Wassim Nashabeh

Capillary zone electrophoresis of proteins with hydrophilic fused-silica capillaries

Abstract Fused-silica capillaries having surface-bound hydroxylated polyether functions were developed for the separation of proteins by capillary zone electrophoresis. In one approach, the hydrophilic coatings consisted of two layers: a glyceropropylpolysiloxane sublayer covalently attached to the inner surface and a polyether top layer. In a second approach, the capillary wall was coated with polysiloxane polyether chains the monomeric units of which at both ends were covalently attached to the capillary inner surface with possible interconnection. These coatings yielded capillaries with different electroosmotic flow characteristics. The relatively long polyether chains of the various coatings were effective in shielding the unreacted surface silanols, thus minimizing solute-wall adsorption. As a consequence, high separation efficiencies were obtained in the pH range 4.0–7.5, which allowed the separation of widely differing proteins, the characterization of heterogeneous proteins and the fingerprinting of crude protein mixtures. The various coatings were stable and exhibited reproducible separations from run-to-run, day-to-day, and column-to-column. Furthermore, a procedure was developed for restoring collapsed capillaries after prolonged use.

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-aminopyridine and 6-aminoquinoline) were utilized for the electrophoresis and sensitive detection 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 of pyridylamino derivatives of maltooligosaccharides

Abstract Maltooligosaccharides derivatized with 2-aminopyridine were separated by capillary zone electrophoresis in the pH range 3.0–4.5 using 0.1 M phosphate solutions as the running electrolyte. The inclusion of small amounts of tetrabutylammonium bromide in the electrolyte solution facilitated the separation at pH 5.0 and yielded high separation efficiency. The separated zones of pyridylamino derivatives of maltooligosaccharides migrated across the fused-silica capillary and passed the detection point in the order of increasing size. The “overall mobility” was a linear function of the number of glucose residues in the homologous series.

Capillary zone electrophoresis of α1-acid glycoprotein fragments from trypsin and endoglycosidase digestions

Capillary zone electrophoresis with fused-silica tubes having hydrophilic coating on the inner walls was evaluated in the separation of peptide and glycopeptide fragments from trypsin digestion of alpha 1-acid glycoprotein. Submapping of glycosylated and nonglycosylated tryptic fragments of the glycoprotein by capillary electrophoresis was facilitated by selective isolation of the glycopeptides on concanavalin A silica-based stationary phases prior to the electrophoretic run. In addition, the electrophoretic map and submaps of the whole tryptic digest and its concanavalin A fractions, respectively, allowed the elucidation of the microheterogeneity of the glycoprotein. Also, capillary zone electrophoresis proved suitable for the mapping of the oligosaccharide chains cleaved from the glycoproteins by endoglycosidase digestion. The oligosaccharides cleaved from human and bovine alpha 1-acid glycoprotein were analyzed after derivatization with 2-aminopyridine, which allowed their sensitive detection by on column UV absorption. The separation was best achieved when 0.1 M phosphate solution, pH 5.0, containing 50 mM tetrabutylammonium bromide was used as the running electrolyte. The effect of the organic salt on separation was attributed to ion-pair formation and/or hydrophobic interaction.

Enzymophoresis of nucleic acids by tandem capillary enzyme reactor-capillary zone electrophoresis☆

Enzymophoresis with coupled heterogeneous capillary enzyme reactor-capillary zone electrophoresis was developed and evaluated in the area of nucleic acids. Ribonuclease T1, hexokinase and adenosine deaminase were successfully immobilized on the inner walls of short fused-silica capillaries through glutaraldehyde attachment. These open-tubular capillary enzyme reactors were quite stable for a prolonged period of use under operation conditions normally used in capillary zone electrophoresis. The capillary enzyme reactors coupled in series with capillary zone electrophoresis served as peak locator on the electropherogram, improved the system selectivity, and facilitated the quantitative determination of the analytes with good accuracy. Also, they allowed the on-line digestion and mapping of minute amounts of transfer ribonucleic acids, and the simultaneous synthesis and separation of nanogram quantities of oligonucleotides.

Chapter 8 High Performance Capillary Electrophoresis of Carbohydrates and Glycoconjugates

Publisher Summary This chapter discusses high-performance capillary electrophoresis (HPCE) of carbohydrates and glycoconjugates. It highlights major progress made in the electrophoretic process, that is, the capillary column. The chapter reviews approaches and concepts that are most useful in the separation of carbohydrates by HPCE and highlights the dynamic behavior of carbohydrate species in the electrophoretic process. Important applications of the HPCE technique are also discussed. The intrinsic high resolving power of electrophoresis is particularly suitable for the separation of carbohydrates, which encompass a wide spectrum of compounds, many of which are isomers or slightly different from each other. In electrophoresis, a charged particle is transported through the conductive medium at a steady state, characterized by a constant electrophoretic migration velocity. Using the same basic instrumentation, HPCE can be performed in several modes to achieve a given separation. The various modes of HPCE include capillary zone electrophoresis (CZE), capillary electrophoresis with sieving matrices including capillary gel electrophoresis (CGE) and capillary electrophoresis with entangled polymer (CEEP), micellar electrokinetic capillary chromatography (MECC), capillary isoelectric focusing (CIEF), and capillary isotachophoresis (CITP).

Fundamental and practical aspects of coupled capillaries for the control of electroosmotic flow in capillary zone electrophoresis of proteins

Abstract This article represents an extension to a new approach, which was introduced very recently by our laboratory, for the control of the magnitude of electroosmotic flow in capillary zone electrophoresis. In this new approach, short fused-silica capillaries having different ξ potentials were coupled in series, and the amount of the electroosmotic flow was conveniently varied by changing the lengths of the individual capillary segments. The different coupled capillary systems evaluated in this study comprised various combinations of untreated fused-silica capillaries and polyether-coated capillaries having various electroosmotic flow characteristics. A general equation relating the average electroosmotic flow velocity in the coupled capillaries to the intrinsic electroosmotic velocity of the connected segments and their corresponding lengths has been derived and verified experimentally. The rate of the electroosmotic flow in a given system of coupled capillaries could be tuned over a range bordered by the lowest and highest intrinsic flow-rates of the connected capillary segments. In addition, a system of coupled capillaries that permitted a stepwise change in the rate of the electroosmotic flow during analysis was introduced and evaluated. These elution schemes were useful in the rapid separation of oppositely charged proteins in a single electrophoretic run and in the rapid analytical determination of the various components of heterogeneous proteins.