Uncoated Capillary Research Articles

Rapid loading of large sample volumes, analyte cleanup, and modified moving boundary transient isotachophoresis conditions for membrane preconcentration-capillary electrophoresis in small diameter capillaries.

Using a removable membrane preconcentration (mPC) cartridge, large sample volumes can be loaded prior to final assembly of the mPC capillary electrophoresis (CE) capillary. For narrow-bore (< or = 25 microns ID) uncoated mPC-CE capillaries, applied to peptide analysis, efficient moving boundary transient isotachphoresis (tITP) conditions at the onset of electrophoresis are described. The enhancement of mPC-CE-mass spectrometry (MS) technology afforded by rapid sample loading and modified moving boundary tITP conditions are demonstrated by analysis of major histocompatibility complex (MHC) class I peptides that were derived from a Kb precipitation of mouse EL-4 cells. Furthermore, we demonstrate the structural characterization of these immunologically significant molecules by mPC-CE-tandem mass spectrometry (mPC-CE-MS/MS).

Analysis of Oligonucleotides by On-Column Transient Capillary Isotachophoresis and Capillary Electrophoresis in Poly(ethylene glycol)-Filled Columns

Capillary electrophoresis in poly(ethylene glycol) (PEG) solution-filled uncoated silica capillaries was demonstrated to be suitable for separation of oligonucleotides in 100 mM ammonium formate (pH 4.5) buffers. Reversed polarity (−30 kV at the injector end) was used, because the electroosmotic flow (EOF) at 4.5 was considerably lower than the mobility of the anionic analytes. On-column transient isotachophoresis (ITP) was developed for preconcentration of oligonucleotide samples in PEG solution-filled capillaries. The ITP focusing step allows injection of 20% of column length without loss of peak resolution. The detection limit of the method was 100 ng/mL of 16-mer oligonucleotide by using UV detection at wavelength 254 nm (signal-to-noise ratio 10).

Comparative analysis of somatostatin analog peptides by capillary electrophoresis and micellar elektrokinetic chromatography.

Capillary electrophoresis (CE) and micellar electrokinetic chromatography (MEKC) methods, utilizing uncoated silica capillary and triethyl ammonium phosphate or sodium borate buffers in the pH range of 2.25-11.0, containing sodium dodecyl sulfate (SDS) (0-100 mM) for analysis of somatostatin-analog peptides were developed. The method presented here was compared with the reversed-phase high performance liquid chromatographic (RP-HPLC) and CE methods developed for analysis of peptides. The peptides investigated in this work can be separated by CE on the basis of their electrophoretic mobility in aqueous buffer of low pH value (pH 2.25) or by MEKC on the basis of their hydrophobicity in SDS containing buffer of high pH value (pH 11.0). Optimal MEKC separation of the investigated peptides has been achieved at pH 11.0 in an Na-borate buffer containing 100 mM SDS. CE at pH 2.25 proved insensitive to the hydrophobicity of the peptides investigated. By contrast, results obtained with MEKC at pH 11.0 proved to be anologous to those obtained by RP-HPLC, with highly hydrophobic peptides-migrating slower than peptides without hydrophobic moieties.

Direct determination of glutathione in human blood by micellar electrokinetic chromatography: simultaneous determination of lipoamide and lipoic acid.

A reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 microns internal diameter (ID), 50 mM Tris-HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10-20 kV. On-line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidize and reduced forms, lipoamide and alpha-lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.

Determination of Tetracycline Residues in Bovine Milk, Serum, and Urine by Capillary Electrophoresis

A method was developed for simultaneous determination of oxytetracycline (OTC), chlortetracycline, tetracycline, and doxycycline levels in cow milk, serum, and urine by capillary electrophoresis (CE). The tetracyclines (TCs) were extracted specifically with a metal-chelating affinity column. Extracts were then applied to C18 cartridges treated with dimethyldichlorosilane, and the TCs were eluted with ethanol. Salt-free sample residues were run on CE with a diode array detector at 370 nm. A 50 cm x 75 micron uncoated capillary at 15 kV and 23 degrees C was used to separate the TCs. The run buffer contained 10 mM sodium dodecyl sulfate, 50 mM borate, and 50 mM phosphate, pH 8.5. TC peaks were identified by migration times, coinjection of standards, and diode array spectra. Limits of detection for TCs were 1.3-5.6 ng/mL, and limits of quantitation were 1.7-8.7 ng/mL in cow fluid samples. OTC appeared consistently at 13.3-13.4 min in milk, serum, and urine samples from a cow treated with OTC. OTC was further identified by coinjection of OTC standard and OTC diode array spectra. The CE method can be used to simultaneously determine 4 TC residues at low parts-per-billion levels.

Effects of alkylamines on electroosmotic flow and protein migration behaviour in capillary electrophoresis

This paper reports the use of four closely related alkylamines as running electrolyte additives in capillary electrophoresis that permit the control of electroosmotic flow and protein migration behaviour in uncoated capillaries. At pH 2.5 the direction of the electroosmotic flow was anodic with all additives and at constant ionic strength its magnitude increased with increasing alkylamine concentration. The observations are in qualitative agreement with a previous reported theoretical model that correlates the electroosmotic mobility with the charge density in the Stern region of the electric double layer, arising from the adsorption of the additive, and the charge density at the capillary wall due to dissociation of silanols.

Determination of impurities in a novel analogue of adenosine 5′-triphosphate by capillary electrophoresis

A capillary electrophoretic method using an uncoated capillary was developed to resolve potential impurities in FPL 67085XX, a novel phosphonate analogue of adenosine 5′-triphosphate. The effects of buffers, buffer concentration, eleunt pH and methanol were investigated. Optimum resolution of the impurities was achieved using an eluent consisting of 7% (v/v) methanol in 25 m M phosphate buffer-2 m M EDTA adjusted to pH 6.6.

Analysis of 3-(N-Oxalyl)-L-2,3-diaminopropanoic Acid and Its .alpha.-Isomer in Grass Pea (Lathyrus sativus) by Capillary Zone Electrophoresis

A method was developed for the simultaneous quantitative determination of the neurotoxic nonprotein amino acid 3-(N-oxalyl)-L-2,3-diaminopropanoic acid (β-ODAP) and its nontoxic n-isomer 2-(N-oxalyl)-L-2,3-diaminopropanoic acid (α-ODAP) in seeds of grass pea (Lathyrus sativus) by capillary zone electrophoresis (CZE). Samples of 0.5 g of seed flour were extracted in 2 volumes of 10 mL of ethanol-water (6:4) by tumbling for 45 min each time. Hippuric acid was used as an internal standard. The separation was performed in a 50 μm i.d. uncoated capillary at 515 V/cm and 40 o C in 20 mM Na 2 HPO 4 buffer at pH 7.8 with direct UV detection at 195 nm. A linear response rvas recorded in the concentration range 0.015-1.8 mM. This corresponds to a detection limit of 0.1 g kg -1 . Comparison between the CZE method and a standard colorimetric method for measuring total ODAP in 38 lines of grass pea gave a coefficient of correlation of 0.994

The use of coated and uncoated capillaries for the electrophoretic separation of DNA in dilute polymer solutions

We show that both uncoated and polyacrylamide-coated capillaries provide separation of large DNA restriction fragments (2.0-23.1 kbp) by capillary electrophoresis in dilute cellulosic polymer solutions. Uncoated capillaries, however, provide significantly better resolution of DNA fragments, particularly when ultra-dilute polymer solutions are used. This is because electroosmotic flow in uncoated capillaries increases the residence time of DNA in the capillary, without significantly contributing to band-broadening. At a given field strength and polymer concentration in the buffer, the electrophoretic mobilities of DNA restriction fragments in coated capillaries are virtually identical to those previously measured in uncoated capillaries. It is concluded that the fused silica surface of the capillary does not play a significant role in the mechanism of DNA separation by capillary electrophoresis in uncrosslinked polymer solutions. Thus, the separation of large DNA which has been observed to occur in ultra-dilute polymer solutions arises primarily from entanglement interactions between the cellulosic polymers and DNA restriction fragments which occur within the bulk of the polymer solution.

Quantitative analysis of major whey proteins by capillary electrophoresis using uncoated capillaries.

A method that allows separation and quantitation of the main whey proteins by capillary electrophoresis using uncoated capillaries is proposed. Separations are performed using 100 mM borate buffer, pH 8.2, containing 30 mM sodium sulfate. The use of high pH and high ionic strength buffer reduces adsorption of proteins on the capillary wall, making their separation possible. Reproducibility of migration times and areas of peaks are improved by optimizing the capillary equilibration protocol and by using an internal standard. Relative standard deviations ranging between 0.74 and 1.03% for migration times and 2.14 to 5.23% for areas of major peaks are obtained. Detection limits equal to or lower than 0.5 mg/100 mL are achieved. Linear relationships of peak area versus concentration have been used to quantitate bovine serum albumin (BSA), alpha-LA (alpha-lactalbumin), beta-LG A (beta-lactoglobulin A) and beta-LG B (beta-lactoglobulin B) in cow's milk subjected to different thermal treatments. Electrophoretic profiles of these milk samples show peaks from other peptides besides those from main proteins. Characteristic patterns for whey from different species are obtained.

Performance of amino-silylated fused-silica capillaries for the separation of enkephalin-related peptides by capillary zone electrophoresis and micellar electrokinetic chromatography.

Enkephalin-related peptides were separated at low pH in a capillary with covalently bonded aminopropyl groups. The peptides are electrostatically repelled from the capillary surface and much higher efficiencies and faster separations were achieved compared to separations using uncoated capillaries. At low pH the amino groups are protonated, which results in reversed electroosmosis. The influence of voltage and ionic strength on the mobility and the separation efficiency was studied. The repeatability of migration times within one day was very good with relative standard deviations of 0.3-0.7%. Increasing the pH decreased the electroosmosis, eventually turning towards the cathode in the pH range 5-6; the separation performance, however, was lower at higher pH. Neutral and anionic micellar agents were added to the background electrolyte at different concentrations; the enkephalins had weak association with the neutral micellar agents but were distributed to the anionic taurodeoxycholic acid (TDC) micelles, giving rise to changes in separation selectivities. Very high efficiencies were obtained for peptides with a low distribution to the TDC micelles, while the efficiencies were impaired for those with a strong association with the micelles, which may indicate a slow mass transfer in the association process.

The role of histamine receptors in the regulation of the oxygen uptake of rat brain cortical and hypothalamic slices

Chemically oversulfated galactosaminoglycans with potential as therapeutic agents (inhibitors of human leukocyte elastase) were tested as chiral selectors in capillary electrophoresis of basic racemates. The high anionic character of these compounds provides them with anodic mobility in acidic buffer; using uncoated capillaries, the enantioresolution of racemic basic drugs was obtained at pH 2.5. Dimethindene, chloroquine and chlorpheniramine were enantioresolved applying negative voltage (−15 kV) while the other analytes (propranolol, pindolol, tetrahydrozoline and cloperastine) exhibited catodic migration. The addition of organic solvents to the running buffer was evaluated in order to increase the resolution; methanol provides the best results and in general, baseline separation of the analytes was reached. The studied oversulfated mucopolysaccharide, shows the same ionic character of heparin but presents different stereochemistry and sites of sulfation. A comparison with heparin, used in the same acidic conditions, may underline the role of ionic, spatial and steric features of glycosaminoglycans in the enantiorecognition.

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.

Trace analysis of organics in aqueous samples by concentration in plastic tubing and multiplex gas chromatography

The inside wall of an uncoated polyethylene capillary traps organic substances from a water sample pumped through it by a nitrogen gas stream. Heating the capillary in a chromatographic oven slowly releases the trapped organic substances from the wall. Nitrogen carrier gas transports sample substances released through a thermal desorption modulator and onto a chromatographic column. Pulsing the temperature of the modulator modulates the concentrations of sample components as they enter the column. Computing the cross correlation of the detector output signal against the applied modulation signal generates the chromatogram. Detection limits below 1 ppb are possible using a flame ionization detector. No sample pretreatment or cold trap is required.

Capillary electrophoretic examination of underivatized O-linked and N-linked oligosaccharide mixtures and immunoglobulin G antibody-released oligosaccharide libraries

A procedure for the analysis of mixtures of underivatized, cleaved O-linked and N-linked oligosaccharides by capillary electrophoresis has been developed. The species of interest are separated by two borate-based buffer systems with an uncoated capillary. The procedure is applied to hydrazinolysis-released oligosaccharides from mouse, rat, sheep and human IgG protein samples which are examined within 6 minutes. Selectivity with respect to sample variation is demonstrated by analysis of thermally stressed samples.

Electrophoresis of proteins in uncoated capillaries with amines and amino sugars as electrolyte additives

The effect of triethylamine and triethanolamine in the running electrolyte on the electroosmotic flow and the migration of four standard basic proteins in bare fused-silica capillaries was examined. At pH 2.5 the direction of the electroosmotic flow was anodal with both additives and at constant ionic strength its magnitude increased with increasing additive concentration. The observations are in qualitative agreement with a theoretical model which is based on the Gouy—Chapman—Stern theory and incorporates a Langmuir-type relationship and also the Von Smoluchowski expression for the electroosmotic mobility, with the approximation to identify the zeta potential with the potential at the Stern plane. From the independence of the electrophoretic mobilities of the additive concentration and type the absence of interactions between the four basic proteins and the two alkylamines is inferred. The utility of glucosamine and galactosamine as additives for the capillary electrophoresis of basic proteins is also demonstrated and their effectiveness is compared with that of the alkylamines.

Neutral and anionic cyclodextrins in capillary zone electrophoresis: enantiomeric separation of ephedrine and related compounds.

Two methods for the enantiomeric separation of chiral ephedrine alkaloids (ephedrine, methylephedrine, methylpseudoephedrine and norephedrine) by capillary zone electrophoresis in uncoated capillaries were developed. Both methods were optimized to more than 100,000 theoretical plate numbers. The first method used a neutral cyclodextrin (CD) derivative: heptakis (2,6-di-O-methyl)-beta-cyclodextrin at an acidic pH of 2.5 (20 mM phosphate buffer) at an 18 mM concentration. The second used a newly developed acidic CD derivative, the tetrakis[6-O-(4-sulfobutyl)]-beta-cyclodextrin sodium salt. The benefits of this new reagent for the chiral separation are a wide range of basic pH available for the enantiomeric resolution. It is also useful for other cases, e.g. preventing adsorption without additives.

Capillary zone electrophoresis of malto-oligosaccharides derivatized with 8-aminonaphthalene-1,3,6-trisulfonic acid

Malto-oligosaccharides were derivatized via their reducing end with 8-aminonaphthalene-1,3,6-trisulfonic acid by reductive amination, and the separation and electrophoretic migration behavior of the labelled sugars were investigated by capillary zone electrophoresis. Series of linear malto-oligosaccharides were found particularly suitable for both the study of the effect of the operating conditions on the separation and the investigation of the relationship between the electrophoretic mobility and the molecular size of the homologues. The electrophoretic mobility of the malto-oligosaccharide conjugates was found to be a linear function of the molecular mass to the negative two-thirds power. The sugar derivatives employed here carry three negative charges due to the presence of the dissociated sulfonic acid groups even at strongly acidic pH. Therefore, the analytes can migrate in the electric field without interference by electroendosmotic flow and/or wall adsorption in uncoated silica capillaries at low pH. As a result, the separation of these carbohydrate conjugates can be carried out under such conditions with high speed and efficiency in free solution, i.e., without an anticonvective medium such as a gel or a viscous polymer solution. Appropriate use of triethylammonium phosphate buffer, pH 2.5, as the background electrolyte improves not only the reproducibility, but also the efficiency and speed of the separation. The labelled sugars allow monitoring of the separation by UV detector or laser-induced fluorescence detector with concomitant enhancement of analytical sensitivity.

Capillary Electrophoresis for Protein Analysis: Separation of Human Growth Hormone and Human Insulin Molecular Forms

The capillary zone electrophoresis (CZE) technique was evaluated for separation and quantification of human growth hormone (hGH), human insulin (hI), and proinsulin. Three different molecular forms of biosynthetic hGH (20K, 22K, 44K), methionyl-hGH, biosynthetic hI, and proinsulin, were studied. The hormones were separated with uncoated capillaries, and various analytical conditions were tested (different buffers, ionic strength, pH). The samples were introduced both at positive pressure and electrokinetically, and the voltage applied was varied in each case. Linearity, repeatibility, and limit of detection (0.8 μM for hGH and 1.3 μM for insulin) were determined. Different purification steps were tried in order to find a suitable preanalytical procedure for hGH and hI purification from plasma. Recovery rates from 65 to 80% were obtained.

Trace determination of volatile organic compounds in water by enrichment in ultra-thick-film capillary traps and gas chromatography

The technique by which volatile organic compounds are enriched in coated or uncoated capillaries, and desorbed for qualitative gas chromatographic determination, was adapted for quantitative analysis by using a 1-m length of ultra-thick-film trap (145-μm film thickness) to extract the volatile compounds flowing through the trap at 0°C. The trapped volatiles are thermally desorbed, cryotrapped on an analytical capillary column and determined by gas chromatography with flame ionization detection. It was found that analytes such as dichloromethane and chloroform did not break through when sampling 1 ml of water. With this sampling volume, relative standard deviations well below ca. 2% and ca. 2–4% were obtained for most of the analytes at levels of 1 ppm (v/v) and 1 ppb (v/v), respectively.