the ESI interface. The characteristic properties of the proposed electrolyte system and of the formed inverse gradient are discussed in detail using calculated diagrams and computer simulations. It is shown that the system is surprisingly robust and allows sensitive analyses of trace amounts of weak acids in the pKa range between approx. 6 and 9. As a first practical application of electrophoretic focusing on inverse EMD gradient, the analysis of several sulfonamides in waters is reported. It demonstrates the potential of the developed methodology for fast and high-sensitivity analyses of ionic trace analytes, with reached LODs around 3 × 10(-9) M (0.8 ng mL(-1)) of sulfonamides in spiked drinking water without any sample pretreatment. Copyright © 2016 Elsevier B.V. All rights reserved.

Separation of large DNA molecules by applying pulsed electric field to size exclusion chromatography-based microchip

NASA Astrophysics Data System (ADS)

Azuma, Naoki; Itoh, Shintaro; Fukuzawa, Kenji; Zhang, Hedong

2018-02-01

Through electrophoresis driven by a pulsed electric field, we succeeded in separating large DNA molecules with an electrophoretic microchip based on size exclusion chromatography (SEC), which was proposed in our previous study. The conditions of the pulsed electric field required to achieve the separation were determined by numerical analyses using our originally proposed separation model. From the numerical results, we succeeded in separating large DNA molecules (λ DNA and T4 DNA) within 1600 s, which was approximately half of that achieved under a direct electric field in our previous study. Our SEC-based electrophoresis microchip will be one of the effective tools to meet the growing demand of faster and more convenient separation of large DNA molecules, especially in the field of epidemiological research of infectious diseases.

PDMS free-flow electrophoresis chips with integrated partitioning bars for bubble segregation.

PubMed

Köhler, Stefan; Weilbeer, Claudia; Howitz, Steffen; Becker, Holger; Beushausen, Volker; Belder, Detlev

2011-01-21

In this work, a microfluidic free-flow electrophoresis device with a novel approach for preventing gas bubbles from entering the separation area is presented. This is achieved by integrating partitioning bars to reduce the channel depth between electrode channels and separation chamber in order to obtain electrical contact and simultaneously prevent bubbles from entering the separation area. The three-layer sandwich chip features a reusable carrier plate with integrated ports for fluidic connection combined with a softlithographically cast microfluidic PDMS layer and a sealing glass slide. This design allows for a straightforward and rapid chip prototyping process. The performance of the device is demonstrated by free-flow zone electrophoretic separations of fluorescent dye mixtures as well as by the separation of labeled amines and amino acids with separation voltages up to 297 V.

Research on the preparation, biocompatibility and bioactivity of magnesium matrix hydroxyapatite composite material.

PubMed

Linsheng, Li; Guoxiang, Lin; Lihui, Li

2016-08-12

In this paper, magnesium matrix hydroxyapatite composite material was prepared by electrophoretic deposition method. The optimal process parameters of electrophoretic deposition were HA suspension concentration of 0.02 kg/L, aging time of 10 days and voltage of 60 V. Animal experiment and SBF immersion experiment were used to test the biocompatibility and bioactivity of this material respectively. The SD rats were divided into control group and implant group. The implant surrounding tissue was taken to do tissue biopsy, HE dyed and organizational analysis after a certain amount of time in the SD rat body. The biological composite material was soaked in SBF solution under homeothermic condition. After 40 days, the bioactivity of the biological composite material was evaluated by testing the growth ability of apatite on composite material. The experiment results showed that magnesium matrix hydroxyapatite biological composite material was successfully prepared by electrophoretic deposition method. Tissue hyperplasia, connective tissue and new blood vessels appeared in the implant surrounding soft tissue. No infiltration of inflammatory cells of lymphocytes and megakaryocytes around the implant was found. After soaked in SBF solution, a layer bone-like apatite was found on the surface of magnesium matrix hydroxyapatite biological composite material. The magnesium matrix hydroxyapatite biological composite material could promot calcium deposition and induce bone-like apatite formation with no cytotoxicity and good biocompatibility and bioactivity.

Physiological responses to environmental factors related to space flight

NASA Technical Reports Server (NTRS)

Pace, N.; Grunbaum, B. W.; Kodama, A. M.; Mains, R. C.; Rahlmann, D. F.

1975-01-01

Physiological procedures and instrumentation developed for the measurement of hemodynamic and metabolic parameters during prolonged periods of weightlessness are described along with the physiological response of monkeys to weightlessness. Specific areas examined include: cardiovascular studies; thyroid function; blood oxygen transport; growth and reproduction; excreta analysis for metabolic balance studies; and electrophoretic separation of creatine phosphokinase isoenzymes in human blood.

Functional integration of PCR amplification and capillary eletrophoresis in a microfabricated DNA analysis device

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

Woolley, A.T.; deMello, A.J.; Mathies, R.A.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10{degree}C/s heating, 2.5{degree}C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an `electrophoretic valve` to couple the PCR and CE devices on-chip. To demonstrate the functionality ofmore » this system, a 15 min PCR amplification of a {Beta}-globin target cloned in m13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. 33 refs., 6 figs.« less

Characterization of complexes between phenethylamine enantiomers and β-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

PubMed

Wahl, Joachim; Furuishi, Takayuki; Yonemochi, Etsuo; Meinel, Lorenz; Holzgrabe, Ulrike

2017-04-01

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, methyl-β-cyclodextrin and 6-O-α-maltosyl-β-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Separation of multiphosphorylated peptide isomers by hydrophilic interaction chromatography on an aminopropyl phase.

PubMed

Singer, David; Kuhlmann, Julia; Muschket, Matthias; Hoffmann, Ralf

2010-08-01

The separation of isomeric phosphorylated peptides is challenging and often impossible for multiphosphorylated isomers using chromatographic and capillary electrophoretic methods. In this study we investigated the separation of a set of single-, double-, and triple-phosphorylated peptides (corresponding to the human tau protein) by ion-pair reversed-phase chromatography (IP-RPC) and hydrophilic interaction chromatography (HILIC). In HILIC both hydroxyl and aminopropyl stationary phases were tested with aqueous acetonitrile in order to assess their separation efficiency. The hydroxyl phase separated the phosphopeptides very well from the unphosphorylated analogue, while on the aminopropyl phase even isomeric phosphopeptides attained baseline separation. Thus, up to seven phosphorylated versions of a given tau domain were separated. Furthermore, the low concentration of an acidic ammonium formate buffer allowed an online analysis with electrospray ionization tandem mass spectrometry (ESI-MS/MS) to be conducted, enabling peptide sequencing and identification of phosphorylation sites.

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip

PubMed Central

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis. PMID:24404011

High-throughput particle manipulation by hydrodynamic, electrokinetic, and dielectrophoretic effects in an integrated microfluidic chip.

PubMed

Li, Shunbo; Li, Ming; Bougot-Robin, Kristelle; Cao, Wenbin; Yeung Yeung Chau, Irene; Li, Weihua; Wen, Weijia

2013-01-01

Integrating different steps on a chip for cell manipulations and sample preparation is of foremost importance to fully take advantage of microfluidic possibilities, and therefore make tests faster, cheaper and more accurate. We demonstrated particle manipulation in an integrated microfluidic device by applying hydrodynamic, electroosmotic (EO), electrophoretic (EP), and dielectrophoretic (DEP) forces. The process involves generation of fluid flow by pressure difference, particle trapping by DEP force, and particle redirect by EO and EP forces. Both DC and AC signals were applied, taking advantages of DC EP, EO and AC DEP for on-chip particle manipulation. Since different types of particles respond differently to these signals, variations of DC and AC signals are capable to handle complex and highly variable colloidal and biological samples. The proposed technique can operate in a high-throughput manner with thirteen independent channels in radial directions for enrichment and separation in microfluidic chip. We evaluated our approach by collecting Polystyrene particles, yeast cells, and E. coli bacteria, which respond differently to electric field gradient. Live and dead yeast cells were separated successfully, validating the capability of our device to separate highly similar cells. Our results showed that this technique could achieve fast pre-concentration of colloidal particles and cells and separation of cells depending on their vitality. Hydrodynamic, DC electrophoretic and DC electroosmotic forces were used together instead of syringe pump to achieve sufficient fluid flow and particle mobility for particle trapping and sorting. By eliminating bulky mechanical pumps, this new technique has wide applications for in situ detection and analysis.

Determination of thermodynamic values of acidic dissociation constants and complexation constants of profens and their utilization for optimization of separation conditions by Simul 5 Complex.

PubMed

Riesová, Martina; Svobodová, Jana; Ušelová, Kateřina; Tošner, Zdeněk; Zusková, Iva; Gaš, Bohuslav

2014-10-17

In this paper we determine acid dissociation constants, limiting ionic mobilities, complexation constants with β-cyclodextrin or heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and mobilities of resulting complexes of profens, using capillary zone electrophoresis and affinity capillary electrophoresis. Complexation parameters are determined for both neutral and fully charged forms of profens and further corrected for actual ionic strength and variable viscosity in order to obtain thermodynamic values of complexation constants. The accuracy of obtained complexation parameters is verified by multidimensional nonlinear regression of affinity capillary electrophoretic data, which provides the acid dissociation and complexation parameters within one set of measurements, and by NMR technique. A good agreement among all discussed methods was obtained. Determined complexation parameters were used as input parameters for simulations of electrophoretic separation of profens by Simul 5 Complex. An excellent agreement of experimental and simulated results was achieved in terms of positions, shapes, and amplitudes of analyte peaks, confirming the applicability of Simul 5 Complex to complex systems, and accuracy of obtained physical-chemical constants. Simultaneously, we were able to demonstrate the influence of electromigration dispersion on the separation efficiency, which is not possible using the common theoretical approaches, and predict the electromigration order reversals of profen peaks. We have shown that determined acid dissociation and complexation parameters in combination with tool Simul 5 Complex software can be used for optimization of separation conditions in capillary electrophoresis. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

PubMed Central

Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

2009-01-01

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

Surface Characteristics and Adhesion Behavior of Escherichia coli O157:H7: Role of Extracellular Macromolecules

USDA-ARS?s Scientific Manuscript database

Surface macromolecule cleavage experiments were conducted on enterohaemorrhagic Escherichia coli O157:H7 cells to investigate the influence of these macromolecules on cell surface properties. Electrophoretic mobility, hydrophobicity, and titration experiments were carried out on proteinase K treate...

Fluid mechanics of electroosmotic flow and its effect on band broadening in capillary electrophoresis.

PubMed

Ghosal, Sandip

2004-01-01

Electroosmotic flow (EOF) usually accompanies electrophoretic migration of charged species in capillary electrophoresis unless special precautions are taken to suppress it. The presence of the EOF provides certain advantages in separations. It is an alternative to mechanical pumps, which are inefficient and difficult to build at small scales, for transporting reagents and analytes on microfluidic chips. The downside is that any imperfection that distorts the EOF profile reduces the separation efficiency. In this paper, the basic facts about EOF are reviewed from the perspective of fluid mechanics and its effect on separations in free solution capillary zone electrophoresis is discussed in the light of recent advances.

Molecular separations using nanostructured porous thin films fabricated by glancing angle deposition

NASA Astrophysics Data System (ADS)

Bezuidenhout, Louis Wentzel

Biomolecular separation techniques are an enabling technology that indirectly in.uence many aspects of our lives. Advances have led to faster analyses, reduced costs, higher specificity, and new analytical techniques, impacting areas such as health care, environmental monitoring, polymer sciences, agriculture, and nutrition. Further development of separations technology is anticipated to follow the path of computing technology such that miniaturization through the development of microfluidics technology, lab-on-a-chip systems, and other integrative, multi-component systems will further extend our analysis capabilities. Creation of new and improvement of existing separation technologies is an integral part of the pathway to miniaturized systems. the work of this thesis investigates molecular separations using porous nanostructured films fabricated by the thin film process glancing angle deposition (GLAD). Structural architecture, pore size and shape, and film density can be finely controlled to produce high-surface area thin films with engineered morphology. The characteristic size scales and structural control of GLAD films are well-suited to biomolecules and separation techniques, motivating investigation into the utility and performance of GLAD films for biomolecular separations. This project consisted of three phases. First, chromatographic separation of dye molecules on silica GLAD films was demonstrated by thin layer chromatography Direct control of film nanostructure altered the separation characteristics; most strikingly, anisotropic structures provided two-dimensional analyte migration. Second, nanostructures made with GLAD were integrated in PDMS microfluidic channels using a sacrificial etching process; DNA molecules (10/48 kbp and 6/10/20 kbp mixtures) were electrophoretically separated on a microfluidic chip using a porous bed of SiO2 vertical posts. Third, mass spectrometry of proteins and drugs in the mass range of 100-1300 m/z was performed using laser desorption/ionization (LDI) on silicon GLAD films, and the influence of film thickness, porosity, structure, and substrate on performance was characterized. The application of GLAD nanostructured thin films to biomolecular separations is demonstrated and validated in this thesis. Chromatographic separation of dye molecules, electrophoretic separation of DNA molecules, and mass spectrometric isolation of small proteins and drug molecules by laser desorption ionization were demonstrated using GLAD films. All three methods yielded promising results and establish GLAD as a potential technology for biomolecular separations.

Population genetic analysis of oral treponemes by multilocus enzyme electrophoresis.

PubMed

Dahle, U R; Olsen, I; Tronstad, L; Caugant, D A

1995-10-01

Seventeen treponemes recently isolated from necrotic pulps, periodontal and periapical infections and 17 previously well characterized oral treponemal strains were analyzed by multilocus enzyme electrophoresis. Ten genetic loci were characterized on the basis of the electrophoretic mobilities of their enzymatic products. All loci were polymorphic. The average number of alleles per locus was 7.8. The genetic diversity among the electrophoretic types at each locus ranged from 0.624 to 0.836 with a mean genetic diversity per locus of 0.751. The 34 strains represented 34 electrophoretic types, constituting 6 main divisions (I-VI) separated at genetic distances greater than 0.75. Several of the previously characterized treponemes revealed multiple bands of enzyme activity at several loci, indicating that they were not pure. The characterized strains usually clustered within established species, whereas fresh clinical isolates overlapped species borders. There was a large genetic difference between some reference and clinical strains, indicating that the latter may contain undescribed species. Treponema socranskii and Treponema denticola strains clustered in distinct divisions (IV and V, respectively), with the exception of T. denticola strain FDC 51B2 and T. socranskii subsp. paredis strain VPI D46CPE1, both previously well described. This indicated that the taxonomic assignment of these 2 strains should be reconsidered.

Analysis of electrophoresis performance

NASA Technical Reports Server (NTRS)

Roberts, Glyn O.

1988-01-01

A flexible efficient computer code is being developed to simulate electrophoretic separation phenomena, in either a cylindrical or a rectangular geometry. The code will computer the evolution in time of the concentrations of an arbitrary number of chemical species, and of the temperature, pH distribution, conductivity, electric field, and fluid motion. Use of nonuniform meshes and fast accurate implicit time-stepping will yield accurate answers at economical cost.

Nanostructured Diamond Device for Biomedical Applications.

PubMed

Fijalkowski, M; Karczemska, A; Lysko, J M; Zybala, R; KozaneckI, M; Filipczak, P; Ralchenko, V; Walock, M; Stanishevsky, A; Mitura, S

2015-02-01

Diamond is increasingly used in biomedical applications because of its unique properties such as the highest thermal conductivity, good optical properties, high electrical breakdown voltage as well as excellent biocompatibility and chemical resistance. Diamond has also been introduced as an excellent substrate to make the functional microchip structures for electrophoresis, which is the most popular separation technique for the determination of analytes. In this investigation, a diamond electrophoretic chip was manufactured by a replica method using a silicon mold. A polycrystalline 300 micron-thick diamond layer was grown by the microwave plasma-assisted CVD (MPCVD) technique onto a patterned silicon substrate followed by the removal of the substrate. The geometry of microstructure, chemical composition, thermal and optical properties of the resulting free-standing diamond electrophoretic microchip structure were examined by CLSM, SFE, UV-Vis, Raman, XRD and X-ray Photoelectron Spectroscopy, and by a modified laser flash method for thermal property measurements.

Continuous electrophoretic purification of individual analytes from multicomponent mixtures.

PubMed

McLaren, David G; Chen, David D Y

2004-04-15

Individual analytes can be isolated from multicomponent mixtures and collected in the outlet vial by carrying out electrophoretic purification through a capillary column. Desired analytes are allowed to migrate continuously through the column under the electric field while undesired analytes are confined to the inlet vial by application of a hydrodynamic counter pressure. Using pressure ramping and buffer replenishment techniques, 18% of the total amount present in a bulk sample can be purified when the resolution to the adjacent peak is approximately 3. With a higher resolution, the yield could be further improved. Additionally, by periodically introducing fresh buffer into the sample, changes in pH and conductivity can be mediated, allowing higher purity (>or=99.5%) to be preserved in the collected fractions. With an additional reversed cycle of flow counterbalanced capillary electrophoresis, any individual component in a sample mixture can be purified providing it can be separated in an electrophoresis system.

Morphology of human embryonic kidney cells in culture after space flight

NASA Technical Reports Server (NTRS)

Todd, P.; Kunze, M. E.; Williams, K.; Morrison, D. R.; Lewis, M. L.; Barlow, G. H.

1985-01-01

The ability of human embyronic kidney cells to differentiate into small epithelioid, large epithelioid, domed, and fenestrated morphological cell types following space flight is examined. Kidney cells exposed to 1 day at 1 g, then 1 day in orbit, and a 12 minute passage through the electrophoretic separator are compared with control cultures. The data reveal that 70 percent of small epithelioid, 16 percent of large epithelioid, 9 percent of dome-forming, and 5 percent of fenestrated cells formed in the space exposed cells; the distributions correlate well with control data. The formation of domed cells from cells cultured from low electrophoretic mobility fractions and small epithelioid cells from high mobility fractions is unaffected by space flight conditions. It is concluded that storage under microgravity conditions does not influence the morphological differentiation of human embryonic kidney cells in low-passage culture.

Formation of diamond nanoparticle thin films by electrophoretic deposition

NASA Astrophysics Data System (ADS)

Goto, Yosuke; Ohishi, Fujio; Tanaka, Kuniaki; Usui, Hiroaki

2016-03-01

Thin films of diamond nanoparticles were prepared by electrophoretic deposition (EPD) using 0.5 wt % dispersions in water, ethanol, and 2-propanol. The film growth rate increased with increasing voltage applied to the electrodes. However, an excessive increase in voltage caused the degradation of film morphology. The optimum voltage was 4 V with an electrode separation of 5 mm. The film growth rate was higher in organic solvents than in water. The deposited film had a smooth surface with an average surface roughness comparable to the size of primary particles of the source material. It is notable that the EPD films had a considerably higher physical stability than spin-coated and cast films. The stability was further improved by thermally annealing the films. IR analysis revealed that the diamond nanoparticles have carboxy and amino groups on their surfaces. It is considered that the stability of the EPD films originate from a chemical reaction between these functional groups.

A stable and convenient protein electrophoresis titration device with bubble removing system.

PubMed

Zhang, Qiang; Fan, Liu-Yin; Li, Wen-Lin; Cong, Feng-Song; Zhong, Ran; Chen, Jing-Jing; He, Yu-Chen; Xiao, Hua; Cao, Cheng-Xi

2017-07-01

Moving reaction boundary titration (MRBT) has a potential application to immunoassay and protein content analysis with high selectivity. However, air bubbles often impair the accuracy of MRBT, and the leakage of electrolyte greatly decreases the safety and convenience of electrophoretic titration. Addressing these two issues a reliable MRBT device with modified electrolyte chamber of protein titration was designed. Multiphysics computer simulation was conducted for optimization according to two-phase flow. The single chamber was made of two perpendicular cylinders with different diameters. After placing electrophoretic tube, the resident air in the junction next to the gel could be eliminated by a simple fast electrolyte flow. Removing the electrophoretic tube automatically prevented electrolyte leakage at the junction due to the gravity-induced negative pressure within the chamber. Moreover, the numerical simulation and experiments showed that the improved MRBT device has following advantages: (i) easy and rapid setup of electrophoretic tube within 20 s; (ii) simple and quick bubble dissipates from the chamber of titration within 2 s; (iii) no electrolyte leakage from the two chambers: and (iv) accurate protein titration and safe instrumental operation. The developed technique and apparatus greatly improves the performance of the previous MRBT device, and providing a new route toward practical application. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

PubMed Central

Belzunces, L P; Toutant, J P; Bounias, M

1988-01-01

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion. Images Fig. 3. Fig. 6. PMID:2849414

nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins - Separation, Detection, and Sampling of Noncovalent Biospecific Complexes

NASA Astrophysics Data System (ADS)

Engel, Nicole Y.; Weiss, Victor U.; Marchetti-Deschmann, Martina; Allmaier, Günter

2017-01-01

In order to better understand biological events, lectin-glycoprotein interactions are of interest. The possibility to gather more information than the mere positive or negative response for interactions brought mass spectrometry into the center of many research fields. The presented work shows the potential of a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (nES GEMMA) to detect weak, noncovalent, biospecific interactions besides still unbound glycoproteins and unreacted lectins without prior liquid phase separation. First results for Sambucus nigra agglutinin, concanavalin A, and wheat germ agglutinin and their retained noncovalent interactions with glycoproteins in the gas phase are presented. Electrophoretic mobility diameters (EMDs) were obtained by nES GEMMA for all interaction partners correlating very well with molecular masses determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the individual molecules. Moreover, EMDs measured for the lectin-glycoprotein complexes were in good accordance with theoretically calculated mass values. Special focus was laid on complex formation for different lectin concentrations and binding specificities to evaluate the method with respect to results obtained in the liquid phase. The latter was addressed by capillary electrophoresis on-a-chip (CE-on-a-chip). Of exceptional interest was the fact that the formed complexes could be sampled according to their size onto nitrocellulose membranes after gas-phase separation. Subsequent immunological investigation further proved that the collected complex actually retained its native structure throughout nES GEMMA analysis and sampling.

The influence of addition of ion-pairing acid and organic modifier of the mobile phase on retention and migration of peptides in pressurized planar electrochromatography system with octadecyl silica-based adsorbent.

PubMed

Gwarda, Radosław Ł; Dzido, Tadeusz H

2018-07-13

In our previous papers we have investigated the influence of the mobile phase composition on mechanism of retention, selectivity and efficiency of peptide separation in various high-performance thin-layer chromatography (HPTLC) systems with commercially available silica-based adsorbents. We have also investigated the influence of pH of the mobile phase buffer on migration and separation of peptides in pressurized planar electrochromatography (PPEC). Here we investigate the influence of concentration of ion-pairing additive, and concentration and type of organic modifier of the mobile phase on migration of peptides in PPEC system with octadecyl silica-based adsorbent, and with the same set of the solutes as before. We compare our current results with the results obtained before for similar HPTLC and PPEC systems, and discuss the influence of particular variables on retention, electrophoretic mobility of solutes and electroosmotic flow of the mobile phase. We show, that the final selectivity of peptide separation results from co-influence of all the three factors mentioned. Concentration of organic modifier of the mobile phase, as well as concentration of ion-pairing additive, affect the retention, the electrophoretic mobility, and the electroosmotic flow simultaneously. This makes independent optimization of these factors rather difficult. Anyway PPEC offers much faster separation of peptides with quite different selectivity, in comparison to HPTLC, with similar adsorbents and similar mobile phase composition. However, we also present and discuss the issue of extensive tailing of peptide zones in the PPEC in comparison to similar HPTLC systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Electro-Optical Platform for the Manipulation of Live Cells

DTIC Science & Technology

2002-10-02

system, other physical forces may play a significant role. In particular, electroosmotic forces that cause fluid movement relative to a surface can...occur due to the mobility of ions in solution. Electroosmotic forces are commonly utilized in capillary electrophoretic separa- tion, where the capillary...fluid motion that acts to entrain particles to be separated.46 Thus, in the chamber presented here, the patterned anode can induce electroosmotic flow

Low-pressure, high-temperature thermal bonding of polymeric microfluidic devices and their applications for electrophoretic separation

NASA Astrophysics Data System (ADS)

Sun, Yi; Chian Kwok, Yien; Nguyen, Nam-Trung

2006-08-01

A new method for thermally bonding poly(methyl methacrylate) (PMMA) substrates has been demonstrated. PMMA substrates are first engraved by CO2-laser micromachining to form microchannels. Both channel width and depth can be adjusted by varying the laser power and scanning speed. Channel depths from 50 µm to 1500 µm and widths from 150 µm to 400 µm are attained. CO2 laser is also used for drilling and dicing of the PMMA parts. Considering the thermal properties of PMMA, a novel thermal bonding process with high temperature and low bonding pressure has been developed for assembling PMMA sheets. A high bonding strength of 2.15 MPa is achieved. Subsequent inspection of the cross sections of several microdevices reveals that the dimensions of the channels are well preserved during the bonding process. Electroosmotic mobility of the ablated channel is measured to be 2.47 × 10-4 cm2 V-1 s-1. The functionality of these thermally bonded microfluidic substrates is demonstrated by performing rapid and high-resolution electrophoretic separations of mixture of fluorescein and carboxyfluorescein as well as double-stranded DNA ladders (ΦX174-Hae III dsDNA digest). The performance of the CO2 laser ablated and thermally bonded PMMA devices compares favorably with those fabricated by other professional means.

Comparative Skeletal Muscle Proteomics Using Two-Dimensional Gel Electrophoresis

PubMed Central

Murphy, Sandra; Dowling, Paul; Ohlendieck, Kay

2016-01-01

The pioneering work by Patrick H. O’Farrell established two-dimensional gel electrophoresis as one of the most important high-resolution protein separation techniques of modern biochemistry (Journal of Biological Chemistry 1975, 250, 4007–4021). The application of two-dimensional gel electrophoresis has played a key role in the systematic identification and detailed characterization of the protein constituents of skeletal muscles. Protein changes during myogenesis, muscle maturation, fibre type specification, physiological muscle adaptations and natural muscle aging were studied in depth by the original O’Farrell method or slightly modified gel electrophoretic techniques. Over the last 40 years, the combined usage of isoelectric focusing in the first dimension and sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in the second dimension has been successfully employed in several hundred published studies on gel-based skeletal muscle biochemistry. This review focuses on normal and physiologically challenged skeletal muscle tissues and outlines key findings from mass spectrometry-based muscle proteomics, which was instrumental in the identification of several thousand individual protein isoforms following gel electrophoretic separation. These muscle-associated protein species belong to the diverse group of regulatory and contractile proteins of the acto-myosin apparatus that forms the sarcomere, cytoskeletal proteins, metabolic enzymes and transporters, signaling proteins, ion-handling proteins, molecular chaperones and extracellular matrix proteins. PMID:28248237

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

PubMed

Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

2016-09-01

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

Double-helix micro-channels on microfluidic chips for enhanced continuous on-chip derivatization followed by electrophoretic separation.

PubMed

Peng, Xianglu; Zhao, Lei; Guo, Jinxiu; Yang, Shenghong; Ding, Hui; Wang, Xiayan; Pu, Qiaosheng

2015-10-15

Micro-channels that contain a special inner structure are critical for efficient mixing and chemical reactions. In this paper, we described the facile fabrication of an integrated microchip with double-helix type micro-channels to improve mixing efficiency and to facilitate multi-step derivatization reactions prior to electrophoretic separation. With a prepared microchip, reagents, samples and reaction products could be driven through micro-channels by siphon, and no other pumping device was necessary. To test its performance, reductive amination of aldehydes with 8-aminonaphthalene-1,3,6-trisulfonate acid disodium (ANTS) was attempted via microchip electrophoresis with laser induced fluorescence (LIF). The effect of the geometry of the reaction micro-channel on the reaction's efficiency was evaluated. Under the selected conditions, successful derivatization of five aldehydes was realized for highly reproducible analysis. The relative standard deviations of the peak areas for 30 consecutive injections were in the range of 0.28-1.61%. The method was applied for the determination of aldehydes in real samples with standard addition recoveries of 87.8-102.8%. Good tolerance of organic solvents was achieved, and the proposed method can potentially be employed for rapid screening of excessively added aldehyde food flavoring. Copyright © 2015 Elsevier B.V. All rights reserved.

Simultaneous separation of water- and fat-soluble vitamins in isocratic pressure-assisted capillary electrochromatography using a methacrylate-based monolithic column.

PubMed

Yamada, Hiroki; Kitagawa, Shinya; Ohtani, Hajime

2013-06-01

A method of simultaneous separation of water- and fat-soluble vitamins using pressure-assisted CEC with a methacrylate-based capillary monolithic column was developed. In the proposed method, water-soluble vitamins were mainly separated electrophoretically, while fat soluble-ones were separated chromatographically by the interaction with a methacrylate-based monolith. A mixture of six water-soluble and four fat-soluble vitamins was separated simultaneously within 20 min with an isocratic elution using 1 M formic acid (pH 1.9)/acetonitrile (30:70, v/v) containing 10 mM ammonium formate as a mobile phase. When the method was applied to a commercial multivitamin tablet and a spiked one, the vitamins were successfully analyzed, and no influence of the matrix contained in the tablet was observed. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Non-aqueous capillary electrophoretic separation of cholesterol and 25-hydroxycholesterol after derivatization with Girard P reagent.

PubMed

Gregus, Michal; Roberg-Larsen, Hanne; Lundanes, Elsa; Foret, Frantisek; Kuban, Petr; Wilson, Steven Ray

2017-10-01

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure. Copyright © 2017 Elsevier B.V. All rights reserved.

21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Electrophoretic hemoglobin analysis system. 864.7440 Section 864.7440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin...

21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Electrophoretic hemoglobin analysis system. 864.7440 Section 864.7440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin...

21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Electrophoretic hemoglobin analysis system. 864.7440 Section 864.7440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin...

21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Electrophoretic hemoglobin analysis system. 864.7440 Section 864.7440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin...

21 CFR 864.7440 - Electrophoretic hemoglobin analysis system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Electrophoretic hemoglobin analysis system. 864.7440 Section 864.7440 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN....7440 Electrophoretic hemoglobin analysis system. (a) Identification. An electrophoretic hemoglobin...

Antibody enhancement of free-flow electrophoresis

NASA Technical Reports Server (NTRS)

Cohly, H. H. P.; Morrison, Dennis R.; Atassi, M. Zouhair

1988-01-01

Specific T cell clones and antibodies (ABs) were developed to study the efficiency of purifying closely associated T cells using Continuous Flow Electrophoresis System. Enhanced separation is accomplished by tagging cells first with ABs directed against the antigenic determinants on the cell surface and then with ABs against the Fc portion of the first AB. This second AB protrudes sufficiently beyond the cell membrane and glycocalyx to become the major overall cell surface potential determinant and thus causes a reduction of electrophoretic mobility. This project was divided into three phases. Phase one included development of specific T cell clones and separation of these specific clones. Phase two extends these principles to the separation of T cells from spleen cells and immunized lymph node cells. Phase three applies this double antibody technique to the separation of T cytotoxic cells from bone marrow.

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

PubMed Central

Gray, A J; Beecher, D E; Olson, M V

1984-01-01

A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

Capillary Electrophoresis Sensitivity Enhancement Based on Adaptive Moving Average Method.

PubMed

Drevinskas, Tomas; Telksnys, Laimutis; Maruška, Audrius; Gorbatsova, Jelena; Kaljurand, Mihkel

2018-06-05

In the present work, we demonstrate a novel approach to improve the sensitivity of the "out of lab" portable capillary electrophoretic measurements. Nowadays, many signal enhancement methods are (i) underused (nonoptimal), (ii) overused (distorts the data), or (iii) inapplicable in field-portable instrumentation because of a lack of computational power. The described innovative migration velocity-adaptive moving average method uses an optimal averaging window size and can be easily implemented with a microcontroller. The contactless conductivity detection was used as a model for the development of a signal processing method and the demonstration of its impact on the sensitivity. The frequency characteristics of the recorded electropherograms and peaks were clarified. Higher electrophoretic mobility analytes exhibit higher-frequency peaks, whereas lower electrophoretic mobility analytes exhibit lower-frequency peaks. On the basis of the obtained data, a migration velocity-adaptive moving average algorithm was created, adapted, and programmed into capillary electrophoresis data-processing software. Employing the developed algorithm, each data point is processed depending on a certain migration time of the analyte. Because of the implemented migration velocity-adaptive moving average method, the signal-to-noise ratio improved up to 11 times for sampling frequency of 4.6 Hz and up to 22 times for sampling frequency of 25 Hz. This paper could potentially be used as a methodological guideline for the development of new smoothing algorithms that require adaptive conditions in capillary electrophoresis and other separation methods.

Capillary Electrophoresis Chips for Fingerprinting Endotoxin Chemotypes and Subclasses.

PubMed

Kocsis, Béla; Makszin, Lilla; Kilár, Anikó; Péterfi, Zoltán; Kilár, Ferenc

2017-01-01

Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among the LPS from different bacterial serotypes and their mutants include variations mainly within the composition and length or missing of their O-polysaccharide chains. Microchip electrophoretic methodology enables the structural characterization of LPS molecules from several bacteria and the quantitative evaluation of components of endotoxin extracts. The improved microchip electrophoretic method is based on the direct labeling of endotoxins by covalent binding of a fluorescent dye. The classification of the S-type LPSs can be done according to their electrophoretic profiles, which are characteristics of the respective bacterial strains. According to the number, distribution, and the relative amounts of components in an endotoxin extract, it is possible to differentiate between the S-type endotoxins from different Gram-negative bacterial strains. The microchip electrophoresis affords high-resolution separation of pure and partially purified (e.g., obtained from whole-cell lysate) S and R endotoxins. This microchip technique provides a new, standardizable, fast, and sensitive method for the detection of endotoxins and for the quantitative evaluation of components of an endotoxin extract.

Methods, microfluidic devices, and systems for detection of an active enzymatic agent

DOEpatents

Sommer, Gregory J; Hatch, Anson V; Singh, Anup K; Wang, Ying-Chih

2014-10-28

Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

Fluid mechanics of continuous flow electrophoresis

NASA Technical Reports Server (NTRS)

Saville, D. A.; Ostrach, S.

1978-01-01

The following aspects of continuous flow electrophoresis were studied: (1) flow and temperature fields; (2) hydrodynamic stability; (3) separation efficiency, and (4) characteristics of wide gap chambers (the SPAR apparatus). Simplified mathematical models were developed so as to furnish a basis for understanding the phenomena and comparison of different chambers and operating conditions. Studies of the hydrodynamic stability disclosed that a wide gap chamber may be particularly sensitive to axial temperature variations which could be due to uneven heating or cooling. The mathematical model of the separation process includes effects due to the axial velocity, electro-osmotic cross flow and electrophoretic migration, all including the effects of temperature dependent properties.

Dual-opposite injection capillary electrophoresis: Principles and misconceptions.

PubMed

Blackney, Donna M; Foley, Joe P

2017-03-01

Dual-opposite injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. The electroosmotic flow (EOF) is suppressed to allow all ions to reach the detector quickly. Depending on the individual electrophoretic mobilities of the analytes of interest and the effective length that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. This review discusses the principles, applications, and limitations of dual-opposite injection capillary electrophoresis. Common misconceptions regarding DOI-CE are clarified. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The early days of blotting.

PubMed

Southern, Edwin

2015-01-01

The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA fragments to a membrane support through capillary action, is also known as Southern blotting. This procedure enables the detection of a specific DNA sequence by hybridization with probes. The term Southern blotting led to a "geographic" naming tradition, with RNA blotting bearing the name Northern blotting and protein transfer to membranes becoming known as Western blotting.

Tellurate and periodate solutions as media for paper electrophoresis of carbohydrates.

PubMed

Alesofie, B M; Popiel, W J

1973-02-01

Electrophoretic separations of sugars and other polyhydroxy compounds may be performed in 0.2M telluric acid media adjusted to pH 10 with sodium hydroxide, and in 0.07M sodium metaperiodate at pH 11. Oxidation by periodate appears to be only slight under these conditions. Migration rates of 21 compounds are reported relative to the movement of d-ribose. In both electrolytes the compounds form anionic complexes.

Liquid and gel electrodes for transverse free flow electrophoresis

DOEpatents

Jung, Byoungsok; Rose, Klint A; Shusteff, Maxim; Persat, Alexandre; Santiago, Juan

2015-04-07

The present invention provides a mechanism for separating or isolating charged particles under the influence of an electric field without metal electrodes being in direct contact with the sample solution. The metal electrodes normally in contact with the sample are replaced with high conductivity fluid electrodes situated parallel and adjacent to the sample. When the fluid electrodes transmit the electric field across the sample, particles within the sample migrate according to their electrophoretic mobility.

Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector.

PubMed

Urban, Pawel L; Goodall, David M; Bergström, Edmund T; Bruce, Neil C

2007-08-31

An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.

Polymer encapsulated inorganic black pigment nanoparticles and their electrophoretic characteristics.

PubMed

Sim, H H; Kim, Y J; Choi, H J

2012-12-01

Black inorganic pigment modified with poly(styrene-co-acrylonitrile) was fabricated via dispersion polymerization, and then the synthesized hybrid nanoparticles were examined by SEM to confirm their morphology, while their density and size were studied using a gas pycnometer and electrophoretic light scattering apparatus, respectively. We also confirmed their chemical structure and coated state via FT-IR and TGA. Electrophoretic characteristics including the zeta potential were examined via an electrophoretic light scattering apparatus, while the movement of particles was directly observed by an optical microscopy under an electric field applied. The hybrid nanoparticles were confirmed to possess an electrophoretic property as a potential candidate for the microcapsule-type electrophoretic display.

Biochemical changes in desmosomes of bovine muzzle epidermis during differentiation.

PubMed

Konohana, A; Konohana, I; Roberts, G P; Marks, R

1987-10-01

Biochemical changes taking place in desmosomes during differentiation have been studied. Bovine muzzle epidermis was sliced horizontally into 6 layers, 0.2 mm thick, and desmosomes were isolated from each layer. These were then analyzed by polyacrylamide gel electrophoresis. The electrophoretic patterns of desmosomal proteins from the 6 layers were found to be qualitatively similar to each other, but there was an increase in the ratio of the amount of 150 kD glycoprotein (desmoglein I) relative to 240 and 210 kD proteins (desmoplakins) in the upper layers of the epidermis. This finding was supported by the similar increase observed in electrophoretic patterns of proteins extracted directly from each layer of the epidermis in electrophoretic sample buffer. In order to study the fate of desmosomal components in the stratum corneum, serial skin surface biopsies were stained with antisera against desmosomal components using indirect immunofluorescence techniques. This experiment showed that desmosomal proteins and glycoproteins persist in the stratum corneum but quantitatively decrease in the outer layers. This decrease may play a significant role in desquamation.

Nanopore detection of DNA molecules in crowded neutral polymer solutions

NASA Astrophysics Data System (ADS)

Sharma, Rajesh Kumar; Dai, Liang; Doyle, Patrick; Garaj, Slaven

Nanopore sensing is a precise technique for analysis of the structure and dynamics of individual biomolecules in different environments, and has even become a prominent technique for next-gen DNA sequencing. In the nanopore sensor, an individual DNA molecule is electrophoretically translocated through a single, nanometer-scaled pore in a solid-state membrane separating two chambers filled with electrolyte. The conformation of the molecule is deduced from modulations in the ionic current through the pore during the translocation event. Using nanopores, we investigated the dynamics of the DNA molecules in a crowded solution of neutral polymers of different sizes and concentrations. The translocation dynamics depends significantly on the size and concentration of the polymers, as different contributions to the electrophoretic and entropic forces on the DNA molecules come into play. This setup offers an excellent, tuneable model-system for probing biologically relevant questions regarding the behaviour of DNA molecules in highly confined and crowded environments. Singapore-MIT Alliance for Research and Technology.

Electrophoretic study of enzymes from cereal aphid populations : 4. Detection of hidden genetic variation within populations of the grain aphid Sitobion avenae (F.) (Hemiptera: Aphididae).

PubMed

Loxdale, H D; Rhodes, J A; Fox, J S

1985-07-01

A study of variation in three peptidases (PEP-3 to -5) in a parthenogenetic S. avenae field population at Rothamsted using serial one-dimensional polyacrylamide gel electrophoresis (involving changes of gel concentration and electrophoretic run-time) increased the overall number of "allozymes" (mobility variants) detected from 10 under standard conditions (6% gels, 2 h run-time) to 22, as well as revealing putative heterozygous banding patterns under some test conditions. However, an examination of another enzyme, 6-phosphogluconate dehydrogenase (6-PGD) in a sample collected at Rothamsted the following year failed, using a combination of serial methods (changes of gel concentration) and isoelectric focusing, to increase the total number of 6-PGD bands separated (seven, none of which appeared to be allelic in origin). Nevertheless, some major bands were split into several bands, whilst other infrequent bands were either gained or lost. The findings are briefly discussed.

Continuous flow electrophoretic separation of proteins and cells from mammalian tissues

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Barlow, Grant H.; Blaisdell, Steven J.; Cleveland, Carolyn; Farrington, Mary Ann; Feldmeier, Mary; Hatfield, J. Michael; Lanham, J. Wayne; Grindeland, Richard; Snyder, Robert S.

1987-01-01

This paper describes an apparatus for continuous flow electrophoresis (CFE), designed to separate macromolecules and cells at conditions of microgravity. In this CFE, buffer flows upward in a 120-cm long flow chamber, which is 16-cm wide x 3.0-mm thick in the microgravity version (and 6-cm wide x 1.5-mm thick in the unit-gravity laboratory version). Ovalbumin and rat serum albumin were separated in space (flight STS-4) with the same resolution of the two proteins achieved at 25 percent total w/v concentration that was obtained in the laboratory at 0.2 percent w/v concentration. Rat anterior pituitary cells, cultured human embryonic kidney cells, and canine Langerhans cells were separated into subpopulations (flight STS-8) more effectively than in unit gravity, with comparable resolution having been achieved at 100 times the concentration possible on earth.

Bioprocessing in Microgravity: Applications of Continuous Flow Electrophoresis to Rat Anterior Pituitary Particles

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Salada, T.; Cenci, R.; Krishnan, K.; Seaman, G. V. F.; Snyder, R.; Matsumiya, H.; Nagaoka, S.

1996-01-01

In this report we describe the results of a continuous flow electrophoresis (CFE) experiment done on STS-65 in which we tested the idea that intracellular growth hormone (GH) particles contained in a cell lysate prepared from cultured rat anterior pituitary cells in microgravity might have different electrophoretic mobilities from those in a synchronous ground control cell lysate. Collectively, the results suggested that CFE processing in microgravity was better than on earth; more samples could be processed at a time (6 x) and more variant forms of GH molecules could be resolved as well. We had also hoped to carry out a pituitary cell CFE experiment, but failure of the hardware required that the actual cell electrophoresis trials be done on earth shortly after Shuttle landing. Data from these experiments showed that space-flown cells possessed a higher electrophoretic mobility than ground control cells, thereby offering evidence for the idea that exposure of cultured cells to microgravity can change their net surface charge-density especially when the cells are fed. Collectively, the results from this pituitary cell experiment document the advantage of using coupled cell culture and CFE techniques in the microgravity environment.

Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough.

PubMed

Dubey, S; Kalia, Y N

2014-06-10

The original aim of the study was to investigate the transdermal iontophoretic delivery of lysozyme and to gain further insight into the factors controlling protein electrotransport. Initial experiments were done using porcine skin. Lysozyme transport was quantified by using an activity assay based on the lysis of Micrococcus lysodeikticus and was corrected for the release of endogenous enzyme from the skin during current application. Cumulative iontophoretic permeation of lysozyme during 8h at 0.5mA/cm(2) (0.7mM; pH6) was surprisingly low (5.37±3.46μg/cm(2) in 8h) as compared to electrotransport of cytochrome c (Cyt c) and ribonuclease A (RNase A) under similar conditions (923.0±496.1 and 170.71±92.13μg/cm(2), respectively) - despite its having a higher electrophoretic mobility. The focus of the study then became to understand and explain the causes of its poor iontophoretic transport. Lowering formulation pH to 5 increased histidine protonation in the protein and decreased the ionisation of fixed negative charges in the skin (pI ~4.5) and resulted in a small but statistically significant increase in permeation. Co-iontophoresis of acetaminophen revealed a significant inhibition of electroosmosis; inhibition factors of 12-16 were indicative of strong lysozyme binding to skin. Intriguingly, lidocaine electrotransport, which is due almost exclusively to electromigration, was also decreased (approximately 2.7-fold) following skin pre-treatment by lysozyme iontophoresis (cf. iontophoresis of buffer solution) - suggesting that lysozyme was also able to influence subsequent cation electromigration. In order to elucidate the site of skin binding, different porcine skin models were tested (dermatomed skin with thicknesses of 250 and 750μm, tape-stripped skin and heat-separated dermis). Although no difference was seen between permeation across 250 and 750μm dermatomed skin (13.57±12.20 and 5.37±3.46μg/cm(2), respectively), there was a statistically significant increase across tape-stripped skin and heat-separated dermis (36.86±7.48 and 43.42±13.11μg/cm(2), respectively) - although transport was still much less than that seen across intact skin for Cyt c or RNase A. Furthermore, electroosmotic inhibition factors fell to 2.2 and 1.0 for tape-stripped skin and heat-separated dermis - indicating that lysozyme affected convective solvent flow through interactions with the epidermis and predominantly the stratum corneum. Finally, cation exchange and hydrophobic interaction chromatography confirmed that although lysozyme had greater positive charge than Cyt c or RNase A under the conditions used for iontophoresis, it also possessed the highest surface hydrophobicity, which may have facilitated the interactions with the transport pathways and encouraged aggregation in the skin microenvironment. Thus, high charge and electrophoretic mobility seem to be inadequate descriptors to predict the transdermal iontophoretic transport of proteins whose complex three dimensional structures can facilitate interactions with cutaneous transport pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Li, Q.; Lu, X.

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Electrophoretic Characteristics of Outer Membrane Proteins of Neisseria meningitidis,

DTIC Science & Technology

1987-07-01

chemo-organotropic aerobic or facultative microbes, producing catalase and cytochrome oxidase (Morello and Bohnoff, 1980; Reyn, 1974). These bacteria...resolution, the porosity, pH , and TRIS and glycine concentrations in the stacking and separating gels were investigated. The UNCLASSIFIED UNCLASSIFIED /5...stacking gels were 1.7 cm in length and contained, 0.375 M Tris-HCl ( pH 8.8) and 0.1% SDS. The electrode buffer ( pH 8.3 ) consisted of 0.025 M Tris-HCI

Chemical characterization of detrital sugar chains with peptides in oceanic surface particulate organic matter

NASA Astrophysics Data System (ADS)

Tsukasaki, A.; Nishida, T.; Tanoue, E.

2016-02-01

For better understanding of the dynamics of organic matter in the ocean interior, particulate organic matter (POM) in oceanic surface water is a key material as a starting material in food chain and biological carbon pump, and the source of dissolved organic matter. POM consists of a mixture of non-living POM (detritus) and small amount of living POM (organisms). Particulate combined amino acids (PCAAs) are one of the major components of POM and the most important source of nitrogen and carbon for heterotrophic organisms in marine environments. In our previous studies of molecular-level characterization of PCAAs using electrophoretic separation (SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis) with specific detection of protein/peptide and sugar chains, we reported that most of PCAAs existed as small-sized peptide chains with carbohydrate-rich remnants. Although carbohydrates are one of the major carbon components of POM, the details of molecular-level structures including sugar chains are unknown. In this study, we applied electrophoretic separation for sugar chains (FACE: fluorophore-assisted carbohydrate electrophoresis) to the POM samples collected from the surface water of the Pacific Ocean. The results showed that sugar chains with various degree of polymerization were detected in POM. The possible roles of such sugar chains in marine biogeochemical cycle of organic matter are discussed in the presentation.

The use of experimental design for the development of a capillary zone electrophoresis method for the quantitation of captopril.

PubMed

Mukozhiwa, S Y; Khamanga, S M M; Walker, R B

2017-09-01

A capillary zone electrophoresis (CZE) method for the quantitation of captopril (CPT) using UV detection was developed. Influence of electrolyte concentration and system variables on electrophoretic separation was evaluated and a central composite design (CCD) was used to optimize the method. Variables investigated were pH, molarity, applied voltage and capillary length. The influence of sodium metabisulphite on the stability of test solutions was also investigated. The use of sodium metabisulphite prevented degradation of CPT over 24 hours. A fused uncoated silica capillary of 67.5cm total and 57.5 cm effective length was used for analysis. The applied voltage and capillary length affected the migration time of CPT significantly. A 20 mM phosphate buffer adjusted to pH 7.0 was used as running buffer and an applied voltage of 23.90 kV was suitable to effect a separation. The optimized electrophoretic conditions produced sharp, well-resolved peaks for CPT and sodium metabisulphite. Linear regression analysis of the response for CPT standards revealed the method was linear (R2 = 0.9995) over the range 5-70 μg/mL. The limits of quantitation and detection were 5 and 1.5 μg/mL. A simple, rapid and reliable CZE method has been developed and successfully applied to the analysis of commercially available CPT products.

Latex samples for RAMSES electrophoresis experiment on IML 2

NASA Technical Reports Server (NTRS)

Seaman, Geoffrey V. F.; Knox, Robert J.

1994-01-01

The objectives of these reported studies were to provide ground based support services for the flight experiment team for the RAMSES experiment to be flown aboard IML-2. The specific areas of support included consultation on the performance of particle based electrophoresis studies, development of methods for the preparation of suitable samples for the flight hardware, the screening of particles to obtain suitable candidates for the flight experiment, and the electrophoretic characterization of sample particle preparations. The first phases of these studies were performed under this contract, while the follow on work was performed under grant number NAG8 1081, 'Preparation and Characterization of Latex Samples for RAMSES Experiment on IML 2.' During this first phase of the experiment the following benchmarks were achieved: Methods were tested for the concentration and resuspension of latex samples in the greater than 0.4 micron diameter range to provide moderately high solids content samples free of particle aggregation which interferred with the normal functioning of the RAMSES hardware. Various candidate latex preparations were screened and two candidate types of latex were identified for use in the flight experiments, carboxylate modified latex (CML) and acrylic acid-acrylamide modified latex (AAM). These latexes have relatively hydrophilic surfaces, are not prone to aggregate, and display sufficiently low electrophoretic mobilities in the flight buffer so that they can be used to make mixtures to test the resolving power of the flight hardware.

Recycling isoelectric focusing with computer controlled data acquisition system. [for high resolution electrophoretic separation and purification of biomolecules

NASA Technical Reports Server (NTRS)

Egen, N. B.; Twitty, G. E.; Bier, M.

1979-01-01

Isoelectric focusing is a high-resolution technique for separating and purifying large peptides, proteins, and other biomolecules. The apparatus described in the present paper constitutes a new approach to fluid stabilization and increased throughput. Stabilization is achieved by flowing the process fluid uniformly through an array of closely spaced filter elements oriented parallel both to the electrodes and the direction of the flow. This seems to overcome the major difficulties of parabolic flow and electroosmosis at the walls, while limiting the convection to chamber compartments defined by adjacent spacers. Increased throughput is achieved by recirculating the process fluid through external heat exchange reservoirs, where the Joule heat is dissipated.

Enantioseparation of rabeprazole and omeprazole by nonaqueous capillary electrophoresis with an ephedrine-based ionic liquid as the chiral selector.

PubMed

Ma, Zheng; Zhang, Lijuan; Lin, Lina; Ji, Ping; Guo, Xingjie

2010-12-01

An ephedrine-based chiral ionic liquid, (+)-N,N-dimethylephedrinium-bis(trifluoromethanesulfon)imidate ([DMP](+) [Tf(2) N](-) ), served as both chiral selector and background electrolyte in nonaqueous capillary electrophoresis. The enantioseparation of rabeprazole and omeprazole was achieved in acetonitrile-methanol (60:40 v/v) containing 60 mm[DMP](+) [Tf(2) N](-) . The influences of separation conditions, including the concentration of [DMP](+) [Tf(2) N](-) , the electrophoretic media and the buffer, on enantioseparation were evaluated. The mechanism of enantioseparation was investigated and discussed. Ion-pair interaction and hydrogen bonding may be responsible for the main separation mechanism. Copyright © 2010 John Wiley & Sons, Ltd.

HB Puerta del Sol [HBA1:c.148A>C], HB Valdecilla [HBA2:c.3G>T], HB Gran Vía [HBA2:c.98T>G], HB Macarena [HBA2:c.358C>T] and HB El Retiro [HBA2:c.364_366dupGTG]: description of five new hemoglobinopathies.

PubMed

de la Fuente-Gonzalo, Félix; Nieto, Jorge M; Velasco, Diego; Cela, Elena; Pérez, Germán; Fernández-Teijeiro, Ana; Escudero, Antonio; Villegas, Ana; González-Fernández, Fernando A; Ropero, Paloma

2016-04-01

Structural hemoglobinopathies do not usually have a clinical impact, but they can interfere with the analytical determination of some parameters, such as the glycated hemoglobin in diabetic patients. Thalassemias represent a serious health problem in areas where their incidence is high. The defects in the post-translational modifications produce hyper-unstable hemoglobin that is not detected by most of electrophoretic or chromatographic methods that are available so far. We studied seven patients who belong to six unrelated families. The first two families were studied because they had peak abnormal hemoglobin (Hb) during routine analytical assays. The other four families were studied because they had microcytosis and hypochromia with normal HbA2 and HbF without iron deficiency. HbA2 and F quantification and abnormal Hb separation were performed by chromatographic and electrophoretic methods. The molecular characterization was performed using specific sequencing. The Hb Puerta del Sol presents electrophoretic mobility and elution in HPLC that is different from HbA and similar to HbS. The electrophoretic and chromatographic profiles of the four other variants are normal and do not show any anomalies, and their identification was only possible with sequencing. Some variants, such as Hb Valdecilla, Hb Gran Vía, Hb Macarena and Hb El Retiro, have significant clinical impact when they are associated with other forms of α-thalassemia, which could lead to more serious forms of this group of pathologies as for HbH disease. Therefore, it is important to maintain an adequate program for screening these diseases in countries where the prevalence is high to prevent the occurrence of severe forms.

Cell Partition in Two Polymer Aqueous Phases

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1985-01-01

In a reduced gravity environment the two polymer phases will not separate via density driven settling in an acceptably short length of time. It is to be expected that a certain amount of phase separation will take place, however, driven by the reduction in free energy gained when the interfacial area is reduced. This stage of separation process will therefore depend directly on the magnitude of the interfacial tension between the phases. In order to induce complete phase separation in a short time, electric field-induced separation which occurs because the droplets of one phase in the other have high electrophoretic mobilities which increase with droplet size was investigated. These mobilities are significant only in the presence of certain salts, particularly phosphates. The presence of such salts, in turn has a strong effect on the cell partition behavior in dextran-poly (ethylene glycol) (PEG) systems. The addition of the salts necessary to produce phase drop mobilities has a large effect on the interfacial tensions in the systems.

Controlling the electrophoretic mobility of single-walled carbon nanotubes: a comparison of theory and experiment.

PubMed

Usrey, Monica L; Nair, Nitish; Agnew, Daniel E; Pina, Cesar F; Strano, Michael S

2007-07-03

The electrophoretic mobilities of single-walled carbon nanotubes (SWNTs) in agarose gels subjected to negatively charged covalent functionalization and noncovalent anionic surfactant adsorption are compared using a simplified hydrodynamic model. Net charges are calculated on the basis of estimated friction coefficients for cylindrical rodlike particles. The effects of functionalization with negatively charged 4-hydroxybenzene diazonium and anionic sodium cholate are quantified and compared with model predictions. The adsorption of Na+ counterions into the nonionic surfactant layer adsorbed on SWNTs (Triton-X-405) is shown to induce a positive charge and reverse the mobility under select conditions. This effect has not been identified or quantified for nanoparticle systems and may be important in the processing of these systems.

Electrophoretic characterization of the Mammalian nuclear matrix proteome, nuclear envelope, nucleoli and covalently bound ADP-ribose polymers: potential applications to cancer.

PubMed

Aranda, Xavier G; Racho, Ronald G; Pacheco-Rodríguez, Gustavo; Alvarez-González, Rafael

2014-01-01

Nucleic acid metabolism is biochemically compartmentalized to the nucleus. Thus, it is necessary to define the proteome of the various macromolecular structures within this organelle. We isolated the nuclear matrix (NM) fraction from rat liver by sequential centrifugation steps at 13,000 rpm, staggered between endogenous nuclease treatment for 2 h at 37°C, followed by high-salt (H.S.; 2.0 M NaCl) and non-ionic detergent extractions (0.1%- or 1.0% Triton X-100) to eliminate the bulk of chromosomal DNA/RNA, histone proteins and the nuclear envelope (NE). Integrity of the NM and NE structures was confirmed by electron microscopy. Next, we analyzed the NM proteome on a 20% polyacrylamide gel using the PhastSystem. We observed the absence of histone proteins and the characteristic presence of the lamins by Coomassie blue staining. By contrast, upon silver staining, following electrophoretic separation with a Tris-Borate-EDTA buffer, we observed the NM-associated nucleic RNA and protein-free ADP-ribose polymers. While polymers are found in much lower concentration than RNA in NM, they were purified by affinity chromatography on boronate resin prior to electrophoresis. We observed the electrophoretic resolution of free ADP-ribose chains (5-25 units) by silver staining. The significance of our observations to cancer studies and carcinogenesis is discussed. Copyright© 2014, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Physical-chemical studies of proteins of squid nerve axoplasm, with special reference to the axon fibrous protein.

PubMed

DAVISON, P F; TAYLOR, E W

1960-03-01

The proteins in the axoplasm of the squid, Dosidicus gigas, have been resolved electrophoretically into a major fraction including the fibrous protein, and possibly its structural subunits, and a minor fraction including at least two proteins with low sedimentation coefficients. A partially reversible change in the structure of the fibrous protein occurs under the action of 0.4 M salt or high pH. These experiments have been interpreted to indicate that in the intact fiber one, or a few, protofibrils are arranged helically or longitudinally along the fiber axis, and linked by electrostatic bonds. On the dissociation of these bonds the separated protofibrils assume a less extended form and sediment more rapidly than the intact fibers. Some material with a lower sedimentation rate is also released on the dissociation. This fraction may comprise smaller chain fragments. The volume fraction and the approximate refractive index of the fibers have been calculated.

Physical-Chemical Studies of Proteins of Squid Nerve Axoplasm, with Special Reference to the Axon Fibrous Protein

PubMed Central

Davison, Peter F.; Taylor, Edwin W.

1960-01-01

The proteins in the axoplasm of the squid, Dosidicus gigas, have been resolved electrophoretically into a major fraction including the fibrous protein, and possibly its structural subunits, and a minor fraction including at least two proteins with low sedimentation coefficients. A partially reversible change in the structure of the fibrous protein occurs under the action of 0.4 M salt or high pH. These experiments have been interpreted to indicate that in the intact fiber one, or a few, protofibrils are arranged helically or longitudinally along the fiber axis, and linked by electrostatic bonds. On the dissociation of these bonds the separated protofibrils assume a less extended form and sediment more rapidly than the intact fibers. Some material with a lower sedimentation rate is also released on the dissociation. This fraction may comprise smaller chain fragments. The volume fraction and the approximate refractive index of the fibers have been calculated. PMID:13814536

Effect of conductivity and concentration on the sample stream in the transverse axis of a continuous flow electrophoresis chamber

NASA Technical Reports Server (NTRS)

Miller, Teresa Y.; Williams, George O.; Snyder, Robert S.

1985-01-01

The resolution of continuous flow electrophoresis systems is generally measured by the spread of the sample bands in the direction of the electrophoretic migration. This paper evaluates the cross section of the sample bands in the plane perpendicular to the flow and shows that the spread in the direction perpendicular to the migration increased significantly with the applied electric field. Concentrated samples of monodisperse latex particles and vinyltoluene T-butylstyrene particles in sample buffers of different electrical conductivities were used to map the shape of the sample bands relative to the zero electric field case. As the electric field was applied, the sample band spread from an initial diameter of only one-third the chamber thickness until it approached the chamber walls where electroosmosis significantly reduced the resolution of separation. It can be shown, however, that it is possible to minimize these distortions by careful sample preparation and experiment design.

Separation of lymphocytes by electrophoresis under terrestrial conditions and at zero gravity

NASA Technical Reports Server (NTRS)

Rubin, A. L.

1977-01-01

Electrophoretic mobility (EPM) of human peripheral lymphocytes were examined with the following objectives: To determine differences in EPM of lymphocytes under immuno-stimulated and immuno-suppressed states. To define the conditions necessary for the separation of lymphocyte sub-populations in normal and pathological conditions; To investigate immunological active, charged chemical groups on lymphocyte surfaces; and to investigate pathophysiological mechanisms of immune responsiveness, as reflected by alterations in EPM. To evaluate the potential of lymphocyte electrophoresis as: (1) a means of monitoring the immune status of kidney transplant recipients, (2) in predicting the outcome of kidney transplants, and (3) as a method for separation of lymphocyte sub-populations, the EPM was studied for unfractionated human peripheral lymphocytes and of populations enriched with T and "B" cells from normal adults, hemodialysis patients and kidney transplant recipients.

Separation of oligopeptides, nucleobases, nucleosides and nucleotides using capillary electrophoresis/electrochromatography with sol-gel modified inner capillary wall.

PubMed

Svobodová, Jana; Kofroňová, Olga; Benada, Oldřich; Král, Vladimír; Mikšík, Ivan

2017-09-29

The aim of this article is to study the modification of an inner capillary wall with sol-gel coating (pure silica sol-gel or silica sol-gel containing porphyrin-brucine conjugate) and determine its influence on the separation process using capillary electrophoresis/electrochromatography method. After modification of the inner capillary surface the separation of analytes was performed using two different phosphate buffers (pH 2.5 and 9.0) and finally the changes in electrophoretic mobilities of various samples were calculated. To confirm that the modification of the inner capillary surface was successful, the parts of the inner surfaces of capillaries were observed using scanning electron microscopy. The analytes used as testing samples were oligopeptides, nucleosides, nucleobases and finally nucleotides. Copyright © 2017 Elsevier B.V. All rights reserved.

Triazine herbicide imprinted monolithic column for capillary electrochromatography.

PubMed

Aşır, Süleyman; Derazshamshir, Ali; Yılmaz, Fatma; Denizli, Adil

2015-12-01

Trietazine was selectively separated from aqueous solution containing the competitor molecule cyanazine, which is similar in size and shape to the template molecule. Structural features of the molecularly imprinted column were figured out by SEM. The influence of the mobile-phase composition, applied electrical field, and pH of the mobile phase on the recognition of trietazine by the imprinted monolithic polymer has been evaluated, and the imprint effect in the trietazine-imprinted monolithic polymer was demonstrated by an imprinting factor. The optimized monolithic column resulted in separation of trietazine from a structurally related competitor molecule, cyanazine. In addition, fast separation was obtained within 6 min by applying higher electrical field, with the electrophoretic mobility of 2.97 × 10(-8) m(2) V(-1) s(-1) at pH 11.0. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of diterpenoic compounds in natural resins applied as binders in museum objects by capillary electrophoresis.

PubMed

Findeisen, Anna; Kolivoska, Viliam; Kaml, Isabella; Baatz, Wolfgang; Kenndler, Ernst

2007-07-20

The exudates of conifers consist mainly of diterpenoic acids of the abietane and pimarane type (abietic, neoabietic, dehydroabietic, palustric, pimaric, isopimaric, levopimaric and sandaracopimaric acid) and larixol acetate. These natural resins were used as adhesives, coatings, varnishes or plasticizers in artistic and historic works since ancient times. For the purpose of conservation and restoration and for art historic examination of such museum objects the identification of the binding media used is undoubtedly of paramount importance. In the present paper, the characterization of these resins based on the pattern of their diterpenoid constituents is carried out by capillary electrophoresis. For separation a background electrolyte which has been initially introduced for the analysis of chlorinated and natural resin acids in waste water was modified and the experimental conditions were adjusted in terms of resolution and analysis time. Separation was carried out in borate buffer at pH 9.25 (ionic strength 20 mmol L(-1)) with methyl-beta-cyclodextrin and sulfobutylether-beta-cyclodextrin as additives to increase selectivity and enhance the solubility of the analytes. With this electrophoretic system the resin acids of interest and larixol acetate--all as anionic cyclodextrin complexes--were separated within 5 min and detected at 200, 250 and 270 nm with a diode array detector. The electrophoretic patterns served for the characterisation of the relevant diterpenoic resins, balsams and copals. Sample pre-treatment was limited to sonication in methanol at 55 degrees C for 30 min. This enables the identification of the resins in mixtures with other binders like plant gums, animal glues or drying oils, even when these media are present in excess. Colophony was identified as resinous constituent of a modelling mass for gilded frames originating from the 19th century.

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

A simple preparative free-flow electrophoresis joined with gratis gravity: I. Gas cushion injector and self-balance collector instead of multiple channel pump.

PubMed

Chen, Su; Palmer, James F; Zhang, Wei; Shao, Jing; Li, Si; Fan, Liu-Yin; Sun, Ren; Dong, Yu-Chao; Cao, Cheng-Xi

2009-06-01

This paper describes a novel free-flow electrophoresis (FFE), which is joined with gratis gravity, gas cushion injector (GCI) and self-balance collector instead of multiple channel pump, for the purpose of preparative purification. The FFE was evaluated by systemic experiments. The results manifest that (i) even though one-channel peristaltic pump is used for the driving of background buffer, there is still stable flow in the FFE chamber; (ii) the stable flow is induced by the gravity-induced pressure due to the difference of buffer surfaces in the GCI and self-balance collector; (iii) the pulse flow of background buffer induced by the peristaltic pump is greatly reduced by the GCI with good compressibility of included air; (iv) the FFE can be well used for zone electrophoretic separation of amino acids; (v) up to 20 inlets simultaneous sample injection and up to five to tenfold condensation of amino acid can be achieved by combining the FFE device with the method of moving reaction boundary. To the best of authors' knowledge, FFE has not been used for such separation and condensation of amino acids. The relevant results achieved in the paper have evident significance for the development of preparative FFE.

High Molecular Weight Forms of Mammalian Respiratory Chain Complex II

PubMed Central

Nůsková, Hana; Holzerová, Eliška; Vrbacký, Marek; Pecina, Petr; Hejzlarová, Kateřina; Kľučková, Katarína; Rohlena, Jakub; Neuzil, Jiri; Houštěk, Josef

2013-01-01

Mitochondrial respiratory chain is organised into supramolecular structures that can be preserved in mild detergent solubilisates and resolved by native electrophoretic systems. Supercomplexes of respiratory complexes I, III and IV as well as multimeric forms of ATP synthase are well established. However, the involvement of complex II, linking respiratory chain with tricarboxylic acid cycle, in mitochondrial supercomplexes is questionable. Here we show that digitonin-solubilised complex II quantitatively forms high molecular weight structures (CIIhmw) that can be resolved by clear native electrophoresis. CIIhmw structures are enzymatically active and differ in electrophoretic mobility between tissues (500 – over 1000 kDa) and cultured cells (400–670 kDa). While their formation is unaffected by isolated defects in other respiratory chain complexes, they are destabilised in mtDNA-depleted, rho0 cells. Molecular interactions responsible for the assembly of CIIhmw are rather weak with the complexes being more stable in tissues than in cultured cells. While electrophoretic studies and immunoprecipitation experiments of CIIhmw do not indicate specific interactions with the respiratory chain complexes I, III or IV or enzymes of the tricarboxylic acid cycle, they point out to a specific interaction between CII and ATP synthase. PMID:23967256

Cobalt complexes as internal standards for capillary zone electrophoresis-mass spectrometry studies in biological inorganic chemistry.

PubMed

Holtkamp, Hannah U; Morrow, Stuart J; Kubanik, Mario; Hartinger, Christian G

2017-07-01

Run-by-run variations are very common in capillary electrophoretic (CE) separations and cause imprecision in both the migration times and the peak areas. This makes peak and kinetic trend identification difficult and error prone. With the aim to identify suitable standards for CE separations which are compatible with the common detectors UV, ESI-MS, and ICP-MS, the Co III complexes [Co(en) 3 ]Cl 3 , [Co(acac) 3 ] and K[Co(EDTA)] were evaluated as internal standards in the reaction of the anticancer drug cisplatin and guanosine 5'-monophosphate as an example of a classical biological inorganic chemistry experiment. These Co III chelate complexes were considered for their stability, accessibility, and the low detection limit for Co in ICP-MS. Furthermore, the Co III complexes are positively and negatively charged as well as neutral, allowing the detection in different areas of the electropherograms. The background electrolytes were chosen to cover a wide pH range. The compatibility to the separation conditions was dependent on the ligands attached to the Co III centers, with only the acetylacetonato (acac) complex being applicable in the pH range 2.8-9.0. Furthermore, because of being charge neutral, this compound could be used as an electroosmotic flow (EOF) marker. In general, employing Co complexes resulted in improved data sets, particularly with regard to the migration times and peak areas, which resulted, for example, in higher linear ranges for the quantification of cisplatin.

Electrophoretic separation of alginic sodium diester and sodium hexametaphosphate in chondroitin sulfate that interfere with the cetylpyridinium chloride titration assay.

PubMed

Weiguo, Zhang; Giancaspro, Gabriel; Adams, Kristie M; Neal-Kababick, James; Hildreth, Jana; Li, Aishan; Roman, Mark C; Betz, Joseph M

2014-01-01

The most commonly used chondroitin sulfate (CS) assay method is cetylpyridinium chloride (CPC) titration. Cellulose acetate membrane electrophoresis (CAME) is the technique used for detection of impurities in the U.S. Pharmacopeia's CS monograph. Because CPC titration is a relatively nonspecific quantitative technique, the apparent amount of CS as determined by CPC titration alone may not reflect the true amount of CS due to possible interference with the CPC assay by impurities that contain CPC titratable functional groups. When CAME is used in conjunction with CPC titration, certain non-CS and adulterants can be visualized and estimated, and a true value for CS can be assigned once the presence of these non-CS impurities has been ruled out. This study examines conjunct application of CPC and CAME in ascertaining CS assay and purity in the presence of certain adulterants. These include propylene glycol alginate sulfate sodium, known in commerce as alginic sodium diester (ASD), and Zero One (Z1), a water-soluble agent newly reported in the CS marketplace and subsequently identified as sodium hexametaphosphate. ASD, Z1, and CS are similar in physical appearance and solubility in water and ethanol. They are also titratable anions and form ionic pairs with CPC, therefore interfering with the CPC titration assay for CS CAME separates these adulterants from each other and from CS by differences in their electrophoretic mobility. CAME is able to detect these impurities in CS at levels as low as 0.66% by weight. Although it is recommended that a method for detecting impurities (e.g., CAME) be used in cormbination with relatively nonspecific assay methods such as CPC titration, this is seldom done in practice. Assay results for CS derived fromn CPC titration may, therefore, be misleading, leaving the CS supply chain vulnerable to adulteration. In this study, the authors investigated ASD and Z1 adulteration of CS and developed an electrophoretic separation of these adulterants in CS and procedures to isolate ASD from CS matrixes containing these adulterants. The authors describe in this paper utilization of an orthogonal approach to establish the identity of Z1 as sodium hexametaphosphate and to confirm the identity of ASD, including ethanol fractionation, FTIR spectroscopy, differential scanning calorimetry, and NMR spectroscopy. The authors suggest that CAME is a cost-effective and easy to use methodfor detecting certain impurities in CS raw ingredients and recommend that CPC and CAME be used in combination by QC laboratories as a means of effectively deterring the practice of adulterating CS raw materials with the known adulterants ASD and Z1 and/or other non-chondroitin substances that can be separated from CSby CAME and that exhibit CPC titration behavior similar to CS.

Electrophoretic fractional elution apparatus employing a rotational seal fraction collector

NASA Technical Reports Server (NTRS)

Bier, M. (Inventor)

1977-01-01

Electrophoretic fractional elution apparatus which has a column with a rotating seal joint is described. A thin jet of eluting buffer is directed across the lumen of the electrophoretic column in a direction perpendicular to that of electrophoretic migration. Either the content of the column is rotated with respect to the stationary jet, or the jet is rotated with respect to the column. The system may employ electrophoresis either in free solution or in packed columns.

A SEROLOGICAL AND ELECTROPHORETIC STUDY OF DIPHTHERIA ANTISERA IRRADIATED WITH STERILIZING DOSES OF $gamma$-RAYS

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

Kaulen, D.R.; Chakhava, O.V.

1958-01-01

The effects of irradiation on the antitoxic, anaphylactic, and electrophoretic properties of diphtheria antisera were studied at the various doses used for sterilization. Both crude and purified diphtheria antitoxic antisera were used. Irradiations were carried out with a cobalt-60 source with a total power of 5 kc. The dosage rate was 600 r/min. Data are tabulated. The results demonstrate considerable changes in the properties of antisera taking place as a result of exposure to large doses of gamma radiation. In all experiments a regular fall in the antitoxin titre was demonstrated. A greater destruction of antitoxin was observed in themore » crude antiserum than in the purified. Possible reaction mechanisms involved are discussed. (C.H.)« less

PBT,PBO-Based Hybrid Polymers with Nonlinear Optical Properties or High Electrical Conductivity

DTIC Science & Technology

1988-08-29

standing. Experiments with stronger oxidizing agents such as nitrosonium salts (e.g., NO+Br4, NO+PF6) and high-potential quinones (e.g., DDQ...several unique possibilities. First, the ionic structure should raise Tg. Second, electrophoretic ion migration under the influence of the poling field

Relationship between luminous fish and symbiosis. I. Comparative studies of lipopolysaccharides isolated from symbiotic luminous bacteria of the luminous marine fish, Physiculus japonicus.

PubMed

Kuwae, T; Andoh, M; Fukasawa, S; Kurata, M

1983-01-01

In order to investigate the relationship between host and symbiosis in the luminous marine fish, Physiculus japonicus, the bacterial lipopolysaccharides (LPS) of symbiotic luminous bacteria were compared serologically and electrophoretically. Five symbiotic luminous bacteria (PJ strains) were separately isolated from five individuals of this fish species caught at three points, off the coasts of Chiba, Nakaminato, and Oharai. LPS preparations were made from these bacteria by Westphal's phenol-water method and highly purified by repeated ultracentrifugation. These LPSs contained little or no 2-keto-3-deoxyoctonate and had powerful mitogenic activity. In sodium dodecylsulfate polyacrylamide gel electrophoresis, these PJ-1 to -5 LPSs were separated by their electrophoretic patterns into three groups; the first group included PJ-1 and PJ-4, the second group PJ-2 and PJ-3, and the third group PJ-5 alone. The results agreed with those of the double immunodiffusion test; precipitin lines completely coalesced within each group but not with other groups. In immunoelectrophoresis, one precipitin line was observed between anti PJ-2 LPS serum and PJ-5 LPS but the electrophoretic mobility of PJ-5 LPS was clearly different from that of the PJ-2 LPS group. Furthermore, in a 50% inhibition test with PJ-2 LPS by the passive hemolysis system, the doses of PJ-2 LPS, PJ-3 LPS, and PJ-5 LPS required for 50% inhibition (ID50) in this system were 0.25, 0.25, and 21.6 micrograms/ml for each alkali-treated LPS, respectively, and the ID50's of both PJ-1 LPS and PJ-4 LPS were above 1,000 micrograms/ml. These results indicate that PJ-5 LPS has an antigenic determinant partially in common with LPS from the PJ-2 group but not with LPS from the PJ-1 group and that the symbiotic luminous bacterium PJ-5 is more closely related to the PJ-2 group than to the PJ-1 group. These results show that the species Physiculus japonicus is symbiotically associated with at least three immunologically different strains of luminous marine bacteria in its specialized light organ.

Linear and Nonlinear Spectroscopic Probing of Solute Interactions with Chemically Modified Silica Surfaces

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

Wirth, Mary J

Solar energy conversion through biology would provide a renewable and nonpolluting abundance of energy. The bacterium Halobacterium salinarum converts solar to electrical energy by virtue of a transmembrane protein, bacteriorhodopsin. This transmembrane protein pumps protons across a nonconducting bilayer upon irradiation with green light. The bacterium evolved to perform this function inefficiently. If we were able to understand this process to engineer this protein for efficiency, then inexpensive energy production could be achieved. There are tens of thousands of different types of halobacteria, giving the opportunity to study different efficiencies and relating these to the protein structures. Technology does notmore » yet exist to perform such screening. The goal of this research is to generate new separation technology that can ultimately enable such screening. This involves creating a method for separating oriented and functional transmembrane proteins that remain in an electrically insulating lipid bilayer, with aqueous solutions on either side of the bilayer. A pH change across the lipid bilayer upon irradiation of a known concentration of proteins would probe function. Differences in proton pumping efficiency for different proteins variants would provide structure-function information for engineering the proteins. A schematic diagram from the original proposal is shown here. The idea is that (a) a lipid bilayer supported on a hydrophilic polymer film will make the bilayer fluid, and (b) applying an electric field will cause electrophoretic migration of the transmembrane proteins. We demonstrated this concept experimentally in a paper that was published just after this new grant period started (Lipid Bilayers on Polyacrylamide Brushes for Inclusion of Membrane Proteins, Emily A. Smith, Jason W. Coym, Scott M. Cowell, Victor J. Hruby, Henry I. Yamamura, Mary J. Wirth, Langmuir, 21, 9644-9650, 2005). The electrophoretic mobility was slow (10{sup -8} cm{sup 2}/Vs), and we project that a two order of magnitude increase would make this a practical tool. We are investigating two ways of improving electrophoretic mobility: better polymer supports, and a novel nanoporous medium that suspends the bilayer over free solution.« less

Electrophoretic characterization of aldehyde-fixed red blood cells, kidney cells, lynphocytes and chamber coatings

NASA Technical Reports Server (NTRS)

1976-01-01

Ground-based electrokinetic data on the electrophoresis flight experiment to be flown on the Apollo-Soyuz Test Project experiment MA-011 are stipulated. Aldehyde-fixed red blood cells, embryonic kidney cells and lymphocytes were evaluated by analytical particle electrophoresis. The results which aided in the interpretation of the final analysis of the MA-011 experiment are documented. The electrophoresis chamber surface modifications, the buffer, and the material used in the column system are also discussed.

Cadmium migration in aerospace nickel cadmium cells

NASA Technical Reports Server (NTRS)

Mcdermott, P. P.

1976-01-01

The effects of temperature, the nature of separator material, charge and discharge, carbonate contamination, and the mode of storage are studied with respect to the migration of active material from the negative toward the positive plate. A theoretical model is proposed which takes into account the solubility of cadmium in various concentrations of hydroxide and carbonate at different temperatures, the generation of the cadmiate ion, Cd(OH)3(-), during discharge, the migration of the cadmiate ion and particulate Cd(OH)2 due to electrophoretic effects and the movement of electrolyte in and out of the negative plate and, finally, the recrystallization of cadmiate ion in the separator as Cd(OH)2. Application of the theoretical model to observations of cadmium migration in cycled cells is also discussed.

Effect of AOT Microemulsion Composition on the Hydrodynamic Diameter and Electrophoretic Mobility of Titanium Oxide Nanoparticles

NASA Astrophysics Data System (ADS)

Shaparenko, N. O.; Beketova, D. I.; Demidova, M. G.; Bulavchenko, A. I.

2018-05-01

The hydrodynamic diameter and electrophoretic mobility of titania nanoparticles in AOT microemulsions are studied depending on their water content (from 0 to 1.5 vol %), chloroform content in n-decane-chloroform mixture (from 0 to 30 vol %) and temperature (from 0 to 60°C). Considerable changes in diameter (from 20 to 400 nm) are detected upon adding water to the microemulsion. The electrophoretic mobility grows by 2-3 times upon adding chloroform, or as the temperature falls. The observed features allow us to halve the time of electrophoretic concentration for 140 nm TiO2 nanoparticles, and to concentrate 14 nm nanoparticles that do not exhibit electrophoretic mobility in the absence of chloroform.

Classification of Spanish white wines using their electrophoretic profiles obtained by capillary zone electrophoresis with amperometric detection.

PubMed

Arribas, Alberto Sánchez; Martínez-Fernández, Marta; Moreno, Mónica; Bermejo, Esperanza; Zapardiel, Antonio; Chicharro, Manuel

2014-06-01

A method was developed for the simultaneous detection of eight polyphenols (t-resveratrol, (+)-catechin, quercetin and p-coumaric, caffeic, sinapic, ferulic, and gallic acids) by CZE with electrochemical detection. Separation of these polyphenols was achieved within 25 min using a 200 mM borate buffer (pH 9.4) containing 10% methanol as separation electrolyte. Amperometric detection of polyphenols was carried out with a glassy carbon electrode (GCE) modified with a multiwalled carbon nanotubes (CNT) layer obtained from a dispersion of CNT in polyethylenimine. The excellent electrochemical properties of this modified electrode allowed the detection and quantification of the selected polyphenols in white wines without any pretreatment step, showing remarkable signal stability despite the presence of potential fouling substances in wine. The electrophoretic profiles of white wines, obtained using this methodology, have proven to be useful for the classification of these wines by means of chemometric multivariate techniques. Principal component analysis and discriminant analysis allowed accurate classification of wine samples on the basis of their grape varietal (verdejo and airén) using the information contained in selected zones of the electropherogram. The utility of the proposed CZE methodology based on the electrochemical response of CNT-modified electrodes appears to be promising in the field of wine industry and it is expected to be successfully extended to classification of a wider range of wines made of other grape varietals. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Triblock copolymer matrix-based capillary electrophoretic microdevice for high-resolution multiplex pathogen detection.

PubMed

Kim, Se Jin; Shin, Gi Won; Choi, Seok Jin; Hwang, Hee Sung; Jung, Gyoo Yeol; Seo, Tae Seok

2010-03-01

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE-SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) triblock copolymer as a sieving matrix for CE-SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO-PPO-PEO copolymers, 255-bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO-PPO-PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan gel. Due to enhanced dynamic coating and sieving ability, PEO-PPO-PEO copolymer displayed fourfold enhancement of resolving power in the CE-SSCP to separate same-sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high-resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO-PPO-PEO triblock copolymer an excellent matrix in the CE-SSCP analysis on the microdevice.

FACTORS AFFECTING THE UPTAKE OF LISSAMINE GREEN BY SERUM PROTEINS

PubMed Central

Brackenridge, C. J.

1960-01-01

Eight physicochemical factors which affect the uptake of lissamine green on filter paper impregnated with serum proteins have been examined, and their relevance to the staining of electrophoretically separated protein fractions is discussed. It is shown that grade of paper, weight of protein applied, separate and combined denaturation and staining time, temperature and concentration of staining solution, concentration of denaturant, and type of protein all influence the weight of dye absorbed per unit weight of applied protein, and must be rigidly standardized if valid quantitative results are to be obtained. Five sets of conditions are obtained for optimal staining and it is found that separation of denaturant from dye yields the best procedure. It is concluded that lissamine green is an excellent dye for the staining and quantitative estimation of separated protein fractions in paper electrophoresis, and that conditions can usually be arranged to produce a linear relation between dye uptake and protein concentration in an experimentally efficient manner. PMID:13803681

Recent advances in capillary electrophoresis separation of monosaccharides, oligosaccharides, and polysaccharides.

PubMed

Mantovani, Veronica; Galeotti, Fabio; Maccari, Francesca; Volpi, Nicola

2018-01-01

This article illustrates the basis and applications of methodologies for the analysis of simple and complex carbohydrates by means of CE. After a description of the most common and novel approaches useful for the analysis and characterization of carbohydrates, this review covers the recent advances in CE separation of monosaccharides, oligosaccharides, and polysaccharides. Various CE techniques are also illustrated for the study of carbohydrates derived from complex glyco-derivatives such as glycoproteins and glycolipids, essential for biopharmaceutical and glycoproteomics applications as well as for biomarker detection. Most glycans have no significant UV absorption, and derivatization with fluorophore groups prior to separation usually results in higher sensitivity and an improved electrophoretic profile. We also discuss the recent applications and separations by CE of derivatized simple and more complex carbohydrates with different chromophoric active tags. Overall, this review aims to give an overview of the most recent state-of-the-art techniques used in carbohydrate analysis by CE. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of chiral electrophoretic separation methods for phenethylamines and application on impurity analysis.

PubMed

Borst, Claudia; Holzgrabe, Ulrike

2010-12-15

A chiral microemulsion electrokinetic chromatography method has been developed for the separation of the enantiomers of the phenethylamines ephedrine, N-methylephedrine, norephedrine, pseudoephedrine, adrenaline (epinephrine), 2-amino-1-phenylethanol, diethylnorephedrine, and 2-(dibutylamino)-1-phenyl-1-propanol, respectively. The separations were achieved using an oil-in-water microemulsion consisting of the oil-component ethyl acetate, the surfactant sodium dodecylsulfate, the cosurfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The method was compared to an already described CZE method, which made use of heptakis(2,3-di-O-diacetyl-6-O-sulfo)-beta-cyclodextrin (HDAS) as chiral selector. Additionally, the developed method was successfully applied to the related substances analysis of noradrenaline, adrenaline, dipivefrine, ephedrine and pseudoephedrine monographed in the European Pharmacopoeia 6. Copyright 2010 Elsevier B.V. All rights reserved.

Chitosan as cationic polyelectrolyte for the modification of electroosmotic flow and its utilization for the separation of inorganic anions by capillary zone electrophoresis.

PubMed

Takayanagi, Toshio; Motomizu, Shoji

2006-09-01

Cationic polyelectrolyte of chitosan was used for the reversal of electroosmotic flow in capillary zone electrophoresis. The chitosan was dissolved in acetic acid solution, and stable electroosmotic flow was obtained at the chitosan concentrations between 50 and 300 microg/mL. Separation of inorganic anions was carried out using the dynamically coated capillary by capillary zone electrophoresis. Nine kinds of anions were separated and detected with the capillary. The electrophoretic mobility of the analyte anions decreased with increasing concentrations of chitosan in the migrating solution through ion-ion interaction, but the migration order of the analyte anions was not changed in the concentration range of the chitosan examined. The signal shape for the analyte anions was developed by using field-enhanced sample stacking with 10 mM sodium sulfate.

Electrophoresis for the analysis of heparin purity and quality.

PubMed

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J

2012-06-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, Richard D. [Richland, WA; Udseth, Harold R. [Richland, WA; Olivares, Jose A. [Los Alamos, NM

1994-10-18

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5-100 KVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., .+-.2-8 KVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit, or by conduction through a sheath electrode discharged in an annular sheath flow about the capillary exit.

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, R.D.; Udseth, H.R.; Olivares, J.A.

1994-10-18

A system and method for analyzing molecular constituents of a composition sample include: forming a solution of the sample, separating the solution by capillary electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5--100 kVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g.,{+-}2--8 kVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit, or by conduction through a sheath electrode discharged in an annular sheath flow about the capillary exit. 21 figs.

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, R.P.; Udseth, H.R.; Olivares, J.A.

1989-12-05

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5--100 kVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., [+-]2--8 kVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit, or by conduction through a sheath electrode discharged in an annular sheath flow about the capillary exit. 21 figs.

Electrokinetic sample preconcentration and hydrodynamic sample injection for microchip electrophoresis using a pneumatic microvalve.

PubMed

Cong, Yongzheng; Katipamula, Shanta; Geng, Tao; Prost, Spencer A; Tang, Keqi; Kelly, Ryan T

2016-02-01

A microfluidic platform was developed to perform online electrokinetic sample preconcentration and rapid hydrodynamic sample injection for zone electrophoresis using a single microvalve. The polydimethylsiloxane microchip comprises a separation channel, a side channel for sample introduction, and a control channel which is used as a pneumatic microvalve aligned at the intersection of the two flow channels. The closed microvalve, created by multilayer soft lithography, serves as a nanochannel preconcentrator under an applied electric potential, enabling current to pass through while preventing bulk flow. Once analytes are concentrated, the valve is briefly opened and the stacked sample is pressure injected into the separation channel for electrophoretic separation. Fluorescently labeled peptides were enriched by a factor of ∼450 in 230 s. This method enables both rapid analyte concentration and controlled injection volume for high sensitivity, high-resolution CE. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrophoresis for the analysis of heparin purity and quality

PubMed Central

Volpi, Nicola; Maccari, Francesca; Suwan, Jiraporn; Linhardt, Robert J.

2012-01-01

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment. PMID:22736353

Combined electrophoresis-electrospray interface and method

DOEpatents

Smith, Richard P.; Udseth, Harold R.; Olivares, Jose A.

1989-01-01

A system and method for analyzing molecular constituents of a composition sample includes: forming a solution of the sample, separating the solution by capillary electrophoresis into an eluent of constituents longitudinally separated according to their relative electrophoretic mobilities, electrospraying the eluent to form a charged spray in which the molecular constituents have a temporal distribution; and detecting or collecting the separated constituents in accordance with the temporal distribution in the spray. A first high-voltage (e.g., 5-100 KVDC) is applied to the solution. The spray is charged by applying a second high voltage (e.g., .+-.2-8 KVDC) between the eluent at the capillary exit and a cathode spaced in front of the exit. A complete electrical circuit is formed by a conductor which directly contacts the eluent at the capillary exit, or by conduction through a sheath electrode discharged in an annular sheath flow about the capillary exit.

Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis.

PubMed

Walz, M; Kück, U

1995-12-01

The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals. The highest transformation frequency was obtained with vector pMW1. On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum. Southern analysis suggests that the vector copies are integrated as tandem repeats into the S. macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis. Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules. Electrophoretic karyotyping was used to further characterize S. macrospora transformants. Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb. Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes. In a few transformants, major rearrangements were detected. Transformants were sexually propagated to analyze the fate of the heterologous vector DNA. Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis. However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations. Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S. macrospora.

Factors affecting the separation performance of proteins in capillary electrophoresis.

PubMed

Zhu, Yueping; Li, Zhenqing; Wang, Ping; Shen, Lisong; Zhang, Dawei; Yamaguchi, Yoshinori

2018-04-15

Capillary electrophoresis (CE) is an effective tool for protein separation and analysis. Compared with capillary gel electrophoresis (CGE), non-gel sieving capillary electrophoresis (NGSCE) processes the superiority on operation, repeatability and automaticity. Herein, we investigated the effect of polymer molecular weight and concentration, electric field strength, and the effective length of the capillary on the separation performance of proteins, and find that (1) polymer with high molecular weight and concentration favors the separation of proteins, although concentrated polymer hinders its injection into the channel of the capillary due to its high viscosity. (2) The resolution between the adjacent proteins decreases with the increase of electric field strength. (3) When the effective length of the capillary is long, the separation performance improves at the cost of separation time. (4) 1.4% (w/v) hydroxyethyl cellulose (HEC), 100 V/cm voltage and 12 cm effective length offers the best separation for the proteins with molecular weight from 14,400 Da to 97,400 Da. Finally, we employed the optimal electrophoretic conditions to resolve Lysozyme, Ovalbumin, BSA and their mixtures, and found that they were baseline resolved within 15 min. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultra-High-Speed DNA Fragment Separations Using Microfabricated Capillary Array Electrophoresis Chips

NASA Astrophysics Data System (ADS)

Woolley, Adam T.; Mathies, Richard A.

1994-11-01

Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of φX174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 μm) have been explored. By use of channels with an effective length of only 3.5 cm, separations of φX174 Hae III DNA fragments from ≈70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQα alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.

Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

ERIC Educational Resources Information Center

Brunauer, Linda S.

2016-01-01

A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size…

Selecting enhancing solutions for electrokinetic remediation of dredged sediments polluted with fuel.

PubMed

Rozas, F; Castellote, M

2015-03-15

In this paper a procedure for selecting the enhancing solutions in electrokinetic remediation experiments is proposed. For this purpose, dredged marine sediment was contaminated with fuel, and a total of 22 different experimental conditions were tested, analysing the influence of different enhancing solutions by using three commercial non-ionic surfactants, one bio-surfactant, one chelating agent, and one weak acid. Characterisation, microelectrophoretic and electrokinetic remediation trials were carried out. The results are explained on the basis of the interactions between the fuel, the enhancing electrolytes and the matrix. For one specific system, the electrophoretic zeta potential, (ζ), of the contaminated matrix in the solution was found to be related to the electroosmotic averaged ζ in the experiment and not to the efficiency in the extraction. This later was correlated to a parameter accounting for both contributions, the contaminant and the enhancing solution, calculated on the basis of differences in the electrophoretic ζ in different conditions which has allowed to propose a methodology for selection of enhancing solutions. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA mobility modifier

DOEpatents

Barron, Annelise

2002-01-01

Polyamides comprising at least one hydrophilic C.sub.1 -C.sub.10 hydrocarbyl substituent on an amide nitrogen atom, and methods for producing and using the same is provided. In particular, polyamides of the formula: ##STR1## and methods for using the same for altering the ratio of charge/translational frictional drag of binding polymers to allow electrophoretic separation of polynucleotides or analogs thereof in a non-sieving liquid medium is provided, where a, q, L.sup.1, P.sup.1, Q.sup.1, R, R.sup.1, R.sup.10 and R.sup.11 are those described herein.

DNA mobility modifier

DOEpatents

Barron, Annelise E.

2004-04-20

Polyamides comprising at least one hydrophilic C.sub.1 -C.sub.10 hydrocarbyl substituent on an amide nitrogen atom, and methods for producing and using the same is provided. In particular, polyamides of the formula: ##STR1## and methods for using the same for altering the ratio of charge/translational frictional drag of binding polymers to allow electrophoretic separation of polynucleotides or analogs thereof in a non-sieving liquid medium is provided, where a, q, L.sup.1, P.sup.1, Q.sup.1, R, R.sup.1, R.sup.10 and R.sup.11 are those described herein.

Separate physiological roles for two isozymes of pyridine nucleotide-linked glycerol-3-phosphate dehydrogenase in chicken.

NASA Technical Reports Server (NTRS)

White, H. B., III; Kaplan, N. O.

1972-01-01

The isozymes considered are designated 'liver type' and 'muscle type' based on the tissue of highest concentration. Electrophoretic analysis shows that the liver type is found in small amounts or is undetectable in all tissues studied except liver. The muscle type is found in skeletal muscles and kidney. Presumptive hybrid enzymes occur at low levels in chicken liver and kidney. The tissue distribution of glyceron-3-P dehydrogenase in several birds capable of sustained flight is different than in chicken.

Capillary electrophoresis-high resolution sector field inductively coupled plasma mass spectrometry.

PubMed

Sonke, Jeroen E; Salters, Vincent J M

2007-08-03

The background and applications of high resolution sector field inductively coupled plasma mass spectrometry (HR-ICP-MS) as a detector for capillary (CE) and gel electrophoretic separations are reviewed. Notable progress has been made in the fields of bioinorganic and environmental (geo-) chemistry. Metallomics, the study of metal species interactions and functions in biological systems, puts substantial technical demands on speciation analysis. The combination of high species resolving power (CE) and high sensitivity-high mass resolving power (HR-ICP-MS) provides a solid base to meet such demands.

A DC electrophoresis method for determining electrophoretic mobility through the pressure driven negation of electro osmosis

NASA Astrophysics Data System (ADS)

Karam, Pascal; Pennathur, Sumita

2016-11-01

Characterization of the electrophoretic mobility and zeta potential of micro and nanoparticles is important for assessing properties such as stability, charge and size. In electrophoretic techniques for such characterization, the bulk fluid motion due to the interaction between the fluid and the charged surface must be accounted for. Unlike current industrial systems which rely on DLS and oscillating potentials to mitigate electroosmotic flow (EOF), we propose a simple alternative electrophoretic method for optically determining electrophoretic mobility using a DC electric fields. Specifically, we create a system where an adverse pressure gradient counters EOF, and design the geometry of the channel so that the flow profile of the pressure driven flow matches that of the EOF in large regions of the channel (ie. where we observe particle flow). Our specific COMSOL-optimized geometry is two large cross sectional areas adjacent to a central, high aspect ratio channel. We show that this effectively removes EOF from a large region of the channel and allows for the accurate optical characterization of electrophoretic particle mobility, no matter the wall charge or particle size.

Ensemble of electrophoretically captured gold nanoparticles as a fingerprint of Boltzmann velocity distribution

NASA Astrophysics Data System (ADS)

Hong, S. H.; Kang, M. G.; Lim, J. H.; Hwang, S. W.

2008-07-01

An ensemble of electrophoretically captured gold nanoparticles is exploited to fingerprint their velocity distribution in solution. The electrophoretic capture is performed using a dc biased nanogap electrode, and panoramic scanning electron microscopic images are inspected to obtain the regional density of the captured gold nanoparticles. The regional density profile along the surface of the electrode is in a quantitative agreement with the calculated density of the captured nanoparticles. The calculated density is obtained by counting, in the Boltzmann distribution, the number of nanoparticles whose thermal velocity is smaller than the electrophoretic velocity.

Capillary electrochromatographic separation of peptides using a macrocyclic polyamine for molecular recognition.

PubMed

Chen, Tse-Hsien; Misra, Tarun Kumar; Liu, Chuen-Ying

2008-04-01

A macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N(8)), in the bonded phase was employed as a molecular receptor for CEC separation of oligopeptides. Parameters affecting the performance of the separations were considered. Baseline separation for the mixture of angiotensin I, angiotensin II, [Sar(1), Thr(8)]-angiotensin II, beta-casomorphin bovine, beta-casomorphin human, oxytocin acetate, tocinoic acid, vasopressin, and FMRF amide could be achieved using phosphate buffer (30 mM, pH 7) as the mobile phase. Column efficiency with average theoretical plate numbers of 69 000 plates/m and RSDs of <1% (n = 6) was achieved. [Met(5)]-enkephalin and [Leu(5)]-enkephalin, which have identical pI values and similar masses could be completely separated using acetate buffer (30 mM) with pH gradient (pH 3 inlet side and pH 4 outlet side). The results suggest that the mechanism for the peptide separation was mediated by a combination of electrophoretic migration and chromatographic retention involving anion coordination and anion exchange. After long-term use, the deviation of the EOF of the column after more than 600 injections was still within 6.0% of that for a freshly prepared column.

Particle sizer and DNA sequencer

DOEpatents

Olivares, Jose A.; Stark, Peter C.

2005-09-13

An electrophoretic device separates and detects particles such as DNA fragments, proteins, and the like. The device has a capillary which is coated with a coating with a low refractive index such as Teflon.RTM. AF. A sample of particles is fluorescently labeled and injected into the capillary. The capillary is filled with an electrolyte buffer solution. An electrical field is applied across the capillary causing the particles to migrate from a first end of the capillary to a second end of the capillary. A detector light beam is then scanned along the length of the capillary to detect the location of the separated particles. The device is amenable to a high throughput system by providing additional capillaries. The device can also be used to determine the actual size of the particles and for DNA sequencing.

Analysis and characterization of heparin impurities.

PubMed

Beni, Szabolcs; Limtiaco, John F K; Larive, Cynthia K

2011-01-01

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations.

Further improvement of hydrostatic pressure sample injection for microchip electrophoresis.

PubMed

Luo, Yong; Zhang, Qingquan; Qin, Jianhua; Lin, Bingcheng

2007-12-01

Hydrostatic pressure sample injection method is able to minimize the number of electrodes needed for a microchip electrophoresis process; however, it neither can be applied for electrophoretic DNA sizing, nor can be implemented on the widely used single-cross microchip. This paper presents an injector design that makes the hydrostatic pressure sample injection method suitable for DNA sizing. By introducing an assistant channel into the normal double-cross injector, a rugged DNA sample plug suitable for sizing can be successfully formed within the cross area during the sample loading. This paper also demonstrates that the hydrostatic pressure sample injection can be performed in the single-cross microchip by controlling the radial position of the detection point in the separation channel. Rhodamine 123 and its derivative as model sample were successfully separated.

Electrophoresis-mass spectrometry probe

DOEpatents

Andresen, Brian D.; Fought, Eric R.

1987-01-01

The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface.

Capillary electrophoresis of chitooligosaccharides in acidic solution: simple determination using a quaternary-ammonium-modified column and indirect photometric detection with crystal violet.

PubMed

Hattori, Toshiaki; Anraku, Nobuhiro; Kato, Ryo

2010-02-01

Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 microM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC. 2009 Elsevier B.V. All rights reserved.

An analytical model for enantioseparation process in capillary electrophoresis

NASA Astrophysics Data System (ADS)

Ranzuglia, G. A.; Manzi, S. J.; Gomez, M. R.; Belardinelli, R. E.; Pereyra, V. D.

2017-12-01

An analytical model to explain the mobilities of enantiomer binary mixture in capillary electrophoresis experiment is proposed. The model consists in a set of kinetic equations describing the evolution of the populations of molecules involved in the enantioseparation process in capillary electrophoresis (CE) is proposed. These equations take into account the asymmetric driven migration of enantiomer molecules, chiral selector and the temporary diastomeric complexes, which are the products of the reversible reaction between the enantiomers and the chiral selector. The solution of these equations gives the spatial and temporal distribution of each species in the capillary, reproducing a typical signal of the electropherogram. The mobility, μ, of each specie is obtained by the position of the maximum (main peak) of their respective distributions. Thereby, the apparent electrophoretic mobility difference, Δμ, as a function of chiral selector concentration, [ C ] , can be measured. The behaviour of Δμ versus [ C ] is compared with the phenomenological model introduced by Wren and Rowe in J. Chromatography 1992, 603, 235. To test the analytical model, a capillary electrophoresis experiment for the enantiomeric separation of the (±)-chlorpheniramine β-cyclodextrin (β-CD) system is used. These data, as well as, other obtained from literature are in closed agreement with those obtained by the model. All these results are also corroborate by kinetic Monte Carlo simulation.

Polyamidoamine dendrimers as sweeping agent and stationary phase for rapid and sensitive open-tubular capillary electrophoretic determination of heavy metal ions.

PubMed

Ge, Ying; Guo, Yujun; Qin, Weidong

2014-04-01

Polyamidoamine (PAMAM) dendrimer generation 2.5 was synthesized and evaluated as sweeping agent for in-column enrichment and as stationary phase for capillary electrochromatographic separation of heavy metal ions, viz., Pb(II), Cu(II), Hg(II), Zn(II) and Co(II), in a running buffer containing 4-(2-pyridylazo)resorcinol (PAR) as a chromogenic reagent. During experiment, a plug of aqueous PAMAM generation 2.5 solution was first introduced to the capillary, followed by electrokinetic injection of the heavy metal ions under a positive voltage. In this step, PAMAM acted as a sweeping agent, stacking the metal ions on the analyte/PAMAM boundary by forming metal ion-PAMAM complexes. The second preconcentration process occurred when PAR, a stronger ligand, moving toward the injection end under the electric field, reached and re-swept the metal ion-PAMAM zone, forming metal ion-PAR complexes. During separation, the neutral PAMAM moved toward the detector with the electroosmotic flow, dynamically coating the capillary wall, forming stationary phases that affected the separation of the metal ions. Due to the function of PAMAM, the detection sensitivity and resolution of the heavy metal ions improved significantly. Under the optimum conditions, the detection limits were 0.299, 0.184, 0.774, 0.182 and 0.047 μg/L for Pb(II), Cu(II), Hg(II), Zn(II) and Co(II), respectively. The method was successfully applied to the determination of heavy metals in snow, tap and rain water samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Exploring the intermolecular interactions acting in solvent-modified MEKC by Molecular Dynamics and NMR: The effect of n-butanol on the separation of diclofenac and its impurities.

PubMed

Caprini, Claudia; Pasquini, Benedetta; Melani, Fabrizio; Del Bubba, Massimo; Giuffrida, Alessandro; Calleri, Enrica; Orlandini, Serena; Furlanetto, Sandra

2018-02-05

An integrated approach involving CE experiments, Molecular Dynamics (MD) simulations and two-dimensional NOE spectroscopy (2D-NOESY) experiments was employed to elucidate the intermolecular interactions and the separation mechanisms involved in a solvent-modified MEKC method for the simultaneous determination of diclofenac sodium and its impurities. The CE findings indicated that the addition of n-butanol (nBuOH) to the SDS micellar solution played a primary role for controlling the partitioning into the mixed micelles and the migration of the analytes and that the presence of nBuOH as cosurfactant was compulsory for achieving the complete separation of the compounds. The different capacity factors of the analytes were calculated and a change in solute association with the mixed micelle when changing the SDS/nBuOH molar ratio was highlighted. The optimal SDS/nBuOH molar ratio for the electrophoretic separation was 1:8. On the other hand, both MD simulations and NMR experiments indicated that the most favorable molar ratio for the formation of mixed SDS/nBuOH micelles was 1:2. These results suggested that probably there is an excess of nBuOH in the background electrolyte, both as free molecules and in form of aggregates, which is able to interact with the analytes, and thus may compete with mixed micelles for the considered compounds. The calculated values of gain in potential energy of the analytes when included in mixed micelles were in agreement with the observed migration order of the compounds. The role of methyl-β-cyclodextrin (MβCyD) in the background electrolyte was also investigated, since the addition of this CyD to the solvent-modified MEKC system was found to be useful to reduce the analysis time. MD simulations and 2D-NOESY spectra highlighted the formation of inclusion complexes with MβCyD not only with the analytes, but also with SDS. MβCyD may lower the availability of both SDS and nBuOH for forming micelles and mostly may compete with the mixed micelle as a second pseudostationary phase. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved Bacterial and Viral Recoveries from 'Complex' Samples using Electrophoretically Assisted Acoustic Focusing

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

Ness, K; Rose, K; Jung, B

2008-03-27

Automated front-end sample preparation technologies can significantly enhance the sensitivity and reliability of biodetection assays [1]. We are developing advanced sample preparation technologies for biowarfare detection and medical point-of-care diagnostics using microfluidic systems with continuous sample processing capabilities. Here we report an electrophoretically assisted acoustic focusing technique to rapidly extract and enrich viral and bacterial loads from 'complex samples', applied in this case to human nasopharyngeal samples as well as simplified surrogates. The acoustic forces capture and remove large particles (> 2 {micro}m) such as host cells, debris, dust, and pollen from the sample. We simultaneously apply an electric fieldmore » transverse to the flow direction to transport small ({le} 2 {micro}m), negatively-charged analytes into a separate purified recovery fluid using a modified H-filter configuration [Micronics US Patent 5,716,852]. Hunter and O'Brien combined transverse electrophoresis and acoustic focusing to measure the surface charge on large particles, [2] but to our knowledge, our work is the first demonstration combining these two techniques in a continuous flow device. Marina et al. demonstrated superimposed dielectrophoresis (DEP) and acoustic focusing for enhanced separations [3], but these devices have limited throughput due to the rapid decay of DEP forces. Both acoustic standing waves and electric fields exert significant forces over the entire fluid volume in microchannels, thus allowing channels with larger dimensions (> 100 {micro}m) and high throughputs (10-100 {micro}L/min) necessary to process real-world volumes (1 mL). Previous work demonstrated acoustic focusing of microbeads [4] and biological species [5] in various geometries. We experimentally characterized our device by determining the biological size-cutoff where acoustic radiation pressure forces no longer transport biological particles. Figure 1 shows images of E.Coli ({approx}1 {micro}m) and yeast ({approx}4-5 {micro}m) flowing in a microchannel (200 {micro}m deep, 500 {micro}m wide) at a flow rate of 10 {micro}L/min. The E.Coli does not focus in the acoustic field while the yeast focuses at the channel centerline. This result suggests the acoustic size-cutoff for biological particles in our device lies between 2 and 3 {micro}m. Transverse electrophoresis has been explored extensively in electric field flow fractionation [6] and isoelectric focusing devices [7]. We demonstrated transverse electrophoretic transport of a wide variety of negatively-charged species, including fluorophores, beads, viruses, E.Coli, and yeast. Figure 2 shows the electromigration of a fluorescently labeled RNA virus (MS2) from the lower half of the channel to the upper half region with continuous flow. We demonstrated the effectiveness of our electrophoretically assisted acoustic focusing device by separating virus-like particles (40 nm fluorescent beads, selected to aid in visualization) from a high background concentration of yeast contaminants (see Figure 3). Our device allows for the efficient recovery of virus into a pre-selected purified buffer while background contaminants are acoustically captured and removed. We also tested the device using clinical nasopharyngeal samples, both washes and lavages, and demonstrated removal of unknown particulates (>2 ?m size) from the sample. Our future research direction includes spiking known amounts of bacteria and viruses into clinical samples and performing quantitative off-chip analysis (real-time PCR and flow cytometry).« less

Countercurrent distribution of biological cells

NASA Technical Reports Server (NTRS)

Brooks, D. E.

1982-01-01

Detailed physiochemical studies of dextran/poly(ethylene glycol) (PEG) two phase systems were carried out to characterize and provide understanding of the properties of the systems which determine cell partition and the electrophoretic behavior of phase drops responsible for electric field driven phase separation. A detailed study of the electrostatic and electrokinetic potentials developed in these systems was carried out. The salt partition was examined both in phase systems and with pure polymer solutions via equilibrium dialysis and mechanism of sulfate, chloride and phosphate partition shown to be exclusion by PEG rather than binding by dextran. Salt partition was shown to have a strong effect on the polymer compositions of the phases as well, an effect which produces large changes in the interfacial tension between them. These effects were characterized and the interfacial tension shown to obey a power law with respect to its dependence on the length of the tie line describing the system composition on a phase diagram. The electrostatic potential differences measured via salt bridges were shown to obey thermodynamic predictions. The electrophoretic mobilities measured were utilized to provide a partial test of Levine's incomplete theory of phase drop electrophoresis. The data were consistent with Levine's expression over a limited range of the variables tested.

Red fluorescent protein responsible for pigmentation in trematode-infected Porites compressa tissues.

PubMed

Palmer, Caroline V; Roth, Melissa S; Gates, Ruth D

2009-02-01

Reports of coral disease have increased dramatically over the last decade; however, the biological mechanisms that corals utilize to limit infection and resist disease remain poorly understood. Compromised coral tissues often display non-normal pigmentation that potentially represents an inflammation-like response, although these pigments remain uncharacterized. Using spectral emission analysis and cryo-histological and electrophoretic techniques, we investigated the pink pigmentation associated with trematodiasis, infection with Podocotyloides stenometre larval trematode, in Porites compressa. Spectral emission analysis reveals that macroscopic areas of pink pigmentation fluoresce under blue light excitation (450 nm) and produce a broad emission peak at 590 nm (+/-6) with a 60-nm full width at half maximum. Electrophoretic protein separation of pigmented tissue extract confirms the red fluorescence to be a protein rather than a low-molecular-weight compound. Histological sections demonstrate green fluorescence in healthy coral tissue and red fluorescence in the trematodiasis-compromised tissue. The red fluorescent protein (FP) is limited to the epidermis, is not associated with cells or granules, and appears unstructured. These data collectively suggest that the red FP is produced and localized in tissue infected by larval trematodes and plays a role in the immune response in corals.

Signal enhancement for peptide analysis in liquid chromatography-electrospray ionization mass spectrometry with trifluoroacetic acid containing mobile phase by postcolumn electrophoretic mobility control.

PubMed

Wang, Nan-Hsuan; Lee, Wan-Li; Her, Guor-Rong

2011-08-15

A strategy based on postcolumn electrophoretic mobility control (EMC) was developed to alleviate the adverse effect of trifluoroacetic acid (TFA) on the liquid chromatography-mass spectrometry (LC-MS) analysis of peptides. The device created to achieve this goal consisted of a poly(dimethylsiloxane) (PDMS)-based junction reservoir, a short connecting capillary, and an electrospray ionization (ESI) sprayer connected to the outlet of the high-performance liquid chromatography (HPLC) column. By apply different voltages to the junction reservoir and the ESI emitter, an electric field was created across the connecting capillary. Due to the electric field, positively charged peptides migrated toward the ESI sprayer, whereas TFA anions remained in the junction reservoir and were removed from the ionization process. Because TFA did not enter the ESI source, ion suppression from TFA was alleviated. Operation of the postcolumn device was optimized using a peptide standard mixture. Under optimized conditions, signals for the peptides were enhanced 9-35-fold without a compromise in separation efficiency. The optimized conditions were also applied to the LC-MS analysis of a tryptic digest of bovine serum albumin.

Two-dimensional electrophoretic analysis of nuclear matrix proteins in human colon adenocarcinoma.

PubMed

Toumpanaki, A; Baltatzis, G E; Gaitanarou, E; Seretis, E; Toumpanakis, C; Aroni, K; Kittas, Christos; Voloudakis-Baltatzis, I E

2009-01-01

The aim of the present study was to observe possible qualitative and quantitative expression differences between nuclear matrix proteins (NMPs) of human colon adenocarcinoma and their mirror biopsies, using the technique of two-dimensional gel electrophoresis, in order to identify the existence of specific NMP fingerprints for colon cancer. Colon tissues were examined ultrastructurally and NMPs were isolated biochemically, by serial extraction of lipids, soluble proteins, DNA, RNA, and intermediate filaments and were separated according to their isoelectric point (pI) and their molecular weight (MW) by high-resolution two-dimensional electrophoresis (2D). By comparing the 2D electropherograms of colon cancer tissues and mirror biopsy tissues we observed qualitative and quantitative expression differences between their NMPs but also a differentiation of NMP composition between the stages of malignancy. Moreover, despite the similarities between mirror biopsy samples, a highlight percentage of exception was observed. Electrophoretic results provided in this study demonstrated that the examined NMPs could be further investigated as potential markers for detection of colorectal cancer in an early stage, for the assessment of the disease progression, as well as useful tools for individual therapy and for preventing a possible recurrence of cancer and metastasis.

On-line capillary electrophoresis-based enzymatic methodology for the study of polymer-drug conjugates.

PubMed

Coussot, G; Ladner, Y; Bayart, C; Faye, C; Vigier, V; Perrin, C

2015-01-09

This work aims at studying the potentialities of an on-line capillary electrophoresis (CE)-based digestion methodology for evaluating polymer-drug conjugates degradability in the presence of free trypsin (in-solution digestion). A sandwich plugs injection scheme with transverse diffusion of laminar profile (TDLFP) mode was used to achieve on-line digestions. Electrophoretic separation conditions were established using poly-l-Lysine (PLL) as reference substrate. Comparison with off-line digestion was carried out to demonstrate the feasibility of the proposed methodology. The applicability of the on-line CE-based digestion methodology was evaluated for two PLL-drug conjugates and for the four first generations of dendrigraft of lysine (DGL). Different electrophoretic profiles presenting the formation of di, tri, and tetralysine were observed for PLL-drug and DGL. These findings are in good agreement with the nature of the linker used to link the drug to PLL structure and the predicted degradability of DGL. The present on-line methodology applicability was also successfully proven for protein conjugates hydrolysis. In summary, the described methodology provides a powerful tool for the rapid study of biodegradable polymers. Copyright © 2014 Elsevier B.V. All rights reserved.

Serum protein electrophoretic pattern in one-humped camels (Camelus dromedarius) in Tripoli, Libya.

PubMed

Abdoslam, Omran; Bayt-Almal, Mahmoud; Almghrbe, Abdullah; Algriany, Omran

2018-01-01

The aim of this study was to characterize serum protein capillary electrophoretic pattern in apparently healthy adult male (age: 3-7 years) dromedary camels and also evaluate total protein and albumin levels using automated analyzer. Blood samples were taken from 20 camels. 5ml of blood was collected from the jugular vein and serum was separated from samples by centrifugation. Capillary electrophoresis of serum proteins identified six protein fractions in adult camels, including albumin, alpha1, alpha2, beta1, beta2 and gamma globulins, serum levels of these parameters were 3.9±0.04 g/dl, 0.16±0.01 g/dl, 0.39±0.03 g/dl, 0.515±0.03 g/dl, 0.205±0.01 g/dl and 0.61±0.04 g/dl, and 65.42±0.62 g/l, respectively. The total protein concentration was 65.42±0.62 g/L, while, the albumin/globulin (A/G) ratio was 2.4±0.14. The present study indicates six peaks with minicapillary electrophoresis and the results obtained were compared and interpreted in the light of finding reported by other investigators in camels.

Capillary electrophoresis separation of neutral organic compounds, pharmaceutical drugs, proteins and peptides, enantiomers, and anions

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

Ding, Wei -Liang

1999-02-12

Addition of a novel anionic surfactant, namely lauryl polyoxyethylene sulfate, to an aqueous-acetonitrile electrolyte makes it possible to separate nonionic organic compounds by capillary electrophoresis. Separation is based on differences in the association between analytes and the surfactant. Highly hydrophobic compounds such as polyaromatic hydrocarbons are well separated by this new surfactant. Migration times of analytes can be readily changed over an unusually large range by varying the additive concentration and the proportion of acetonitrile in the electrolyte. Several examples are given, including the separation of four methylbenz[a]anthracene isomers and the separation of normal and deuterated acetophenone. The effect ofmore » adding this new surfactant to the acidic electrolyte was also investigated. Incorporation of cetyltrimethylammonium bromide in the electrolyte is shown to dynamically coat the capillary and reverse electroosmotic flow. Chiral recognition mechanism is studied using novel synthetic surfactants as chiral selectors, which are made from amino acids reacting with alkyl chloroformates. A satisfactory separation of both inorganic and organic anions is obtained using electrolyte solutions as high as 5 M sodium chloride using direct photometric detection. The effect of various salts on electrophoretic and electroosmotic mobility is further discussed. Several examples are given under high-salt conditions.« less

Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

PubMed

Tanaka, Yoshihide

2002-07-01

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.

Static adsorptive coating of poly(methyl methacrylate) microfluidic chips for extended usage in DNA separations.

PubMed

Du, Xiao-Guang; Fang, Zhao-Lun

2005-12-01

A simple and robust static adsorptive (dynamic) coating process using 2% hydroxyethylcellulose was developed for surface modification of poly(methyl methacrylate) (PMMA) microfluidic chips for DNA separations, suitable for usage over extended periods, involving hundreds of runs. The coating medium was also used as a sieving matrix for the DNA separations following the coating process. Four consecutive static treatments, by simply filling the PMMA chip channels with sieving matrix once every day, were required for obtaining a stable coating and optimum performance. The performance of the coated chips at different phases of the coating process was studied by consecutive gel electrophoretic separations with LIF detection using a PhiX-174/HaeIII DNA digest sample. The coated chip, with daily renewal of the sieving matrix, showed high stability in performance during a 25-day period of systematic study, involving more than 100 individual runs. The performance of the coated chip also remained almost the same after 3 months of continuous usage, during which over 200 separations were performed. The average precision of migration time for the 603-bp fragment was 1.31% RSD (n = 6) during the 25-day study, with a separation efficiency of 6.5 x 10(4) plates (effective separation length 5.4 cm).

Modern Approach to Medical Diagnostics - the Use of Separation Techniques in Microorganisms Detection.

PubMed

Chylewska, Agnieszka; Ogryzek, M; Makowski, Mariusz

2017-10-23

New analytical and molecular methods for microorganisms are being developed on various features of identification i.e. selectivity, specificity, sensitivity, rapidity and discrimination of the viable cell. The presented review was established following the current trends in improved pathogens separation and detection methods and their subsequent use in medical diagnosis. This contribution also focuses on the development of analytical and biological methods in the analysis of microorganisms, with special attention paid to bio-samples containing microbes (blood, urine, lymph, wastewater). First, the paper discusses microbes characterization, their structure, surface, properties, size and then it describes pivotal points in the bacteria, viruses and fungi separation procedure obtained by researchers in the last 30 years. According to the above, detection techniques can be classified into three categories, which were, in our opinion, examined and modified most intensively during this period: electrophoretic, nucleic-acid-based, and immunological methods. The review covers also the progress, limitations and challenges of these approaches and emphasizes the advantages of new separative techniques in selective fractionating of microorganisms. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

DNA Extraction by Isotachophoresis in a Microfluidic Channel

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

Stephenson, S J

Biological assays have many applications. For example, forensics personnel and medical professionals use these tests to diagnose diseases and track their progression or identify pathogens and the host response to them. One limitation of these tests, however, is that most of them target only one piece of the sample - such as bacterial DNA - and other components (e.g. host genomic DNA) get in the way, even though they may be useful for different tests. To address this problem, it would be useful to extract several different substances from a complex biological sample - such as blood - in anmore » inexpensive and efficient manner. This summer, I worked with Maxim Shusteff at Lawrence Livermore National Lab on the Rapid Automated Sample Prep project. The goal of the project is to solve the aforementioned problem by creating a system that uses a series of different extraction methods to extract cells, bacteria, and DNA from a complex biological sample. Biological assays can then be run on purified output samples. In this device, an operator could input a complex sample such as blood or saliva, and would receive separate outputs of cells, bacteria, viruses, and DNA. I had the opportunity to work this summer with isotachophoresis (ITP), a technique that can be used to extract nucleic acids from a sample. This technique is intended to be the last stage of the purification device. Isotachophoresis separates particles based on different electrophoretic mobilities. This technique is convenient for out application because free solution DNA mobility is approximately equal for DNA longer than 300 base pairs in length. The sample of interest - in our case DNA - is fed into the chip with streams of leading electrolyte (LE) and trailing electrolyte (TE). When an electric field is applied, the species migrate based on their electrophoretic mobilities. Because the ions in the leading electrolyte have a high electrophoretic mobility, they race ahead of the slower sample and trailing electrolyte ions. Conversely, the trailing electrolyte ions have a slow electrophoretic mobility, so they lag behind the sample, thus trapping the species of interest between the LE and TE streams. In a typical isotachophoresis configuration, the electric field is applied in a direction parallel to the direction of flow. The species then form bands that stretch across the width of the channel. A major limitation of that approach is that only a finite amount of sample can be processed at once, and the sample must be processed in batches. For our purposes, a form of free-flow isotachophoresis is more convenient, where the DNA forms a band parallel to the edges of the channel. To achieve this, in our chip, the electric field is applied transversely. This creates a force perpendicular to the direction of flow, which causes the different ions to migrate across the flow direction. Because the mobility of the DNA is between the mobility of the leading and the trailing electrolyte, the DNA is focused in a tight band near the center of the channel. The stream of DNA can then be directed to a different output to produce a highly concentrated outlet stream without batch processing. One hurdle that must be overcome for successful ITP is isolating the electrochemical reactions that result from the application of high voltage for the actual process of isotachophoresis. The electrochemical reactions that occur around metal electrodes produce bubbles and pH changes that are detrimental to successful ITP. The design of the chips we use incorporates polyacrylamide gels to serve as electrodes along the central channel. For our design, the metal electrodes are located away from the chip, and high conductivity buffer streams carry the potential to the chip, functioning as a 'liquid electrode.' The stream then runs alongside a gel barrier. The gel electrode permits ion transfer while simultaneously isolating the separation chamber from any contaminants in the outer, 'liquid electrode' streams. The difference in potential from one side of the chip to the other creates an electric field. This field traverses the inner, separation channel, containing the leading electrolyte, the trailing electrolyte, and the sample of interest (DNA). To increase the ease of use of the chips, a newer chip design has been fabricated. This design has wire electrodes integrated on the chip, rather than elsewhere. To keep the pH changes and bubbling isolated from the separation channel, the chip contains deeper wells near the electrodes so that the flowing buffer can wash away any gases that form around the electrode. This design is significantly more compact because it eliminates the cumbersome electrode boxes. Eliminating the electrode boxes also decreases the required voltage, making the experiments safer. This happens because when the 'liquid electrode' streams travel through small diameter tubing, they lose much of their voltage due to the electrical resistance of the fluid in the tubing.« less

A chiral enantioseparation generic strategy for anti-Alzheimer and antifungal drugs by short end injection capillary electrophoresis using an experimental design approach.

PubMed

Abdel-Megied, Ahmed M; Hanafi, Rasha S; Aboul-Enein, Hassan Y

2018-02-01

The present study describes a generic strategy using capillary electrophoretic (CE) method for chiral enantioseparation of anti-Alzheimer drugs, namely, donepezil (DON), rivastigmine (RIV), and antifungal drugs, namely, ketoconazole (KET), Itraconazole (ITR), fluconazole (FLU), and sertaconazole (SRT) in which these drugs have different basic and acidic properties. Several modified cyclodextrins (CDs) were applied for enantioseparation of racemates such as highly sulfated α, γ CDs, hydroxyl propyl-β-CD, and Sulfobutyl ether-β-CD. The starting screening conditions consist of 50-mM phosphate-triethanolamine buffer at pH 2.5, an applied voltage of 15 kV, and a temperature of 25°C. The CE strategy implemented in the separation starts by screening prior to the optimization stage in which an experimental design is applied. The design of experiment (DOE) was based on a full factorial design of the crucial two factors (pH and %CD) at three levels, to make a total of nine (3 2 ) experiments with high, intermediate, and low values for both factors. Evaluation of the proposed strategy pointed out that best resolution was obtained at pH 2.5 for five racemates using low percentages of HS-γ-CD, while SBE-β-CD was the most successful chiral selector offering acceptable resolution for all the six racemates, with the best separation at low pH values and at higher %CD within 10-min runtime. Regression study showed that the linear model shows a significant lack of fit for all chiral selectors, anticipating that higher orders of the factors are most likely to be present in the equation with possible interactions. © 2017 Wiley Periodicals, Inc.

FAST TRACK COMMUNICATION: High material efficiency found in electrophoretic deposition of conjugated polymer

NASA Astrophysics Data System (ADS)

Tada, Kazuya; Onoda, Mitsuyoshi

2009-09-01

The material efficiency of electrophoretic deposition of a fluorene-based conjugated polymer, poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}] (PDOF-MEHPV), from suspensions with a mixture of acetonitrile and toluene as dispersant is studied. It has been found that the recovery rate of the electrophoretic deposition from a suspension containing 90% of the poor solvent acetonitrile reaches 98%. Although the recovery rate decreases with decreasing acetonitrile content, almost 70% of the polymer can be deposited on the substrates from the suspension containing equivalent volumes of the good and poor solvents by electrophoretic deposition, from which smooth and transparent films suitable for electronic devices are obtained.

Geometrical separation method for lipoproteins using bioformulated-fiber matrix electrophoresis: size of high-density lipoprotein does not reflect its density.

PubMed

Tabuchi, Mari; Seo, Makoto; Inoue, Takayuki; Ikeda, Takeshi; Kogure, Akinori; Inoue, Ikuo; Katayama, Shigehiro; Matsunaga, Toshiyuki; Hara, Akira; Komoda, Tsugikazu

2011-02-01

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases.

Modern bioanalysis of proteins by electrophoretic techniques.

PubMed

Krizkova, Sona; Ryvolova, Marketa; Masarik, Michal; Zitka, Ondrej; Adam, Vojtech; Hubalek, Jaromir; Eckschlager, Tomas; Kizek, Rene

2014-01-01

In 1957, protein rich in cysteine able to bind cadmium was isolated from horse kidney and named as metallothionein according to its structural properties. Further, this protein and metallothionein-like proteins have been found in tissues of other animal species, yeasts, fungi and plants. MT is as a potential cancer marker in the focus of interest, and its properties, functions, and behavior under various conditions are intensively studied. Our protocol describes separation of two major mammalian isoforms of MT (MT-1 and MT-2) using capillary electrophoresis (CE) coupled with UV detector. This protocol enables separation of MT isoforms and studying of their basic behavior as well as their quantification with detection limit in units of ng per μL. Sodium borate buffer (20 mM, pH 9.5) was optimized as a background electrolyte, and the separation was carried out in fused silica capillary with internal diameter of 75 μm and electric field intensity of 350 V/cm. Optimal detection wavelength was 254 nm.

Fully packed capillary electrochromatographic microchip with self-assembly colloidal silica beads.

PubMed

Park, Jongman; Lee, Dami; Kim, Won; Horiike, Shigeyoshi; Nishimoto, Takahiro; Lee, Se Hwan; Ahn, Chong H

2007-04-15

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.

Dopamine-imprinted monolithic column for capillary electrochromatography.

PubMed

Aşır, Süleyman; Sarı, Duygu; Derazshamshir, Ali; Yılmaz, Fatma; Şarkaya, Koray; Denizli, Adil

2017-11-01

A dopamine-imprinted monolithic column was prepared and used in capillary electrochromatography as stationary phase for the first time. Dopamine was selectively separated from aqueous solution containing the competitor molecule norepinephrine, which is similar in size and shape to the template molecule. Morphology of the dopamine-imprinted column was observed by scanning electron microscopy. The influence of the organic solvent content of mobile phase, applied pressure and pH of the mobile phase on the recognition of dopamine by the imprinted monolithic column has been evaluated, and the imprinting effect in the dopamine-imprinted monolithic polymer was verified. Developed dopamine-imprinted monolithic column resulted in excellent separation of dopamine from structurally related competitor molecule, norepinephrine. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 5.81 × 10 -5  m 2 V -1 s -1 at pH 5.0 and 500 mbar pressure. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fractionation of mineral species by electrophoresis

NASA Technical Reports Server (NTRS)

Dunning, J. D.; Herren, B. J.; Tipps, R. W.; Snyder, R. S.

1982-01-01

The fractionation of fine-grained aggregates into their major components is a problem in many scientific areas including earth and planetary science. Electrophoresis, the transport of electrically charged particles, immersed in a suspension medium, by a direct current field (Bier, 1959), was employed in this study as a means of separating simulated lunar soil into its constituent minerals. In these tests, conducted in a static analytical cylindrical microelectrophoresis apparatus, samples of simulated lunar soil and samples of pure mineral constituents were placed in the chamber; the electrophoretic mobilities of the lunar soil and the individual mineral constituents were measured. In most of the suspension buffers employed separability was indicated, on the basis of differences in mobility, for all the constituent mineral species except ilmenite and pyroxene, which were not efficiently separable in any of the buffers. Although only a few suspension media were employed, the success of this initial study suggests that electrophoresis may be an important mineral fractionation option in fine-grained aggregate processing.

Concurrent DNA Preconcentration and Separation in Bipolar Electrode-Based Microfluidic Device

PubMed Central

Song, Hongjun; Wang, Yi; Garson, Charles; Pant, Kapil

2015-01-01

This paper presents a bipolar electrode (BPE) device in a microfluidic dual-channel design for concurrent preconcentration and separation of composite DNA containing samples. The novelty of the present effort relies on the combination of BPE-induced ion concentration polarization (ICP) and end-labeled free-solution electrophoresis (ELFSE). The ion concentration polarization effect arising from the faradaic reaction on the BPE is utilized to exert opposing electrophoretic and electroosmotic forces on the DNA samples. Meanwhile, end-labeled free-solution electrophoresis alters the mass-charge ratio to enable simultaneous DNA separation in free solution. The microfluidic device was fabricated using standard and soft lithography techniques to form gold-on-glass electrode capped with a PDMS microfluidic channel. Experimental testing with various DNA samples was carried out over a range of applied electric field. Concentration ratios up to 285× within 5 minutes for a 102-mer DNA, and concurrent preconcentration and free-solution separation of binary mixture of free and bound 102-mer DNA within 6 minutes was demonstrated. The effect of applied electric field was also interrogated with respect to pertinent performance metrics of preconcentration and separation. PMID:26005497

Development of a temperature gradient focusing method for in situ extraterrestrial biomarker analysis.

PubMed

Danger, Grégoire; Ross, David

2008-08-01

Scanning temperature gradient focusing (TGF) is a recently described technique for the simultaneous concentration and separation of charged analytes. It allows for high analyte peak capacities and low LODs in microcolumn electrophoretic separations. In this paper, we present the application of scanning TGF for chiral separations of amino acids. Using a mixture of seven carboxyfluorescein succinimidyl ester-labeled amino acids (including five chiral amino acids) which constitute the Mars7 standard, we show that scanning TGF is a very simple and efficient method for chiral separations. The modulation of TGF separation parameters (temperature window, pressure scan rate, temperature range, and chiral selector concentration) allows optimization of peak efficiencies and analyte resolutions. The use of hydroxypropyl-beta-CD at low concentration (1-5 mmol/L) as a chiral selector, with an appropriate pressure scan rate ( -0.25 Pa/s) and with a low temperature range (3-25 degrees C over 1 cm) provided high resolution between enantiomers (Rs >1.5 for each pair of enantiomers) using a short, 4 cm long capillary. With these new results, the scanning TGF method appears to be a viable method for in situ trace biomarker analysis for future missions to Mars or other solar system bodies.

Photovoltaic cell assembly

DOEpatents

Beavis, Leonard C.; Panitz, Janda K. G.; Sharp, Donald J.

1990-01-01

A photovoltaic assembly for converting high intensity solar radiation into lectrical energy in which a solar cell is separated from a heat sink by a thin layer of a composite material which has excellent dielectric properties and good thermal conductivity. This composite material is a thin film of porous Al.sub.2 O.sub.3 in which the pores have been substantially filled with an electrophoretically-deposited layer of a styrene-acrylate resin. This composite provides electrical breakdown strengths greater than that of a layer consisting essentially of Al.sub.2 O.sub.3 and has a higher thermal conductivity than a layer of styrene-acrylate alone.

Interaction and Aggregation of Colloidal Biological Particles and Droplets in Electrically-Driven Flows

NASA Technical Reports Server (NTRS)

Davis, Robert H.; Loewenberg, Michael

1997-01-01

The primary objective of this research was to develop a fundamental understanding of aggregation and coalescence processes during electrically-driven migration of cells, particles and droplets. The process by which charged cells, particles, molecules, or drops migrate in a weak electric field is known as electrophoresis. If the migrating species have different charges or surface potentials, they will migrate at different speeds and thus may collide and aggregate or coalesce. Aggregation and coalescence are undesirable, if the goal is to separate the different species on the basis of their different electrophoretic mobilities.

Methods and apparatus for analysis of chromatographic migration patterns

DOEpatents

Stockham, Thomas G.; Ives, Jeffrey T.

1993-01-01

A method and apparatus for sharpening signal peaks in a signal representing the distribution of biological or chemical components of a mixture separated by a chromatographic technique such as, but not limited to, electrophoresis. A key step in the method is the use of a blind deconvolution technique, presently embodied as homomorphic filtering, to reduce the contribution of a blurring function to the signal encoding the peaks of the distribution. The invention further includes steps and apparatus directed to determination of a nucleotide sequence from a set of four such signals representing DNA sequence data derived by electrophoretic means.

Electrophoretic separation of proteins in space

NASA Technical Reports Server (NTRS)

Brown, R. K.

1976-01-01

Commercially available and synthetic wide range and short range ampholytes used in the isoelectric focusing of proteins was analyzed by ion exchange chromatography. A pH gradient over the pH range 3.8 to 11.0 was used to elute the ampholytes from a column of a sulfonated polystyrene resin. The wide range ampholytes were resolved into some 60 to 70 ninhydrin positive components. The recovery obtained with the method was quantitative. Acid short range ampholytes have approximately 35 components which elute readily from the ion exchange resin. Basic short range ampholytes gave about 50 components, most of which eluted at alkaline pH.

Electrophoretic mobilities of cultured human embryonic kidney cells in various buffers

NASA Technical Reports Server (NTRS)

1985-01-01

Data on the electrophoretic mobility distributions of cells in the new D-1 buffer and the interlaboratory standardization of urokinase assay methods are presented. A table of cell strains and recent data on cell dispersal methods are also included. It was decided that glycerol in A-1 electrophoretic mobility data on cultured human embryonic kidney cells subjected to electrophoresis in this buffer. The buffer composition is presented.

Electrophoresis-mass spectrometry probe

DOEpatents

Andresen, B.D.; Fought, E.R.

1987-11-10

The invention involves a new technique for the separation of complex mixtures of chemicals, which utilizes a unique interface probe for conventional mass spectrometers which allows the electrophoretically separated compounds to be analyzed in real-time by a mass spectrometer. This new chemical analysis interface, which couples electrophoresis with mass spectrometry, allows complex mixtures to be analyzed very rapidly, with much greater specificity, and with greater sensitivity. The interface or probe provides a means whereby large and/or polar molecules in complex mixtures to be completely characterized. The preferred embodiment of the probe utilizes a double capillary tip which allows the probe tip to be continually wetted by the buffer, which provides for increased heat dissipation, and results in a continually operating interface which is more durable and electronically stable than the illustrated single capillary tip probe interface. 8 figs.

Chaotropic salts: novel modifiers for the capillary electrophoretic analysis of benzodiazepines.

PubMed

Su, Hsiu-Li; Lan, Min-Tsu; Lin, Kuan-Wen; Hsieh, You-Zung

2008-08-01

This paper describes a CE method for analyzing benzodiazepines using the chaotropic salts lithium trifluoromethanesulfonate (LiOTf), lithium hexafluorophosphate (LiPF(6)), and lithium bis(trifluoromethanesulfonyl)imide (LiNTf(2)) as modifiers in the running buffer. Although adequate resolution of seven benzodiazepine analytes occurred under the influence of each of the chaotropic anions, the separation efficiency was highest when bis(trifluoromethanesulfonyl)imide (Tf(2)N(-)) was the modifier. We applied affinity CE in conjunction with linear analysis to determine the association constants for the formation of complexes between the Tf(2)N(-) anion and the benzodiazepines. According to the estimated Gibbs free energies, the interactions between this chaotropic anion and the benzodiazepines were either ion-dipole or ion-induced dipole interactions. Adding chaotropic salts as modifiers into CE buffers is a simple and reproducible technique for separating benzodiazepines.

Separation of 1-23-kb complementary DNA strands by urea-agarose gel electrophoresis.

PubMed

Hegedüs, Eva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

2009-09-01

Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea-agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of approximately 1-20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-dependent conformation impinging on the electrophoretic mobility of the ss molecules. This phenomenon can be exploited for the efficient preparation of strand-specific probes and for the separation of the complementary DNA strands for subsequent analysis, offering a new tool for various cell biological research areas.

Purification and cultivation of human pituitary growth hormone secreting cells

NASA Technical Reports Server (NTRS)

Hymer, W. C.

1984-01-01

A multiphase study was conducted to examine the properties of growth hormone cells. Topics investigated included: (1) to determine if growth hormone (GH) cells contained within the rat pituitary gland can be separated from the other hormone producing cell types by continuous flow electrophoresis (CFE); (2) to determine what role, if any, gravity plays in the electrophoretic separation of GH cells; (3) to compare in vitro GH release from rat pituitary cells previously exposed to microgravity conditions vs release from cells not exposed to microgravity; (4) to determine if the frequency of different hormone producing pituitary cell types contained in cell suspensions can be quantitated by flow cytometry; and (5) to determine if GH contained within the human post mortem pituitary gland can be purified by CFE. Specific experimental procedures and results are included.

Photothermal trace detection in capillary electrophoresis for biomedical diagnostics and toxic materials (invited)

NASA Astrophysics Data System (ADS)

Faubel, Werner; Heissler, Stefan; Pyell, Ute; Ragozina, Natalia

2003-01-01

Two applications of a near-field thermal lens capillary electrophoresis detector in the deep ultraviolet region (pump beam 257 nm wavelength) will be presented: (1) Capillary electrophoretic determination of the pharmaceuticals Tramadol, Verapamil, and Papaverin. Direct separation techniques were used for the different classes of substances with characteristic absorbance spectra. The combination of capillary electrophoresis and the highly sensitive detection with thermal lens spectroscopy permits the analysis of nanoliter volume samples common in biomedical diagnostics without any preconcentration step. (2) The determination of (nonfluorescent) nitro aromatic explosives in contaminated soil. These compounds are detected with the laboratory built thermal lens detector after their separation by micellar electrokinetic chromatography. Its shown that this type of detection makes it possible to obtain limits of detection 1-2 orders of magnitude lower than those obtained with classical absorption spectrometric detection.

Three-dimensional fluorescence analysis of chernozem humic acids and their electrophoretic fractions

NASA Astrophysics Data System (ADS)

Trubetskoi, O. A.; Trubetskaya, O. E.

2017-09-01

Polyacrylamide gel electrophoresis in combination with size-exclusion chromatography (SEC-PAGE) has been used to obtain stable electrophoretic fractions of different molecular size (MS) from chernozem humic acids (HAs). Three-dimensional fluorescence charts of chernozem HAs and their fractions have been obtained for the first time, and all fluorescence excitation-emission maxima have been identified in the excitation wavelength range of 250-500 nm. It has been found that fractionation by the SEC-PAGE method results in a nonuniform distribution of protein- and humin-like fluorescence of the original HA preparation among the electrophoretic fractions. The electrophoretic fractions of the highest and medium MSs have only the main protein-like fluorescence maximum and traces of humin-like fluorescence. In the electrophoretic fraction of the lowest MS, the intensity of protein-like fluorescence is low, but the major part of humin-like fluorescence is localized there. Relationships between the intensity of protein-like fluorescence and the weight distribution of amino acids have been revealed, as well as between the degree of aromaticity and the intensity of humin-like fluorescence in electrophoretic fractions of different MSs. The obtained relationships can be useful in the interpretation of the spatial structural organization and ecological functions of soil HAs.

Enhanced sludge dewatering by electrofiltration. A feasibility study.

PubMed

Saveyn, H; Huybregts, L; Van der Meeren, P

2001-01-01

Sludge treatment is a major issue in today's waste water treatment. One of the problems encountered is the limiting dewaterability of mainly biological sludges, causing high final treatment costs for incineration or landfill. Although during recent years, improvements are realised in the field of dewatering, the actual dry solids content after dewatering remains at a maximum value of about 35%. In order to increase the dry solids content, the technique of electrofiltration was investigated. Electrofiltration is the combination of two known techniques, traditional pressure filtration and electroosmotic/electrophoretic dewatering. Pressure filtration is based on pressure as the driving force for dewatering a sludge. Limitations hereby lie in the clogging of the filter cloth due to the build-up of the filtercake. Electroosmotic/electrophoretic dewatering is based on an electric field to separate sludge colloid particles from the surrounding liquid by placing the sludge liquor between two oppositely charged electrodes. In this case, mobile sludge particles will move to one electrode due to their natural surface charge, and the liquid phase will be collected at the oppositely charged electrode. Combination of both techniques makes it possible to create a more homogeneous filter cake and prevent the filter from clogging, resulting in higher cake dry solids contents and shorter filtration cycles. To investigate the feasibility of this technique for the dewatering of activated sludge, a filter unit was developed for investigations on lab scale. Multiple dewatering tests were performed in which the electric parameters for electrofiltration were varied. It was derived from these experiments that very high filter cake dry solids contents (to more than 60%), and short filtration cycles were attainable by using a relatively small electric DC field. The power consumption was very low compared to the power needed to dewater sludge by thermal drying techniques. For this reason, this technique seems very promising for the dewatering of biological sludges.

Electrophoretic and Electrolytic Deposition of Ceramic Particles on Porous Substrates

DTIC Science & Technology

1990-08-30

hydrodynamic drag force exerted on the particle due to the electroosmotic flow of the solvent inside the pore, the electrophoretic force exerted on the...8217 - electrophoretic velocity UN - electroosmotic velocity b - pore mean radius D - diffusion coefficient k - local deposition rate Large Peclet numbers and small...experimentally as the charge is acquired spontaneously on mixing the particles with the solvent and it may be reversed upon addition ot ionic compounds. The

Electrophoretic and Electrolytic Deposition of Ceramic Particles on Porous Substrates

DTIC Science & Technology

1992-09-30

particle penetration is facilitated by the electrophoretic force exerted on it and the electroosmotic flow of the fluid into the pores. 1 2 The...skeleton showed that the whole cross--section of the graphite was impregnated. - The existence of an electroosmotic effect was demonstrated by the...Pe) and the Damkohler number (A): Pe ((U" + Us)b -kb where U" - electrophoretic velocity Um - electroosmotic velocity b - pore mean radius D

A macrocyclic polyamine as an anion receptor in the capillary electrochromatographic separation of carbohydrates.

PubMed

Liu, Chuen-Ying; Chen, Tse-Hsien; Misra, Tarun Kumar

2007-06-22

An analytical approach of the 32-membered macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N8) was described for the capillary electrochromatographic (CEC) separation of derivatized mono- and disaccharides. The column displayed reversal electroosmotic flow (EOF) at pH below 7.0, while a cathodic EOF was shown at pH above 7.0. The reductive amination of saccharides was carried out with p-aminobenzoic acid. Some parameters that affect the CEC separations were investigated. Several competitive ligands, such as Tris, EDTA and phosphate were also examined for the effect on the performance. We achieved a complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side). The relative standard deviation of the retention time measured for each sample was less than 4% in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. With the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.

Microfluidic devices fabricated in poly(methyl methacrylate) using hot-embossing with integrated sampling capillary and fiber optics for fluorescence detection.

PubMed

Qi, Shize; Liu, Xuezhu; Ford, Sean; Barrows, James; Thomas, Gloria; Kelly, Kevin; McCandless, Andrew; Lian, Kun; Goettert, Jost; Soper, Steven A

2002-05-01

High-aspect-ratio microstructures have been prepared using hot-embossing techniques in poly(methyl methacrylate) (PMMA) from Ni-based molding dies prepared using LIGA (Lithographie, Galvanoformung, Abformung). Due to the small amount of mask undercutting associated with X-ray lithography and the high energy X-ray beam used during photoresist patterning, deep structures with sharp and smooth sidewalls have been prepared. The Ni-electroforms produced devices with minimal replication errors using hot-embossing at a turn around time of approximately 5 min per device. In addition, several different polymers (with different glass transition temperatures) could be effectively molded with these Ni-electroforms and many devices (>300) molded with the same master without any noticeable degradation. The PMMA devices consisted of deep and narrow channels for insertion of a capillary for the automated electrokinetic loading of sample into the microfluidic device and also, a pair of optical fibers for shuttling laser light to the detection zone and collecting the resulting emission for fluorescence analysis. Electrophoretic separations of double-stranded DNA ladders Phi X174 digested with Hae III) were performed with fluorescence detection accomplished using near-IR excitation. It was found that the narrow width of the channels did not contribute significantly to electrophoretic zone broadening and the plate numbers generated in the extended length separation channel allowed sorting of the 271/281 base pair fragments associated with this sizing ladder when electrophoresed in methylcellulose entangled polymer solutions. The dual fiber detector produced sub-attomole detection limits with the entire detector, including laser source, electronics and photon transducer, situated in a single box measuring 3'' x 10" x 14".

Mass spectrometric analysis of electrophoretically separated allergens and proteases in grass pollen diffusates

PubMed Central

Raftery, Mark J; Saldanha, Rohit G; Geczy, Carolyn L; Kumar, Rakesh K

2003-01-01

Background Pollens are important triggers for allergic asthma and seasonal rhinitis, and proteases released by major allergenic pollens can injure airway epithelial cells in vitro. Disruption of mucosal epithelial integrity by proteases released by inhaled pollens could promote allergic sensitisation. Methods Pollen diffusates from Kentucky blue grass (Poa pratensis), rye grass (Lolium perenne) and Bermuda grass (Cynodon dactylon) were assessed for peptidase activity using a fluorogenic substrate, as well as by gelatin zymography. Following one- or two-dimensional gel electrophoresis, Coomassie-stained individual bands/spots were excised, subjected to tryptic digestion and analysed by mass spectrometry, either MALDI reflectron TOF or microcapillary liquid chromatography MS-MS. Database searches were used to identify allergens and other plant proteins in pollen diffusates. Results All pollen diffusates tested exhibited peptidase activity. Gelatin zymography revealed high Mr proteolytic activity at ~ 95,000 in all diffusates and additional proteolytic bands in rye and Bermuda grass diffusates, which appeared to be serine proteases on the basis of inhibition studies. A proteolytic band at Mr ~ 35,000 in Bermuda grass diffusate, which corresponded to an intense band detected by Western blotting using a monoclonal antibody to the timothy grass (Phleum pratense) group 1 allergen Phl p 1, was identified by mass spectrometric analysis as the group 1 allergen Cyn d 1. Two-dimensional analysis similarly demonstrated proteolytic activity corresponding to protein spots identified as Cyn d 1. Conclusion One- and two-dimensional electrophoretic separation, combined with analysis by mass spectrometry, is useful for rapid determination of the identities of pollen proteins. A component of the proteolytic activity in Bermuda grass diffusate is likely to be related to the allergen Cyn d 1. PMID:14577842

Differentiation of highly virulent strains of Streptococcus suis serotype 2 according to glutamate dehydrogenase electrophoretic and sequence type.

PubMed

Kutz, Russell; Okwumabua, Ogi

2008-10-01

The glutamate dehydrogenase (GDH) enzymes of 19 Streptococcus suis serotype 2 strains, consisting of 18 swine isolates and 1 human clinical isolate from a geographically varied collection, were analyzed by activity staining on a nondenaturing gel. All seven (100%) of the highly virulent strains tested produced an electrophoretic type (ET) distinct from those of moderately virulent and nonvirulent strains. By PCR and nucleotide sequence determination, the gdh genes of the 19 strains and of 2 highly virulent strains involved in recent Chinese outbreaks yielded a 1,820-bp fragment containing an open reading frame of 1,344 nucleotides, which encodes a protein of 448 amino acid residues with a calculated molecular mass of approximately 49 kDa. The nucleotide sequences contained base pair differences, but most were silent. Cluster analysis of the deduced amino acid sequences separated the isolates into three groups. Group I (ETI) consisted of the seven highly virulent isolates and the two Chinese outbreak strains, containing Ala(299)-to-Ser, Glu(305)-to-Lys, and Glu(330)-to-Lys amino acid substitutions compared with groups II and III (ETII). Groups II and III consisted of moderately virulent and nonvirulent strains, which are separated from each other by Tyr(72)-to-Asp and Thr(296)-to-Ala substitutions. Gene exchange studies resulted in the change of ETI to ETII and vice versa. A spectrophotometric activity assay for GDH did not show significant differences between the groups. These results suggest that the GDH ETs and sequence types may serve as useful markers in predicting the pathogenic behavior of strains of this serotype and that the molecular basis for the observed differences in the ETs was amino acid substitutions and not deletion, insertion, or processing uniqueness.

Micelle to solvent stacking of organic cations in micellar electrokinetic chromatography with sodium dodecyl sulfate.

PubMed

Quirino, Joselito P; Aranas, Agnes T

2011-10-14

The on-line sample concentration technique, micelle to solvent stacking (MSS), was studied for small organic cations (quaternary ammonium herbicides, β-blocker drugs, and tricyclic antidepressant drugs) in reversed migration micellar electrokinetic chromatography. Electrokinetic chromatography was carried out in fused silica capillaries with a background solution of sodium dodecyl sulfate (SDS) in a low pH phosphate buffer. MSS was performed using anionic SDS micelles in the sample solution for analyte transport and methanol or acetonitrile as organic solvent in the background solution for analyte effective electrophoretic mobility reversal. The solvent also allowed for the separation of the analyte test mixtures. A model for focusing and separation was developed and the mobility reversal that involved micelle collapse was experimentally verified. The effect of analyte retention factor was observed by changing the % organic solvent in the background solution or the concentration of SDS in the sample matrix. With an injection length of 31.9 cm (77% of effective capillary length) for the 7 test drugs, the LODs (S/N=3) of 5-14 ng/mL were 101-346-fold better when compared to typical injection. The linearity (R(2), range=0.025-0.8 μg/mL), intraday and interday repeatability (%RSD, n=10) were ≥0.988, <6.0% and <8.5%, respectively. In addition, analysis of spiked urine samples after 10-fold dilution with the sample matrix yielded LODs=0.02-0.10 μg/mL. These LODs are comparable to published electrophoretic methods that required off-line sample concentration. However, the practicality of the technique for more complex samples will rely on dedicated sample preparation schemes. Copyright © 2011 Elsevier B.V. All rights reserved.

THE EFFECTS OF HIGH VOLTAGE CATHODE RAY IONIZING RADIATION ON SOME OF THE PHYSICAL AND CHEMICAL PROPERTIES OF WHEAT FLOUR PROTEIN

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

Alsup, E.B.

1959-01-01

The effect of irradiation on some of the physical and chemical properties of wheat flour protein was determined. A study of the baking quality and palatability of an irradiated flour product, yields of crude gluten from irradiated flour, and electrophoretic analyses of some of the protein fractions of irradiated flour were studied. Triangle taste tests indicated that 50000 rep was the dosage level at which the irradiation treatment of flour could be detected in baking powder biscuits. Taste panel scores for color, odor, and baking characteristics of biscuits made with flour treated with 100000 rep or less of ionizing radiation,more » however, were not significantly different from the scores for the control biscuits. An off-odor and darkening in color of the biscuits were readily apparent at a dosage of 500000 rep. The weight of crude gluten extracted from flour was greater from flour exposed to dosages of 1000000 rep or less than from the control flour, but no differences were found in the volumes of the baked balls of crude gluten. This indicates a greater water retention with the irradiated flour. Very small yields of crude gluten at dosages of 3000000 rep or more indicated that definite changes had occurred in the protein structure. Electrophoretic analyses by the moving boundary method were performed on acetic acid extracts of whole flour in a citric acid-disodium phos, phate buffer pH 2.2, ionic strength 0.024. Electrophoretic patterns obtained from the non-irradiated flour indicated that there were at least six separate protein components present, including one which was fast-moving and high in relative concentration, one which was slow-moving and medium high in relative concentration, and four relatively small components. Irradiation of the flour with 300000 rep produced no apparent changes in the electrophoretic patterns obtained, but it 1000000 rep or more, the relative concentrations of the two largest components were changed (the slow-moving one becoming larger and the fastermoving one smaller with each increase in dosage). Also with increased radiation dosages, the peaks of the smaller components became less distinct until only the two main peaks were distinguishable at 10000000 rep. (Dissertition Abstr., 23: No. 4, 1962)« less

Fabrication and kinetics study of nano-Al/NiO thermite film by electrophoretic deposition.

PubMed

Zhang, Daixiong; Li, Xueming

2015-05-21

Nano-Al/NiO thermites were successfully prepared as film by electrophoretic deposition (EPD). For the key issue of this EPD, a mixture solvent of ethanol-acetylacetone (1:1 in volume) containing 0.00025 M nitric acid was proved to be a suitable dispersion system for EPD. The kinetics of electrophoretic deposition for both nano-Al and nano-NiO were investigated; the linear relation between deposition weight and deposition time in short time and parabolic relation in prolonged time were observed in both EPDs. The critical transition time between linear deposition kinetics and parabolic deposition kinetics for nano-Al and nano-NiO were 20 and 10 min, respectively. The theoretical calculation of the kinetics of electrophoretic deposition revealed that the equivalence ratio of nano-Al/NiO thermites film would be affected by the behavior of electrophoretic deposition for nano-Al and nano-NiO. The equivalence ratio remained steady when the linear deposition kinetics dominated for both nano-Al and nano-NiO. The equivalence ratio would change with deposition time when deposition kinetics for nano-NiO changed into parabolic kinetics dominated after 10 min. Therefore, the rule was suggested to be suitable for other EPD of bicomposites. We also studied thermodynamic properties of electrophoretic nano-Al/NiO thermites film as well as combustion performance.

Preparation and application of microcapsule-encapsulated color electrophortic fluid in Isopar M system for electrophoretic display

NASA Astrophysics Data System (ADS)

Sun, Cui; Feng, Ya-Qing; Zhang, Bao; Li, Xiang-Gao; Shao, Ji-Zhou; Han, Jing-Jing; Chen, Xu

2013-05-01

The use of Isopar M as a liquid suspending fluid for electrophoretic display was studied. The dispersion stability and chargeability of pigments suspended in Isopar M were investigated. Polyisobutylene monosuccinimide (T-151) as the charge control additive in Isopar M electrophoretic fluid can provide a good electrophoretic mobility to the particles. The wall materials of a series of blue-white, red-white and yellow-white dual-particle microcapsules were prepared by in situ polymerization of urea and formaldehyde. The mass ratio of wall/core material was a key factor in influencing the yield of microcapsules. The concentration of resorcinol has an impact on the surface morphology and mechanical strength of microcapsule wall. Microcapsules' surface morphologies were characterized by optical microscopy and scanning electron microscopy. The performance of the microcapsules with different binder materials and adhesive layers were investigated. Contrast ratio of microcapsules display device were tested every 10 days for a period of 90 days. The compatibility of Isopar M with both the electrophoretic particles and bounding capsule was studied.

Electrophoretic kinetics of concentrated TiO2 nanoparticle suspensions in aprotic solvent

NASA Astrophysics Data System (ADS)

Lee, So-Yeon; Yim, Jung-Ryoul; Lee, Se-Hee; Choi, In-Suk; Nam, Ki Tae; Joo, Young-Chang

2018-01-01

We studied the dependences of the concentration of additive and particle size on the electrophoretic mobility of TiO2 nanoparticles. A high concentration of TiO2 nanoparticles was dispersed in aprotic solvent, which is similar to the operating conditions of electrophoretic applications. Because spectroscopy has limits to measuring the electrophoretic mobility of concentrated suspensions in aprotic solvents, we developed a new measurement to determine the electrophoretic mobility of particles using the reflectance change according to the motion of the particles. TiO2 nanoparticles with sizes of 31 nm to 164 nm were synthesized by hydrolysis and were dispersed in cyclohexanone with a dye (Sudan Black B) for use in the new measurement method. In a concentrated suspension in aprotic solvent, the mobility of the particles was proportional to the dye concentration and was inversely proportional to the size of the particles. This infers that the particle size influences the drag force rather than the surface charge, and therefore, to increase the mobility by changing the surface charge, an additive is effective. [Figure not available: see fulltext.

Simultaneous sizing and electrophoretic mobility measurement of sub-micron particles using Brownian motion

PubMed Central

Palanisami, Akilan; Miller, John H.

2011-01-01

The size and surface chemistry of micron scale particles are of fundamental importance in studies of biology and air particulate pollution. However, typical electrophoretic measurements of these and other sub-micron scale particles (300 nm – 1 μm) cannot resolve size information within heterogeneous mixtures unambiguously. Using optical microscopy, we monitor electrophoretic motion together with the Brownian velocity fluctuations—using the latter to measure size by either the Green-Kubo relation or by calibration from known size standards. Particle diameters are resolved to ±12% with 95% confidence. Strikingly, the size resolution improves as particle size decreases due to the increased Brownian motion. The sizing ability of the Brownian assessed electrophoresis method described here complements the electrophoretic mobility resolution of traditional capillary electrophoresis. PMID:20882556

Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

NASA Technical Reports Server (NTRS)

Kunze, M. E.

1985-01-01

A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

Insight into nanoparticle charging mechanism in nonpolar solvents to control the formation of Pt nanoparticle monolayers by electrophoretic deposition

DOE PAGES

Cernohorsky, Ondrej; Grym, Jan; Yatskiv, Roman; ...

2016-08-13

We report on the formation of Pt nanoparticle monolayers by electrophoretic deposition from nonpolar solvents. First, the growth kinetics of Pt nanoparticles prepared by the reverse micelle technique are described in detail. Second, a model of nanoparticle charging in nonpolar media is discussed and methods to control the nanoparticle charging are proposed. Lastly, essential parameters of the electrophoretic deposition process to control the deposition of nanoparticle monolayers are discussed and mechanisms of their formation are analyzed.

Insight into nanoparticle charging mechanism in nonpolar solvents to control the formation of Pt nanoparticle monolayers by electrophoretic deposition

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

Cernohorsky, Ondrej; Grym, Jan; Yatskiv, Roman

We report on the formation of Pt nanoparticle monolayers by electrophoretic deposition from nonpolar solvents. First, the growth kinetics of Pt nanoparticles prepared by the reverse micelle technique are described in detail. Second, a model of nanoparticle charging in nonpolar media is discussed and methods to control the nanoparticle charging are proposed. Lastly, essential parameters of the electrophoretic deposition process to control the deposition of nanoparticle monolayers are discussed and mechanisms of their formation are analyzed.

Contact-lens type of micromachined hydrogenated amorphous Si fluorescence detector coupled with microfluidic electrophoresis devices

NASA Astrophysics Data System (ADS)

Kamei, Toshihiro; Wada, Takehito

2006-09-01

A 5.8-μm-thick SiO2/Ta2O5 multilayer optical interference filter was monolithically integrated and micromachined on a hydrogenated amorphous Si (a-Si :H) pin photodiode to form a fluorescence detector. A microfluidic electrophoresis device was mounted on a detection platform comprising a fluorescence-collecting half-ball lens and the micromachined fluorescence detector. The central aperture of the fluorescence detector allows semiconductor laser light to pass up through the detector and to irradiate an electrophoretic separation channel. The limit of detection is as low as 7nM of the fluorescein solution, and high-speed DNA fragment sizing can be achieved with high separation efficiency. The micromachined a-Si :H fluorescence detector exhibits high sensitivity for practical fluorescent labeling dyes as well as integration flexibility on various substances, making it ideal for application to portable microfluidic bioanalysis devices.

Method for enhanced accuracy in predicting peptides using liquid separations or chromatography

DOEpatents

Kangas, Lars J.; Auberry, Kenneth J.; Anderson, Gordon A.; Smith, Richard D.

2006-11-14

A method for predicting the elution time of a peptide in chromatographic and electrophoretic separations by first providing a data set of known elution times of known peptides, then creating a plurality of vectors, each vector having a plurality of dimensions, and each dimension representing the elution time of amino acids present in each of these known peptides from the data set. The elution time of any protein is then be predicted by first creating a vector by assigning dimensional values for the elution time of amino acids of at least one hypothetical peptide and then calculating a predicted elution time for the vector by performing a multivariate regression of the dimensional values of the hypothetical peptide using the dimensional values of the known peptides. Preferably, the multivariate regression is accomplished by the use of an artificial neural network and the elution times are first normalized using a transfer function.

CAPILLARY ELECTROPHORETIC BEHAVIOR OF SEVEN SULFONYLUREAS

EPA Science Inventory

The electrophoretic behavior of bensulfuron Me, sulfometuron Me, nicosulfuron (Accent), chlorimuron Et, thifensulfuron Me (Harmony), metsulfuron Me, and chlorsulfuron was studied under capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) conditio...

Accelerated SDS depletion from proteins by transmembrane electrophoresis: Impacts of Joule heating.

PubMed

Unterlander, Nicole; Doucette, Alan Austin

2018-02-08

SDS plays a key role in proteomics workflows, including protein extraction, solubilization and mass-based separations (e.g. SDS-PAGE, GELFrEE). However, SDS interferes with mass spectrometry and so it must be removed prior to analysis. We recently introduced an electrophoretic platform, termed transmembrane electrophoresis (TME), enabling extensive depletion of SDS from proteins in solution with exceptional protein yields. However, our prior TME runs required 1 h to complete, being limited by Joule heating which causes protein aggregation at higher operating currents. Here, we demonstrate effective strategies to maintain lower TME sample temperatures, permitting accelerated SDS depletion. Among these strategies, the use of a magnetic stir bar to continuously agitate a model protein system (BSA) allows SDS to be depleted below 100 ppm (>98% removal) within 10 min of TME operations, while maintaining exceptional protein recovery (>95%). Moreover, these modifications allow TME to operate without any user intervention, improving throughput and robustness of the approach. Through fits of our time-course SDS depletion curves to an exponential model, we calculate SDS depletion half-lives as low as 1.2 min. This promising electrophoretic platform should provide proteomics researchers with an effective purification strategy to enable MS characterization of SDS-containing proteins. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of vanillin in vanilla perfumes and air by capillary electrophoresis.

PubMed

Minematsu, Saaya; Xuan, Guang-Shan; Wu, Xing-Zheng

2013-12-01

The present study investigated capillary electrophoretic detection of vanillin in vanilla perfume and air. An UV-absorbance detector was used in a home-made capillary electrophoretic instrument. A fused silica capillary (outer diameter: 364 μm, inner diameter: 50 μm) was used as a separation capillary, and a high electric voltage (20 kV) was applied across the two ends of the capillary. Total length of the capillary was 70 cm, and the effective length was 55 cm. Experimental results showed that the vanillin peak was detected at about 600, 450, and 500 seconds when pH of running buffers in CE were 7.2, 9.3, and 11.5, respectively. The peak area of vanillin was proportional to its concentration in the range of 0-10(-2) mol/L. The detection limit was about 10(-5) mol/L. Vanillin concentration in a 1% vanilla perfume sample was determined to be about 3×10(-4) mol/L, agreed well with that obtained by a HPLC method. Furthermore, determination of vanillin in air by combination of CE and active carbon adsorption method was investigated. Copyright © 2013 The Research Centre for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V. All rights reserved.

Dibutyryl cyclic AMP stimulates expression of ependymin mRNA and the synthesis and release of the protein into the culture medium by neuroblastoma cells (NB2a/d1).

PubMed

Shashoua, V E; Nolan, P M; Shea, T B; Milinazzo, B

1992-06-01

Northern blot, immunoprecipitation, and gel electrophoretic data demonstrate that the mouse neuroblastoma NB2a/d1 cells express ependymin mRNA and synthesize and release into the culture medium a protein with immunoreactivity and electrophoretic mobility properties identical to ependymin. This is a brain extracellular glycoprotein that has been implicated in the consolidation process of memory formation and neuronal regeneration. In labeling experiments with 35S-methionine, dibutyrylcyclic3',5'-adenosine-monophosphate (dbcAMP) was found to stimulate the expression of ependymin mRNA and the enhanced synthesis and release of ependymin into the culture medium at the same time that dbcAMP stimulation of neurite outgrowth takes place. These results are consistent with the proposed role of the protein in the mechanism of neuronal regeneration and synaptogenesis. The data indicate that the NB2a/d1 cell line is a good model system for studies of the functional properties of ependymin.

Electrophoretic mobility shift scanning using an automated infrared DNA sequencer.

PubMed

Sano, M; Ohyama, A; Takase, K; Yamamoto, M; Machida, M

2001-11-01

Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which includes cooling the gel plates with a reduced well-to-read length, has made it possible to detect shifted bands within 1 h. Further, we have developed a rapid ligation-based method for generating IRDye-labeled probes with an approximately 60% cost reduction. This method has the advantages of real-time scanning, stability of labeled probes, and better safety associated with nonradioactive methods of detection. Analysis of a promoter from an industrially important filamentous fungus, Aspergillus oryzae, in a prototype experiment revealed that the method we describe has potential for use in systematic scanning and identification of the functionally important elements to which cellular factors bind in a sequence-specific manner.

Electrophoretic deposition (EPD): Mechanisms, kinetics, and application to ceramics

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

Sarkar, P.; Nicholson, P.S.

1996-08-01

The mechanisms of electrophoretic deposition (EPD) are discussed and their shortcomings identified. The kinetics of the processes involved are analyzed for constant-current and constant-voltage conditions. A method of determining the Hamaker constant of suspended particles is developed by modeling the relationship between the particle interaction energy and the suspension stability. A three-probe dc technique is used to map the voltage profile around the depositing electrode, and the results are used to explain discrepancies between the calculated and experimentally observed voltage drops during deposition. A mechanism of deposition is proposed based on DLVO theory and particle double-layer distortion/thinning on application ofmore » a dc field to the suspension. Kinetic equations are developed for constant-current and constant-voltage EPD using mass balance conditions; these are verified by experiments. After the phenomenon is introduced and discussed, a critique of the application of EPD to the synthesis of ceramic shapes and coatings is given.« less

Methods and apparatus for analysis of chromatographic migration patterns

DOEpatents

Stockham, T.G.; Ives, J.T.

1993-12-28

A method and apparatus are presented for sharpening signal peaks in a signal representing the distribution of biological or chemical components of a mixture separated by a chromatographic technique such as, but not limited to, electrophoresis. A key step in the method is the use of a blind deconvolution technique, presently embodied as homomorphic filtering, to reduce the contribution of a blurring function to the signal encoding the peaks of the distribution. The invention further includes steps and apparatus directed to determination of a nucleotide sequence from a set of four such signals representing DNA sequence data derived by electrophoretic means. 16 figures.

Purification of plant viral and satellite double-stranded RNAs on DEAE monoliths.

PubMed

Krajacić, Mladen; Ivancic-Jelecki, Jelena; Forcic, Dubravko; Vrdoljak, Anto; Skorić, Dijana

2007-03-09

Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens' presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.

Indirect photometric detection of boron cluster anions electrophoretically separated in methanol.

PubMed

Vítová, Lada; Fojt, Lukáš; Vespalec, Radim

2014-04-18

3,5-Dinitrobenzoate and picrate are light absorbing anions pertinent to indirect photometric detection of boron cluster anions in buffered methanolic background electrolytes (BGEs). Tris(hydroxymethyl)aminomethane and morpholine have been used as buffering bases, which eliminated baseline steps, and minimized the baseline noise. In methanolic BGEs, mobilities of boron cluster anions depend on both ionic constituents of the BGE buffer. This dependence can be explained by ion pair interaction of detected anions with BGE cations, which are not bonded into ion pairs with the BGE anions. The former ion pair interaction decreases sensitivity of the indirect photometric detection. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrophoretic manipulation of multiple-emulsion droplets

NASA Astrophysics Data System (ADS)

Schoeler, Andreas M.; Josephides, Dimitris N.; Chaurasia, Ankur S.; Sajjadi, Shahriar; Mesquida, Patrick

2014-02-01

Electrophoretic manipulation of multiple-emulsion oil-in-water-in-oil (O/W)/O and water-in-oil-in-water-in-oil (W/O/W)/O core-shell droplets is shown. It was found that the electrophoretic mobility of the droplets is determined solely by the outer water shell, regardless of size or composition of the inner droplets. It was observed that the surface charge of the outer water shell can be changed and the polarity can be reversed through contact with a biased electrode in a similar way as with simple W/O droplets. Furthermore, addition of the anionic surfactant, sodium dodecyl sulfate to the outer water shell reverses the initial polarity and hence, electrophoretic mobility of the core-shell droplets before contact with an electrode. The results have practical implications for the manipulation of oil droplets in a continuous oil phase.

An Investigation of Traveling-Wave Electrophoresis using a Trigonometric Potential

NASA Astrophysics Data System (ADS)

Vopal, James

Traveling-wave electrophoresis, a technique for microfluidic separations in lab-on-achip devices, is investigated using a trigonometric model that naturally incorporates the spatial periodicity of the device. Traveling-wave electrophoresis can be used to separate high-mobility ions from low-mobility ions in forensic and medical applications, with a separation threshold that can be tuned for specific applications by simply choosing the traveling wave frequency. Our simulations predict plateaus in the average ion velocity verses the mobility, plateaus that correspond to Farey fractions and yield Devil's staircases for non-zero discreteness values. The plateaus indicate that ions with different mobilities can travel with the same average velocity. To determine the conditions for chaos, Lyapunov exponents and contact maps are employed. Through the use of contact maps, the chaotic trajectories are determined to be either narrowband or broadband. Narrowband chaotic trajectories are exhibited in the plateaus of the average velocity, while broadband chaotic trajectories are exhibited where the average velocity varies nonmonotonically with the mobility. Narrowband chaos will be investigated in future work incorporating the role of diffusion. The results of this and future work can be used to develop new tools for electrophoretic separation.

Capillary electrophoretic study of dibasic acids of different structures: Relation to separation of oxidative intermediates in remediation

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

Yu, Z.; Cocke, D.L.

Dicarboxylic acids are important in environmental chemistry because they are intermediates in oxidative processes involved in natural remediation and waste management processes such as oxidative detoxification and advanced oxidation. Capillary electrophoresis (CE), a promising technique for separating and analyzing these intermediates, has been used to examine a series of dibasic acids of different structures and conformations. This series includes malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, fumaric acid, phthalic acid, and trans, trans-muconic acid. The CE parameters as well as structural variations (molecular structure and molecular isomers, buffer composition, pH, applied voltage, injection mode, current,more » temperature, and detection wavelength) that affect the separations and analytical results have been examined in this study. Those factors that affect the separation have been delineated. Among these parameters, the pH has been found to be the most important, which affects the double-layer of the capillary wall, the electro-osmotic flow and analyte mobility. The optimum pH for separating these dibasic acids, as well as the other parameters are discussed in detail and related to the development of methods for analyzing oxidation intermediates in oxidative waste management procedures.« less

Development anmd testing of electrophoresis solutions. Task I.1: Development of optimal buffer system

NASA Technical Reports Server (NTRS)

1985-01-01

Two buffers were explored for testing: low ionic strength electrophoresis buffer with and without density gradient material. It was found that the electrophoresis routine was better tolerated when Ficoll was present. The results of a viability study of primary human fetal kidney (HFK-1) cells at the first passage are shown. Cell strain HFK-1 was used in several experiments at the first and second passage. The HFK consisted mainly of fibroblasts, and HFK-1 has a high epithelioid cell content. The chromosomes of HFK were examined and found to be euploid. The stock medium for cell electrophoresis is described. In this solution density gradient solutes such as sucrose and Ficoll are dissolved to bring the osmolarity to 0.30. Its ionic strength is less than 0.01M, and its conductivity is usually 0.0011 mho/cm. Methods for viability determination included direct microscopic counting of the percent cells attached and spread within 24 hr of plating test cultures or electrophoretically separated fractions. The Cytograf viability assay concept was tested, and shown that blue stained cells scatter less light into the 0.8 to 3.3 deg angular interval than do unstained cells.

Inhibition of human low-density lipoprotein oxidation in vitro by Maharishi Ayur-Veda herbal mixtures.

PubMed

Sharma, H M; Hanna, A N; Kauffman, E M; Newman, H A

1992-12-01

In this study, we examined the effect of the Maharishi Ayur-Veda herbal mixtures (MAHMs) Maharishi Amrit Kalash-4 and -5 (M-4 and M-5), MA-631, and Maharishi Coffee Substitute (MCS) on low-density lipoprotein (LDL) oxidation and compared the potency of these mixtures to ascorbic acid, alpha-tocopherol, and probucol. LDL was incubated in 95% air and 5% CO2, with or without 3 microM Cu(+2), in the presence or absence of MAHMs, for 6 or 24 h. In a separate experiment, LDL was incubated as above except MAHMs were added at 0, 1.5, and 3.5 h after incubation started to assess their effect on initiation and propagation of LDL oxidation. Our results demonstrate that MAHMs caused concentration-dependent inhibition of LDL oxidation as assessed by thiobarbituric acid-reactive substances and electrophoretic mobility. The MAHM showed more antioxidant potency in preventing LDL oxidation than ascorbic acid, alpha-tocopherol, or probucol. Also, MAHMs inhibited both initiation and propagation of cupric ion-catalyzed LDL oxidation. These results suggest the importance of further research on these herbal mixtures in the investigation of atherosclerosis and free radical-induced injury.

On-chip Micro- and Nanofluidic Electrokinetic Injection and Separation for PEGylation Analysis

NASA Astrophysics Data System (ADS)

Shelton, Elijah; Baum, Mary; Morse, Dan; Pennathur, Sumita; Pennathur Nanofluidics Laboratory Collaboration; Morse Laboratory Collaboration

2012-11-01

We present an experimental study of micro- and nanofluidic electrokinetic injection and separation in borosilcate channels as a method for characterizing size and zeta potential of biomolecules-specifically polyethlylene glycol (PEG), keyhole limpet hemocyanine (KLH), and pegylated KLH. While pegylation (the conjugation of proteins with PEG) is an established technique for enhancing a protein's therapeutic properties, reliable characterization of these conjugations by traditional analysis techniques (i.e. gel-electrophoresis, zetasizer) remains a challenge. Using a three-step electrokinetic sequence (load, gate, and inject), FITC labeled species and a fluorescein tracer dye are injected into a channel where they separate according to differences in electrophoretic mobility. We find the average absolute mobility of pegylated subunit KLH in 1 micron channels to be 56% that of unpegylated subunit KLH. In a 250 nm channel, we measure a 33% shift in the average absolute mobility of PEG dendrimers as compared to measurements in a 1 micron channel. These results begin to demonstrate how a micro- and nanofluidic-based approach might address the demand for effective and accessible nanoparticle characterization platforms. Supported by the Institute for Collaborative Biotechnologies.

Carbon nanotube/poly(ethylene-co-vinyl acetate) composite electrode for capillary electrophoretic determination of esculin and esculetin in Cortex Fraxini.

PubMed

Chen, Zhi; Zhang, Luyan; Chen, Gang

2009-10-01

In this report, a novel carbon nanotube/poly(ethylene-co-vinyl acetate) (CNT/EVA) composite electrode was developed for the amperometric detection in CE. The composite electrode was fabricated by packing a mixture of CNTs and melted EVA in a piece of fused-silica capillary under heat. It was coupled with CE for the separation and detection of esculin and esculetin in Cortex Fraxini, a traditional Chinese medicine, to demonstrate its feasibility and performance. Esculin and esculetin have been well separated within 9 min in a 40 cm long capillary at a separation voltage of 12 kV using a 50 mM borate buffer (pH 9.2). The new CNT-based CE detector offered significantly lower detection potentials, yielded enhanced S/N characteristics, and exhibited high resistance to surface fouling and enhanced stability. It showed long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n=15) and should also find a wide range of applications in other microfluidic analysis systems.

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip.

PubMed

Tsai, Shuo-Wen; Loughran, Michael; Suzuki, Hiroaki; Karube, Isao

2004-02-01

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.

Sub-minute method for simultaneous determination of aspartame, cyclamate, acesulfame-K and saccharin in food and pharmaceutical samples by capillary zone electrophoresis.

PubMed

Vistuba, Jacqueline Pereira; Dolzan, Maressa Danielli; Vitali, Luciano; de Oliveira, Marcone Augusto Leal; Micke, Gustavo Amadeu

2015-05-29

This paper reports the development of a sub-minute separation method by capillary zone electrophoresis for the determination of aspartame, cyclamate, acesulfame-K and saccharin in food products and pharmaceutical samples. Separations were performed in a fused uncoated silica capillary with UV detection at 220nm. Samples and standards were injected hydrodynamically using the short-end injection procedure. The electrophoretic system was operated under constant voltage of -30kV. The background electrolyte was composed of 45mmolL(-1) 2-amino-2-(hydroxymethyl)-1,3-propanediol and 15mmolL(-1) benzoic acid at pH 8.4. The separation time for all analytes was less than 1min. Evaluation of analytical parameters of the method showed good linearity (r(2)>0.9972), limit of detection of 3.3-6.4mgL(-1), intermediate precision better than 9.75% (peak area of sample) and recovery in the range of 91-117%. Copyright © 2015 Elsevier B.V. All rights reserved.

Separation of alpha-lactalbumin grafted- and non-grafted maghemite core/silica shell nanoparticles by capillary zone electrophoresis.

PubMed

Petr, Jan; Teste, Bruno; Descroix, Stéphanie; Siaugue, Jean-Michel; Gareil, Pierre; Varenne, Anne

2010-08-01

The use of nanoparticles (NPs) in immunodiagnostics is a challenging task for many reasons, including the need for miniaturization. In view of the development of an assay dedicated to an original, miniaturized and fully automated immunodiagnostics which aims to mimic in vivo interactions, magnetic zwitterionic bifunctional amino/polyethyleneoxide maghemite core/silica shell NPs functionalized with allergenic alpha-lactalbumin were characterized by CE. Proper analytical performances were obtained through semi-permanent capillary coating with didodecyldimethylammonium bromide (DDAB) or permanent capillary wall modification by hydroxypropylcellulose. The influence of experimental conditions (e.g. buffer component nature, pH, ionic strength, and electric field strength) on sample stability, electrophoretic mobility, and dispersion was investigated using either DDAB- or hydroxypropylcellulose-coated capillaries. Adsorption to the capillary wall and aggregation phenomena were evaluated according to the CE conditions. The proper choice of experimental conditions, i.e. separation under -10 kV in a 25 mM ionic strength MES/NaOH (pH 6.0) with a DDAB-coated capillary, allowed the separation of the grafted and the non-grafted NPs.

Simultaneous analysis of seven oligopeptides in microbial fuel cell by micro-fluidic chip with reflux injection mode.

PubMed

Wang, Wei; Wang, Zijian; Lin, Xiuli; Wang, ZongWen; Fu, FengFu

2012-10-15

In this work, a reflux injection mode for the cross form micro-fluidic chip was studied. This injection mode could flexibly control the length of sample plug from less than one channel width (<83 μm) to tens of channel widths (millimeter-sized) by adjusting the injection time. Namely, the separation resolution or sample detection sensitivity could be selectively improved by changing injection time. Composed of four steps, the reflux injection mode alleviated the electrophoretic sampling bias and prevented sample leakage successfully. On a micro-fluidic chip coupled with laser induced fluorescence (LIF) detector, the injection mode was applied to separate seven oligopeptides, namely GG, GL, RPP, KPV, VKK, WYD and YWS. All analytes were completely separated and detected within 12 min with detection limits of 25-625 nmol/L. At last, the proposed method had been successfully applied to detect oligopeptides consumed by bacillus licheniformis in anode chamber of microbial fuel cell (MFC) to study the effect of oligopeptides on the MFC running. Copyright © 2012 Elsevier B.V. All rights reserved.

A Rapid Method for Optimizing Running Temperature of Electrophoresis through Repetitive On-Chip CE Operations

PubMed Central

Kaneda, Shohei; Ono, Koichi; Fukuba, Tatsuhiro; Nojima, Takahiko; Yamamoto, Takatoki; Fujii, Teruo

2011-01-01

In this paper, a rapid and simple method to determine the optimal temperature conditions for denaturant electrophoresis using a temperature-controlled on-chip capillary electrophoresis (CE) device is presented. Since on-chip CE operations including sample loading, injection and separation are carried out just by switching the electric field, we can repeat consecutive run-to-run CE operations on a single on-chip CE device by programming the voltage sequences. By utilizing the high-speed separation and the repeatability of the on-chip CE, a series of electrophoretic operations with different running temperatures can be implemented. Using separations of reaction products of single-stranded DNA (ssDNA) with a peptide nucleic acid (PNA) oligomer, the effectiveness of the presented method to determine the optimal temperature conditions required to discriminate a single-base substitution (SBS) between two different ssDNAs is demonstrated. It is shown that a single run for one temperature condition can be executed within 4 min, and the optimal temperature to discriminate the SBS could be successfully found using the present method. PMID:21845077

Electrophoretic study of the genome of human rotavirus from Maceió, Brazil.

PubMed

Houly, C A; Uchoa, M M; Zaidan, A M; Gomes-Neto, A; de-Oliveira, F M; Athayde, M A; Almeida, M F; Pereira, H G

1986-01-01

Rotaviruses were detected by enzyme immunoassay (EIA) in 53 (13.3%) of 397 fecal samples from children with acute gastroenteritis in the city of Maceió, Alagoas, Brazil. Polyacrylamide gel electrophoretic (PAGE) patterns characteristic of rotavirus double-stranded RNA were detected in 51 (96.2%) of the 53 EIA-positive samples. Of the RNA-positive samples, 1 (2%) was classified as subgroup 1 (short profile), 49 (96%) as subgroup 2 (long profile) and 1 (2%) could not be classified because of the absence of bands 10 and 11. The strains of subgroup 2 showed a great degree of electrophoretic heterogeneity and could be divided into several subcategories. Two samples showed splitting of one of the genome segments. PAGE, a very sensitive method capable of identifying rotavirus RNA genomes, has demonstrated that human rotaviruses detected in Maceió present many differences in RNA electrophoretic patterns.

Electrophoresis of fd-virus particles: experiments and an analysis of the effect of finite rod lengths.

PubMed

Buitenhuis, Johan

2012-09-18

The electrophoretic mobility of rodlike fd viruses is measured and compared to theory, with the theoretical calculations performed according to Stigter (Stigter, D. Charged Colloidal Cylinder with a Gouy Double-Layer. J. Colloid Interface Sci. 1975, 53, 296-306. Stigter, D. Electrophoresis of Highly Charged Colloidal Cylinders in Univalent Salt- Solutions. 1. Mobility in Transverse Field. J. Phys. Chem. 1978, 82, 1417-1423. Stigter, D. Electrophoresis of Highly Charged Colloidal Cylinders in Univalent Salt Solutions. 2. Random Orientation in External Field and Application to Polyelectrolytes. J. Phys. Chem. 1978, 82, 1424-1429. Stigter, D. Theory of Conductance of Colloidal Electrolytes in Univalent Salt Solutions. J. Phys. Chem. 1979, 83, 1663-1670), who describes the electrophoretic mobility of infinite cylinders including relaxation effects. Using the dissociation constants of the ionizable groups on the surfaces of the fd viruses, we can calculate the mobility without any adjustable parameter (apart from the possible Stern layer thickness). In addition, the approximation in the theoretical description of Stigter (and others) of using a model of infinitely long cylinders, which consequently is independent of the aspect ratio, is examined by performing more elaborate numerical calculations for finite cylinders. It is shown that, although the electrophoretic mobility of cylindrical particles in the limit of low ionic strength depends on the aspect ratio much more than "end effects", at moderate and high ionic strengths the finite and infinite cylinder models differ only to a degree that can be attributed to end effects. Furthermore, the range of validity of the Stokes regime is systematically calculated.

Improved design of electrophoretic equipment for rapid sickle-cell-anemia screening

NASA Technical Reports Server (NTRS)

Reddick, J. M.; Hirsch, I.

1974-01-01

Effective mass screening may be accomplished by modifying existing electrophoretic equipment in conjunction with multisample applicator used with cellulose-acetate-matrix test paper. Using this method, approximately 20 to 25 samples can undergo electrophoresis in 5 to 6 minutes.

Comparing nanostructured hydroxyapatite coating on AZ91 alloy samples via sol-gel and electrophoretic deposition for biomedical applications.

PubMed

Rojaee, Ramin; Fathi, Mohammadhossein; Raeissi, Keyvan

2014-12-01

Magnesium is one of the most critical elements in hard tissues regeneration and therefore causes speeding up the restoration of harmed bones, while high deterioration rate of magnesium in body fluid restricts it to be used as biodegradable implants. Alloying magnesium with some relatively nobler metals such as aluminium, zinc, rare earth elements, magnesium-bioceramics composites, and surface modification techniques are some of the routes to control magnesium corrosion rate. In this study AZ91 magnesium alloy had been coated by nanostructured hydroxyapatite via sol-gel dip coating and electrophoretical methods to survey the final barricade properties of the obtained coatings. In order to perform electrophoretic coating, powders were prepared by sol-gel method, and then the powders deposited on substrates utilizing direct current electricity. Zeta potentials of the electrophoresis suspensions were measured to determine a best mode for good quality coatings. Transmission Electron Microscopy (TEM), and Scanning Electron Microscopy (SEM) were used to confirm nanoscale dimension, and the uniformity of the nanostructured hydroxyapatite coating, respectively. Fourier Transform-Infrared and X-ray diffraction analysis were utilized for functional group and phase structure evaluation of the prepared coatings, correspondingly. Electrochemical corrosion tests were performed in SBF at 37±1 (°)C which revealed considerable increase in corrosion protection resistivity and corrosion current density for electrophoretic coated specimens versus sol-gel coated specimens. Results showed that both sol-gel and electrophoretical techniques seem to be suitable to coat magnesium alloys for biomedical applications but electrophoretic coating technique is a better choice due to the more homogeneity and more crystalline structure of the coating.

Modulation-frequency encoded multi-color fluorescent DNA analysis in an optofluidic chip.

PubMed

Dongre, Chaitanya; van Weerd, Jasper; Besselink, Geert A J; Vazquez, Rebeca Martinez; Osellame, Roberto; Cerullo, Giulio; van Weeghel, Rob; van den Vlekkert, Hans H; Hoekstra, Hugo J W M; Pollnau, Markus

2011-02-21

We introduce a principle of parallel optical processing to an optofluidic lab-on-a-chip. During electrophoretic separation, the ultra-low limit of detection achieved with our set-up allows us to record fluorescence from covalently end-labeled DNA molecules. Different sets of exclusively color-labeled DNA fragments-otherwise rendered indistinguishable by spatio-temporal coincidence-are traced back to their origin by modulation-frequency-encoded multi-wavelength laser excitation, fluorescence detection with a single ultrasensitive, albeit color-blind photomultiplier, and Fourier analysis decoding. As a proof of principle, fragments obtained by multiplex ligation-dependent probe amplification from independent human genomic segments, associated with genetic predispositions to breast cancer and anemia, are simultaneously analyzed.

Molecular mass determination and assay of venom hyaluronidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

PubMed

Cevallos, M A; Navarro-Duque, C; Varela-Julia, M; Alagon, A C

1992-08-01

We describe a procedure for molecular mass determination of hyaluronidases present in animal venoms from different families. Hyaluronidases can be revealed, following their electrophoretic separation in sodium dodecyl sulfate-polyacrylamide gel containing hyaluronic acid, by incubating the gel in Triton X-100 to remove sodium dodecyl sulfate and restore in situ enzyme activity. This method allows the detection of as little as 0.025 turbidity-reducing units after 2 hr incubation. All the hyaluronidases from the analyzed invertebrate venoms had a mass below 50,000 and showed only one component, while those from vertebrate venoms were more than 60,000 and in many instances contained more than one form.

Recent approaches for enhancing sensitivity in enantioseparations by CE.

PubMed

Sánchez-Hernández, Laura; García-Ruiz, Carmen; Luisa Marina, María; Luis Crego, Antonio

2010-01-01

This article reviews the latest methodological and instrumental improvements for enhancing sensitivity in chiral analysis by CE. The review covers literature from March 2007 until May 2009, that is, the works published after the appearance of the latest review article on the same topic by Sánchez-Hernández et al. [Electrophoresis 2008, 29, 237-251]. Off-line and on-line sample treatment techniques, on-line sample preconcentration strategies based on electrophoretic and chromatographic principles, and alternative detection systems to the widely employed UV/Vis detection in CE are the most relevant approaches discussed for improving sensitivity. Microchip technologies are also included since they can open up great possibilities to achieve sensitive and fast enantiomeric separations.

Fast and Versatile Fabrication of PMMA Microchip Electrophoretic Devices by Laser Engraving

PubMed Central

Gabriel, Ellen Flávia Moreira; Coltro, Wendell Karlos Tomazelli; Garcia, Carlos D.

2014-01-01

This paper describes the effects of different modes and engraving parameters on the dimensions of microfluidic structures produced in PMMA using laser engraving. The engraving modes included raster and vector while the explored engraving parameters included power, speed, frequency, resolution, line-width and number of passes. Under the optimum conditions, the technique was applied to produce channels suitable for CE separations. Taking advantage of the possibility to cut-through the substrates, the laser was also used to define solution reservoirs (buffer, sample, and waste) and a PDMS-based decoupler. The final device was used to perform the analysis of a model mixture of phenolic compounds within 200 s with baseline resolution. PMID:25113407

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

PubMed

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Interpretive Reporting of Protein Electrophoresis Data by Microcomputer

PubMed Central

Talamo, Thomas S.; Losos, Frank J.; Kessler, G. Frederick

1982-01-01

A microcomputer based system for interpretive reporting of protein electrophoretic data has been developed. Data for serum, urine and cerebrospinal fluid protein electrophoreses as well as immunoelectrophoresis can be entered. Patient demographic information is entered through the keyboard followed by manual entry of total and fractionated protein levels obtained after densitometer scanning of the electrophoretic strip. The patterns are then coded, interpreted, and final reports generated. In most cases interpretation time is less than one second. Misinterpretation by computer is uncommon and can be corrected by edit functions within the system. These discrepancies between computer and pathologist interpretation are automatically stored in a data file for later review and possible program modification. Any or all previous tests on a patient may be reviewed with graphic display of the electrophoretic pattern. The system has been in use for several months and is presently well accepted by both laboratory and clinical staff. It also allows rapid storage, retrieval and analysis of protein electrophoretic datab.

Tridodecylamine, an efficient charge control agent in non-polar media for electrophoretic inks application

NASA Astrophysics Data System (ADS)

Noel, Amélie; Mirbel, Déborah; Cloutet, Eric; Fleury, Guillaume; Schatz, Christophe; Navarro, Christophe; Hadziioannou, Georges; CyrilBrochon

2018-01-01

In order to obtain efficient electrophoretic inks, Tridodecylamine (Dod3N), has been studied as charge control agent (CCA) in a non-polar paraffin solvent (Isopar G) for various inorganic pigments (TiO2 and Fe2O3). All hydrophobic mineral oxides, i.e. treated with octyltrimethoxysilane (C8) or dodecyltrimethoxysilane (C12), were found to be negatively charged in presence of Dod3N. The electrophoretic mobilities of inorganic pigments seemed to be strongly dependent of their isoelectric point (IEP) and also of the concentration of dod3N with an optimum range between 10 and 20 mM depending on the pigments. Finally, an electrophoretic ink constituted of hydrophobic mineral oxides in presence of Dod3N was tested in a device. Its efficiency as charge control agent to negatively charge hydrophobic particles was confirmed through good optical properties and fast response time (220 ms at 200 kV m-1).

Electrophoretic characterisation of the outer membrane proteins of Yersinia pestis isolated in north-east Brazil.

PubMed Central

Abath, F. G.; Almeida, A. M.; Ferreira, L. C.

1989-01-01

The outer membrane proteins of 38 Yersinia pestis isolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts among Y. pestis strains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wild Y. pestis isolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed. Images Fig. 2 PMID:2606164

Recent highlights in electro-driven separations- selected applications of alkylthiol gold nanoparticles in capillary electrophoresis and capillary electro-chromatography.

PubMed

Guihen, Elizabeth

2017-09-01

To date, alkylthiol gold nanoparticles (AuNPs) have been widely used in electro-chromatographic separation techniques as a viable alternative to traditional stationary phases. This is mainly due to their stability, chemical inertness, ease of functionality, increased phase ratio, ability to form self-assembled monolayers. They also yield versatile stationary phases with highly specific targeted functionalities. At the nanoscale region, the chemical and physical properties of a molecule display different attributes to that of the parent molecules or material, hence these features can be harnessed in electro-driven chromatographic separations. Application areas illustrating the use of AuNPs in separation science continue to grow and expand to cover many different kinds of analysis. The last decade has witnessed a successful trend in miniaturisation of chemical separation systems toward the micro and nanoscale ranges. Nanoparticle-based stationary phases fit well with performing chemical separations on microfluidic and capillary platforms. In this review the theory of the use of alkylthiol gold nanoparticles in electro-chromatographic driven separation methods will be discussed. This will be followed by details of recent and selected applications showing alkylthiol gold nanoparticles in capillary electrophoretic and open-tubular electro-chromatographic separations. This review will focus solely on alkylthiol based gold nanoparticles, therefore other kinds of chemical moieties bonded to gold nanoparticles are outside the scope of this review. Finally the future outlook of this exciting technology will be outlined in some detail in the final section. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris.

PubMed

Mayrhofer, G; Andrews, R H; Ey, P L; Chilton, N B

1995-07-01

Giardia that infect humans are known to be heterogeneous but they are assigned currently to a single species, Giardia intestinalis (syn. G. lamblia). The genetic differences that exist within G. intestinalis have not yet been assessed quantitatively and neither have they been compared in magnitude with those that exist between G. intestinalis and species that are morphologically similar (G. duodenalis) or morphologically distinct (e.g. G. muris). In this study, 60 Australian isolates of G. intestinalis were analysed electrophoretically at 27 enzyme loci and compared with G. muris and a feline isolate of G. duodenalis. Isolates of G. intestinalis were distinct genetically from both G. muris (approximately 80% fixed allelic differences) and the feline G. duodenalis isolate (approximately 75% fixed allelic differences). The G. intestinalis isolates were extremely heterogeneous but they fell into 2 major genetic assemblages, separated by fixed allelic differences at approximately 60% of loci examined. The magnitude of the genetic differences between the G. intestinalis assemblages approached the level that distinguished the G. duodenalis isolate from the morphologically distinct G. muris. This raises important questions about the evolutionary relationships of the assemblages with Homo sapiens, the possibility of ancient or contemporary transmission from animal hosts to humans and the biogeographical origins of the two clusters.

Three-dimensional electrodes for dye-sensitized solar cells: synthesis of indium-tin-oxide nanowire arrays and ITO/TiO2 core-shell nanowire arrays by electrophoretic deposition

NASA Astrophysics Data System (ADS)

Wang, Hong-Wen; Ting, Chi-Feng; Hung, Miao-Ken; Chiou, Chwei-Huann; Liu, Ying-Ling; Liu, Zongwen; Ratinac, Kyle R.; Ringer, Simon P.

2009-02-01

Dye-sensitized solar cells (DSSCs) show promise as a cheaper alternative to silicon-based photovoltaics for specialized applications, provided conversion efficiency can be maximized and production costs minimized. This study demonstrates that arrays of nanowires can be formed by wet-chemical methods for use as three-dimensional (3D) electrodes in DSSCs, thereby improving photoelectric conversion efficiency. Two approaches were employed to create the arrays of ITO (indium-tin-oxide) nanowires or arrays of ITO/TiO2 core-shell nanowires; both methods were based on electrophoretic deposition (EPD) within a polycarbonate template. The 3D electrodes for solar cells were constructed by using a doctor-blade for coating TiO2 layers onto the ITO or ITO/TiO2 nanowire arrays. A photoelectric conversion efficiency as high as 4.3% was achieved in the DSSCs made from ITO nanowires; this performance was better than that of ITO/TiO2 core-shell nanowires or pristine TiO2 films. Cyclic voltammetry confirmed that the reaction current was significantly enhanced when a 3D ITO-nanowire electrode was used. Better separation of charge carriers and improved charge transport, due to the enlarged interfacial area, are thought to be the major advantages of using 3D nanowire electrodes for the optimization of DSSCs.

Separating large microscale particles by exploiting charge differences with dielectrophoresis.

PubMed

Polniak, Danielle V; Goodrich, Eric; Hill, Nicole; Lapizco-Encinas, Blanca H

2018-04-13

Dielectrophoresis (DEP), the migration of particles due to polarization effects under the influence of a nonuniform electric field, was employed for characterizing the behavior and achieving the separation of larger (diameter >5 μm) microparticles by exploiting differences in electrical charge. Usually, electrophoresis (EP) is the method of choice for separating particles based on differences in electrical charge; however, larger particles, which have low electrophoretic mobilities, cannot be easily separated with EP-based techniques. This study presents an alternative for the characterization, assessment, and separation of larger microparticles, where charge differences are exploited with DEP instead of EP. Polystyrene microparticles with sizes varying from 5 to 10 μm were characterized employing microdevices for insulator-based dielectrophoresis (iDEP). Particles within an iDEP microchannel were exposed simultaneously to DEP, EP, and electroosmotic (EO) forces. The electrokinetic behavior of four distinct types of microparticles was carefully characterized by means of velocimetry and dielectrophoretic capture assessments. As a final step, a dielectropherogram separation of two distinct types of 10 μm particles was devised by first characterizing the particles and then performing the separation. The two types of 10 μm particles were eluted from the iDEP device as two separate peaks of enriched particles in less than 80 s. It was demonstrated that particles with the same size, shape, surface functionalization, and made from the same bulk material can be separated with iDEP by exploiting slight differences in the magnitude of particle charge. The results from this study open the possibility for iDEP to be used as a technique for the assessment and separation of biological cells that have very similar characteristics (shape, size, similar make-up), but slight variance in surface electrical charge. Copyright © 2018 Elsevier B.V. All rights reserved.

Chiral separation of benzoporphyrin derivative mono- and diacids by laser induced fluorescence-capillary electrophoresis.

PubMed

Peng, Xuejun; Sternberg, Ethan; Dolphin, David

2002-01-01

A method for the separation of benzoporphyrin derivative mono- and diacid (BPDMA, BPDDA) enantiomers by laser induced fluorescence-capillary electrophoresis (LIF-CE) has been developed. By using 300 mM borate buffer, pH 9.2, 25 mM sodium cholate and 10% acetronitrile as electrolyte, +10 kV electrokinetic sampling injection of 2 s and an applied +20 kV voltage across the ends of a 37 cm capillary (30 cm to the detector, 50 microm ID), all six BPD stereoisomers were baseline-separated within 20 min. Formation constants, free electrophoretic and complexation mobilities with borate and cholate were determined based on dynamic complexation capillary electrophoresis theory. The BPD enantiomers can be quantitatively determined in the range of 10(-2)-10(-5) mg mL(-1). The correlation coefficients (r2) of the least-squares linear regression analysis of the BPD enantiomers are in the range of 0.9914-0.9997. Their limits of detection are 2.18-3.5 x 10(-3) mg mL(-1). The relative standard deviations for the separation were 2.90-4.64% (n = 10). In comparison with high-performance liquid chromatography (HPLC), CE has better resolution and efficiency. This separation method was successfully applied to the BPD enantiomers obtained from a matrix of bovine serum and from liposomally formulated material as well as from studies with rat, dog and human microsomes.

Preconcentration and Separation of Mixed-Species Samples Near a Nano-Junction in a Convergent Microchannel

PubMed Central

Chiu, Ping-Hsien; Weng, Chen-Hsun; Yang, Ruey-Jen

2015-01-01

A fluidic microchip incorporating a convergent microchannel and a Nafion-nanoporous membrane is proposed for the preconcentration and separation of multi-species samples on a single platform. In the device, sample preconcentration is achieved by means of the ion concentration polarization effect induced at the micro/nano interface under the application of an external electric field, while species separation is achieved by exploiting the different electrophoretic mobilities of the sample components. The experimental results show that the device is capable of detecting C-reactive protein (CRP) with an initial concentration as low as 9.50 × 10−6 mg/L given a sufficient preconcentration time and driving voltage. In addition, it is shown that a mixed-species sample consisting of three negatively-charged components (bovine serum albumin (BSA), tetramethylrhodamine(TAMRA) isothiocyanate-Dextran and fluorescent polymer beads) can be separated and preconcentrated within 20 min given a driving voltage of 100 V across 1 cm microchannel in length. In general, the present results confirm the feasibility of the device for the immunoassay or detection of various multi-species samples under low concentration in the biochemical and biomedical fields. The novel device can therefore improve the detection limit of traditional medical facilities. PMID:26690167

Model creation of moving redox reaction boundary in agarose gel electrophoresis by traditional potassium permanganate method.

PubMed

Xie, Hai-Yang; Liu, Qian; Li, Jia-Hao; Fan, Liu-Yin; Cao, Cheng-Xi

2013-02-21

A novel moving redox reaction boundary (MRRB) model was developed for studying electrophoretic behaviors of analytes involving redox reaction on the principle of moving reaction boundary (MRB). Traditional potassium permanganate method was used to create the boundary model in agarose gel electrophoresis because of the rapid reaction rate associated with MnO(4)(-) ions and Fe(2+) ions. MRB velocity equation was proposed to describe the general functional relationship between velocity of moving redox reaction boundary (V(MRRB)) and concentration of reactant, and can be extrapolated to similar MRB techniques. Parameters affecting the redox reaction boundary were investigated in detail. Under the selected conditions, good linear relationship between boundary movement distance and time were obtained. The potential application of MRRB in electromigration redox reaction titration was performed in two different concentration levels. The precision of the V(MRRB) was studied and the relative standard deviations were below 8.1%, illustrating the good repeatability achieved in this experiment. The proposed MRRB model enriches the MRB theory and also provides a feasible realization of manual control of redox reaction process in electrophoretic analysis.

Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

EPA Science Inventory

The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

Application of partition technology to particle electrophoresis

NASA Technical Reports Server (NTRS)

Van Alstine, James M.; Harris, J. Milton; Karr, Laurel J.; Bamberger, Stephan; Matsos, Helen C.; Snyder, Robert S.

1989-01-01

The effects of polymer-ligand concentration on particle electrophoretic mobility and partition in aqueous polymer two-phase systems are investigated. Polymer coating chemistry and affinity ligand synthesis, purification, and analysis are conducted. It is observed that poly (ethylene glycol)-ligands are effective for controlling particle electrophoretic mobility.

Enhanced specific capacitance of an electrophoretic deposited MnO2-carbon nanotube supercapacitor

NASA Astrophysics Data System (ADS)

Tagsin, Patin; Klangtakai, Pawinee; Harnchana, Viyada; Amornkitbamrung, Vittaya; Pimanpang, Samuk; Kumnorkaew, Pisist

2017-12-01

MnO2 and MnO2-carbon nanotubes (CNT) composite films were grown directly on stainless- steel substrates using an electrophoretic process employing supercapacitor electrodes. An electrophoretic MnO2 film with a nanoplate-like structure was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Supercapacitor performance was studied using cyclic voltammetry (CV), charge-discharge (CD) and electrochemical impedance spectroscopy (EIS). The specific capacitance (SC) of the electrophoretic MnO2 film was 60 F/g at 1 A/g, with a 38.33% retention of the initial SC values after 1000 cycles. The low SC value of the MnO2 films was attributed to the high series and charge-transfer resistances of 1.70 Ω and 3.20, respectively. The MnO2-CNT composites with the addition of 0.04, 0.06 and 0.08 g CNT to the electrophoretic MnO2 film were found to greatly increase the SC to 300, 206 and 169 F/g at 1 A/g, respectively. The series and charge-transferred resistances of MnO2-CNT composite films decreased to 1.38 - 1.52 Ω and 2.62 - 2.86 Ω, respectively. The SC improvement of the composite electrodes was attributed to presence of two active storage materials (MnO2 and CNT), a high film specific surface area and electrical conductivity.

Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins

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

Preston, D.A.; Wu, C.Y.; Blaszczak, L.C.

Radiolabeled penicillin G is widely used as the imaging agent in penicillin-binding protein (PBP) assays. The disadvantages of most forms of labeled penicillin G are instability on storage and the long exposure times usually required for autoradiography or fluorography of electrophoretic gels. We investigated the utility of radioiodinated penicillin V as an alternative reagent. Radioiodination of p-(trimethylstannyl)penicillin V with ({sup 125}I)Na, using a modification of the chloramine-T method, is simple, high yielding, and site specific. We demonstrated the general equivalence of commercially obtained ({sup 3}H)penicillin G and locally synthesized ({sup 125}I)penicillin V (IPV) in their recognition of bacterial PBPs. Profilesmore » of PBPs in membranes from Bacteroides fragilis, Escherichia coli, Providencia rettgeri, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, and Enterococcus faecium labeled with IPV or (3H)penicillin G were virtually identical. Use of IPV as the imaging agent in competition experiments for determination of the affinities of various beta-lactam antibiotics for the PBPs of E. coli yielded results similar to those obtained in experiments with ({sup 3}H)penicillin G. Dried electrophoretic gels from typical PBP experiments, using IPV at 37.3 Ci/mmol and 30 micrograms/ml, exposed X-ray film in 8 to 24 h. The stability of IPV on storage at 4{degrees}C was inversely proportional to specific activity. At 37.3 Ci/mmol and 60 micrograms/ml, IPV retained useful activity for at least 60 days at 4{degrees}C. IPV represents a practical and stable reagent for rapid PBP assays.« less

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Monitoring the enrichment of virgin olive oil with natural antioxidants by using a new capillary electrophoresis method.

PubMed

Nevado, Juan José Berzas; Robledo, Virginia Rodríguez; Callado, Carolina Sánchez-Carnerero

2012-07-15

The enrichment of virgin olive oil (VOO) with natural antioxidants contained in various herbs (rosemary, thyme and oregano) was studied. Three different enrichment procedures were used for the solid-liquid extraction of antioxidants present in the herbs to VOO. One involved simply bringing the herbs into contact with the VOO for 190 days; another keeping the herb-VOO mixture under stirring at room temperature (25°C) for 11 days; and the third stirring at temperatures above room level (35-40°C). The efficiency of each procedure was assessed by using a reproducible, efficient, reliable analytical capillary zone electrophoresis (CZE) method to separate and determine selected phenolic compounds (rosmarinic and caffeic acid) in the oil. Prior to electrophoretic separation, the studied antioxidants were isolated from the VOO matrix by using an optimised preconcentration procedure based on solid phase extraction (SPE). The CZE method was optimised and validated. Copyright © 2012 Elsevier Ltd. All rights reserved.

A sub-minute electrophoretic method for simultaneous determination of naphazoline and zinc.

PubMed

Ribeiro, Michelle M A C; Oliveira, Thiago C; Batista, Alex D; Muñoz, Rodrigo A A; Richter, Eduardo M

2016-11-11

This paper reports for the first time, a method for simultaneous determination of naphazoline (NPZ) and zinc (Zn) using an analytical separation technique (capillary electrophoresis with capacitively coupled contactless conductivity detection -CE-C 4 D). A single run is possible every 55s (sampling rate=65h -1 ). The separation by CE-C 4 D was achieved on a fused silica capillary (50cm length - 10cm effective, 50μm i.d.) with a background electrolyte (BGE) composed by 20mmolL -1 of 2-(morpholin-4-yl)ethane-1-sulfonic acid (MES) and 20mmolL -1 of histidine (HIS) (pH 6.0). Detection limits were estimated at 20 and 30μmolL -1 and recovery values for spiked samples were 98 and 102% for NPZ and Zn, respectively. The developed procedure was compared to HPLC (NPZ) and FAAS (Zn) and no statistically significant differences were observed (95% confidence level). Copyright © 2016 Elsevier B.V. All rights reserved.

ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

EPA Science Inventory

The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms isolated from clinical and environmental sources were measured in 9.15 mM KH2PO4 buffered water. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V^-1 ...

ELECTROPHORETIC MOBILITY OF MYCOBACTERIUM AVIUM COMPLEX ORGANISMS

EPA Science Inventory

The electrophoretic mobilities (EPMs) of thirty Mycobacterium avium Complex (MAC) organisms were measured. The EPMs of fifteen clinical isolates ranged from -1.9 to -5.0 µm cm V^-1s^-1, and the EPMs of fifteen environmental isolates ranged from -1...

ELECTROPHORETIC MOBILITIES OF ESCHERICHIA COLI 0157:H7 AND WILD-TYPE ESCHERICHIA COLI STRAINS

EPA Science Inventory

The electrophoretic mobility (EPM) of a number of human-virulent and "wild-type" Escherichia coli strains in phosphate buffered water was measured. The impact of pH, ionic strength, cation type (valence) and concentration, and bacterial strain on the EPM was investigated. Resul...

A simple method using two-step hot embossing technique with shrinking for fabrication of cross microchannels on PMMA substrate and its application to electrophoretic separation of amino acids in functional drinks.

PubMed

Wiriyakun, Natta; Nacapricha, Duangjai; Chantiwas, Rattikan

2016-12-01

This work presents a simple hot embossing method with a shrinking procedure to produce cross-shape microchannels on poly(methyl methacrylate) (PMMA) substrate for the fabrication of an electrophoresis chip. The proposed method employed a simple two-step hot embossing technique, carried out consecutively on the same piece of substrate to make the crossing channels. Studies of embossing conditions, i.e. temperature, pressure and time, were carried out to investigate their effects on the dimension of the microchannels. Applying a simple shrinking procedure reduced the size of the channels from 700±20µm wide×150±5µm deep to 250±10µm wide×30±2µm deep, i.e. 80% and 64% reduction in the depth and width, respectively. Thermal fusion was employed to bond the PMMA substrate with a PMMA cover plate to produce the microfluidic device. Replication of microchip was achieved by precise control of conditions in the fabrication process (pressure, temperature and time), resulting in lower than 7% RSD of channel dimension, width and depth (n =10 devices). The method was simple and robust without the use of expensive equipment to construct the microstructure on a thermoplastic substrate. The PMMA microchip was used for demonstration of amine functionalization on the PMMA surface, measurement of electroosmotic flow and for electrophoretic separation of amino acids in functional drink samples. The precision of migration time and peak area of the amino acids, Lys, Ile and Phe at 125μM to 500μM, were in the range 3.2-4.2% RSD (n=9 devices) and 4.5-5.3% RSD (n=9 devices), respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfluidic [correction of microfludic] network.

PubMed

Li, Yan; Buch, Jesse S; Rosenberger, Frederick; DeVoe, Don L; Lee, Cheng S

2004-02-01

An integrated protein concentration/separation system, combining non-native isoelectric focusing (IEF) with sodium dodecyl sulfate (SDS) gel electrophoresis on a polymer microfluidic chip, is reported. The system provides significant analyte concentration and extremely high resolving power for separated protein mixtures. The ability to introduce and isolate multiple separation media in a plastic microfluidic network is one of two key requirements for achieving multidimensional protein separations. The second requirement lies in the quantitative transfer of focused proteins from the first to second separation dimensions without significant loss in the resolution acquired from the first dimension. Rather than sequentially sampling protein analytes eluted from IEF, focused proteins are electrokinetically transferred into an array of orthogonal microchannels and further resolved by SDS gel electrophoresis in a parallel and high-throughput format. Resolved protein analytes are monitored using noncovalent, environment-sensitive, fluorescent probes such as Sypro Red. In comparison with covalently labeling proteins, the use of Sypro staining during electrophoretic separations not only presents a generic detection approach for the analysis of complex protein mixtures such as cell lysates but also avoids additional introduction of protein microheterogeneity as the result of labeling reaction. A comprehensive 2-D protein separation is completed in less than 10 min with an overall peak capacity of approximately 1700 using a chip with planar dimensions of as small as 2 cm x 3 cm. Significant enhancement in the peak capacity can be realized by simply raising the density of microchannels in the array, thereby increasing the number of IEF fractions further analyzed in the size-based separation dimension.

High pH instability of quaternary ammonium surfactant coatings in capillary electrophoresis.

PubMed

Shulman, Lisa; Pei, Lei; Bahnasy, Mahmoud F; Lucy, Charles A

2017-06-12

The two-tailed cationic surfactant dioctadecyldimethyl ammonium bromide (DODAB) produces semi-permanent coatings that yield strongly reversed electroosmotic flow (EOF), for example -0.31 ± 0.01 cm 2 kV -1 s -1 at pH 3.5. Moreover, these coatings are easy to prepare, regenerable, cost effective, and yield high efficiency (520 000-900 000 plates per m) separations of cationic proteins over many runs under acidic (pH 3.5) conditions. Given the quaternary amine functionality of DODAB, we were surprised to observe that DODAB coatings become unstable at pH > 7. At pH 7.2, the EOF of a DODAB coated capillary drifted from reversed to cathodic over only 5 runs, and protein separations became severely compromised. By pH 12, no EOF reversal was observed. Electrophoretic and mass spectrometric studies demonstrate that the coating decomposition involves a surface conversion of the quaternary amine in DODAB to a variety of products, although the exact mechanism remains elusive. Regardless, the results herein demonstrate that semi-permanent coatings based on cationic two-tailed surfactants such as DODAB are limited to separations using acidic buffers.

Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination.

PubMed

Chiem, N H; Harrison, D J

1998-03-01

A glass microchip is described in which reagents and serum samples for competitive immunoassay of serum theophylline can be mixed, reacted, separated, and analyzed. The device functions as an automated microfluidic immunoassay system, creating a lab-on-a-chip. Electroosmotic pumping was used to control first the mixing of 50-fold-diluted serum sample with labeled theophylline tracer in a 1:1 ratio, followed by 1:1 mixing and reaction with anti-theophylline antibody. The 51-nL on-chip mixer gave the same concentration as dilution performed off-chip, within 3%. A 100-pL plug of the reacted solution was then injected into an electrophoresis separation channel integrated within the same chip. Measurements of free and bound tracer by fluorescence detection gave linear calibration curves of signal vs log[theophylline] between 0 and 40 mg/L, with a slope of 0.52 +/- 0.03 and an intercept of -0.04 +/- 0.04 after a 90-s reaction time. A detection limit of 0.26 mg/L in serum (expressed before the dilution step, actual concentration of 1.3 micrograms/L at the detector) was obtained. Recovery values were 107% +/- 8% for 15 mg/L serum samples.

Determination of hydrolyzable tannins in the fruit of Terminalia chebula Retz. by high-performance liquid chromatography and capillary electrophoresis.

PubMed

Juang, Lih-Jeng; Sheu, Shuenn-Jyi; Lin, Ta-Chen

2004-06-01

A RP-HPLC method for determining fourteen components (gallic acid, chebulic acid, 1,6-di-O-galloyl-D-glucose, punicalagin, 3,4,6-tri-O-galloyl-D-glucose, casuarinin, chebulanin, corilagin, neochebulinic acid, terchebulin, ellagic acid, chebulagic acid, chebulinic acid, and 1,2,3,4,6-penta-O-galloyl-D-glucose) in the fruit of Terminalia chebula Retz. is described. The separation was achieved within 80 min using a binary gradient with mobile phases consisting of a pH 2.7 phosphoric acid solution and an 80% CH3CN solution. Capillary electrophoretic analyses were also attempted, and it was found that CZE (25 mM Na2B4O7, 5 mM NaH2PO4, pH 7.0) was an efficient method for the separation of gallotannins, while an MEKC method (25 mM Na2B4O7, 5 mM NaH2PO4, 20 mM SDS, pH 7.0, and 10% acetonitrile) provided a better separation for most of the tannins examined. The HPLC and CE methods developed were both successfully applied to the assay of tannins in commercial samples of Chebulae Fructus.

Purity Determination by Capillary Electrophoresis Sodium Hexadecyl Sulfate (CE-SHS): A Novel Application For Therapeutic Protein Characterization.

PubMed

Beckman, Jeff; Song, Yuanli; Gu, Yan; Voronov, Sergey; Chennamsetty, Naresh; Krystek, Stanley; Mussa, Nesredin; Li, Zheng Jian

2018-02-20

Capillary gel electrophoresis using sodium dodecyl sulfate (CE-SDS) is used commercially to provide quantitative purity data for therapeutic protein characterization and release. In CE-SDS, proteins are denatured under reducing or nonreducing conditions in the presence of SDS and electrophoretically separated by molecular weight and hydrodynamic radius through a sieving polymer matrix. Acceptable performance of this method would yield protein peaks that are baseline resolved and symmetrical. Nominal CE-SDS conditions and parameters are not optimal for all therapeutic proteins, specifically for Recombinant Therapeutic Protein-1 (RTP-1), where acceptable resolution and peak symmetry were not achieved. The application of longer alkyl chain detergents in the running buffer matrix substantially improved assay performance. Matrix running buffer containing sodium hexadecyl sulfate (SHS) increased peak resolution and plate count 3- and 8-fold, respectively, compared to a traditional SDS-based running gel matrix. At Bristol-Myers Squibb (BMS), we developed and qualified a viable method for the characterization and release of RTP-1 using an SHS-containing running buffer matrix. This work underscores the potential of detergents other than SDS to enhance the resolution and separation power of CE-based separation methods.

Analysis of optical purity and impurity of synthetic D-phenylalanine products using sulfated beta-cyclodextrin as chiral selector by reversed-polarity capillary electrophoresis.

PubMed

Zhao, Yan; Yang, Xing-Bin; Jiang, Ru; Sun, Xiao-Li; Li, Xiao-Ye; Liu, Wen-Min; Zhang, Sheng-Yong

2006-02-01

A new capillary electrophoresis (CE) method has been achieved for simultaneous separation and quantification of phenylalanine, N-acetylphenylalanine enantiomers, and prochiral N-acetylaminocinnamic acid, possibly co-existent in reaction systems or synthesized products of D-phenylalanine. The separation was carried out in an uncoated capillary under reversed-electrophoretic mode. Among the diverse charged cyclodextrins (CDs) examined, highly sulfated (HS)-beta-CD as the chiral selector exhibited the best enantioselectivity. The complete separation of the analytes was obtained under the optimum conditions of pH 2.5, 35 mM Tris buffer containing 4% HS-beta-CD, applied voltage -15 kV, and capillary temperature 25 degrees C. Furthermore, the proposed method was applied to the determination of optical purity and trace impurities in three batches of the asymmetric synthetic samples of D-phenylalanine, and satisfactory results were obtained. The determination recoveries of the samples were in the range of 97.8-103.8%, and precisions fell within 2.3-5.0% (RSD). The results demonstrate that this CE method is a useful, simple technique and is applicable to purity assays of D-phenylalanine. (c) 2005 Wiley-Liss, Inc.

Simultaneous determination of multiclass preservatives including isothiazolinones and benzophenone-type UV filters in household and personal care products by micellar electrokinetic chromatography.

PubMed

Lopez-Gazpio, Josu; Garcia-Arrona, Rosa; Millán, Esmeralda

2015-04-01

In this work, a simple and reliable micellar electrokinetic chromatography method for the separation and quantification of 14 preservatives, including isothiazolinones, and two benzophenone-type UV filters in household, cosmetic and personal care products was developed. The selected priority compounds are widely used as ingredients in many personal care products, and are included in the European Regulation concerning cosmetic products. The electrophoretic separation parameters were optimized by means of a modified chromatographic response function in combination with an experimental design, namely a central composite design. After optimization of experimental conditions, the BGE selected for the separation of the targets consisted of 60 mM SDS, 18 mM sodium tetraborate, pH 9.4 and 10% v/v methanol. The MEKC method was checked in terms of linearity, LODs and quantification, repeatability, intermediate precision, and accuracy, providing appropriate values (i.e. R(2) ≥ 0.992, repeatability RSD values ˂9%, and accuracy 90-115%). Applicability of the validated method was successfully assessed by quantifying preservatives and UV filters in commercial consumer products. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microencapsulation and Electrostatic Processing Device

NASA Technical Reports Server (NTRS)

Morrison, Dennis R. (Inventor); Mosier, Benjamin (Inventor); Cassanto, John M. (Inventor)

2001-01-01

A microencapsulation and electrostatic processing (MEP) device is provided for forming microcapsules. In one embodiment, the device comprises a chamber having a filter which separates a first region in the chamber from a second region in the chamber. An aqueous solution is introduced into the first region through an inlet port, and a hydrocarbon/ polymer solution is introduced into the second region through another inlet port. The filter acts to stabilize the interface and suppress mixing between the two immiscible solutions as they are being introduced into their respective regions. After the solutions have been introduced and have become quiescent, the interface is gently separated from the filter. At this point, spontaneous formation of microcapsules at the interface may begin to occur, or some fluid motion may be provided to induce microcapsule formation. In any case, the fluid shear force at the interface is limited to less than 100 dynes/sq cm. This low-shear approach to microcapsule formation yields microcapsules with good sphericity and desirable size distribution. The MEP device is also capable of downstream processing of microcapsules, including rinsing, re-suspension in tertiary fluids, electrostatic deposition of ancillary coatings, and free-fluid electrophoretic separation of charged microcapsules.

A method for determining electrophoretic and electroosmotic mobilities using AC and DC electric field particle displacements.

PubMed

Oddy, M H; Santiago, J G

2004-01-01

We have developed a method for measuring the electrophoretic mobility of submicrometer, fluorescently labeled particles and the electroosmotic mobility of a microchannel. We derive explicit expressions for the unknown electrophoretic and the electroosmotic mobilities as a function of particle displacements resulting from alternating current (AC) and direct current (DC) applied electric fields. Images of particle displacements are captured using an epifluorescent microscope and a CCD camera. A custom image-processing code was developed to determine image streak lengths associated with AC measurements, and a custom particle tracking velocimetry (PTV) code was devised to determine DC particle displacements. Statistical analysis was applied to relate mobility estimates to measured particle displacement distributions.

Stabilization of green bodies via sacrificial gelling agent during electrophoretic deposition

DOEpatents

Worsley, Marcus A.; Kuntz, Joshua D.; Rose, Klint A.

2016-03-22

In one embodiment, a method for electrophoretic deposition of a three-dimensionally patterned green body includes suspending a first material in a gelling agent above a patterned electrode of an electrophoretic deposition (EPD) chamber, and gelling the suspension while applying a first electric field to the suspension to cause desired patterning of the first material in a resulting gelation. In another embodiment, a ceramic, metal, or cermet includes a plurality of layers, wherein each layer includes a gradient in composition, microstructure, and/or density in an x-y plane oriented parallel to a plane of deposition of the plurality of layers along a predetermined distance in a z-direction perpendicular to the plane of deposition.

Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives.

PubMed

Sanchez-Moreno, M; Ortega, J E; Valero, A

1989-12-01

High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat (Capra hircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

Effect of pH on the Electrophoretic Mobility of Spores of Bacillus anthracis and Its Surrogates in Aqueous Solutions

EPA Science Inventory

Electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates were measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here prov...

Electrophoretic Process For Purifying Wastewater

NASA Technical Reports Server (NTRS)

Sammons, David W.; Twitty, Garland E.; Sharnez, Rizwan; Egen, Ned B.

1992-01-01

Microbes, poisonous substances, and colloidal particles removed by combination of electric fields. Electrophoretic process removes pathogenicorganisms, toxins, toxic metals, and cooloidal soil particles from wastewater. Used to render domestic, industrial, and agricultural wastewater streams potable. Process also useful in bioregenerative and other closed systems like in space stations and submarines, where water must be recycled.

[Teaching design and practice of human blood type traits in genetics comprehensive laboratory course].

PubMed

Zhao, Jian; Hu, Dong-mei; Yu, Da-de; Dong, Ming-liang; Li, Yun; Fan, Ying-ming; Wang, Yan-wei; Zhang, Jin-feng

2016-05-01

Comprehensive laboratory courses, which enable students to aptly apply theoretic knowledge and master experiment skills, play an important role in the present educational reform of laboratory courses. We utilized human ABO blood type as the experimental subject, and designed the experiment--"Molecular Genotyping of Human ABO Blood Type and Analysis of Population Genetic Equilibrium". In the experiment, DNA in mucosal cells is extracted from students' saliva, and each student's genotype is identified using a series of molecular genetics technologies, including PCR amplification of target fragments, enzymatic digestion, and electrophoretic separation. Then, taking the whole class as an analogous Mendel population, a survey of genotype frequency of ABO blood type is conducted, followed with analyses of various population genetic parameters using Popgene. Through the open laboratory course, students can not only master molecular genetic experimental skills, but also improve their understanding of theoretic knowledge through independent design and optimization of molecular techniques. After five years of research and practice, a stable experimental system of molecular genetics has been established to identify six genotypes of ABO blood types, namely I(A)I(A), I(A)i, I(B)I(B), I(B)i, I(A)I(B) and ii. Laboratory courses of molecular and population genetics have been integrated by calculating the frequencies of the six genotypes and three multiple alleles and testing population genetic equilibrium. The goal of the open laboratory course with independent design and implementation by the students has been achieved. This laboratory course has proved effective and received good reviews from the students. It could be applied as a genetics laboratory course for the biology majors directly, and its ideas and methods could be promoted and applied to other biological laboratory courses.

Determination of the microenvironment-pH and charge and size characteristics of amino acids through their electrophoretic mobilities determined by CZE.

PubMed

Piaggio, Maria V; Peirotti, Marta B; Deiber, Julio A

2007-10-01

Effective electrophoretic mobility data of 20 amino acids reported in the literature are analyzed and interpreted through simple physicochemical models, which are able to provide estimates of coupled quantities like hydrodynamic shape factor, equivalent hydrodynamic radius (size), net charge, actual pK values of ionizing groups, partial charges of ionizing groups, hydration number, and pH near molecule (microenvironment-pH of the BGE). It is concluded that the modeling of the electrophoretic mobility of these analytes requires a careful consideration of hydrodynamic shape coupled to hydration. In the low range of pH studied here, distinctive hydrodynamic behaviors of amino acids are found. For instance, amino acids with basic polar and ionizing side chain remain with prolate shape for pH values varying from 1.99 to 3.2. It is evident that as the pH increases from low values, amino acids get higher hydrations as a consequence each analyte total charge also increases. This result is consistent with the monotonic increase of the hydrodynamic radius, which accounts for both the analyte and the quite immobilized water molecules defining the electrophoretic kinematical unit. It is also found that the actual or effective pK value of the alpha-carboxylic ionizing group of amino acids increases when the pH is changed from 1.99 to 3.2. Several limitations concerning the simple modeling of the electrophoretic mobility of amino acids are presented for further research.

Reliable simultaneous zymographic method of characterization of cellulolytic enzymes from fungal cellulase complex.

PubMed

Dojnov, Biljana; Grujić, Marica; Vujčić, Zoran

2015-08-01

A method for zymographic detection of specific cellulases in a complex (endocellulase, exocellulase, and cellobiase) from crude fermentation extracts, after a single electrophoretic separation, is described in this paper. Cellulases were printed onto a membrane and, subsequently, substrate gel. Cellobiase isoforms were detected on the membrane using esculine as substrate, endocellulase isoforms on substrate gel with copolymerized carboxymethyl cellulose (CMC), while exocellulase isoforms were detected in electrophoresis gel with 4-methylumbelliferyl-β-d-cellobioside (MUC). This can be a useful additional tool for monitoring and control of fungal cellulase production in industrial processes and fundamental research, screening for particular cellulase producers, or testing of new lignocellulose substrates. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

PubMed

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Hormone Purification by Isoelectric Focusing

NASA Technical Reports Server (NTRS)

Bier, M.

1985-01-01

Various ground-based research approaches are being applied to a more definitive evaluation of the natures and degrees of electroosmosis effects on the separation capabilities of the Isoelectric Focusing (IEF) process. A primary instrumental system for this work involves rotationally stabilized, horizontal electrophoretic columns specially adapted for the IEF process. Representative adaptations include segmentation, baffles/screens, and surface coatings. Comparative performance and development testing are pursued against the type of column or cell established as an engineering model. Previously developed computer simulation capabilities are used to predict low-gravity behavior patterns and performance for IEF apparatus geometries of direct project interest. Three existing mathematical models plus potential new routines for particular aspects of simulating instrument fluid patterns with varied wall electroosmosis influences are being exercised.

Simultaneous determination of five tetracycline and macrolide antibiotics in feeds using HPCE.

PubMed

Tong, Jing; Rao, Qinxiong; Zhu, Kui; Jiang, Zhigang; Ding, Shuangyang

2009-12-01

This work demonstrates the potential of HPCE in the analysis of antibiotics in a complex matrix such as feedstuffs. Using 20 mM citric acid-40 mM Na(2)HPO(4) buffer (pH 2.65), the five antibiotics, tetracycline, oxytetracycline, doxycycline, tilmicosin, and tylosin were successfully separated at 30 kV in a 64.5 cm x 75 microm id capillary. Good repeatability, stability, and reliability of the method were supported by <10% CV with mean recoveries of >70%, and the limit of detection of the five analytes was 0.5-1 mg/kg. It was for the first time that a capillary electrophoretic method was employed to simultaneously detect five tetracycline and macrolide antibiotics in animal feeds.

Fast and versatile fabrication of PMMA microchip electrophoretic devices by laser engraving.

PubMed

Moreira Gabriel, Ellen Flávia; Tomazelli Coltro, Wendell Karlos; Garcia, Carlos D

2014-08-01

This paper describes the effects of different modes and engraving parameters on the dimensions of microfluidic structures produced in PMMA using laser engraving. The engraving modes included raster and vector, while the explored engraving parameters included power, speed, frequency, resolution, line-width, and number of passes. Under the optimum conditions, the technique was applied to produce channels suitable for CE separations. Taking advantage of the possibility to cut-through the substrates, the laser was also used to define solution reservoirs (buffer, sample, and waste) and a PDMS-based decoupler. The final device was used to perform the analysis of a model mixture of phenolic compounds within 200 s with baseline resolution. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Method for using magnetic particles in droplet microfluidics

NASA Technical Reports Server (NTRS)

Shah, Gaurav Jitendra (Inventor); Kim, Chang-Jin (Inventor)

2012-01-01

Methods of utilizing magnetic particles or beads (MBs) in droplet-based (or digital) microfluidics are disclosed. The methods may be used in enrichment or separation processes. A first method employs the droplet meniscus to assist in the magnetic collection and positioning of MBs during droplet microfluidic operations. The sweeping movement of the meniscus lifts the MBs off the solid surface and frees them from various surface forces acting on the MBs. A second method uses chemical additives to reduce the adhesion of MBs to surfaces. Both methods allow the MBs on a solid surface to be effectively moved by magnetic force. Droplets may be driven by various methods or techniques including, for example, electrowetting, electrostatic, electromechanical, electrophoretic, dielectrophoretic, electroosmotic, thermocapillary, surface acoustic, and pressure.

Particle Aggregation During Fe(III) Bioreduction in Nontronite

NASA Astrophysics Data System (ADS)

Jaisi, D. P.; Dong, H.; Hi, Z.; Kim, J.

2005-12-01

This study was performed to evaluate the rate and mechanism of particle aggregation during bacterial Fe (III) reduction in different size fractions of nontronite and to investigate the role of different factors contributing to particle aggregation. To achieve this goal, microbial Fe(III) reduction experiments were performed with lactate as an electron donor, Fe(III) in nontronite as an electron acceptor, and AQDS as an electron shuttle in bicarbonate buffer using Shewanella putrefaceins CN32. These experiments were performed with and without Na- pyrophosphate as a dispersant in four size fractions of nontronite (0.12-0.22, 0.41-0.69, 0.73-0.96 and 1.42-1.8 mm). The rate of nontronite aggregation during the Fe(III) bioreduction was measured by analyzing particle size distribution using photon correlation spectroscopy (PCS) and SEM images analysis. Similarly, the changes in particle morphology during particle aggregation were determined by analyses of SEM images. Changes in particle surface charge were measured with electrophoretic mobility analyzer. The protein and carbohydrate fraction of EPS produced by cells during Fe(III) bioreduction was measured using Bradford and phenol-sulfuric acid extraction method, respectively. In the presence of the dispersant, the extent of Fe(III) bioreduction was 11.5-12.2% within the first 56 hours of the experiment. There was no measurable particle aggregation in control experiments. The PCS measurements showed that the increase in the effective diameter (95% percentile) was by a factor of 3.1 and 1.9 for particle size of 0.12-0.22 mm and 1.42-1.80 mm, respectively. The SEM image analyses also gave the similar magnitude of increase in particle size. In the absence of the dispersant, the extent of Fe(III) bioreduction was 13.4-14.5% in 56 hours of the experiment. The rate of aggregation was higher than that in the presence of the dispersant. The increase in the effective diameter (95% percentile) was by a factor of 13.6 and 4.1 for the particles size of 0.12-0.22 and 1.42-1.8 mm, respectively. The particle aggregation was limited in control experiment to the factor of 2.8 and 2.1 for these two size fractions, respectively. The measured electrophoretic mobility decreased with increase in the extent of bioreduction and aggregation, but the rate of decrease was greatest in the finest size fraction. The EPS measurements showed the increase in the carbohydrate and protein fractions as a result of bioreduction. Separate experiments were performed to understand the relative contribution of Fe(III) reduction and EPS production in controlling nontronite particle aggregation The rate of particle aggregation was measured for nontronite that was chemically pre-reduced by dithionite to various extents, both with and without addition of dextran, a neutral and pure EPS. The aggregation rate was greater in the nontronite that were pre-reduced to a higher extent than those with a lower extent of reduction. The relative contribution to particle aggregation due to Fe(III) reduction and polysaccharide bridging was about 4:1. However, in the real system where bacterial cells are involved, and amount of EPS production and extent of Fe(III) bioreduction increase with time, the relative contribution may be different than in this simple system. In summary, we conclude that both Fe(III) reduction and microbial production of EPS contribute to the observed nontronite particle aggregation with Fe(III) reduction playing more dominant role.

Solid oxide fuel cell electrolytes produced via very low pressure suspension plasma spray and electrophoretic deposition

NASA Astrophysics Data System (ADS)

Fleetwood, James D.

Solid oxide fuel cells (SOFCs) are a promising element of comprehensive energy policies due to their direct mechanism for converting the oxidization of fuel, such as hydrogen, into electrical energy. Both very low pressure plasma spray and electrophoretic deposition allow working with high melting temperature SOFC suspension based feedstock on complex surfaces, such as in non-planar SOFC designs. Dense, thin electrolytes of ideal composition for SOFCs can be fabricated with each of these processes, while compositional control is achieved with dissolved dopant compounds that are incorporated into the coating during deposition. In the work reported, sub-micron 8 mole % Y2O3-ZrO2 (YSZ) and gadolinia-doped ceria (GDC), powders, including those in suspension with scandium-nitrate dopants, were deposited on NiO-YSZ anodes, via very low pressure suspension plasma spray (VLPSPS) at Sandia National Laboratories' Thermal Spray Research Laboratory and electrophoretic deposition (EPD) at Purdue University. Plasma spray was carried out in a chamber held at 320 - 1300 Pa, with the plasma composed of argon, hydrogen, and helium. EPD was characterized utilizing constant current deposition at 10 mm electrode separation, with deposits sintered from 1300 -- 1500 °C for 2 hours. The role of suspension constituents in EPD was analyzed based on a parametric study of powder loading, powder specific surface area, polyvinyl butyral (PVB) content, polyethyleneimine (PEI) content, and acetic acid content. Increasing PVB content and reduction of particle specific surface area were found to eliminate the formation of cracks when drying. PEI and acetic acid content were used to control suspension stability and the adhesion of deposits. Additionally, EPD was used to fabricate YSZ/GDC bilayer electrolyte systems. The resultant YSZ electrolytes were 2-27 microns thick and up to 97% dense. Electrolyte performance as part of a SOFC system with screen printed LSCF cathodes was evaluated with peak power densities as high as 520 mW/cm2 at 800 °C for YSZ and 350 mW/cm 2 at 800 °C for YSZ/GDC bilayer electrolytes.

Separation and detection of VX and its methylphosphonic acid degradation products on a microchip using indirect laser-induced fluorescence.

PubMed

Heleg-Shabtai, Vered; Gratziany, Natzach; Liron, Zvi

2006-05-01

The application of indirect LIF (IDLIF) technique for on-chip electrophoretic separation and detection of the nerve agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and its major phosphonic degradation products, ethyl methylphosphonic acid (EMPA) and methylphosphonic acid (MPA) was demonstrated. Separation and detection of MPA degradation products of VX and the nerve agent isopropyl methylphosphonofluoridate (GB) are presented. The negatively charged dye eosin was found to be a good fluorescent marker for both the negatively charged phosphonic acids and the positively charged VX, and was chosen as the IDLIF visualization fluorescent dye. Separation and detection of VX, EMPA, and MPA in a simple-cross microchip were completed within less than a minute, and consumed only a 50 pL sample volume. A characteristic system peak that appeared in all IDLIF electropherograms served as an internal standard that increased the reliability of peak identification. The negative peak of both VX and the MPAs is in agreement with indirect detection theory and with previous reports in the literature. The LOD of VX and EMPA by IDLIF was 30 and 37 microM, respectively. Despite the fact that the detection sensitivity is relatively low, the rapid simultaneous on-chip analysis of both VX and its degradation products as well as the separation and detection of the MPA degradation products of both VX and GB, increases detection reliability and may present a choice when sensitivity is not critical compared with speed and simplicity of the assay.

DNA analysis using an integrated microchip for multiplex PCR amplification and electrophoresis for reference samples.

PubMed

Le Roux, Delphine; Root, Brian E; Reedy, Carmen R; Hickey, Jeffrey A; Scott, Orion N; Bienvenue, Joan M; Landers, James P; Chassagne, Luc; de Mazancourt, Philippe

2014-08-19

A system that automatically performs the PCR amplification and microchip electrophoretic (ME) separation for rapid forensic short tandem repeat (STR) forensic profiling in a single disposable plastic chip is demonstrated. The microchip subassays were optimized to deliver results comparable to conventional benchtop methods. The microchip process was accomplished in sub-90 min compared with >2.5 h for the conventional approach. An infrared laser with a noncontact temperature sensing system was optimized for a 45 min PCR compared with the conventional 90 min amplification time. The separation conditions were optimized using LPA-co-dihexylacrylamide block copolymers specifically designed for microchip separations to achieve accurate DNA size calling in an effective length of 7 cm in a plastic microchip. This effective separation length is less than half of other reports for integrated STR analysis and allows a compact, inexpensive microchip design. This separation quality was maintained when integrated with microchip PCR. Thirty samples were analyzed conventionally and then compared with data generated by the microfluidic chip system. The microfluidic system allele calling was 100% concordant with the conventional process. This study also investigated allelic ladder consistency over time. The PCR-ME genetic profiles were analyzed using binning palettes generated from two sets of allelic ladders run three and six months apart. Using these binning palettes, no allele calling errors were detected in the 30 samples demonstrating that a microfluidic platform can be highly consistent over long periods of time.

Predicting tensorial electrophoretic effects in asymmetric colloids

NASA Astrophysics Data System (ADS)

Mowitz, Aaron J.; Witten, T. A.

2017-12-01

We formulate a numerical method for predicting the tensorial linear response of a rigid, asymmetrically charged body to an applied electric field. This prediction requires calculating the response of the fluid to the Stokes drag forces on the moving body and on the countercharges near its surface. To determine the fluid's motion, we represent both the body and the countercharges using many point sources of drag known as Stokeslets. Finding the correct flow field amounts to finding the set of drag forces on the Stokeslets that is consistent with the relative velocities experienced by each Stokeslet. The method rigorously satisfies the condition that the object moves with no transfer of momentum to the fluid. We demonstrate that a sphere represented by 1999 well-separated Stokeslets on its surface produces flow and drag force like a solid sphere to 1% accuracy. We show that a uniformly charged sphere with 3998 body and countercharge Stokeslets obeys the Smoluchowski prediction [F. Morrison, J. Colloid Interface Sci. 34, 210 (1970), 10.1016/0021-9797(70)90171-2] for electrophoretic mobility when the countercharges lie close to the sphere. Spheres with dipolar and quadrupolar charge distributions rotate and translate as predicted analytically to 4% accuracy or better. We describe how the method can treat general asymmetric shapes and charge distributions. This method offers promise as a way to characterize and manipulate asymmetrically charged colloid-scale objects from biology (e.g., viruses) and technology (e.g., self-assembled clusters).

Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis.

PubMed

d'Orlyé, Fanny; Varenne, Anne; Georgelin, Thomas; Siaugue, Jean-Michel; Teste, Bruno; Descroix, Stéphanie; Gareil, Pierre

2009-07-01

In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3-(aminopropyl)triethoxysilane and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV-visible detection were obtained through semi-permanent capillary coating with didodecyldimethyl-ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge-dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaiser's original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.

Susceptibilities of Norwegian Candida albicans strains to fluconazole: emergence of resistance. The Norwegian Yeast Study Group.

PubMed Central

Sandven, P; Bjørneklett, A; Maeland, A

1993-01-01

All Candida albicans isolates in Norwegian microbiological laboratories in 1991 judged clinically important (except vaginal isolates) were collected. The isolates were tested for susceptibility to fluconazole with an agar dilution test and a commercially available agar diffusion test. A total of 212 strains (95%) were susceptible to fluconazole, and MICs for most of the strains (92%) were < or = 1.56 micrograms/ml. The agar diffusion test using a 15-micrograms tablet and a 48-h incubation period separated resistant from susceptible strains with a wide margin. The only exception was a strain for which the MIC was 6.25 micrograms/ml. The difference in zone size between the resistant and the susceptible populations of strains was 11 mm. Accordingly, it appears that the agar diffusion test is an appropriate method for detecting fluconazole resistance. The 12 fluconazole-resistant isolates originated from eight AIDS patients with oral or esophageal Candida infections. Seven of the patients had been given fluconazole for 1 month or more, often as self medication. Four had infections that were clinically resistant to fluconazole; one additional patient responded only when the dose was increased. All isolates recovered from these patients were analyzed by multilocus enzyme electrophoresis. The 12 C. albicans isolates belonged to five electrophoretic types, but three of four patients attending one hospital had isolates belonging to one electrophoretic type. One possible explanation for this finding could be that a nosocomial spread of resistant strains has occurred. PMID:8285631

Fast evaluation of enantioselective drug metabolism by electrophoretically mediated microanalysis: application to fluoxetine metabolism by CYP2D6.

PubMed

Asensi-Bernardi, Lucía; Martín-Biosca, Yolanda; Escuder-Gilabert, Laura; Sagrado, Salvador; Medina-Hernández, María José

2013-12-01

In this work, a capillary electrophoretic methodology for the enantioselective in vitro evaluation of drugs metabolism is applied to the evaluation of fluoxetine (FLX) metabolism by cytochrome 2D6 (CYP2D6). This methodology comprises the in-capillary enzymatic reaction and the chiral separation of FLX and its major metabolite, norfluoxetine enantiomers employing highly sulfated β-CD and the partial filling technique. The methodology employed in this work is a fast way to obtain a first approach of the enantioselective in vitro metabolism of racemic drugs, with the additional advantage of an extremely low consumption of enzymes, CDs and all the reagents involved in the process. Michaelis-Menten kinetic parameters (Km and Vmax ) for the metabolism of FLX enantiomers by CYP2D6 have been estimated by nonlinear fitting of experimental data to the Michaelis-Menten equation. Km values have been found to be 30 ± 3 μM for S-FLX and 39 ± 5 μM for R-FLX. Vmax estimations were 28.6 ± 1.2 and 34 ± 2 pmol·min(-1) ·(pmol CYP)(-1) for S- and R-FLX, respectively. Similar results were obtained using a single enantiomer (R-FLX), indicating that the use of the racemate is a good option for obtaining enantioselective estimations. The results obtained show a slight enantioselectivity in favor of R-FLX. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The influence of hydrodynamic slip on the electrophoretic mobility of a spherical colloidal particle

NASA Astrophysics Data System (ADS)

Khair, Aditya S.; Squires, Todd M.

2009-04-01

Recent theoretical studies have suggested a significant enhancement in electro-osmotic flows over hydrodynamically slipping surfaces, and experiments have indeed measured O(1) enhancements. In this paper, we investigate whether an equivalent effect occurs in the electrophoretic motion of a colloidal particle whose surface exhibits hydrodynamic slip. To this end, we compute the electrophoretic mobility of a uniformly charged spherical particle with slip length λ as a function of the zeta (or surface) potential of the particle ζ and diffuse-layer thickness κ-1. In the case of a thick diffuse layer, κa ≪1 (where a is the particle size), simple arguments show that slip does lead to an O(1) enhancement in the mobility, owing to the reduced viscous drag on the particle. On the other hand, for a thin-diffuse layer κa ≫1, the situation is more complicated. A detailed asymptotic analysis, following the method of O'Brien [J. Colloid Interface Sci. 92, 204 (1983)], reveals that an O(κλ) increase in the mobility occurs at low-to-moderate zeta potentials (with ζ measured on the scale of thermal voltage kBT /e≈25 mV). However, as ζ is further increased, the mobility decreases and ultimately becomes independent of the slip length—the enhancement is lost—which is due to the importance of nonuniform surface conduction within the thin-diffuse layer, at large ζ and large, but finite, κa. Our asymptotic calculations for thick and thin-diffuse layers are corroborated and bridged by computation of the mobility from the numerical solution of the full electrokinetic equations (using the method of O'Brien and White [J. Chem. Soc., Faraday Trans. 2 74, 1607 (1978)]). In summary, then, we demonstrate that hydrodynamic slip can indeed produce an enhancement in the electrophoretic mobility; however, such enhancements will not be as dramatic as the previously studied κa →∞ limit would suggest. Importantly, this conclusion applies not only to electrophoresis but also to electro-osmosis over highly charged surfaces, wherein any inhomogeneities (e.g., due to curvature, roughness, charge patterning, or a variation in slip length) will drive nonuniform surface conduction, which prevents the significant slip-driven flow enhancements predicted for a uniform highly charged surface.

A weak cation-exchange monolith as stationary phase for the separation of peptide diastereomers by CEC.

PubMed

Ludewig, Ronny; Nietzsche, Sandor; Scriba, Gerhard K E

2011-01-01

A CEC weak cation-exchange monolith has been prepared by in situ polymerization of acrylamide, methylenebisacrylamide and 4-acrylamidobutyric acid in a decanol-dimethylsulfoxide mixture as porogen. The columns were evaluated by SEM and characterized with regard to the separation of diastereomers and α/β-isomers of aspartyl peptides. Column preparation was reproducible as evidenced by comparison of the analyte retention times of several columns prepared simultaneously. Analyte separation was achieved using mobile phases consisting of acidic phosphate buffer and ACN. Under these conditions the peptides migrated due to their electrophoretic mobility but the EOF also contributed as driving force as a function of the pH of the mobile phase due to increasing dissociation of the carboxyl groups of the polymer. Raising the pH of the mobile phase also resulted in deprotonation of the peptides reducing analyte mobility. Due to these mechanisms each pair of diastereomeric peptides displayed the highest resolution at a different pH of the buffer component of the mobile phase. Comparing the weak-cation exchange monolith to an RP monolith and a strong cation-exchange monolith different elution order of some peptide diastereomers was observed, clearly illustrating that interactions with the stationary phase contribute to the CEC separations. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Semianalytical Analysis of Compressible Electrophoretic Cake Formation

NASA Astrophysics Data System (ADS)

Kambham, Kiran K. R.; Tuncay, Kagan; Corapcioglu, M. Yavuz

1995-05-01

Leaks in geomembrane liners of waste landfills and liquid impoundments cause chemical contaminants to leak into the subsurface environment. A mathematical model is presented to simulate electrophoretic sealing of impoundment leaks. The model describes the formation of a compressible clay cake because of electrical and gravitational forces. The model includes mass balance equations for the solid particles and liquid phase, modified Darcy's law in an electrical field, and Terzaghi's definition of effective stress. The formulation is presented in the Eulerian coordinates. The resulting second-order, nonlinear partial differential equation and the lower boundary condition are linearized to obtain an analytical solution for time-dependent settlement. After discretizing in time the analytical solution is applied to simulate compression of an accreting sediment. In the simulation of an accreting sediment, solid fluxes on either side of suspension/sediment interface are coupled using a no-jump condition. The velocity of a discrete particle in the suspension zone is assumed to be equal to the algebraic sum of electrophoretic and Stoke's settling velocities. An empirical relationship available in the literature is used to account for the effect of concentration on the velocity of solid particles in the suspension zone. The validity of the semianalytical approach is partially verified using an exact steady state solution for self-weight consolidation. The simulation results obtained for a set of material parameters are presented graphically. It is noted that the electrokinetic consolidation of sediment continues even after the completion of electrophoretic settling of all clay particles. An analysis reveals that the electrophoretic cake formation process is quite sensitive to voltage gradient and the coefficient of compressibility.

Features of electrophoretic deposition process of nanostructured electrode materials for planar Li-ion batteries

NASA Astrophysics Data System (ADS)

Melkozyorova, N. A.; Zinkevich, K. G.; Lebedev, E. A.; Alekseyev, A. V.; Gromov, D. G.; Kitsyuk, E. P.; Ryazanov, R. M.; Sysa, A. V.

2017-11-01

The features of electrophoretic deposition process of composite LiCoO2-based cathode and Si-based anode materials were researched. The influence of the deposition process parameters on the structure and composition of the deposit was revealed. The possibility of a local deposition of composites on a planar lithium-ion battery structure was demonstrated.

Numerical study of the influence of solid polarization on electrophoresis at finite Debye thickness.

PubMed

Bhattacharyya, Somnath; De, Simanta

2015-09-01

The influence of solid polarization on the electrophoresis of a uniformly charged dielectric particle for finite values of the particle-to-fluid dielectric permittivity ratio is analyzed quantitatively without imposing the thin Debye length or weak-field assumption. Present analysis is based on the computation of the coupled Poisson-Nernst-Planck and Stokes equations in the fluid domain along with the Laplace equation within the solid. The electrophoretic velocity is determined through the balance of forces acting on the particle. The solid polarization of the charged particle produces a reduction on its electrophoretic velocity compared to a nonpolarizable particle of the same surface charge density. In accordance with the existing thin-layer analysis, our computed results for thin Debye layer shows that the solid polarization is important only when the applied electric field is strong. When the Debye length is in the order of the particle size, the electrophoretic velocity decreases with the rise of the particle permittivity and attains a saturation limit at large values of the permittivity. Our computed solution for electrophoretic velocity is in agreement with the existing asymptotic analyses based on a thin Debye layer for limiting cases.

Nickel foam-supported polyaniline cathode prepared with electrophoresis for improvement of rechargeable Zn battery performance

NASA Astrophysics Data System (ADS)

Xia, Yang; Zhu, Derong; Si, Shihui; Li, Degeng; Wu, Sen

2015-06-01

Porous nickel foam is used as a substrate for the development of rechargeable zinc//polyaniline battery, and the cathode electrophoresis of PANI microparticles in non-aqueous solution is applied to the fabrication of Ni foam supported PANI electrode, in which the corrosion of the nickel foam substrate is prohibited. The Ni foam supported PANI cathode with high loading is prepared by PANI electrophoretic deposition, and followed by PANI slurry casting under vacuum filtration. The electrochemical charge storage performance for PANI material is significantly improved by using nickel foam substrate via the electrophoretic interlayer. The specific capacity of the nickel foam-PANI electrode with the electrophoretic layer is higher than the composite electrode without the electrophoretic layer, and the specific capacity of PANI supported by Ni foam reaches up to 183.28 mAh g-1 at the working current of 2.5 mA cm-2. The present electrophoresis deposition method plays the facile procedure for the immobilization of PANI microparticles onto the surface of non-platinum metals, and it becomes feasible to the use of the Ni foam supported PANI composite cathode for the Zn/PANI battery in weak acidic electrolyte.

Electrophoretic deposited TiO 2 pigment-based back reflectors for thin film solar cells

DOE PAGES

Bills, Braden; Morris, Nathan; Dubey, Mukul; ...

2015-01-16

Highly reflective coatings with strong light scattering effect have many applications in optical components and optoelectronic devices. This paper reports titanium dioxide (TiO 2) pigment-based reflectors that have 2.5 times higher broadband diffuse reflection than commercially produced aluminum or silver based reflectors and result in efficiency enhancements of a single-junction amorphous Si solar cell. Electrophoretic deposition is used to produce pigment-based back reflectors with high pigment density, controllable film thickness and site-specific deposition. Electrical conductivity of the pigment-based back reflectors is improved by creating electrical vias throughout the pigment-based back reflector by making holes using an electrical discharge / dielectricmore » breakdown approach followed by a second electrophoretic deposition of conductive nanoparticles into the holes. While previous studies have demonstrated the use of pigment-based back reflectors, for example white paint, on glass superstrate configured thin film Si solar cells, this work presents a scheme for producing pigment-based reflectors on complex shape and flexible substrates. Finally, mechanical durability and scalability are demonstrated on a continuous electrophoretic deposition roll-to-roll system which has flexible metal substrate capability of 4 inch wide and 300 feet long.« less

Influence of collector surface composition and water chemistry on the deposition of cerium dioxide nanoparticles: QCM-D and column experiment approaches.

PubMed

Liu, Xuyang; Chen, Gexin; Su, Chunming

2012-06-19

The deposition behavior of cerium dioxide (CeO(2)) nanoparticles (NPs) in dilute NaCl solutions was investigated as a function of collector surface composition, pH, ionic strength, and organic matter (OM). Sensors coated separately with silica, iron oxide, and alumina were applied in quartz crystal microbalance with dissipation (QCM-D) to examine the effect of these mineral phases on CeO(2) deposition in NaCl solution (1-200 mM). Frequency and dissipation shift followed the order: silica > iron oxide > alumina in 10 mM NaCl at pH 4.0. No significant deposition was observed at pH 6.0 and 8.5 on any of the tested sensors. However, ≥ 94.3% of CeO(2) NPs deposited onto Ottawa sand in columns in 10 mM NaCl at pH 6.0 and 8.5. The inconsistency in the different experimental approaches can be mainly attributed to NP aggregation, surface heterogeneity of Ottawa sand, and flow geometry. In QCM-D experiments, the deposition kinetics was found to be qualitatively consistent with the predictions based on the classical colloidal stability theory. The presence of low levels (1-6 mg/L) of Suwannee River humic acid, fulvic acid, alginate, citric acid, and carboxymethyl cellulose greatly enhanced the stability and mobility of CeO(2) NPs in 1 mM NaCl at pH 6.5. The poor correlation between the transport behavior and electrophoretic mobility of CeO(2) NPs implies that the electrosteric effect of OM was involved.

Cobalt ferrite nanoparticles with improved aqueous colloidal stability and electrophoretic mobility

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

Munjal, Sandeep, E-mail: drsandeepmunjal@gmail.com; Khare, Neeraj, E-mail: nkhare@physics.iitd.ernet.in

We have synthesized CoFe{sub 2}O{sub 4} (CFO) nanoparticles of size ∼ 12.2 nm by hydrothermal synthesis method. To control the size of these CFO nanoparticles, oleic acid was used as a surfactant. The inverse spinel phase of the synthesized nanoparticles was confirmed by X-ray diffraction method. As synthesized oleic acid coated CFO (OA@CFO) nanoparticles has very less electrophoretic mobility in the water and are not water dispersible. These OA@CFO nanoparticles were successfully turned into water soluble phase with a better colloidal aqueous stability, through a chemical treatment using citric acid. The modified citric acid coated CFO (CA@CFO) nanoparticles were dispersible inmore » water and form a stable aqueous solution with high electrophoretic mobility.« less

The electrophoretic deposition of ZnO on highly oriented pyrolytic graphite

NASA Astrophysics Data System (ADS)

Ghalamboran, Milad; Jahangiri, Mojtaba; Yousefiazari, Ehsan

2017-12-01

Intensive research has been conducted on ZnO thin and thick films in recent years. Such layers, used in different electronic devices, are deposited utilizing various methods, but electrophoretic deposition (EPD) has been chosen because of the advantages like low energy consumption, economical superiority, ecofriendliness, controllability, and high deposition rate. Here, we report electrophoretically depositing ZnO layers onto highly oriented pyrolytic graphite. Well-dispersed and stable ZnO suspensions are used for the deposition of continuous and even layers of ZnO on the substrate. ZnO powder is dispersed in acetone. The electric field applied is in the 250 V/cm to 2000 V/cm range. The morphology of the deposits are studied by SEM at the different stages of the deposition process.

Method for Single-Cell Mass and Electrophoretic Mobility Measurement

DTIC Science & Technology

2010-02-01

Staphylococcus Aureus . The species of the contaminant was determined by catalase test and visual comparison with cultured S. Epidermidis. In this experiment...mnicron’crtrVs)) Corrected EPM Hatogam for teratior 2 d) p 0.3 10 I EPM ((rii n*cmy(V**)) CO, ?rz A ) WO Buoyart Mass vs. EPM forSuspected S. Aureus . 583 V...2 -1 E PM ((n cror*crnYC*) Cxnected EPM His-ograrr for Suspected S. Aureus at - - 332 V/cm EPM ((rnicron*cmY(V’s)) Figure 5-3: Integrated measurements

Single-molecule detection: applications to ultrasensitive biochemical analysis

NASA Astrophysics Data System (ADS)

Castro, Alonso; Shera, E. Brooks

1995-06-01

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.

Western blotting: an introduction.

PubMed

Kurien, Biji T; Scofield, R Hal

2015-01-01

Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

Design Modification of Electrophoretic Equipment

NASA Technical Reports Server (NTRS)

Reddick, J. M.; Hirsch, I.

1973-01-01

The improved design of a zone electrophoretic sampler is reported that can be used in mass screening for hemoglobin S, the cause of sickle cell anemia. Considered is a high voltage multicell cellulose acetate device that requires 5 to 6 minutes electrophoresis periods; cells may be activitated individually or simultaneously. A multisample hemoglobin applicator standardizes the amount of sample applied and transfers the homolysate to the electrical wires.

Simple and effective label-free capillary electrophoretic analysis of sugars by complexation using quinoline boronic acids.

PubMed

Kubo, Takuya; Kanemori, Koichi; Kusumoto, Risa; Kawai, Takayuki; Sueyoshi, Kenji; Naito, Toyohiro; Otsuka, Koji

2015-01-01

An effective separation and detection procedure for sugars by capillary electrophoresis (CE) using a complexation between quinolineboronic acid (QBA) and multiple hydroxyl structure of sugar alcohol is reported. We investigated the variation of fluorescence spectra of a variety of QBAs with sorbitol at a wide range of pH conditions and then found that 5-isoQBA strongly enhanced the fluorescence intensity by the complexation at basic pH conditions. The other sugar alcohols having multiple hydroxyls also revealed the enhancement of the fluorescence intensity with 5-isoQBA, whereas the alternation of the intensity was not found in the sugars such as glucose. After optimization of the 5-isoQBA concentration and pH of the buffered solution in CE analysis, 6 sugar alcohols were successfully separated in the order based on the formation constants with 5-isoQBA, which were calculated from the variation of the fluorescence intensity with each sugar alcohol and 5-isoQBA. Furthermore, the limits of detection for sorbitol and xylitol by the CE method were estimated at 15 and 27 μM, respectively.

Separation of a chemically modified DNA oligomer bound by the carcinogen 2-Amino-1-methy-6-phenylimidazo [4,5-{beta}]pyridine using capillary gel electrophoresis

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

Nguyen, T.N.

1994-05-06

We have optimized the reaction conditions under which unactivated metabolite of the food borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-{beta}]pyridine (PhIP) is covalently bound to the oligodeoxynucleotide d(CCTACGCATCC). Capillary electrophoresis (CE) was used to separate and characterize this DNA oligomer bound by PhIP. We observed 2 major and several minor PhIP adduct species. The 2 major adducts had different absorbance maxima; the major adduct eluates with faster and slower mobilities had absorbance maxima of 360 and 340 nm, respectively. One of the two major PhIP adduct species was resolvable but the peak was broad. Using detection at 260 nm, the other major PhIPmore » adduct with fastest electrophoretic mobility was not resolvable, but coelute with the huge broad unmodified DNA oligomer peak. However, at higher wavelengths (>320 nm) where DNA does not absorb, electropherograms generated by detection at these higher wavelengths showed very heterogeneous binding by PhIP to the DNA oligomer with no interfering absorbance by the DNA.« less

Liquid-phase-based separation systems for depletion, prefractionation and enrichment of proteins in biological fluids and matrices for in-depth proteomics analysis – An update covering the period 2011-2014

PubMed Central

Puangpila, Chanida; Mayadunne, Erandi; Rassi, Ziad El

2015-01-01

This review article expands on the previous one (S. Selvaraju and Z. El Rassi, Electrophoresis 2012, 33, 74-88) by reviewing pertinent literature in the period extending from early 2011 to present. As the previous review article, the present one is concerned with proteomic sample preparation (e.g., depletion of high abundance proteins, reduction of the protein dynamic concentration range, enrichment of a particular sub-proteome), and the subsequent chromatographic and/or electrophoretic pre-fractionation prior to peptide separation and identification by LC-MS/MS. This review article is distinguished from its second version published in Electrophoresis 2012, 33, 74-88 by expanding on capturing/enriching sub-phosphoproteomes by immobilized metal affinity chromatography and metal oxide affinity chromatography. Seventy-seven papers published in the period extending from mid 2011 to the present have been reviewed. By no means this review article is exhaustive, given the fact that its aim is to give a concise treatment of the latest developments in the field. PMID:25287967

Chromatofocusing and isoelectric focusing in immobilized pH gradients compared for characterization of human hemoglobin variants.

PubMed

Paleari, R; Arcelloni, C; Paroni, R; Fermo, I; Mosca, A

1989-03-01

We compared the performance of two highly resolving methods, chromatofocusing (CRF) and isoelectric focusing in immobilized pH gradients (IPGF), for the separation of human hemoglobin variants. Lysates containing 13 different hemoglobins, including variants of clinical and geographical importance, and four electrophoretically "silent" variants (Hb Brockton, Hb Cheverly, Hb Köln, and Hb Waco) were analyzed. Both techniques showed a good intrarun precision (CV = 0.87% for CRF, 0.27% for IPGF) and high and similar resolving power (0.010 pH units, with the pH gradients used in this work). The use of an ultranarrow IPGF range (pH 7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution between Hb Brockton, Hb Köln, and Hb A. In some cases (Hb D-Los Angeles, Hb F, Hb Waco), the variants were separated from Hb A in different orders, depending on which technique was used, probably because of the different analytical principles of the two methods. As a second-level test, both procedures are informative for characterization of human hemoglobin variants.

One-step zymogram method for the simultaneous detection of cellulase/xylanase activity and molecular weight estimation of the enzyme.

PubMed

Cano-Ramírez, Claudia; Santiago-Hernández, Alejandro; Rivera-Orduña, Flor Nohemí; Pineda-Mendoza, Rosa María; Zúñiga, Gerardo; Hidalgo-Lara, María Eugenia

2017-02-01

Here, we describe a zymographic method for the simultaneous detection of enzymatic activity and molecular weight (MW) estimation, following a single electrophoresis step. This involved separating cellulase and xylanase activities from bacteria and fungi, obtained from different sources, such as commercial extracts, crude extract and purified proteins, under denaturing conditions, by 10% polyacrylamide gel electrophoresis, using polyacrylamide gels copolymerized with 1% (w/v) carboxymethylcellulose or beechwood xylan as substrates. Then, enzymes were refolded by treatment with 2.5% Triton X-100 in an appropriate buffer for each enzymatic activity, and visualized by Coomassie blue staining for MW estimation. Finally, Congo red staining revealed bio-active cellulase and xylanase bands after electrophoretic separation of the proteins in the preparations. This method may provide a useful additional tool for screening of particular cellulase and xylanase producers, identification and MW estimation of polypeptides that manifest these activities, and for monitoring and control of fungal and bacterial cellulase and xylanase production. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enabling environmental metagenomics and extremophile discovery through SCODA DNA purification

NASA Astrophysics Data System (ADS)

Lum, T.; Maydan, J.

2016-12-01

A major challenge in nucleic acid preparation from environmental samples is in the ability to separate DNA and RNA from contaminants that often co-purify with methods commonly used. This becomes even more challenging when nucleic acids are in low abundance or when enriching for high molecular weight fragments. Many column- and bead-based methods rely upon selective chemical affinity which is insufficient in dealing with similarly charged contaminants, and also often result in over fragmentation nucleic acids and substantial sample loss. Here we present a unique and alternative parameter for the separation nucleic acids based on the nonlinear response of long, charged polymers to electrophoretic fields. The synchronous coefficient of drag alteration (SCODA) technology is capable of purifying nucleic acids from highly contaminated sample matrices, with molecular weight ranges from 300 bp to over 1 Mbp, and from very low biomass origins. Using a combination of rotating dipole and quadrupole electric fields, SCODA technology concentrates ultrapure nucleic acids that enable PCR, NGS, and optical mapping applications on sample types that are otherwise difficult or impossible to analyze.

Heterogeneity in the growth hormone pituitary gland system of rats and humans: Implications to microgravity based research

NASA Technical Reports Server (NTRS)

Hymer, W. C.; Grindeland, R.; Hayes, C.; Lanham, J. W.; Cleveland, C.; Todd, P.; Morrison, Dennis R.

1988-01-01

The cell separation techniques of velocity sedimentation, flow cytometry and continuous flow electrophoresis were used to obtain enriched populations of growth hormone (GH) cells. The goal was to isolate a GH cell subpopulation which releases GH molecules which are very high in biological activity, it was important to use a method which was effective in processing large numbers of cells over a short time span. The techniques based on sedimentation are limited by cell density overlaps and streaming. While flow cytometry is useful in the analytical mode for objectively establishing cell purity, the numbers of cells which can be processed in the sort mode are so small as to make this approach ineffective in terms of the long term goals. It was shown that continuous flow electrophoresis systems (CFES) can separate GH cells from other cell types on the basis of differences in surface charge. The bioreactive producers appear to be more electrophoretically mobile than the low producers. Current ground based CFES efforts are hampered by cell clumping in low ionic strength buffers and poor cell recoveries from the CFES device.

Zeta-potential Analyses using Micro Electrical Field Flow Fractionation with Fluorescent Nanoparticles

PubMed Central

Chang, Moon-Hwan; Dosev, Dosi; Kennedy, Ian M.

2007-01-01

Increasingly growing application of nanoparticles in biotechnology requires fast and accessible tools for their manipulation and for characterization of their colloidal properties. In this work we determine the zeta-potentials for polystyrene nanoparticles using micro electrical field flow fractionation (μ–EFFF) which is an efficient method for sorting of particles by size. The data obtained by μ–EFFF were compared to zeta potentials determined by standard capillary electrophoresis. For proof of concept, we used polystyrene nanoparticles of two different sizes, impregnated with two different fluorescent dyes. Fluorescent emission spectra were used to evaluate the particle separation in both systems. Using the theory of electrophoresis, we estimated the zeta-potentials as a function of size, dielectric permittivity, viscosity and electrophoretic mobility. The results obtained by the μ–EFFF technique were confirmed by the conventional capillary electrophoresis measurements. These results demonstrate the applicability of the μ–EFFF method not only for particle size separation but also as a simple and inexpensive tool for measurements of nanoparticles zeta potentials. PMID:18542710

Quantitative analysis of synthetic dyes in lipstick by micellar electrokinetic capillary chromatography.

PubMed

Desiderio, C; Marra, C; Fanali, S

1998-06-01

The separation of synthetic dyes, used as color additives in cosmetics, by micellar electrokinetic capillary chromatography (MEKC) is described in this study. The separation of seven dyes, namely eosine, erythrosine, cyanosine, rhodamine B, orange II, chromotrope FB and tartrazine has been achieved in about 3 min in an untreated fused silica capillary containing as background electrolyte a 25 mM tetraborate/phosphate buffer, pH 8.0, and 30 mM sodium dodecyl sulfate. The electrophoretic method exhibits precision and relatively high sensitivity. A detection limit (LOD, signal/noise = 3) in the range of 5-7.5 X 10(-7) M of standard compounds was recorded. Intra-day repeatability of all the studied dye determinations (8 runs) gave the following results (limit values), % standard deviation: 0.24-1.54% for migration time, 0.99-1.24% for corrected peak areas, 0.99-1.24% for corrected peak area ratio (analyte/internal standard) and 1.56-2.74% for peak areas. The optimized method was successfully applied to the analysis of a lipstick sample where eosine and cyanosine were present.

Studies on aqueous two phase polymer systems useful for partitioning of biological materials

NASA Technical Reports Server (NTRS)

Brooks, D. E.; Bamberger, S.

1982-01-01

The two phase systems that result when aqueous solutions of dextran and poly(ethylene glycol) (PEG) are mixed above a critical concentration of a few percent provide a useful medium for the separation of biological cell subpopulations via partition between the top, PEG-rich phase and the liquid-liquid phase boundary. Interfacial tensions of such systems have been measured by the rotating drop technique and found to range between 0.1-100 micro-N/m. The tension was found to depend on the length of the tie line describing the system on a phase diagram, via a power law relationship which differed depending on the concentration of Na phosphate buffer present. The electrokinetic properties of drops of one phase suspended in the other were studied for a variety of systems. It was found that the droplet electrophoretic mobility increased monotonically with phosphate concentration and drop diameter but exhibited the opposite sign from that anticipated from phosphate partition measurements. It was possible to take advantage of these electrokinetic properties and dramatically enhance the speed of phase separation through application of relatively small electric fields.

Characterization of a single-isomer carboxymethyl-beta-cyclodextrin in chiral capillary electrophoresis.

PubMed

Fejős, Ida; Varga, Erzsébet; Benkovics, Gábor; Malanga, Milo; Sohajda, Tamás; Szemán, Julianna; Béni, Szabolcs

2017-08-01

In this work, the synthesis, characterization, and chiral capillary electrophoretic study of heptakis-(2,3-di-O-methyl-6-O-carboxymethyl)-β-CD (HDMCM), a single-isomer carboxymethylated CD, are presented. The pH-dependent and selector concentration-dependent enantiorecognition properties of HDMCM were investigated and discussed herein. The enantioseparation was assessed applying a structurally diverse set of noncharged, basic, and zwitterionic racemates. The increase in the selector concentration and gross negative charge of HDMCM improved the enantioseparation that could be observed in the majority of the cases. HDMCM was also successfully applied as BGE additive in NACE using a methanol-based system in order to prove the separation selectivity features and to highlight the broad applicability of HDMCM. Over 25 racemates showed partial or baseline separation with HDMCM under the conditions investigated, among which optimal enantiomer migration order was found for the four stereoisomers of tadalafil, tapentadol, and dapoxetine, offering the possibility of a chiral CE method development for chiral purity profiling of these drugs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

PubMed Central

Birdsall, Robert E.; Koshel, Brooke M.; Hua, Yimin; Ratnayaka, Saliya N.; Wirth, Mary J.

2013-01-01

Sieving of proteins in silica colloidal crystals of mm dimensions is characterized for particle diameters of nominally 350 and 500 nm, where the colloidal crystals are chemically modified with a brush layer of polyacrylamide. A model is developed that relates the reduced electrophoretic mobility to the experimentally measurable porosity. The model fits the data with no adjustable parameters for the case of silica colloidal crystals packed in capillaries, for which independent measurements of the pore radii were made from flow data. The model also fits the data for electrophoresis in a highly ordered colloidal crystal formed in a channel, where the unknown pore radius was used as a fitting parameter. Plate heights as small as 0.4 μm point to the potential for miniaturized separations. Band broadening increases as the pore radius approaches the protein radius, indicating that the main contribution to broadening is the spatial heterogeneity of the pore radius. The results quantitatively support the notion that sieving occurs for proteins in silica colloidal crystals, and facilitate design of new separations that would benefit from miniaturization. PMID:23229163

High-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding.

PubMed

Dang, Fuquan; Tabata, Osamu; Kurokawa, Masaya; Ewis, Ashraf A; Zhang, Lihua; Yamaoka, Yoshihisa; Shinohara, Shouji; Shinohara, Yasuo; Ishikawa, Mitsuru; Baba, Yoshinobu

2005-04-01

We have developed a novel technique for mass production of microfabricated capillary array electrophoresis (mu-CAE) plastic chips for high-speed, high-throughput genetic analysis. The mu-CAE chips, containing 10 individual separation channels of 50-microm width, 50-microm depth, and a 100-microm lane-to-lane spacing at the detection region and a sacrificial channel network, were fabricated on a poly(methyl methacrylate) substrate by injection molding and then bonded manually using a pressure-sensitive sealing tape within several seconds at room temperature. The conditions for injection molding and bonding were carefully characterized to yield mu-CAE chips with well-defined channel and injection structures. A CCD camera equipped with an image intensifier was used to monitor simultaneously the separation in a 10-channel array with laser-induced fluorescence detection. High-performance electrophoretic separations of phiX174 HaeIII DNA restriction fragments and PCR products related to the human beta-globin gene and SP-B gene (the surfactant protein B) have been demonstrated on mu-CAE plastic chips using a methylcellulose sieving matrix in individual channels. The current work demonstrated greatly simplified the fabrication process as well as a detection scheme for mu-CAE chips and will bring the low-cost mass production and application of mu-CAE plastic chips for genetic analysis.

Active microbial soil communities in different agricultural managements

NASA Astrophysics Data System (ADS)

Landi, S.; Pastorelli, R.

2009-04-01

We studied the composition of active eubacterial microflora by RNA extraction from soil (bulk and rhizosphere) under different environmental impact managements, in a hilly basin in Gallura (Sardinia). We contrasted grassy vineyard, in which the soil had been in continuous contact with plant roots for a long period of time, with traditional tilled vineyard. Moreover, we examined permanent grassland, in which plants had been present for some years, with temporary grassland, in which varying plants had been present only during the respective growing seasons. Molecular analysis of total population was carried out by electrophoretic separation by Denaturing Gradient Gel Electrophoresis (DGGE) of amplified cDNA fragments obtained from 16S rRNA. In vineyards UPGMA (Unweighted Pair Group Mathematical Average) analysis made up separate clusters depending on soil management. In spring both clusters showed similarity over 70%, while in autumn the similarity increased, 84% and 90% for grassy and conventional tilled vineyard respectively. Permanent and temporary grassland joined in a single cluster in spring, while in autumn a partial separation was evidenced. The grassy vineyard, permanent and temporary grassland showed higher richness and diversity Shannon-Weiner index values than vineyard with conventional tillage although no significant. In conclusion the expected effect of the rhizosphere was visible: the grass cover influenced positively the diversity of active microbial population.

Potential role of gold nanoparticles for improved analytical methods: an introduction to characterizations and applications.

PubMed

Wu, Chung-Shu; Liu, Fu-Ken; Ko, Fu-Hsiang

2011-01-01

Nanoparticle-based material is a revolutionary scientific and engineering venture that will invariably impact the existing analytical separation and preconcentration for a variety of analytes. Nanoparticles can be regarded as a hybrid between small molecule and bulk material. A material on the nanoscale produces considerable changes on various properties, making them size- and shape-dependent. Gold nanoparticles (Au NPs), one of the wide variety of core materials available, coupled with tunable surface properties in the form of inorganic or inorganic-organic hybrid have been reported as an excellent platform for a broad range of analytical methods. This review aims to introduce the basic principles, examples, and descriptions of methods for the characterization of Au NPs by using chromatography, electrophoresis, and self-assembly strategies for separation science. Some of the latest important applications of using Au NPs as stationary phases toward open-tubular capillary electrochromatography, gas chromatography, and liquid chromatography as well as roles of run buffer additive to enhance separation and preconcentration in the field of chromatographic, electrophoretic and in chip-based systems are reviewed. Additionally, we review Au NPs-assisted state-of-the-art techniques involving the use of micellar electrokinetic chromatography, an online diode array detector, solid-phase extraction, and mass spectrometry for the preconcentration of some chemical compounds and biomolecules.

[Fabrications of a poly (methyl methacrylate) (PMMA) microfluidic chip-based DNA analysis device].

PubMed

Du, Xiao-Guang

2009-12-01

A DNA analysis device based on poly(methyl methacrylate) (PMMA) microfluidic chips was developed. A PMMA chip with cross microchannels was fabricated by a simple hot embossing. Microchannels were modified with a static adsorptive coating method using 2% hydroxyethyl cellulose. A high-voltage power unit, variable in the range 0-1 800 V, was used for on-chip DNA sample injection and gel electrophoretic separation. High speed, high resolution DNA analysis was obtained with the home-built PMMA chip in a sieving matrix containing 2% hydroxyethyl cellulose with a blue intercalating dye, TO-PRO-3 (TP3), by using diode laser induced fluorescence detection based on optical fibers with a 670 nm long-pass filter. The DNA analysis device was applied for the separation of phiX-174/HaeIII DNA digest sample with 11 fragments ranging from 72 to 1 353 bp. A separation efficiency of 1.14 x 10(6) plates/m was obtained for the 603 bp fragments, while the R of 271/281 bp fragments was 1.2. The device was characterized by simple design, low cost for fabrication and operation, reusable PMMA chips, and good reproducibility. A portable microfluidic device for DNA analysis can be developed for clinical diagnosis and disease screening.

Mobility of long-chain DNA in two-dimensional artificial gels

NASA Astrophysics Data System (ADS)

Turner, Stephen W. P.; Han, Jongyoon; Craighead, Harold G.

2000-03-01

In this study, a two-dimensional array of nanofabricated obstacles is used as an artificial gel to study the electrophoretic mobility dependence of DNA as a function of pore size, molecule length and electric field. Limitations in feature size have prevented previous studies from testing the crossover from the separating to the non-separating regime predicted by the biased reptation model of Lumpkin, Dejardin and Zimm[1] and the modified model of Duke, Semenov and Viovy.[2] That limitation is overcome in this work with the use of electron beam lithography to define features as small as 30 nm. Attainment of these feature sizes was made possible by the use of a sacrificial-layer-based technique for fluidics fabrication.[3] A novel band-launching strategy is used to provide band separation data for the first time in this system. Molecule lengths between 5 and 150 kilobases are studied for electric field strengths from 0.1 to 20 Volts per meter. [1] O. Lumpkin, P. Dejardin and B. Zimm, Biopolymers, Vol. 24, 1573-1593 (1985) [2] T. Duke, A. Semenov and J. Viovy, Phys. Rev. Lett. Vol. 69, No. 22, 3260-3263 (1992) [3] S. Turner, A. Perez, A. Lopez, and H. Craighead, J. Vac. Sci. Technol. B 16(6) 3835-3840 (1998)

Rapid separation and sensitive determination of banned aromatic amines with plastic microchip electrophoresis.

PubMed

Li, Ruina; Wang, Lili; Gao, Xiaotong; Du, Gangfeng; Zhai, Honglin; Wang, Xiayan; Guo, Guangsheng; Pu, Qiaosheng

2013-03-15

Rapid analysis of trace amount of aromatic amines in environmental samples and daily necessities has attracted considerable attentions because some of them are strongly toxic and carcinogenic. In this study, fast and efficient electrophoretic separation and sensitive determination of 5 banned aromatic amines were explored for practical analysis using disposable plastic microchips combined with a low-cost laser-induced fluorescence detector. The effect of running buffer and its additive was systematically investigated. Under the selected condition, 5 fluorescein isothiocyanate labeled aromatic amines could be baseline separated within 90s by using a 10mmol/L borate buffer containing 2% (w/v) hydroxypropyl cellulose. Calibration curves of peak areas vs. concentrations were linear up to 40 or 120μmol/L for different analytes and limits of detection were in a range of 1-3nmol/L. Theoretical plate numbers of 6.8-8.5×10(5)/m were readily achieved. The method exhibited good repeatability, relative standard deviations (n=5) of peak areas and migration times were no more than 4.6% and 0.9%, respectively. The established method was successfully applied in the quantitative analysis of these banned aromatic amines in real samples of waste water and textile, recoveries of added standards were 85-110%. Copyright © 2013 Elsevier B.V. All rights reserved.

Isoelectric focusing of small non-covalent metal species from plants.

PubMed

Köster, Jessica; Hayen, Heiko; von Wirén, Nicolaus; Weber, Günther

2011-03-01

IEF is known as a powerful electrophoretic separation technique for amphoteric molecules, in particular for proteins. The objective of the present work is to prove the suitability of IEF also for the separation of small, non-covalent metal species. Investigations are performed with copper-glutathione complexes, with the synthetic ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) and respective metal complexes (Fe, Ga, Al, Ni, Zn), and with the phytosiderophore 2'-deoxymugineic acid (DMA) and its ferric complex. It is shown that ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid and DMA species are stable during preparative scale IEF, whereas copper-glutathione dissociates considerably. It is also shown that preparative scale IEF can be applied successfully to isolate ferric DMA from real plant samples, and that multidimensional separations are possible by combining preparative scale IEF with subsequent HPLC-MS analysis. Focusing of free ligands and respective metal complexes with di- and trivalent metals results in different pIs, but CIEF is usually needed for a reliable estimation of pI values. Limitations of the proposed methods (preparative IEF and CIEF) and consequences of the results with respect to metal speciation in plants are discussed. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preparative free-flow electrophoresis as a method of fractionation of natural organic materials

USGS Publications Warehouse

Leenheer, J.A.; Malcolm, R.L.

1973-01-01

Preparative free-flow electrophoresis was found to be an efficient method of conducting large-scale fractionations of the natural organic polyelectrolytes occurring in many surface waters and soils. The method of free-flow electrophoresis obviates, the problem of adsorption upon a supporting medium and permits the use of high potential gradients and currents because of an efficient cooling system. Separations were monitored by determining organic carbon concentration with a dissolved carbon analyzer, and color was measured by absorbance at 400 nanometers. Organic materials from waters and soils were purified by filtration, hydrogen exchange, and dialysis and were concentrated by freeze drying or freeze concentration. In electrophoretic fractionations of natural organic materials typically found in surface waters and soils, color was found to increase with the charge of the fraction.

Application of antibiotics as chiral selectors for capillary electrophoretic enantioseparation of pharmaceuticals: a review.

PubMed

Dixit, Shuchi; Park, Jung Hag

2014-01-01

Recent years have witnessed several new trends in chiral separation, for example, the enantiorecognition ability of several new antibiotics has been explored using capillary electrophoresis (CE) prior to HPLC; antibiotics have been employed as chiral selectors (CSs) in a nonaqueous CE (NACE) mode; and several new detection techniques (namely, capacitively coupled contactless conductivity detection) have been used in combination with CE for quantification of enantiomers. On account of these emerging trends, this article aims to review the application of various classes of antibiotics for CE enantioseparation of pharmaceuticals. A detailed account of the basic factors affecting enantioseparation, certain limitations of antibiotics as CSs and strategies to mitigate them, and advantages of NACE while using antibiotics as CSs has also been presented. Copyright © 2013 John Wiley & Sons, Ltd.

Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

PubMed

Robinson, Nicholas P

2013-01-01

Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

Evaluation of a Portable Microchip Electrophoresis Fluorescence Detection System for the Analysis of Amino Acid Neurotransmitters in Brain Dialysis Samples

PubMed Central

OBORNY, Nathan J.; COSTA, Elton E. Melo; SUNTORNSUK, Leena; ABREU, Fabiane C.; LUNTE, Susan M.

2016-01-01

A portable fluorescence detection system for use with microchip electrophoresis was developed and compared to a benchtop system. Using this system, six neuroactive amines commonly found in brain dialysate—arginine, citrulline, taurine, histamine, glutamate, and aspartate—were derivatized offline with naphthalene-2,3-dicarboxaldehyde/cyanide, separated electrophoretically, and detected by fluorescence. Limits of detection for the analytes of interest were 50nM – 250nM for the benchtop system and 250 nM – 1.3 μM for the portable system, both of which were adequate for analyte determination in brain microdialysis samples. The portable system was then demonstrated for the detection of the same six amines in a rat brain microdialysis sample. PMID:26753703

Winnowing DNA for Rare Sequences: Highly Specific Sequence and Methylation Based Enrichment

PubMed Central

Thompson, Jason D.; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue. PMID:22355378

Determination of psilocybin in Psilocybe semilanceata by capillary zone electrophoresis.

PubMed

Pedersen-Bjergaard, S; Sannes, E; Rasmussen, K E; Tønnesen, F

1997-07-04

A capillary zone electrophoretic (CZE) method was developed for the rapid determination of psilocybin in Psilocybe semilanceata. Following a simple two step extraction with 3.0+2.0 ml methanol, the hallucinogenic compound was effectively separated from matrix components by CZE utilizing a 10 mM borate-phosphate running buffer adjusted to pH 11.5. The identity of psilocybin was confirmed by migration time information and by UV spectra, while quantitation was accomplished utilizing barbital as internal standard. The calibration curve for psilocybin was linear within 0.01-1 mg/ml, while intra-day and inter-day variations of quantitative data were 0.5 and 2.5% R.S.D., respectively. In addition to psilocybin, the method was also suitable for the determination of the structurally related compound baeocystin.

Association of electrophoretic karyotype of Candida stellatoidea with virulence for mice

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

Kwon-Chung, K.J.; Wickes, B.L.; Merz, W.G.

1988-07-01

Seven isolates of Candida stellatoidea were studied for their electrophoretic karyotype, virulence for mice, sensitivity to UV radiation, growth rate in vitro, reaction on cycloheximide-indicator medium, and proteinase activity. The isolates exhibited one of two distinct electrophoretic karyotypes as determined by orthogonal field alternating gel electrophoresis (OFAGE). Four isolates, including the type culture of C. stellatoidea, belonged to electrophoretic karyotype type I by OFAGE, showing eight to nine bands of which at least two bands were less than 1,000 kilobases in size as estimated by comparison with the DNA bands of Saccharomyces cerevisiae. These isolates failed to produce fatal infectionmore » in mice within 20 days when 5 X 10(5) cells were injected intravenously. The yeasts were cleared from the kidneys of two of three mice tested by day 30. Type I showed proteinase activity on bovine serum albumin agar at pH 3.8 and produced a negative reaction on cycloheximide-bromcresol green medium within 48 h. The three grouped in type II by OFAGE showed banding patterns similar to those of a well-characterized isolate of Candida albicans. The isolates of type II had an electrophoretic karyotype of six to seven bands approximately 1,200 kilobases or greater in size. All three type II isolates were highly virulent for mice, producing fatality curves similar to those of a previously studied C. albicans isolate. From 80 to 90% of the mice injected with 5 X 10(5) cells intravenously died within 20 days. The type II isolates produced a positive reaction on cycloheximide-bromcresol green agar and showed no proteinase activity on bovine serum albumin agar at the low pH. In addition, the type II isolates grew faster and were significantly more resistant to UV irradiation than the type I isolates.« less

Electrophoretic properties of BSA-coated quantum dots.

PubMed

Bücking, Wendelin; Massadeh, Salam; Merkulov, Alexei; Xu, Shu; Nann, Thomas

2010-02-01

Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn(2+)/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn(2+)/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.

Electrophoretic-deposited CNT/MnO2 composites for high-power electrochemical energy storage/conversion applications

NASA Astrophysics Data System (ADS)

Xiao, Wei; Xia, Hui; Fuh, Jerry Y. H.; Lu, Li

2010-05-01

CNT/MnO2 (birnessite-type) composite films have been successfully deposited on Ni-foil substrate via electrophoretic deposition (EPD). The unique EPD CNT/MnO2 composite film electrode shows enhanced electrical conductivity, good contact between composite films and the substrate and open porous structure, which makes the EPD composite films a promising electrode for high-power supercapacitors and lithium ion batteries.

Fibre reinforced ceramic matrix composite fabrication by electrophoretic infiltration

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

Kooner, S.; Campaniello, J.J.; Pickering, S.

Electrophoretic infiltration is a novel technique for the fabrication of fibre reinforced composites. The fibres are arranged as one of the electrodes such that deposition of the colloidal ceramic occurs in the fibre preform. This method has been investigated for the composite system of carbon fibre reinforced Si{sub 3}N{sub 4} and has produced green composite microstructures with good infiltration uniformity and fibre distribution and few macro defects.

Microflora on explanted silicone rubber voice prostheses: taxonomy, hydrophobicity and electrophoretic mobility.

PubMed

Neu, T R; Verkerke, G J; Herrmann, I F; Schutte, H K; Van der Mei, H C; Busscher, H J

1994-05-01

Silicone rubber voice prostheses are implants which are inserted in a non-sterile environment and therefore become quickly colonized by micro-organisms. The micro-organisms exist on the medical grade silicone rubber as mixed biofilms of bacteria and yeasts. A total of 79 bacterial and 39 yeast strains were isolated from these biofilms by soft ultrasonic treatment. Gram-positive/catalase-negative and Gram-positive/catalase-positive cocci represented the dominant bacterial strains. The yeasts were mainly Candida species. Further characterization of cell surface properties such as hydrophobicity by microbial adhesion to hexadecane and electrophoretic mobility showed a distinct difference when the bacterial strains were compared with the yeasts. The bacterial hydrophobicities ranged from 0 to 100% adhesion to hexadecane, whereas the yeast strains, especially the Candida albicans strains, all had markedly hydrophilic cell surfaces. A comparison of the electrophoretic mobilities showed also differences between bacteria and yeast. The values for the bacteria were found to be between -2.5 to -0.5 (10(-8) m2 V-1 s-1), whereas for the yeasts electrophoretic mobilities were more positive. Based on the adhesive properties of the isolated micro-organisms, strategies can now be developed to modify the properties of the silicone rubber to reduce biofilm formation on such prostheses.