Journal of Chromatography B (v.841, #1-2)

Foreword by I. Miksik (1-2).

Capillary electromigration methods for the study of collagen by Ivan Mikšík; Pavla Sedláková; Kateřina Mikulíková; Adam Eckhardt (3-13).
This review paper gives an overview of capillary electromigration methods used in the analysis of collagen. Analyses of the parent chains as well as of the bromcyane and collagenase fragments of collagens are presented. Methods include capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography as well as combinations of HPLC and capillary electrophoresis, and capillary electrophoresis with mass spectrometry.
Keywords: Collagen; Capillary electrophoresis; Review;

The present review touches on a long-lasting debate on possible artefacts (i.e. generation of spurious spots, not belonging to the biological sample under analysis) induced by the separation technique (in this case, two-dimensional mapping) per se. It is shown here that some of the biggest offenders, always blamed in the past (at least since 1970, i.e. since the inception of gel-base isoelectric focusing protocols), namely deamidation (of Asn and Gln residues) and carbamylation (due to cyanate produced in urea solution), simply do not occur in properly handled samples and have never indeed been demonstrated in real samples, except when forced in purpose. Conversely, two unexpected major artefacts have been recently shown to plague 2D mapping. One is formation of homo- and hetero-oligomers in samples that have been reduced but not alkylated prior to entering the electric field. The phenomenon is highly aggravated in alkaline pH regions and can lead to an impressive number of spurious spots not existing in the original sample. Thus, alkylation (best if performed with acrylamide or vinylpyridines) is a must for avoiding such spurious spots, as well as sample streaking and smearing in the alkaline gel region, and for maintaining sample integrity. In fact, the other unexpected artefact is desulfuration (β-elimination) by which, upon prolonged electrophoresis, the sample looses an ―SH group fro Cys residues. This loss, in the long run, is accompanied by massive protein degradation due to lysis of a C―N bond along the polypeptide chain. Here too, alkylation of ―SH groups of Cys almost completely prevents this noxious degradation phenomenon.
Keywords: Two-dimensional maps; Artefacts; Carbamylation; Beta elimination;

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.
Keywords: Biocatalysts; Enzymes; Ribozymes; Enzymatic activity; Pre-capillary assay; On-capillary assay; Post-capillary assay; EMMA;

Analysis of liposomes by capillary electrophoresis and their use as carrier in electrokinetic chromatography by Gerhard Bilek; Leopold Kremser; Dieter Blaas; Ernst Kenndler (38-51).
This contribution reviews work about liposomes in the context of electrically driven separation methods in the capillary format. The discussion covers four topics. The one broaches the application of liposomes as pseudo-stationary phases or carriers in vesicle or liposome electrokinetic chromatography (EKC) in the way as microemulsions and micelles are used; it includes the chromatographic use of liposomal bilayers as stationary phases attached to the wall for capillary electrochromatography (CEC). The second topic is the characterization and separation of liposomes as analytes by capillary electrophoresis (CE). Then the determination of distribution coefficients and binding constants between liposomes and ligands is discussed, and finally work dealing with peptides and proteins are reviewed with lipid bilayers as constituents of the electrically driven separation system.
Keywords: Liposome; Phospholipids; Capillary electrochromatography; Capillary electrophoresis; Vesicles; Pseudostationary phase; Proteins; Peptides;

Monolithic materials are finding their place in a variety of fields. While liquid chromatography is the most emphasized use of this new category of porous media, some other just as important applications are eclipsed by the success of monolithic columns. This review article describes all current facets of use of monoliths in preconcentration and solid-phase extraction. In addition to the typical off line use that does not seem to be the main stream application for the monolithic materials, in-line connection of the preconcentration with HPLC, electrochromatography, electrophoresis, enzymatic digestion, as well as its applications in microfluidics are presented.
Keywords: Monolith; Solid-phase extraction; Silica; Polymer; Capillary; Microchip;

Miniaturized separation techniques in glycomic investigations by Yehia Mechref; Milos V. Novotny (65-78).
High-sensitivity glycomic analyses are becoming of a great interest in modern biomedical and clinical research, as well as in the development of recombinant protein products. The evolution of separation techniques for glycomic analysis at high sensitivity is highlighted in this review. These methodologies include capillary liquid chromatography, capillary electrophoresis (CE) and capillary electrochromatography (CEC). The potential of such methodologies in glycomic analysis is demonstrated for model glycoproteins as well as total glycomes derived from biological samples.
Keywords: Glycomics; Oligosaccharides; Capillary liquid chromatography; Capillary electrophoresis; Capillary electrochromatography; Hydrophilic nano-LC; Graphitized carbon packings; Permethylation;

Monolithic organic polymeric columns for capillary liquid chromatography and electrochromatography by Karel Štulík; Věra Pacáková; Jana Suchánková; Pavel Coufal (79-87).
This review briefly summarizes the present state of the preparation and use of capillary monolithic columns for liquid chromatography (LC) and electrochromatography (EC). Most important approaches to the preparation of monolithic stationary phases based on organic polymers are outlined and the properties of the monoliths obtained are compared with those of classical particulate phases. A few selected applications of monolithic columns are shown to demonstrate the most important advantages of monolithic capillary columns. It is concluded that both the monolithic and particulate capillary columns are important and that judicious choice of the type suitable for a particular application requires careful consideration of the purpose of the separation and the properties of the solutes to be separated. Monolithic columns are substantially younger than packed ones and thus will require further theoretical and experimental study to further improve their preparation and to enable reliable prediction of their properties and applicability; nevertheless, they are very promising for the future.
Keywords: Monolithic capillary columns; Capillary liquid chromatography; Electrochromatography; Packed capillary columns; Comparison; Review;

Compared to chromatography-based techniques, the concentration limits of detection (CLOD) associated with capillary electrophoresis are worse, and these have largely precluded their use in many practical applications. To overcome this limitation, researchers from various disciplines have exerted tremendous efforts toward developing strategies for increasing the concentration sensitivities of capillary electrophoresis (CE) systems, via the so-called sample enrichment techniques. This review highlights selected developments and advances in this area as applied to the analyses of proteins and peptides in the last 5 years.
Keywords: Sample enrichment; Focusing; Concentration sensitivity; Proteins; Peptides;

The diagnosis of alcohol abuse based on objective data is a necessary requirement in both clinical and forensic environments. Among the different biomarkers of chronic alcohol abuse, carbohydrate-deficient transferrin (CDT) is world wide recognized as the most reliable indicator. However, several problems about the real meaning of CDT and the reliability of its use for the diagnosis of alcohol abuses are still open, as reported by numerous research articles and reviews. The present article presents a critical review of the literature on CDT appeared in the period from 2001 to 2005 (included). The article is organized in the following sections: (1) introduction, (2) definition and structure of human serum CDT, (3) pathomechanisms of the ethanol-induced CDT increase, (4) preanalysis, (5) analysis, (6) data interpretation, (7) review papers, (8) conclusions. As many as 127 papers appeared in the international literature and retrieved by the search engines PubMed and Scopus are quoted.
Keywords: Carbohydrate deficient transferrin; Alcohol abuse; Biomarkers; Analysis;

Almost all proteins are expressed in several variants, also known as isoforms. Individual protein variants differ by modifications of the individual amino acid side chains, or the N- or C-terminus. Typical modifications are glycosylation, phosphorylation, acetylation, methylation, deamidation or oxidation. It is of utmost interest to either get a quantitative picture of the variants of a particular protein or to separate the variants in order to be able to identify their molecular structure. Protein variants are present in native as well as in recombinant proteins. In the case of protein production it is interesting, how variants are generated during fermentation, purification processes, storage, and how present individual variants influence the biological activity. This review provides a comparison of chromatographic and electrophoretic separation methods to analyze and to prepare protein variants.
Keywords: Recombinant protein; Variant; Isoforms; Microheterogeneity; Chromatography; Electrophoresis;

Microchip electrophoresis has a great potential to improve the speed and throughput of chemical and biochemical analyses. Conventional electrophoresis microchip fabrication methods comprise the main steps of channel formation, cover plate binding and access hole construction. While the fabrication of appropriate cover plates and their bonding process are quite essential to the creation of closed microfluidic networks, connection means of microchips to the macro-world is one of the most important parts of microchip fabrication. In this paper the most commonly used approaches are discussed for cover plate connector fabrication in conjunction with high and low temperature glue-less binding processes. The microchannels in the glass substrate were fabricated by sawing and powder blasting under regular laboratory settings, i.e., not necessitating the use of a clean-room environment, making in this way broader availability for electrophoresis microchip technology.
Keywords: Microfabrication; Cover plate binding; Connections; General laboratory setting;

The dependence of the effective electrophoretic mobility on pH of the background electrolyte was experimentally determined by capillary zone electrophoresis (CZE) for cationic forms of amino acids. The pH of the background electrolytes was in the highly acidic range, 1.6–2.6 pH units, to ensure a high degree of protonation of the amino acids. Poly(vinyl alcohol) was added to the background electrolytes to avoid possible adsorption of the analytes at the inner capillary wall. Non-linear regression of the experimental data was applied to obtain the parameters of the relevant regression functions—the actual mobilities and mixed dissociation constants corresponding to the actual ionic strength. The extended Onsager and Debye–Hückel law was used to calculate the limiting mobilities and thermodynamic dissociation constants. The comparison of the experimental electropherogram with the computer prediction by PeakMaster using the determined data is presented for the selected sample of amino acids.
Keywords: Amino acids; Capillary electrophoresis; Conductivity; Mobility; Dissociation constant;

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis by P. Sedláková; J. Svobodová; I. Mikšík; H. Tomás (135-139).
The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 μm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0–6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.
Keywords: PAMAM dendrimers; Dynamic coating; Capillary wall; Capillary electrophoresis;

Separation of microcystins by capillary electrochromatography in monolithic columns by Marta Zeisbergerová; Vratislav Košťál; Markéta Šrámková; Pavel Babica; Luděk Bláha; Zdeněk Glatz; Vladislav Kahle (140-144).
Contribution on microcystin variant analysis by capillary electrochromatography (CEC) with easily affordable spectrophotometric detection is presented. Two types of reversed-phase capillary columns formed by inorganic or organic polymer monoliths were prepared for this purpose. The analyses were performed isocratically by means of tris(hydroxymethyl) aminomethane (TRIS) buffers of mildly alkaline pH containing 30% (v/v) acetonitrile as the mobile phases. The samples were injected electrokinetically and the analyses were done at the same separation field strength of 500 V/cm. Microcystins were detected at 238 nm. Although both column types differ not only in monolith quality (inorganic versus organic) but also in the length of the aliphatic moiety (C8 versus C12) similar results were achieved. The on-column preconcentration as the encouraging prospect of electrochromatographic technique was also tested. Consequently 5% of column volume was injected in contrast with 0.5% at standard injection scheme resulting in the six times enrichment of the low concentrated cyanobacterial extract at the top of the separation column. From these preliminary results can be seen that the CEC method is fully applicable for rapid microcystin screening.
Keywords: Microcystins; Separation; HPLC; Capillary electrochromatography; Monolithic columns;

Determination of purity degree and counter-ion content in lecirelin by capillary zone electrophoresis and capillary isotachophoresis by Petra Sázelová; Václav Kašička; Veronika Šolínová; Dušan Koval (145-151).
Capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) were applied for the determination of peptide purity degree and counter-ion content in lecirelin, the synthetic analogue of luteinizing hormone-releasing hormone (LHRH). CZE analyses were carried out in acidic background electrolyte (100 mM H3PO4, 50 mM Tris, pH 2.25) in bare fused silica capillary using UV-absorption detection at 206 nm. CITP analyses were performed in the electrophoretic analyzer with column coupling, equipped with contactless conductivity detectors both in preseparation capillary and in analytical capillary, and with UV-absorption detector (220 and 254 nm) in analytical capillary. Determinations of peptide purity were carried out in cationic mode with leading electrolyte (LE), 10 mM KOH/AcOH, pH 4.5, and terminating electrolyte (TE), 10 mM β-alanine (BALA)/AcOH, pH 4.4. Degree of peptide purity determined by both CZE and CITP was in the range 60.1–80.9% for crude preparations of lecirelin and in the range 96.4–99.9% for HPLC purified batches. Concentrations of contaminating counter-ions, the anions of trifluoromethanesulfonic acid (TFMSA), trifluoroacetic acid (TFA) and acetic acid (AcOH), were determined by CITP analyses in anionic mode with LE 10 mM HCl/His, pH 6.0, and TE 10 mM 2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.0, by the calibration curve method. Mass percentages of the counterion contents in the analyzed lecirelin batches varied from zero to ca. 9% (TFMSA), 3% (TFA) and 11% (AcOH), respectively.
Keywords: Capillary zone electrophoresis; Capillary isotachophoresis; Lecirelin; Trifluoromethanesulfonic acid; Trifluoroacetic acid; Counterion content;

Capillary isoelectric focusing of proteins and microorganisms in dynamically modified fused silica with UV detection by Marie Horká; Filip Růžička; Jaroslav Horký; Veronika Holá; Karel Šlais (152-159).
We suggest a method for the reproducible and efficient capillary isoelectric focusing of proteins and microorganisms in the pH gradient 3–10. The method involves the segmental injection of the simple ampholytes, the solution of the selected electrolytes, and the sample mixture of bioanalytes and carrier ampholytes to the fused silica capillaries dynamically modified by poly(ethylene glycol), PEG 4000, which is added to the catholyte, the anolyte and injected solutions. In order to receive the reproducible results, the capillaries were rinsed by the mixture of acetone/ethanol between analyses. For the tracing of the pH gradients the low-molecular-mass pI markers were used. The simple proteins and the mixed cultures of microorganisms, Saccharomyces cerevisiae CCM 8191, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Staphylococcus aureus, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224, Staphylococcus epidermidis CCM 4418 and Stenotrophomonas maltophilia, were focused and separated by the method suggested. The minimum detectable number of microbial cells was 5 × 102 to 1 × 103 with on-column UV detection at 280 nm.
Keywords: CIEF; Dynamically modified FS; Spacers; pI markers; Proteins and microorganisms; UV detection;

Digestion studies constitute a functional tool for allergen characterisation. This strategy for investigating allergenic proteins relates to the observation of increased proteolytic resistance of some proteins recognised to exhibit allergenic potential. β-Lactoglobulin (βLG) is one of the major whey proteins, a potent milk allergen and shows a high stability against peptic hydrolysis in its native form. In order to study the impact of milk fermentation process on its digestibility, two complementary analytical methods were applied: capillary zone electrophoresis (CZE) to quantitatively study proteolytic degradation of βLG isolated from different fermented bovine milk products, and enzyme linked immunosorbent assay (ELISA) to assess differences in immunoreactivity. βLG, isolated from either raw or pasteurised cow's milk (CM), as expected, showed only minimal digestibility (less than 10% in 2 h). However, when raw milk or pasteurised milk was fermented, the rate of peptic digestion of the protein significantly increased (up to 45% in 2 h). In accordance with changes in digestibility, the immunochemical response for all fermented samples was lower than that of non-fermented references. Raw and pasteurised milk “naturally” fermented in our laboratory only resulted in a slight reduction (βLG detected is still in the range of milligrams per gram sample), whereas the industrially manufactured sour milk as well as the “Acidophilus milk” reflected a remarkably lower level of immunoreactivity (55–56 μg/g sample).
Keywords: β-Lactoglobulin; Milk; Fermentation; Allergenicity; Peptic digestibility; Complementary analytical methods;