Andreas Rizzi

Fundamental aspects of chiral separations by capillary electrophoresis

A review is presented that surveys the basic theory of direct separation of enantiomers by capillary electrophoretic (CE) techniques. These separations are based on the formation of diastereomeric complexes between the enantiomeric analytes and a chiral selector added to the electrolyte solution. The review covers a comprehensive treatment of the equations needed for optimization of selectivity coefficients, resolution and analysis time in the zone electrophoretic mode. In this context, it takes into account combined equilibria of complexation and protonation/deprotonation as well as complexation and paritition into micelles. On the basis of these equations, the benefits of charged selectors and the optimization potential inherent to pH tuning can be documented. In addition, the review deals with some basic aspects of chiral isoelectric focusing and briefly discusses indirect enantioseparation. In a subsequent section a survey is given on particular features of the various types of chiral selectors. Finally, the recent developments in preparative enantioseparation in continuous free‐flow system and by use of isoelectric membranes are discussed.

Glycosylation analysis of glycoproteins and proteoglycans using capillary electrophoresis-mass spectrometry strategies

This review highlights recent developments in glycosylation analysis by modern MS in combination with CE based preseparation. Focused on CE‐MS strategies aimed for glycotyping, the review addresses the detailed glycoform analysis of glycoproteins, glycopeptides, and proteoglycans. Glycoform analysis in the context of modern glycoproteomics is briefly addressed, as well. CZE, CIEF, and frontal analysis CE approaches hyphenated to high‐resolution multistage MS for the detailed analysis of protein‐linked glycan structures are overviewed in a comprehensive way. Advantages and limitations of various methodological approaches and techniques as well as mass spectrometric instrumentation are discussed in the particular context of glycoanalysis.

pKa shift‐associated effects in enantioseparations by cyclodextrin‐mediated capillary zone electrophoresis

Enantioselective migration of dansylated (Dns) amino acids in the presence of hydroxypropylated‒β‒cyclodextrin under acidic conditions near the pI value of the analytes was investigated by means of capillary zone electrophoresis. Based on the migration data, the pH dependence of the complexation constants was evaluated, as well as the variation of the complex mobilities with pH. As a result of these data, the migration behavior in the pH region near the pI could be understood, which, in some instances, includes the reversal of migration order upon variation of selector concentration. The enantioselective pKa shifts upon complexation could be quantitated for the carboxylic and the amino group separately. pKa shifts were found in the order of 0.8 pI units, the differences between the enantiomers being up to 0.25 pH units. These data were in agreement with the pI shifts reported from isoelectric focusing experiments. The accurate determination of the pI values of the Dns amino acids makes it possible to calibrate the pI scale in isoelectric focusing in the presence of chiral selectors.

Enantiomeric separation of amphetamine related drugs by capillary zone electrophoresis using native and derivatized β-cyclodextrin as chiral additives

Abstract Amphetamine, methamphetamine and several ring-substituted analogs which are under governmental regulations have been separated by capillary zone electrophoresis employing native and various substituted β-cyclodextrins as additives to the background electrolyte. The following chiral selectors were used: native β-cyclodextrin, heptakis(2,6-di-O-methyl)-β-cyclodextrin, heptakis-(2,3,6-tri-O-methyl) β-cyclodextrin, (2-hydroxy)propyl-β-cyclodextrin and carboxymethyl-β-cyclodextrin. The amphetamines were separated without derivatization. Separations are reported with respect to the kind of chiral selector. Native β-cyclodextrin and carboxymethyl-β-cyclodextrin turned out to give optimal resolutions within only a few minutes. This direct method is compared with the indirect method separating the diastereomeric Marfeys derivatized amphetamines by means of non-chiral sodium dodecylsulfate micelles.

Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions

The use of strong promoter systems for recombinant protein production generates high product yields, but also overburdens the host cell metabolism and compromises production. Escherichia coli has highly developed regulatory pathways that are immediately responsive to adverse conditions. To gain insight into stress response mechanisms and to detect marker genes and proteins for stress specific monitoring time course analysis of controlled chemostat cultivations was performed using E. coli total microarray and difference gel electrophoresis (Ettan DIGE). In order to detect differences and consistencies of stress response as well as the impact of the inducer isopropyl-beta-d-thiogalactopyranosid on cells, expression of two recombinant proteins (hSOD and GFPmut3.1) was investigated. Genes involved in aerobic metabolism under control of the ArcB/ArcA two component system were found to be down-regulated, and the interplay of the psp operon, ArcA system and guanosine tetraphosphate is suggested to be involved in stress regulatory mechanisms. A distinct impact of the two recombinant proteins was observed, particularly on levels of known stress regulatory genes and proteins, as well as on the response associated with ArcA and psp. Altogether, 62 genes as well as seven proteins showed consistent expression levels due to recombinant gene expression, and are therefore suggested to be appropriate monitoring targets.

Enantiomeric separation of amphetamine, methamphetamine and ring substituted amphetamines by means of a β-cyclodextrin-chiral stationary phase

SummaryThe enantioseparation of amphetamine, methamphetamine and various ring-substituted amphetamines by use of a chiral stationary phase carrying immobilized native β-cyclodextrin (β-CyD) selectors is reported. The system is evaluated for resolving the specified compounds directly without any derivatization and after derivatization with phenyl isothiocyanate (PITC), naphthyl isothiocyanate (NITC) and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). This direct enantioseparation is compared with the features of indirect separation of diasteromeric derivatives after reaction with the optically pure Marfeys reagent employing a simple non-chiral alkyl-silica (RP-8) column. A selection of those methods best suited for each single amphetamine is given.Seventeen different samples of amphetamine, confiscated by the Swedisch police, were analyzed with respect to their enantiomeric composition. Within this set of samples synthesized by the same method no significant deviation from a racemic ratio could be observed.

Linear polymers applied as pseudo-phases in capillary zone electrophoresis of azo compounds used as textile dyes

Abstract Nine synthetic organic dyes, including seven azo compounds, used as colouring matter for textiles were separated as anions by capillary zone electrophoresis. As most of the solutes are sulfonic acids, the separation could not be effected by varying the pH of the buffer solution. Therefore, two methods were applied to adjust the electrophoretic mobility in a specific way: complexation by bis-tris-propane and interaction with linear polymers added to the buffer and acting as pseudo-phases. A buffer system containing polyethylene glycol and polyvinylpyrrolidone permits the separation of all analytes. Retention of the dyes caused by the polymeric additives was related to the solutes structure. It was demonstrated by cluster analysis that the relative decrease in the electrophoretic mobility of the dyes correlates with the number of benzoaromatic rings in the solute molecules.

High‐performance separation techniques hyphenated to mass spectrometry for ganglioside analysis

Gangliosides, sialic‐acid‐containing glycosphingolipids are involved in numerous biological processes and play essential roles in severe pathologies, with predilection in those of the central nervous system. Formerly, ganglioside composition and quantity were assessed exclusively by thin‐layer chromatographic (TLC), immunochemical, and immunohistochemical methods, which have limited effectiveness being unable to detect minor components in mixtures of high heterogeneity. Increased awareness of the biological importance of gangliosides stimulated the development of analytical methods that are better amenable to complex ganglioside mixtures. More recently, MS in online conjunction with high‐performance separation techniques brought a significant progress to the field. This review highlights the state‐of‐the‐art development and application of separation methods online coupled to MS for ganglioside analysis. Most original and successful protocols based on GC‐MS, LC‐MS, and CE‐MS are presented here together with the special instrumental and sample preparation requirements to be met for effective ganglioside separation, detection, and structural identification. Finally, the advantages and downsides of each methodology as well as the perspectives for simplification, standardization, and upgrading are assessed.

Separation of diastereomers by capillary zone electrophoresis in free solution with polymer additive and organic solvent component Effect of pH and solvent composition

Abstract Diastereomeric derivatives of enantiomers generally exhibit differences in their physico-chemical properties. This fact allows the separation of these compounds in non-chiral separation systems. Although charged diastereomers have been reported to be resolved in certain instances by capillary electrophoresis without any additives, a significant enhancement of stereoselectivity can be achieved by means of polymeric pseudophases. The present paper focuses on the influence of the pH and of the organic solvent component acetonitrile on the resolution of diastereomeric compounds in the absence and presence of a polymeric network of polyvinylpyrrolidone. Experimental data are presented for diastereomeric derivatives of α -amino acids as well as for three anticoagulant drugs obtained by reaction with optically pure (+)-O,O′-dibenzoyl- l -tartaric anhydride.

High level protein-purification allows the unambiguous polypeptide determination of latent isoform PPO4 of mushroom tyrosinase

Graphical abstract Mushroom tyrosinase diversity – six precursor isoforms, all capable of being activated and generating protein purification interfering compounds – is taking one step further to reconnaissance. This innovative method for protein purification provides access to latent tyrosinase from natural sources and enabled experiments revealing insights in a hitherto uncharacterized isoform.