András Guttman

Analysis of monosaccharide composition by capillary electrophoresis.

The monosaccharide composition analysis described in this paper employs capillary electrophoretic separation of sugar monomers liberated from glycoproteins or oligosaccharides, by high temperature acidic hydrolysis. Trifluoroacetic acid was used for sialo- and neutral-sugar hydrolysis, and hydrochloric acid was used for amino-sugar hydrolysis. The neutral- and amino-sugars in the hydrolyzates were then labeled with a charged fluorophore, 8-aminopyrene-1,3,6-trisulfonate, while sialic acids were labeled with 9-aminoacridone. The stoichiometry of labeling was such that only one fluorophore molecule was attached to each monosaccharide molecule. The labeled monosaccharides were then separated by high-performance capillary electrophoresis with laser induced fluorescence detection. The acidic hydrolysis and fluorophore labeling conditions described in this paper are suitable for monosaccharide composition analysis of a wide variety of complex carbohydrates from glycoprotein and/or oligosaccharide samples using capillary electrophoresis.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins

Abstract In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass. In this paper we report a new improved and automated electrophoretic method in the form of high-performance capillary SDS gel electrophoresis. Rapid separations of protein molecules in the molecular mass range of 20 000–200 000 daltons were demonstrated with excellent linearity and intra- and inter-day reproducibility of the migration time. Monomer—dimer forms of the recombinant human ciliary neurotrophic factor were examined by the use of this method under reducing and non-reducing conditions.

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.

Practical aspects of chiral separations of pharmaceuticals by capillary electrophoresis. I: Separation optimization

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. In our model system hydroxypropyl-β-cyclodextrin was used as chiral selector for the separation of racemic propranolol. Results are presented regarding the effect of different operational variables such as buffer pH, concentration of chiral selector, applied electric field and temperature on the chiral separation. Based on the experimental data, the operational variables were optimized to attain maximum resolving power with minimal analysis time.

Reductive amination of N-linked oligosaccharides using organic acid catalysts

Abstract The reductive amination of oligosaccharides with 8-aminopyrene-1,3,6-trisulfonate in several organic acids of varying strength was examined by capillary electrophoresis using laser-induced fluorescence detection. The relationship between the derivatization yield and the pKa of the catalyst (organic acids) is in agreement with the general acid catalysis of the hemiacetal ring opening and the Shiff base formation, one of which is considered to be the rate-determining step of the reductive amination reaction. Derivatization in the presence of organic acids having higher acidity than acetic acid, the most commonly used catalyst, resulted in significantly higher derivatization yield and the highest yield was attained with the use of citric or malic acid as catalysts. This effect was even more prominent for oligosaccharides having N-acetylglucosamine at the reducing end compared to the similar size linear glucose oligomers. Sialylated oligosaccharides were derivatized at 37°C for 16 h with only less than 10% loss of the sialic acid residues. The derivatization procedures were tested on the N-linked oligosaccharides released enzymatically (peptide-N-glycanase F) from bovine fetuin and ribonuclease B.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins I. Reproducibility and stability

Abstract This paper investigates the use of a non-cross-linked polymer gel as a separation matrix for sodium dodecyl sulfate (SDS) capillary gel electrophoresis of proteins. The method employs a polyacrylamide coated capillary filled with a polyethylene oxide-gel buffer solution. A standard seven-protein mixture was chosen for the evaluation of coating stability and reproducibility. It was found that the coating is stable for more than 400 runs with a 1 M HCl wash between each run. The hydrophilic nature of polyethylene oxide also allows high resolution and high efficiency for all protein peaks. The linear plot of log Mr vs. mobility demonstrates a pure sieving mechanism of polyethylene oxide matrices. The molecular masses of twenty-nine standard proteins determined by SDS capillary gel electrophoresis are in good agreement with those obtained from the SDS polyacrylamide gel electrophoresis (PAGE) slab gel method. The capability to replace the gel after each run allows improved run-to-run and batch-to-batch reproducibility. The relative standard deviation (R.S.D.) in migration time of 19 injections of the seven-protein standard mixture, with the 190 injections of crude fetal calf serum in between, falls in the range of 0.351 to 0.453%. The application of this technique for the separation of proteins in chicken egg white and bovine milk is demonstrated.

Capillary sodium dodecyl sulfate gel electrophoresis of proteins II. On the Ferguson method in polyethylene oxide gels

Abstract Capillary sodium dodecyl sulfate (SDS) gel electrophoresis is demonstrated to be a powerful new analytical method for the separation of protein molecules based on their molecular mass. Standard curves of logarithm molecular mass versus reciprocal relative migration time give the estimated molecular mass of sample proteins within the acceptable 10% error for most proteins. Larger errors are possible, however, when special groups such as carbohydrates (glycoproteins) or lipids (lipoproteins) are present due to a different ratio of binding SDS molecules. An automated Ferguson method is shown that corrects for this non-ideal behavior in capillary SDS-polyethylene oxide gel electrophoresis.

Ultra-fast chiral separation of basic drugs by capillary electrophoresis

Abstract Chiral separation-methods development is usually very time-consuming, due to the diversity in chemical structures of pharmaceutical drug substances as well as the suitable separation conditions and the problem to choose the appropriate chiral selector. This paper shows an ultra-fast, capillary electrophoresis based screening procedure which was developed for chiral separation of several basic pharmaceuticals using dimethyl-β-cyclodextrin as chiral selector. Complete enantiomeric separations of basic drugs (metaproterenol and isoproterenol) were achieved as fast as in 40–50 s, with an R.S.D. for the absolute migration time reproducibility of less than 0.75%. The peak efficiency of the separations was usually over one million theoretical plates per meter, which correspond to an efficiency generation rate above 30 000 plates/m s.

Effects of sample matrix and injection plug on dsDNA migration in capillary gel electrophoresis

Reproducible DNA migration times are required for accurate basepair assignment in restriction fragment mapping and polymerase chain reaction product identification. Our data shows DNA migration time shifts with changes in sample ionic strength. Secondly, loss of resolution with replaceable polyacrylamide gels was observed when increasing the length of the sample plug with pressure injection. An easy way to correct for the migration time shifts is to incorporate an internal DNA standard directly into the separation process by consecutively injecting the DNA sample and the DNA standard. This allows for compensation of any possible migration time variation caused by high ionic strength sample matrices. Also high-resolution separations can be maintained with large injection volumes (long injection plug) by using consecutive injections of 0.1 M Tris-acetate buffer and the DNA sample.

Capillary gel electrophoretic separation of DNA restriction fragments in a discontinuous buffer system

Abstract Separation of single and double stranded DNA molecules by capillary gel electrophoresis has a rapidly growing interest. Similar to the polycrylamide slab gel based separation methods, in capillary gel electrophoresis, the paek/bans spacing usually decreases with the increasing size/length of the DNA molecule. Additionally, employing the regularly used Tris-borate buffer system, fronting peaks are often obtained. By the application of an electrolyte step gradient during capillary gel electrophoretic separation of dsDNA molecules, the apparent peak shape can be improved and the required analysis time decreased.