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PubMed1990

Characterization of protein behavior in high-performance capillary electrophoresis using a novel capillary system.

Original calculations of over a million theoretical plate efficiency for macromolecular solutes in the open tubular high-performance capillary electrophoresis experiment considered axial diffusion to be the efficiency limiting factor. In practice, interactions of biopolymers, such as proteins, with the capillary wall has had a significant impact on readily achieving high efficiencies for a wide variety of proteins. This paper reports a capillary system in which protein-surface interactions have been minimized, resulting in high efficiencies (greater than or equal to 300,000 theoretical plates). This system allows the analysis of a set of protein standards over a wide pI range at neutral pH and moderate ionic strength. The characterization of the behavior of those protein standards in this capillary system is described.

Drug Storage↗
PubMed1991

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries.

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Blood Proteins↗
PubMed1993

Pharmacokinetic studies using micellar electrokinetic capillary chromatography with in vivo capillary ultrafiltration probes.

Micellar electrokinetic capillary chromatography (MECC) is shown to be a quantitative method for the determination of theophylline in capillary ultrafiltrates of biological systems. MECC exhibits reproducibility in migration times of 1.3% relative standard deviation (R.S.D.) (n = 31) and peak heights of 3.0% R.S.D. (n = 28). MECC and reversed-phase liquid chromatography (LC) are shown to be complementary techniques for the determination of theophylline in ultrafiltrate samples. In vivo sampling of awake, freely moving rats is achieved using capillary ultrafiltration probes implanted in subcutaneous tissue. The ability of MECC coupled with in vivo capillary ultrafiltration to determine theophylline pharmacokinetics is demonstrated. The half-life of elimination for a 15 mg/kg intraperitoneal dose of theophylline was determined to be 3.1 +/- 0.4 h for MECC and 3.2 +/- 0.4 h for LC (n = 4, mean +/- standard error of the mean). Concurrent results for derived pharmacokinetic parameters (area under the curve, volume of distribution, concentration at time zero and clearance) were obtained for MECC and LC.

Animals↗
PubMed1991

High-performance capillary electrophoresis of proteins. Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins.

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained. Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D. of ca. 3-7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound (ca. 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.

Animals↗
PubMed2000

Characterization by atomic force microscopy of fused-silica capillaries chemically modified for capillary electrokinetic chromatography

Fused-silica capillary tubes with 50-microm bores have been chemically modified to yield octadecyl-derivatized surfaces with increased surface area. Alkylation is achieved in three steps: etching, silane adlayering, and olefin hydrosilation. Surface roughness, surface area, surface-tip attractive forces, and topographic images of the native, etched, silane-adlayered, and alkylated capillary bores as measured by atomic force microscopy are presented here. Significant roughening occurs during the etching process, but the most dramatic change in roughness and surface adhesion forces occurs in the hydrided (silane-adlayered) capillary surface. Topographic smoothing and passivation of surface activity are observed for the alkylated capillary surface.

Journal Article↗
PubMed2003

Nanoinjector for capillary electrophoresis and capillary electrochromatography.

A rotary valve nanoinjector was devised for use in capillary electrophoresis (CE) and capillary electrochromatography (CEC). A fused-silica capillary tip was inserted in a small through-hole in the rotor. The narrow and short capillary tip, with an inner volume of 6-24 nL, was embedded in the hole using epoxy resin. The injection volume was confirmed chromatographically by comparing the peak areas obtained with the nanoinjector to those of a conventional injector. In addition, both the rotor and stator of the injector were made of a nonconducting material, polyimide resin, to be utilized for CE and CEC. The application of the nanoinjector for CE was demonstrated.

Journal Article↗
PubMed1999

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis.

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2002

Comparison of different setups for one- and two-dimensional capillary high-performance liquid chromatography and pressurized capillary electrochromatography coupled on-line with mass spectrometry.

A comparison of different separation methods (high-performance liquid chromatography (HPLC), capillary HPLC (CHPLC) and pressurized capillary electrochromatography (pCEC)) coupled on-line with mass spectrometry (MS) is undertaken using the separation of a crude extract of ergot fungus (secalis cornuti) as an example. New and simple setups for a two-dimensional CHPLC coupled on-line with electrospray ionization (ESI)-MS (2D-CHPLC-MS) as well as for capillary size-exclusion chromatography performed under pCEC conditions and coupled on-line with ESI-MS (CSEC-pCEC-MS) are shown. In addition, an improved method for column packing is presented.

Chromatography, High Pressure Liquid↗
PubMed1998

Analysis of fluorescein isothiocyanate derivatized amphetamine and analogs in human urine by capillary electrophoresis in chip-based and fused-silica capillary instrumentation.

Amines can easily be derivatized with fluorescein isothiocyanate isomer I (FITC) and analyzed by capillary electrophoresis (CE) using alkaline buffers with or without dodecyl sulfate micelles. This paper reports the CE analysis of FITC-derivatized amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine and beta-phenylethylamine in human urine using chip-based and fused-silica capillary instrumentation with laser-induced fluorescence detection. Data obtained via direct labeling of fortified urine are compared to those generated after FITC labeling of urinary extracts that were prepared by solid-phase extraction using a copolymer phase. For a urine volume of 5 mL with a "spiked amine": FITC ratio of 1:250, the latter approach was found to provide a sensitivity that is relevant for toxicological drug screening and confirmation (about 200 ng/mL urine). With direct labeling of 10 microL urine that was alkalinized and diluted for derivatization, the limit of identification was determined to be about 10 microg/mL, a value that is too high for practical purposes. Compared to fused-silica capillaries, electrophoresis in microstructures is shown to provide faster separations and higher efficiencies without loss of accuracy and precision.

Amphetamines↗
PubMed1991

Histochemical studies on the mechanism of macromolecule leakage across the glomerular capillary wall. Barrier effect of the anionic groups of the capillary wall against the serum protein leakage.

For the purpose of revealing the barrier effect of the anionic groups of glomerular capillary wall against the serum protein leakage, morphologic and histochemical observations were made on the rat kidney perfused in situ with three kinds of cationic macromolecules different in chemical characteristics followed by blood flow restoration. The polyethyleneimine perfusion resulted in the complete disapperance of ionized anionic groups of glomerular capillary and the massive protein leakage through glomeruli by blood flow restoration. Cationic ferric colloid perfusion induced moderate protein leakage, and avidin perfusion was less in neutralization effect of anionic groups and the protein leakage was of least. The protein leakage from glomeruli, however, was stopped or markedly suppressed soon after the blood flow restoration by the newly formed functioning anionic barrier probably by some particular serum protein deposition. The findings indicate that the deionization of the glomerular capillary wall will not be responsible for the persistent albuminuria.

Animals↗
PubMed2002

Metal complex-substituted polysiloxanes as novel coatings for capillary electrophoresis and capillary electrochromatography.

Two novel polysiloxane-based polymers, which contain metal complexes, have been prepared. To prepare the Co(TACN)3+(2) (TACN= 1,4,7-triazacyclononane) based polymers, an orthoamide derivative of TACN was added to bromobutane-substituted methylpolysiloxane and hydrolyzed with base. Co(II) was then coordinated to the TACN, followed by cobalt oxidation to make polymer A or followed by N-octyl TACN coordination and cobalt oxidation to make polymer B. In both materials, TACN forms thermodynamically and kinetically stable Co(TACN)3+(2) complexes in which the six coordination sites of the Co(III) are occupied by nitrogens from the TACN. The polymers were coated on fused-silica capillary columns and spherical silica particles, which were used for capillary electrophoresis and capillary electrochromatography, respectively. The open and packed columns showed strong and pH-independent reversed electroosmotic flow.

Chromatography, Micellar Electrokinetic Capillary↗
PubMedDate not supplied

Advantages in the analysis of UDP-sugars by capillary electrophoresis-comparison of the conventional HPLC method with two new capillary electrophoretic micro-procedures.

Two different capillary electrophoretic separation modes for the analysis of UDP-sugars have been tested. In comparison to the traditionally used HPLC method the micellar electrokinetic capillary chromatography (MECC) as well as the capillary zone electrophoresis (CZE) results in an improvement of the separation profile and the speed of the analysis.

Chromatography, High Pressure Liquid↗
PubMed1999

Theory of the BOLD effect in the capillary region: an analytical approach for the determination of T2 in the capillary network of myocardium.

This article presents an analytical approach for the quantification of the blood oxygen level dependent (BOLD) effect in the capillary region. The capillary geometry of myocardium is considered. The relaxation rate R*2 is determined as a function of the capillary radius Rc, the intracapillary volume fraction RBV, and the diffusion coefficient D. When the intracapillary volume fraction is small, the approximation R*2 = RBV x tau(-1) x (square root of (1+(taudeltaomega)2)-1) is valid, with the correlation time tau = (Rc2/4D) x (absolute value (ln RBV)/(1 - RBV)). The predictions of this model agree well with numerical simulations and experimental data of others and with data recently measured by our group.

Animals↗
PubMed1999

A drying step in the protocol to pack capillary columns by centripetal forces for capillary electrochromatography.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.

2-Propanol↗
PubMed2000

Single-cell analysis using capillary electrophoresis: influence of surface support properties on cell injection into the capillary.

Capillary electrophoresis (CE) is an important tool of chemical cytometry. Whole-cell analysis using CE starts with cell injection into the capillary by either siphoning or electroosmosis. However, strong adherence of the cell to the support surface can prevent efficient cell injection and lead to irreproducible analysis. Here we evaluated several surfaces as potential cell supports for HT29 cells (human colon adenocarcinoma). These cells strongly adhered to the surface of untreated glass or polystyrene. Hydrophobic coating with dimethyldichlorosilane (DMS) or Sigmacote did not significantly reduce cell adhesion. In contrast, cell adhesion was reduced significantly when the surface was modified with hydrophilic polymers (hydrogels) such as poly(2-hydrohyethyl methacrylate) (PHEMA) and polyvinyl alcohol (PVA). In addition to their pronounced antiadhesive properties, PHEMA and PVA coatings were the most biocompatible (had highest survival of cells in contact with surface). Hydrogel-coated polystyrene plates were tested as a commercial alternative to hydrogel-coated glass slides. The cell adhesive properties of such plates were similar to those of PHEMA and PVA. However, the biocompatibility of the plates was lower than that of the other surfaces tested. Moreover, in contrast to PHEMA- and PVA-coated glass slides, the plates were sensitive to UV light and therefore should not be used when fluorescent image microscopy with UV excitation precedes CE. The analyses of the data obtained showed that PHEMA- and PVA-coated glass slides were the most suitable cell supports for cell injection into the capillary.

Adenocarcinoma↗
PubMed2000

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis.

Preparation and characterization of calixarene-coated capillaries for capillary electrophoresis (CE) were exemplified with p-allylcalix[4]arene (pACX4) which was immobilized to the fused silica surface using gamma-methacryloxypropyl-trimethoxysilane (gamma-MAPS) as linking agent. Successful gamma-ACX4 coating was suggested by the greatly decreased electroosmotic flow (EOF), due to the introduction of phenolic hydroxyl groups on the inner surface of the capillaries. A slight slope of EOF versus pH at pH <8 would help make the separation reproducible. The coated columns also featured a low ultraviolet (UV) absorption background and long lifetime (> 6 months at 4<pH<9). Moreover, their special selectivity on phenolic compounds indicated a certain extent of selective interactions between solutes and the calixarene coating, which effected separations partially based on an electrochromatographic mechanism.

Calixarenes↗
PubMed2000

Detection of neuropeptides using on-capillary copper complexation and capillary electrophoresis with electrochemical detection.

Capillary electrophoresis with electrochemical detection using a carbon fiber electrode in conjunction with on-capillary copper complexation was evaluated for the determination of peptides in standard and biological matrices. Peptides composed of 2-10 amino acids were investigated. A comparison was made between the responses obtained for peptides containing the oxidizable residue tyrosine and those obtained for their respective copper complexes. Electrochemical detection of non-tyrosine-containing peptides and a cyclic peptide was also demonstrated. A separation of leucine (Leu)-enkephalin and five metabolites was developed and then used for the investigation of Leu-enkephalin metabolism in plasma. The appearance of the des-tyrosine (des-Tyr) Leu-enkephalin, which cannot be detected directly at a carbon electrode, was monitored using the on-capillary complexation technique. Direct injection of the plasma sample was possible using this methodology.

Angiotensins↗
PubMed2001

Separation of basic, acidic and neutral compounds by capillary electrochromatography using uncharged monolithic capillary columns modified with anionic and cationic surfactants.

A mode of capillary electrochromatography (CEC), based on the dynamical adsorption of surfactants on the uncharged monolithic stationary phases has been developed. The monolithic stationary phase, obtained by the in situ polymerization of butyl methacrylate with ethylene dimethacrylate, was dynamically modified with an ionic surfactant such as the long-chain quaternary ammonium salt of cetyltrimethylammonium bromide (CTAB) and long-chain sodium sulfate of sodium dodecyl sulfate (SDS). The ionic surfactant was adsorbed on the surface of polymeric monolith by hydrophobic interaction, and the ionic groups used to generate the electroosmotic flow (EOF). The electroosmotic mobility through these capillary columns increased with increasing the content of ionic surfactants in the mobile phase. In this way, the synthesis of the monolithic stationary phase with binary monomers can be controlled more easily than that with ternary monomers, one of which should be an ionic monomer to generate EOF. Furthermore, it is more convenient to change the direction and magnitude of EOF by changing the concentration of cationic or anionic surfactants in this system. An efficiency of monolithic capillary columns with more than 140000 plates per meter for neutral compounds has been obtained, and the relative standard deviations observed for to and retention factors of neutral solutes were about 0.22% and less than 0.56% for ten consecutive runs, respectively. Effects of mobile phase composition on the EOF of the column and the retention values of the neutral solutes were investigated. Simultaneous separation of basic, neutral and acidic compounds has been achieved.

Anions↗