Free Zone Capillary Electrophoresis Research Articles

Separation and determination of glycosaminoglycan disaccharides by micellar electrokinetic capillary chromatography for studies of pelt glycosaminoglycans

Capillary electrophoresis based on cetyltrimethylammonium bromide micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of glycosaminoglycan (GAG) disaccharide units without derivatization. The influence of changes in several separation conditions was studied, and the separation mechanisms are discussed. Tests of repeatability and linearity were performed for qualitative and quantitative evaluation of the method. The described procedure gives a rapid and efficient determination of GAG disaccharides. Samples of chondroitin sulphates and mink skin were treated with proteases, and the extent of protein cleavage was followed by free zone capillary electrophoresis. The result of the chondroitinase ABC treatment following the protease treatment was evaluated by the MECC method.

Capillary electrophoresis of abnormal hemoglobins associated with α-thalassemias

Capillary electrophoresis was evaluated for separation of hemoglobin species associated with α-thalassemias, and for identification of hemoglobin variants commonly found in the same human populations. Separation of hemoglobins was achieved using capillary isoelectric focusing with chemical mobilization; visible-wavelength absorbance detection was used to identify hemoglobins against a background of nonheme-containing proteins. This technique could easily differentiate hemoglobins Bart's and H (associated with α-thalassemias) from hemoglobin variants. Analysis of globin chains derived from intact hemoglobins was performed by free zone capillary electrophoresis under denaturing conditions. This technique was useful for distinguishing Hb Bart's and Hb H, and for confirming the identity of hemoglobin variants.

Characterization by capillary electrophoresis of the surface glycoproteins of ovine lentiviruses before and after treatment with glycosidic enzymes

Ovine lentiviruses are a group of viruses that infect sheep and goats. These viruses contain a surface glycoprotein (SU) that is very similar among the viral strains. Sera from infected animals react equally well with SU from each strain. Monoclonal antibodies produced to SU can distinguish among some of the viral strains. In order to delineate these differences we treated SU from several viral strains with the glycosidic enzymes. These enzymes included a mixture of exoglycosidases, β-N-acetyl glucosaminidase, neuraminidase and endoglcyosidases D, F and H. After these treatments we observed changes in the reactivities of the monoclonal antibodies that were directed to SU. In order to characterize these changes on the surface epitopes, SU from the different viral strains were subjected to free zone capillary electrophoresis (CZE) using an 0.02 M phosphate buffer at pH 9.0 at a running voltage of 5 kV. Differences were readily seen between SU that had not been treated and SU that had been treated with the glycosidic enzymes. Each viral strain had a characteristic electropherogram. The electropherograms indicated that the heterogeneity of the charge on SU was increased after the enzyme treatments. From these results we have concluded that the carbohydrate moieties play an important role in contributing to the surface charge of SU. This charge affects the nature of its surface epitopes and has an impact on its biological function.

Capillary Electrophoretic Separation of Human Recombinant Erythropoietin (r-HuEPO) Glycoforms

Free zone capillary electrophoresis was investigated for the separation of glycoforms in recombinant erythropoietin (r-HuEPO). Reduction of electroendoosmotic flow through the addition of 1,4-diaminobutane and in the presence of urea resulted in optimum separation of all of the major glycoforms. Incubation of r-HuEPO with neuraminidase caused the slowest migrating species to diminish first in response, indicating that these contained the highest numbers of sialic acids. Additional evidence was obtained from spiking r-HuEPO with individual glycoforms isolated by preparative isoelectric focusing. These results indicate that the separation occurs in a predictable manner in order of increasing number of sialic acids contained in the glycoforms. As a consequence, the electropherograms provide the same information as that obtained from conventional gel isoelectric focusing, although separating by a different mechanism.

Capillary electrophoretic separation of recombinant granulocyte-colony-stimulating factor glycoforms

Free zone capillary electrophoresis separated recombinant human granulocyte-colony-stimulating factor, expressed in Chinese hamster ovary cells, into two well-resolved species. Following incubation with neuraminidase, these species comigrated, eluting earlier than either of the original two species. This indicated that the observed heterogeneity was caused by different amounts of sialic acid present on the carbohydrate portion of the protein. It was determined that optimum separation occurred in the buffer pH range 7–9. Evidence is also presented to show that these glycoforms migrate in order of increasing numbers of sialic acids present.

Borate complexation of flavonoid-O-glycosides in capillary electrophoresis: I. Separation of flavonoid-7-O-glycosides differing in their flavonoid aglycone

Capillary electrophoresis was found to give significantly greater efficiency, selectivity and speed compared with high-performance liquid chromatography for the separation of mixtures of flavonoid-O-glycosides. The migration behaviour of selected flavonoid-O-glycosides and their flavonoid aglycones in free solution capillary electrophoresis and micellar electrokinetic capillary chromatography (MECC) was investigated. A mixture of flavonoid-7-O-glycosides and their flavonoid aglycones was resolved by MECC. A 70 cm × 50 μm I.D. fused-silica capillary column and a 50 m M sodium dodecyl sulphate-20 m M Tris-CHl running buffer (pH 7.1) were used for these electrophoretic separations. Under these neutral conditions, the retention mechanism is based on hydrophobic interactions betweeen flavonoids and the hydrophobic core of the micelles. Free zone capillary electrophoresis of borate complexes was more appropriate for the separation of a mixture of flavonoid-O-glycosides. In that case, the migration behaviour of these selected flavonoid-O-glycosides was explained by ionization of some hydroxyl groups and mainly by borate complexation of the sugar moiety. The identity of each flavonoid-O-glycoside was determined by using a fast-scanning multiple-wavelength UV detector and by recording its on-column UV spectrum in the range of 190–360 nm.

Capillary electrophoresis of hemoglobins and globin chains

Capillary isoelectric focusing (cIEF) and free zone capillary electrophoresis were evaluated for separation of native hemoglobins and globin chains. High-resolution separations of adult human hemoglobin A, fetal human hemoglobin F, and hemoglobin variants S and C were obtained using cIEF with cathodic mobilization. Absorbance detection in the UV and visible regions were compared, and on-line fast UV or visible-wavelength scanning detection was used to obtain spectral information on separated components. Globin chain analysis was performed on the same hemoglobin species by free zone capillary electrophoresis following precipitation of the protein with acidic acetone. Free zone separations were carried out at low pH in the presence of 7 M urea.

Determination of quaternary ammonium compounds by capillary electrophoresis using direct and indirect UV detection

The determination of alkyl and alkylbenzyl quaternary ammonium compounds can be difficult owing to the polarity of the compounds, the formation of micelles by longer chain compounds and the lack of a chromophoric substituent for detection by standard separation techniques. The development of a free zone capillary electrophoresis method necessitated the use of organic modifiers in order to disrupt the formation of micelles by the longer chain (C 12) surfactants. The selectivity obtained by using various levels of tetrahydrofuran as an organic modifier allowed the separation of a mixture of alkylbenzyl and alkylethylbenzyl quaternary compounds. The separation of alkyltrimethyl and dialkyldimethylammonium compounds was accomplished by the addition of a chromophoric cationic compound to the buffer system in order to allow the detection of the compounds with a standard UV absorbance detector. By varying the electrophoretic mobility of the indirect detection reagent, a variety of alkyl quaternary compounds, ranging from tetra- methylammonium chloride to stearyltrimethylammonium chloride, can be detected with good peak shapes. The use of these methods for the determination of quaternary ammonium compounds will also be discussed. The use of internal standard methods and the linearity and linear range of the technique are considered.

Rugged method for the determination of deamidation products in insulin solutions by free zone capillary electrophoresis using an untreated fused-silica capillary

Addition of zwitterions and acetonitrile to the running buffer provided a successful separation of insulin and deamidation products in an untreated fused-silica capillary. A high efficiency and reproducible migration times were obtained without a base wash between each run and without thermostating of the capillary. A comparison was made of results achieved using capillary zone electrophoresis (CZE), ion-exchange chromatography (IEC) and reversed-phase high-performance liquid chromatography. A good correlation was observed between CZE and IEC.

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monotoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and apperance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.

Determination of berberine chloride in pharmaceutical preparations by capillary electrophoresis.

Determination of berberine chloride in pharmaceutical preparations was investigated by capillary electrophoresis. Free zone capillary electrophoresis in phosphate buffer at pH 5.0 gave complete separation of berberine from accompanying substances within 20 min. A sample solution was introduced by hydrostatic method and the on-column detection was performed at 214 nm. The calibration curves of berberine chloride gave good linearity over the concentration range of 10100 μg ml-1. Berberine chloride in various preparations was determined with good reproducibility.

Electrophoretic mobility modeling of proteins in free zone capillary electrophoresis and its application to monoclonal antibody microheterogeneity analysis

A semi-empirical model for describing the electrophoretic mobility of proteins in free solution is derived. Protein mobility is found to be influenced, as dictated by the Debye-Hückel-Henry theory, by protein valence, size and shape, and by solution ionic strength, pH, viscosity and temperature. Protein valence, the most important mobility determining parameter intrinsic to the protein, is calculated for a given pH from its amino acid content using the Henderson-Hesselbalch equation. Electrostatic charge suppression causes actual valence to be less than that calculated. To equate the two an experimentally determined proportionality constant ( F Z ) is introduced. Consequently, F Z can be applied to the calculated valence and mobility-pH titration curve for a protein, resulting in the actual mobility of the protein at any given pH. The model further predicts that the molecular weight ( M) dependency of mobility should be a continuous function of M −1/3 to M −2/3, depending on the magnitude of the protein molecular weight and buffer ionic strength under investigation. Many aspects of the model are demonstrated by its application to the resolution of immunoglobulin G isoelectrotypes, normally only resolved using isoelectric focusing.

Analysis of a vaccine purification process by capillary electrophoresis

Free-zone capillary electrophoresis (CZE) was applied to the analysis of samples from the individual purification steps in the production of a licensed vaccine product. With this technique, real-time analysis can be performed to ensure that purification parameters are being met before proceeding to the next step. In addition to monitoring the product peak, a unique pattern of associated host cell contaminants also is displayed. This unique “fingerprint” at each step of the purification process may have practical utility for production in that it demonstrates lot-to-lot consistency in the manufacturing process of the vaccine.

Rapid protein analysis by capillary electrophoresis

Rapid protein analysis by free-zone capillary electrophoresis in an untreated fused-silica column is presented. Separation of model proteins by the proposed method shows that the retention times of these proteins correlate well with each of their isoelectric points. By using a high voltage gradient, most proteins could be separated in 200 s with excellent reproducibility. Similarly, human serum proteins could be resolved and determined in less than 100 s.

Purification and characterization of rat ovarian 20α-hydroxysteroid dehydrogenase

To investigate the regulatory mechanism of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) (EC 1.1.1.149) activity in ovarian tissue, the enzyme was purified from ovaries of normal mature female rats. Column chromatography of the cytosolic fraction from ovaries on DEAE-Toyopearl 650M revealed two peaks of the 20 alpha-HSD activity at different ionic strengths. These peaks were designated HSD1 and HSD2, respectively. Each of the active fractions was further purified to homogeneity by dye-affinity chromatography using Matrex Green A and AF Red-Toyopearl. Both the fractions appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (at Mr = 33,000 under reducing conditions). Under non-reducing conditions, similar values were obtained on gel-exclusion HPLC, indicating that the enzyme fractions were single-stranded, monomeric polypeptides. Homogeneous HSD1 and HSD2 were purified 361-fold and 509-fold, respectively, and differed in their substrate preference. The two enzyme fractions had Km values of 4.75 microM and 5.16 microM for 20 alpha-dihydroprogesterone, respectively, and showed almost the same RF values on reverse-phase HPLC and free-zone capillary electrophoresis. However, amino acid composition was slightly different, i.e. lysin content was higher in HSD1 than HSD2. Thus, it was clarified that two types of 20 alpha-HSD with very similar molecular structures are present in the rat ovary.