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PubMed1997

Dynamics of capillary electrochromatography experimental study on the electrosmotic flow and conductance in open and packed capillaries.

For capillary electrochromatography (CEC) to become an analytical separation technique of high speed and resolution the factors determining the conductivity of the column as well as the generation and control of electrosmotic flow (EOF) in porous media have to be understood. In the present study the conductance of capillaries packed with a variety of stationary phases was evaluated with respect to the conductance of the open capillary and the data were interpreted in the light of the Tobias equation. However, the consistently observed reduction of the EOF when a capillary having a charged inner wall is packed with particles having charges of the same sign and the dependence of the EOF velocity on the particle size needs further explanation. The data suggests that, due to the employment of relatively long columns packed with small particles, CEC may offer peak capacities much higher than HPLC or micro-HPLC. The CEC columns are unique as they consist of a packed and an open capillary segment having different conductances and consequently different voltage gradients and electrical field strengths. Therefore, any sufficiently detailed study on CEC systems requires also the characterization of the individual column segments. EOF velocities of 6-7 mm/s could be realized at 60 kV applied voltage with a 23/32 cm x 50 microns raw fused-silica capillary packed with 6-micron Zorbax ODS particles. The current was a linear function of the field strength up to 1.8 kV/cm, but at high field strengths the EOF increased with squared field strength. Data on band spreading indicate that with a given column the plate height at high EOF velocities is smaller in CEC than in micro-HPLC and it is weakly dependent on the velocity.

Acetonitriles↗
PubMed1999

Direct detection of endogenous histamine in rat peritoneal mast cells by in-capillary derivatization high-performance capillary electrophoresis.

A simple method for the detection of endogenous histamine in rat peritoneal mast cells was evaluated using on-line mode in-capillary derivatization high-performance capillary electrophoretic (ICD-HPCE) techniques, which were previously developed by our group [S. Oguri et al., J. Chromatogr. A, 787 (1997) 253-260]. The method involves a suspension of peritoneal mast cells (1 x 10(6) cells/ml of saline) collected from a male Wistar rat (eight weeks of age), which are directly introduced into the capillary tube from the anodic end by hydrostatic injection (at 25 cm height, for 2-20 s). When a high-voltage potential (25 kV) is applied to the capillary, which is already filled with the run buffer containing both a lysing reagent (SDS, sodium dodecyl sulfate,) and a derivatizing reagent (OPA, o-phthalaldehyde; NAC, N-acetylcysteine), histamine in the mast cells was detected at high-sensitivity level without further procedures. During ICD-HPCE, the mast cells injected in the capillary were lysed with the lysing reagent, free histamine released from the cell was labeled with the derivatizing reagent, and its derivative was electromigrated, separated and detected with a fluorescence detector (excitation wavelength at 340 nm, emission wavelength at 450 nm) in a fused-silica capillary (75 cm x effective length x 50 microm I.D.). The run buffer used was a 20 mM phosphate-borate buffer (pH 10) containing 20 mM SDS, 2 mM OPA and 2 mM NAC. This method was also examined with regard to the possibility of its use for determination of histamine at the single mast cell level.

Animals↗
PubMed2003

On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface.

An on-line two-dimensional (2D) capillary electrophoresis (CE) system consisting of capillary isoelectric focusing (CIEF) and capillary gel electrophoresis (CGE) was introduced. To validate this 2D system, a dialysis interface was developed by mounting a hollow fiber on a methacrylate resin plate to hyphenate the two CE modes. The two dimensions of capillary shared a cathode fixated into a reservoir in the methacrylate plate; thus, with three electrodes and only one high-voltage source, a 2D CE framework was successfully established. A practical 2D CIEF-CGE experiment was carried out to deal with a target protein, hemoglobin (Hb). After the Hb variants with different isoelectric points (pIs) were focused in various bands in the first-dimension capillary, they were chemically mobilized one after another and fed to the second-dimension capillary for further separation in polyacrylamide gel. During this procedure, a single CIEF band was separated into several peaks due to different molecular weights. The resulting electrophoregram is quite different from that of either CIEF or CGE; therefore, more information about the studied Hb sample can be obtained.

Animals↗
PubMed2003

Absence of OX-43 antigen expression in invasive capillary sprouts: identification of a capillary sprout-specific endothelial phenotype.

Endothelial cells exhibit a number of unique phenotypes, some of which are angiogenesis dependent. To identify a capillary sprout-specific endothelial phenotype, we labeled angiogenic rat mesentery tissue using a microvessel and capillary sprout marker (laminin), selected endothelial cell markers (CD31, tie-2, and BS-I lectin), and the OX-43 monoclonal antibody, which recognizes a 90-kDa membrane glycoprotein of unknown function. In tissues that were stimulated through wound healing and compound 48/80 application, double-immunolabeling experiments with an anti-laminin antibody revealed that the OX-43 antigen was expressed strongly in all microvessels. However, the OX-43 antigen was completely absent from a large percentage (>85%) of the capillary sprouts that were invading the avascular tissue space. In contrast, sprouts that were introverting back into the previously vascularized tissue retained high levels of OX-43 antigen expression. Double-labeling experiments with endothelial markers indicated that the OX-43 antigen was expressed by microvessel endothelium but was absent from virtually all invasive capillary sprout endothelial cells. We conclude that the absence of OX-43 antigen expression marks a novel, capillary sprout-specific, endothelial cell phenotype. Endothelial cells of this phenotype are particularly abundant in capillary sprouts that invade avascular tissue during angiogenesis.

Animals↗
PubMed1977

Capillary lengths, anastomoses, and estimated capillary transit times in skeletal muscle.

Total capillary length, capillary segment length, and number of anastomoses per capillary were measured in rat gracilis muscle at rest and after 2 min of phasic contraction. Mean values of the foregoing variables at rest (+/-SD) were, respectively, 1,012 micronm +/- 484, 409 micron +/- 274, and 0.83 +/- 1.09. Total capillary lengths are well described by the gamma distribution, number of anastomoses by the negative binomial distribution, and segment length by the Weibull distribution. Contraction has no significant effect on the means or the frequency distributions, indicating that: 1) pressure gradients between adjacent capillaries are small, and 2) intercapillary anastomoses do not improve flow distribution in exercise. Erythrocyte velocities observed in resting muscle (Burton, K. S., and P. C. Johnson. Am J. Physiol. 223: 517-524, 1972) were shown to be adequately characterized by the gamma distribution. From these velocities and the observed distribution of path lengths, we computed an estimated distribution of capillary transit times. Mean transit time was 4.29 s. The median was 2.45 s, and 11% of values exceeded 8 s. The range was 90 ms-43 s. This heterogeneity of transit times should profoundly affect calculations of O2 transport and the shape of indicator dilution curves.

Animals↗
PubMed1997

[The technical construction of a computer-supported measuring unit for in-vivo capillary pressure measurement in human nail fold capillaries].

Capillary pressure (CP) is an important determinant of microvascular function. According to Frank Starling, it governs not only the viscosity of the blood and the permeability of the capillary membrane, but also filtration and reabsorption in the microvascular bed. Despite its great significance, the technique of CP measurement is employed in only a few laboratories throughout the world owing to the technical difficulties involved in carrying out such measurements. The first attempts were made by Carrier and Rehberg in 1923. In 1979 Mahler carried out the first dynamic recordings of capillary pressure. The present paper explains the physical basis for direct capillary measurement, and recent progress in this technique is discussed in a review of the literature. We also introduce a newly developed computer-aided system that enables in vivo determination and evaluation of pressure in the nailfold capillaries of the hands and feet. The device permits the simultaneous recording of pressure and bloodflow in the capillaries of the skin, and thus provides additional information on important functions of the cutaneous microcirculation.

Blood Pressure↗
PubMedDate not supplied

Separation of benzodiazepines using cholesterol-modified fused-silica capillaries in capillary electrochromatography.

This paper describes the use of cholesteryl-10-undecenoate as a capillary column modifier for capillary electrochromatography. This bonded phase has shown an entirely different selectivity as compared to octadecyl silica (ODS) phases in reversed-phase liquid chromatography, specifically in the analysis of the benzodiazepines. This difference serves as the basis for the continuing study as well as the present focus on electrochromatography as a relatively new separation technique. To significantly increase the surface area, etching of the inner wall of a 75-micron capillary is performed using ammonium hydrogen difluoride and is then subsequently modified via a silation reaction with triethoxysilane and subjected to hydrosilation with cholesteryl-10-undecenoate to form the bonded phase. The performance of this capillary is compared with unetched cholesterol-modified and bare, fused-silica capillaries. Efficient resolution was noted for the etched capillary showing the effectiveness of the etching process as well as the selective property of the bonded phase.

Benzodiazepines↗
PubMed1986

Physiological studies of macromolecular transport across capillary walls. Studies on continuous capillaries in rat skeletal muscle.

Studies of the transcapillary exchange of fluid and solutes have provided experimental evidence for the following description of the capillary wall: Transport can be adequately described by passive phenomena such as filtration and diffusion across the permeable structures of the capillary barrier. Hydrophilic solutes are progressively restricted in their transcapillary passage with increasing molecular radius in a bimodal manner. The simplest membrane model compatible with these properties is the two-pore model (Grotte 1956), for which there now is massive documentation (Taylor & Granger 1984). Thus, small hydrophilic solutes (radius less than 30A) are transported mainly by diffusion through small equivalent pores (40-65A), which also represent almost 90% of the hydraulic conductivity. Moreover, larger solutes pass mainly through large equivalent pores (250-350A) by convection. Hence, diffusion is of minor importance for macromolecular transport also during conditions of no net fluid flux across the capillary walls, when there is a circulation of fluid between small and large pores and a net filtration of macromolecules at the large pores. The functional small pores are most probably identical to the interendothelial junctions, while the large pore system is more difficult to define. In addition, solutes are subjected to electrostatic charge interactions at the capillary wall. Thus, there is overwhelming evidence of the presence of high densities of negative charges at the capillary endothelium (glycocalyx), the basement membrane and in the interstitium. This suggests the presence of a negative capillary charge barrier restricting anionic solutes, which also could be experimentally verified (study I & II), but the importance of this barrier is not yet fully clear. A simplistic theoretical two-pore model including effects of charge (Munch et al. 1979) was found to describe our experimental data. According to this model, the charge effect will be most important for solutes with molecular radii of 20-40 A, while charges probably are of no importance for the transport of larger solutes. For anionic macromolecules, e.g. albumin, the effective pore radius will be 40-45A, but the steric small pore radius (i.e. for neutral solutes) will be around 65A. Hereby, the area of diffusion calculated from CFC and the small pore radius (64A) will be lower and it will actually approach the values determined by indicator dilution technique (cf. Haraldsson & Rippe 1986).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

Analysis of anxiolytic drugs by capillary electrophoresis with bare and coated capillaries.

The analysis of anxiolytic drugs, including barbiturates and benzodiazepines, by CE with bare and coated capillaries was investigated. Two types of CE modes, capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were employed for the separation of the solutes. Charged, structurally similar compounds exemplified by hypnotic drugs such as barbiturates were separated by both CZE and MEKC. Optimal separation conditions were established by varying the pH, the concentration of surfactant, the strength of the electric field, and the addition of the organic solvent. CZE provided different selectivities than those obtained by MEKC. For the separation of uncharged compounds such as benzodiazepines, MEKC was the method of choice because the structural difference conferred differences in hydrophobicity, leading to the different interaction with the micelle. Separations by CZE and MEKC with plain, bare fused-silica capillaries were compared to those with C8 and C18 coated capillaries. Although for these solutes, resolution and analysis time were comparable in the three types of capillaries, successful separation of more than 20 kinds of anxiolytic drugs as the mixture was achieved by using C8 and C18 coated capillaries as the separation medium.

Anti-Anxiety Agents↗
PubMed1990

Capillary electrophoretic determination of S-carboxymethyl-L-cysteine and its major metabolites in human urine: feasibility investigation using on-column detection of non-derivatized solutes in capillaries with minimal electroosmosis.

The capillary electrophoretic analysis of S-carboxymethyl-L-cysteine and some of its metabolites in human urine is reported using (i) on-column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE-100 (Bio-Rad), featuring coated fused silica capillaries of 25 microns i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 microns i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.

Carbocysteine↗
PubMed1995

High-performance capillary electrophoretic separation of proteins and peptides using a bonded hydrophilic phase capillary.

High performance capillary electrophoresis (HPCE) was applied to the separation of protein and peptide mixtures with molecular masses ranging from 1300 to 96000 Da using a new bonded hydrophilic phase capillary, CElect-P150. This coated capillary reduces the interaction between proteins and silanol groups in capillary walls, allowing a complete recovery of the proteins and peptides of interest. HPCE was also used for the analysis of a complex mixture of tryptic fragments and to monitor the process of enzymatic digestion. Moreover, using a CElect-P150 capillary, highly reproducible analysis was possible without preconditioning the capillary with acid or basic solutions before each new analysis.

Animals↗
PubMed1995

Capillary electrophoretic resolution of phosphorylated peptide isomers using micellar solutions and coated capillaries.

The addition of sodium dodecyl sulfate (SDS) micelles in the running buffer can be used to resolve mono- and diphosphorylated isomers of the insulin receptor peptide by capillary electrophoresis. The effect of SDS on peptide resolution is very dependent on pH. Complete resolution of three monophosphorylated isomers is achieved in uncoated capillaries filled with phosphate buffer containing 25 mM SDS and buffered at pH 6.1. Resolution of the diphosphorylated isomers can be significantly improved by using polyacrylamide coated capillaries. In coated capillaries electroosmotic flow is negligible and the migration order of the isomers is reversed. This allows for a longer period of interaction between the diphosphorylated isomers and the micelle and therefore selectivity is improved. Efficiency of all peptide isomers was also improved in coated capillaries due to reduced adsorption to the capillary wall.

Acrylic Resins↗
PubMed1994

Preparation and characterization of a stable polyacrylamide sieving matrix-filled capillary for high-performance capillary electrophoresis.

A stable sieving matrix of polyacrylamide filled in a capillary was developed. The inner wall of a fused-silica capillary was covalently bonded with a linear polyacrylamide through Si-C linkages, in which cross-linked polyacrylamide gel or linear polyacrylamide solution was filled. The stability of the coating was examined by exposure of the capillaries to alkaline buffer (pH 8) for up to 30 days. Compared with the coatings with linear polyacrylamide bonded through siloxane linkages, the present capillary markedly reduced the electroosmotic flow. Thus, the sieving matrix in the capillary was stabilized, resulting in a prolonged lifetime of the capillary and good reproducibility of separations. The migration behaviours of oligonucleotides were compared for the cross-linked gel and linear polyacrylamide solution at the same concentration.

Base Sequence↗
PubMed1994

Application of a polyamine-coated capillary to the separation of metallothionein isoforms by capillary zone electrophoresis.

In this study a fused-silica capillary treated internally with a polyamine coating which reverses electroosmotic flow in the direction of the anode was evaluated for its ability to resolve metallothionein (MT) isoforms. Analysis of different MTs purified from liver and kidney tissue revealed the following numbers of putative isoform peaks resolved: rabbit (3-6); horse (3-5); rat (2-3), chicken (1); human MT-1 (5-6); sheep (4-5) and pig (4-5). The greater degree of MT isoform heterogeneity detected in this study using the polyamine-coated capillary suggested a higher resolving capacity for capillary zone electrophoresis conducted with this capillary compared to an uncoated one. Using the single isoform of chicken MT (cMT) as a reference standard, relative standard deviations of 2.53, 1.85 and 2.21% for peak migration time, area and height, respectively, were observed for eight consecutive runs. A standard curve for cMT established linearity (r2 = 0.99) for integrated peak area over three log units of cMT concentration with a lower limit of detection estimated to be < or = 5 micrograms/ml. Acetonitrile extracts of chick liver tissue homogenates were successfully analyzed for the presence of MT isoforms from both control and zinc-injected animals. Based on our initial evaluation, capillary zone electrophoresis using the polyamine-coated capillary appears to be a very useful analytical method for the separation and quantification of individual MT isoforms.

Animals↗
PubMed2005

Sheathless capillary electrophoresis/electrospray ionization mass spectrometry using a pulled bare fused-silica capillary as the electrospray emitter.

It has always been assumed that electrical contact at the capillary outlet is a necessary requirement when coupling capillary electrophoresis (CE) with electrospray ionization mass spectrometry (ESI-MS). In this study, we used a pulled bare-capillary tip as the ESI emitter, but neither was it coated with any electrically conductive materials nor was a high external voltage applied on its outlet. In this paper, we demonstrate that this straightforward approach may be used to generate multiply charged ions of proteins and peptides through electrospray ionization. Our results indicate that peptides and proteins, including bradykinin, cytochrome c, myoglobin, and tryptic digest products that elute from a pulled bare-capillary tip can be detected directly by ESI-MS using the tapered bare-capillary interface. Thus, we have demonstrated that CE and ESI-MS may be combined successfully without the need to modify the outlet of the capillary tip with an electrically contacting material.

Journal Article↗
PubMed2005

Flow counterbalanced capillary electrophoresis using packed capillary columns: resolution of enantiomers and isotopomers.

A method with the ability to increase greatly both the resolution and efficiency of a given capillary electrophoretic system is described. This method differs from traditional capillary electrophoresis (CE) in that a counterflow is induced in the direction opposite to the electrokinetic migration of the analyte. This has the effect of extending not only the time the analytes migrate in the electric field but also the effective length and the effective applied voltage of the system. Previous work in our group with flow counterbalanced capillary electrophoresis has utilized an open tube of small inner diameter to reduce peak broadening caused by hydrodynamic flow. Narrow-diameter capillaries (5-10 microm) restricted analysis to fluorescent analytes and laser-induced fluorescence detection. The method described here uses a capillary of much larger inner diameter (75 microm) that has been packed with nonporous silica particles. The packing material reduces the amount of band broadening caused by pressure-induced flow relative to that experienced in an open tube. A larger diameter capillary allows the detection of analytes by UV absorption, not only eliminating the need to tag analytes with fluorescent tags but also allowing for the detection of a much broader range of analytes. The system was evaluated by studying the separations of several enantiomers using only beta-cyclodextrin as the chiral selector. The system was also used to resolve the two naturally occurring isotopes of bromine and to resolve phenylalanine from phenylalanine-d8. Relative to traditional CE, large improvements in resolution and separation efficiency have been achieved with this method.

Journal Article↗
PubMed2003

Dynamic capillary coatings with zwitterionic surfactants for capillary isoelectric focusing.

The use of surfactants as additives was demonstrated for the first time in capillary isoelectric focusing (CIEF) to dynamically modify the surfaces of bare fused silica capillaries. These surfactants were zwitterionic sulfobetaines: dodecyldimethyl (3-sulfopropyl) ammonium hydroxide (C12N3SO3), hexadecyldimethyl (3-sulfopropyl) ammonium hydroxide (C16N3SO3) and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U). They were added directly to the protein-ampholyte mixture, and remained in the capillary during isoelectric focusing and mobilization. The C16N3SO3 and CAS U coatings were shown effective in CEF. Separation of seven IEF protein standards was obtained, with significantly improved resolution compared to that from an uncoated silica capillary. The effect of these surfactants on the electroosmotic flow (EOF) in CIEF was determined. CAS U was effective in suppressing the EOF at neutral and alkaline pH conditions, C16N3SO3 was effective in suppressing EOF at acidic and neutral pH conditions. C12N3SO3 however had little effect on the EOF. The pH gradients formed inside these surfactant coated capillaries were recta-linear at pH 6 to 9 (R2 approximately equal to 0.99). Reproducibility of migration time and peak area was determined. For all three coatings, the migration time standard deviations were less than 1.6 min, and the relative standard deviations of area were below 10%. The protein recovery in the CAS U-modified capillary was quantitative or near-quantitative for five of the seven proteins studied.

Journal Article↗
PubMed1993

Sodium dodecyl sulfate-polyacrylamide solution-filled capillary electrophoresis of proteins using stable linear polyacrylamide-coated capillary.

Sodium dodecyl sulfate (SDS)-linear polyacrylamide solution-filled capillary electrophoresis using a stable coated fused silica capillary has been developed for the separation and molecular weight determination of proteins. A fused silica capillary was coated with linear polyacrylamide through Si-C bond which suppressed the electro-osmotic flow and reduced the adsorption of proteins. Acrylamide solution containing SDS was filled into the coated capillary and polymerized. Compared with a conventional fused silica capillary coated with 3-methacryloxypropyltrimethoxysilane through siloxane linkage (Si-O-Si-C), the developed capillary was much more stable, even in the alkaline condition. Consequently, the durability of the polymer matrices and the reproducibility of separation were markedly improved. An excellent linear relationship was obtained between the mobility and the logarithm of the protein molecular weight. The relative standard deviation of migration times was below 1%.

Electrophoresis, Polyacrylamide Gel↗