Capillary zone electrophoresis of linear and branched oligosaccharides

The electrophoretic behavior of derivatized linear and branched oligosaccharides from various sources was examined in capillary zone electrophoresis with polyether-coated fused-silica capillaries. Two UV-absorbing (also fluorescent) derivatizing agents (2-amino-pyridine and 6-aminoquinoline) were utilized for the electrophoresis and sensitive dtection of neutral oligosaccharides, e.g., N-acetylchitooligosaccharides, high-mannose glycans and xyloglucan oligosaccharides. The oligosaccharides labelled with 6-aminoquinoline yielded eight times higher signal than those tagged with 2-aminopyridine. Plots of logarithmic electrophoretic mobilities of labelled N-acetylchitooligosaccharides with 6-aminoquinoline or 2-aminopyridine versus the number of sugar residues in the homologous series yielded straight lines in the size range studied, the slopes of which were independent of the tagging functions. The slopes of these lines are referred to as the N-acetylglucosaminyl group mobility decrement. The oligosaccharides were better resolved in the presence of tetrabutylammonium bromide in the running electrolyte. Furthermore, the electrophoretic mobilities of branched oligosaccharides were indexed with respect to linear homooligosaccharides, an approach that may prove valuable in correlating and predicting the mobilities of complex oligosaccharides.     

Capillary zone electrophoresis: Effect of physical parameters on separation efficiency and quantitation

Separation efficiency in capillary zone electrophoresis was examined as a function of capillary length and diameter as well as solute concentration for a borosilicate glass capillary/phosphate buffer system. Electroosmotic flow coefficients in borosilicate, fused silica, and teflon capillaries were measured over the useful operating pH range of 3–8 at constant applied power (0.333 W). Sample introduction by a simple, low-dispersion electromigration method was found to be a reliable procedure for solute quantitation.     

Capillary electrophoresis for the analysis of glycosaminoglycans and glycosaminoglycan-derived oligosaccharides

Glycosaminoglycans are a family of polydisperse, highly sulfated complex mixtures of linear polysaccharides that are involved in many life processes. Defining the structure of glycosaminoglycans is an important factor in elucidating their structure-activity relationship. Capillary electrophoresis has emerged as a highly promising technique consuming an extremely small amount of sample and capable of rapid, high-resolution separation, characterization and quantitation of analytes. Numerous capillary electrophoresis methods for analysis of intact glycosaminoglycans and glycosaminoglycan-derived oligosaccharides have been developed. These methods allow for both qualitative and quantitative analysis with a high level of sensitivity. This review is concerned with separation methods of capillary electrophoresis, detection methods and applications to several aspects of research into glycosaminoglycans and glycosaminoglycan-derived oligosaccharides. The importance of capillary electrophoresis in biological and pharmaceutical samples in glycobiology and carbohydrate biochemistry and its possible applications in disease diagnosis and monitoring chemical synthesis are described.     

Capillary zone electrophoresis for polymide composition analysis

Capillary zone electrophoresis (CZE) was employed in polyimide composition analysis. Polymide was decomposed to its corresponding aromatic diamine and aromatic acid monomers by an alkali fusion reaction. Sample treatment is much simpler than published methods, and electropherograms show a good separation of decomposed products under the proper conditions.     

Capillary zone electrophoresis for the characterization of latex particles

Capillary zone electrophoresis (CZE) was applied to the separation of acrylic styrene copolymer emulsion particles. Fast separations could be performed on samples containing chemically identical latex particles of different size, as well as on samples with particles of the same size but differing in chemical composition. The developed method was also used for the analysis of water soluble fractions of urethane dispersions. Additionally, the physical interaction between different particles (e.g., acrylic and urethane particles) could be studied using this method. The separation mechanism is based on the zeta potential of the particles and the relaxation effect under the applied analytical conditions.     

Capillary electrophoresis of acidic oligosaccharides from human milk

Interest in defining the array of oligosaccharides of human milk has been increasing. Pathogens that bind glycans on their host mucosal surfaces may be inhibited by human milk oligosaccharides. It has been postulated that acidic oligosaccharides in human milk may inhibit binding by pathogens that bind acidic glycans in the gut, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides of human milk have been quantified by HPLC and CE. A recent CE technique uses the MEKC mode with direct detection at 205 nm to resolve and quantify, in the native form, the 12 most dominant sialyloligosaccharides of human milk in a single 35-min run. The method gives a linear response from 39 to 2500 microg/mL with a coefficient of variation between 2 to 9% and accuracy from 93 to 109%. This was used to detect variation in expression of specific sialyloligosaccharides in milk. Individual sialyloligosaccharide concentrations in milk differ among individual donors and between less and more mature milk. Thus, CE can be used to measure variation in sialyloligosaccharide expression in milk, and thereby test the relationship of this variation-to-variation in risk of specific diseases in breastfed infants.     

Capillary zone electrophoresis of histidine-containing compounds

Capillary zone electrophoresis has been tested for the separation of angiotensins, cationic heptapeptides and model histidine derivatives. Good separation efficiencies are seen for peptides and model compounds with negative to small positive net charges. For net charge greater than +2, addition of putrescine to pH 6 buffer greatly suppresses ion exchange at anionic sites on fused silica. When operating at pH values where histidine groups are neutral, addition of Zn2+ allows separations based on metal, rather than proton, binding. Separation efficiencies and relative migration times are dependent on capillary length when ion-exchange behavior occurs.     

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.     

Capillary zone electrophoresis for the determination of light-absorbing anions in environmental samples.

A rapid and simple method for separation and determination of inorganic anions by capillary zone electrophoresis was described. The detection was carried out directly with a diode array detector. The experimental conditions, such as concentration of carrier electrolyte, capillary length, voltage, and temperature were optimized. In order to improve selectivity, different organic modifiers were also investigated. The baseline separation of 10 light-absorbing anions was accomplished within 3.5 min with a background electrolyte consisting of 50 mM sodium tetraborate containing 5% MeOH. Linear plots were obtained in the concentration range of 0.1-10 microg/ml. With sample stacking injection, the quantitation limits of the anions were found to be in the range of 0.02-0.1 microg/ml. The proposed method was successfully applied to the determination of inorganic anions in environmental samples and in effluents of a power plant.     

Capillary zone electrophoresis and polymer dynamics

Capillary electrophoresis with a polymer solution support medium is a well-established powerful analytical tool. This paper discusses a possible alternative application of capillary electrophoresis, namely a path for studying the dynamics of polymer solutions. The key to the approach is an inversion of perspective. Instead of treating the migrating species as the object of experimental importance and the support medium as being of importance only because it facilitates separations, one treats the polymer solution as being of experimental importance, and the choice of migrating species as being of interest only because different species may probe different aspects of polymer dynamics.     

Capillary zone electrophoresis of derivatized acidic monosaccharides

A new and specific precolumn derivatization reaction for acidic monosaccharides was introduced and evaluated in the separation and sensitive detection of carbohydrates by capillary electrophoresis. The derivatization reaction involved the attachment of sulfanilic acid (a UV absorbing tag) or 7-aminonaphthalene-1,3-disulfonic acid (a UV absorbing and fluorescing tag) via a condensation reaction between the amino group of the derivatizing agent and the carboxyl group of the sugar in the presence of a water-soluble carbodiimide. The derivatization reaction replaced the weak carboxylic acid of the sugar by a strong sulfonic acid, which is fully ionized at all pH. This allowed the electrophoresis of the sugar derivatives over a wide pH range and permitted the determination of acidic carbohydrates at very low femtomole levels by UV and fluorescence detection.     

Capillary zone electrophoresis of natural organic matter

This paper presents an in-depth look at the use of capillary electrophoretic (CE) techniques for the fingerprinting and characterization of humic substances and natural organic matter. These materials are highly heterogeneous in structure and show all characteristics of mixtures unliked in analytical chemistry. The electrophoretic approach, however, allows the determination of mobility distributions in different solution conditions, representative of the effective charge and size distribution status of the components present. A tabulated review covers over 50 references on the subject and highlights the possibilities and problems encountered in the analysis of such polydisperse materials with CE methods. In a second part of the article the consequences of experimental and buffer parameters on the behavior of humic materials in CE are presented.     

Capillary zone electrophoresis separation of enantiomers of lisuride

Lisuride is an ergot alkaloid derivative with dopaminergic activity. It is used for treatment of Parkinsonism and some other diseases associated with high level of prolactine. Lisuride is a chiral compound derived from natural ergot alkaloids. A new capillary zone electrophoresis (CZE) method capable of separating the enantiomers of lisuride was developed. Using the optimized conditions (acidic electrolyte with the addition of gamma-cyclodextrin (gamma-CD)) as low as 0.02% of undesirable L-lisuride can be detected. Selected method characteristics, i.e., linearity (0-20 mg/l), precision (2.0% at 5 mg/I), and accuracy (101 +/- 4% at 5 mg/l) were evaluated. The optimized method was applied for the analysis of real batches of Lisuride hydrogenmaleate and Lisuride base manufactured by IVAX Pharmaceuticals. It was found that they contain less than 0.02% of undesirable L-enantiomer.     

Capillary zone electrophoresis of lipoarabinomannan by multi-layered concentration

The present article describes a capillary zone electrophoresis method which relies on a multilayered water-alkali solvent stacking with online ionization to enhance detection of mannose, arabinose, and their oligosaccharides, which are used as the migration profile standards but are also the distinctive structural components of lipoarabinomannan. Lipoarabinomannan is detected in patients having tuberculosis. The capillary electrophoresis method with ionization of the whole saccharides without degradation in alkaline solution inside the capillary is based on the structural deprotonation of the molecules under ultrahigh pH conditions. The validation of the capillary electrophoresis parameters revealed that the 15-fold electrolyte–water-injection plug allowed detection of one-third lower concentrations than detected without online concentration. For the first time, the better detectability was seen especially for highly polymerized, but otherwise poorly ionized, arabinooctaose. The applicability of the method for detecting whole large biological saccharide complexes was confirmed by the glycolipid lipoarabinomannan. For the first time also, the migration of the indestructible lipoarabinomannan was detected together with oligosaccharides used representing the capping units, namely mannose, mannobiose, and mannotriose. The myo-inositol-phosphate-lipid unit was seen to migrate separately from the arabinomannan, since it was hydrolyzed from one lipoarabinomannan product under alkaline conditions in capillary electrophoresis.     

Capillary zone electrophoresis method for the simultaneous determination of cations in honey

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 min with only a dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). The running voltage was + 25 kV at 25 degree C. Indirect UV detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84 and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. These cations were identified by their relative migration times with regard to Ba2+ migration time used as reference standard and they were quantified by using an external standard calibration. Twenty-five honey samples were analysed to test the proposed method. Mean contents of 1.22 x 10(3), 93, 85, 54, 11, 1.9 and 2.3 mg/kg were found, respectively, for K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+ cations in analysed honeys. These results were similar than the obtained by other authors.     

Capillary zone electrophoresis of humic acids from the American continent

A multicomponent background electrolyte (BGE) was developed and its composition optimized using artificial neural networks (ANN). The optimal BGE composition was found to be 90 mM boric acid, 115 mM Tris, and 0.75 mM EDTA (pH 8.4). A separation voltage of 20 kV, 20 degrees C and detection at 210 nm were used. The method was applied to characterize several humic acids originating from various countries of the American continent: soil (Argentina), peat (Brazil), leonardite (Guatemala and Mexico) and coal (United States). Comparison with humic acids of International Humic Substances Society (IHSS) standard samples was also done. Well reproducible electropherograms showing a relatively high number of peaks were obtained. Characterization of the samples by elemental analysis and UV spectrophotometry was also done. In spite of the very different origins, the similarities between humic acids are high and by matrix assisted desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry it was shown that most of the m/z patterns are the same in all humic acids. This means that humic acids of different origin have the same structural units or that they contain the same components.     

Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum

Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.     

Capillary zone electrophoresis study of cyclodextrin--lipoic acid host-guest interaction

Lipoic acid is a naturally occurring compound which is being widely investigated for its therapeutic effects in the treatment or prevention of a variety of diseases associated with oxidative injury, particularly diabetes. The diversity of therapeutic applications of lipoic acid requires an appropriate formulation to control its bioavailability, site-targeting delivery and to overcome its inherent chemical instability. In this regard, cyclodextrins (CDs) are ideally suitable due to their well-documented ability to include in their cavity proper guest molecules and protect them from physical or chemical damages. Lipoic acid forms 1:1 inclusion complexes with betaCD as shown in a previous report of an extended investigation that also indicated the suitability of capillary zone electrophoresis (CZE) for the study of such host-guest interactions. In view of these possible applications, we extended the CZE analysis to determine the strength of binding, in a pH 9 phosphate buffer, of lipoic acid with other CD derivatives such as alphaCD, gammaCD and the alkylated derivatives of betaCD, namely (2-hydroxypropyl)-beta-CD (HPbetaCD), and heptakis(2,3,6-tri-O-methyl)-beta-CD (TMbetaCD). Once established that the easily available betaCD is the most suitable receptor for lipoic acid, we set up and here describe a simple and reliable procedure for the quantitative determination of lipoic acid in commercial dietary supplement tablets containing also other active substances and excipients.     

Capillary zone electrophoresis for the determination of average hydrodynamic velocity and diffusion coefficient

The determination of the average hydrodynamic velocity and diffusion coefficient by capillary zone electrophoresis is described. The present simple method only requires basic experimental data, such as peak area, peak height and migration time to complete the task of determining the diffusion coefficient and the average hydrodynamic velocity.