Isotachophoretic analysis of peptides. Selection of electrolyte systems and determination of purity

Capillary isotachophoresis (ITP) was applied to the qualitative and quantitative analysis of both natural and synthetic oligo- and polypeptides. Based on the mathematical model of acid-base equilibria for a general ampholyte, a procedure and a computer program for the calculation of the pH dependence of the effective and specific charge and effective mobility of peptides with known amino acid sequence were developed which allow the selection of electrolyte systems for peptide isotachophoretic analysis to be rationalized. Basic peptides (bovine pancreatic trypsin inhibitor, bull seminal isoinhibitors of trypsin, arginine vasopressin and adamantylamide-alanylisoglutamine) were analysed with a cationic ITP system at acidic pH. Neutral and acidic peptides (insulin, proinsulin, bull seminal isoinhibitors of trypsin, cow colostrum isoinhibitors of trypsin) were analysed with an anionic ITP system, mostly at alkaline pH. Peptide purity (electrophoretic homogeneity) was determined from the ITP degree of purity defined by a peptide itself and the zone length ratio of its admixtures. Enrichment of peptide in the sample during the purification procedure was measured by its zone length relative to unit mass of the amount of sample analysed.     

Investigation of the effect of ionic strength of Tris-acetate background electrolyte on electrophoretic mobilities of mono-, di-, and trivalent organic anions by capillary electrophoresis

The effect of ionic strength of the background electrolyte (BGE) composed of tris(hydroxymethyl)aminomethane (Tris) and acetic acid on the electrophoretic mobility of mono-, di- and trivalent anions of aliphatic and aromatic carboxylic and sulfonic acids was investigated by capillary zone electrophoresis (CZE). Actual ionic mobilities of the above anions were determined from their CZE separations in Tris-acetate BGEs of pH 8.1 to 8.2 in the 3 to 100 mM ionic strength interval at constant temperature (25 degrees C). It was found that the ionic strength dependence of experimentally determined actual ionic mobilities does not follow the course supposed by the classical Onsager theory. A steeper decrease of actual ionic mobilities with the increasing ionic strength of BGE and a higher estimated limiting mobility of the anions than that found in the literature could be attributed to the specific behavior of the Tris-acetate BGEs. Presumably, not only a single type of interaction of anionic analytes with BGE constituents but rather the combination of effects, such as ion association or complexation equilibria, seems to be responsible for the observed deviation of the concentration dependence of the actual ionic mobilities from the Onsager theory. Additionally, several methods for the determination of limiting ionic mobilities from CZE measured actual ionic mobilities were evaluated. It turned out that the determined limiting ionic mobilities significantly depend on the calculation procedure used.     

Separation and investigation of structure-mobility relationships of insect oostatic peptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been applied to qualitative analysis, separation, and physicochemical characterization of synthetic insect oostatic peptides (IOPs) and their derivatives and fragments. Series of homologous IOPs were separated in three acidic background electrolytes (BGEs; pH 2.25, 2.30, 2.40) and an alkaline BGE (pH 8.1). Best separation was achieved in acid BGE composed of 100 mM H3PO4, 50 mM Tris, pH 2.25. The effective electrophoretic mobilities, mu(ep), of all IOPs in four BGEs were determined and several semiempirical models correlating effective mobility with charge-to-size ratio (mu(ep) versus q/Mr k) were tested to describe the migration behavior of IOP in CZE. None of models was found to be unambiguously applicable for the whole set of 20 IOPs differing in size (dipeptide - decapeptide) and charge (-2 to +0.77 elementary charges). However, a high coefficient of correlation, 0.9993, was found for the subset of homologous series of IOPs with decreasing number of proline residues at C-terminus, H-Tyr-Asp-Pro-Ala-Prox-OH, x = 6 - 0, for the dependence of mu(ep) on q/Mr k with k = 0.5 for IOPs as anions in alkaline BGE and with k = 2/3 for IOPs as cations in optimized acidic Tris-phosphate BGE. From these dependences the probable structure of IOPs in solution could be predicted.     

Seasonal Variations of Polycyclic Aromatic Hydrocarbons in Air Particulate Extracts

Aroma-active volatile organic compounds (VOCs) present in brandies produced in the Slovak Republic have been identified by gas chromatography–olfactometry (GC–O) and GC–MS. GC–O data treatment was based on a concept of detection frequency. Direct injection of samples was used due to its simplicity and the fact that determined composition is that of the original samples. This is especially important for correlation of results obtained by sensory evaluation and gas chromatographic measurements. Among 200 organic compounds found by GC only 71 showed olfactive properties at the given concentration levels. In order to identify aroma-active substances, linear retention indices (LRI) have been calculated and compared with available LRI libraries. The most aroma-active compounds have been found in samples of Frucon brandy, while Vinovica showed their minimal content. The results obtained by gas chromatography have been compared with sensory evaluation of brandy odor.     

Determination of stability constants of valinomycin complexes with ammonium and alkali metal ions by capillary affinity electrophoresis

Capillary affinity electrophoresis (CAE) has been employed to investigate quantitatively the interactions of valinomycin, macrocyclic depsipeptide antibiotic ionophore, with univalent cations, ammonium and alkali metal ions, K(+), Cs(+), Na(+), and Li(+), in methanol. The study involved measuring the change in effective electrophoretic mobility of valinomycin while the cation concentrations in the BGE were increased. The corresponding apparent stability (binding) constants of the valinomycin-univalent cation complexes were obtained from the dependence of valinomycin effective mobility on the cation concentration in BGE using a nonlinear regression analysis. The calculated apparent stability constants of the above-mentioned complexes show the substantially higher selectivity of valinomycin for K(+) and Cs(+) ions over Li(+), Na(+), and NH(4)(+) ions. CAE proved to be a suitable method for the investigation of both weak and strong interactions of valinomycin with small ions.     

Capillary zone electrophoresis with electroosmotic flow controlled by external radial electric field.

A new way of regulation of electroosmotic flow (EOF) in capillary zone electrophoresis (CZE) by external electric field has been developed. A set of three high-voltage power supplies is used to form a radial electric field across the capillary wall. One power supply is applied in the usual way as a driving force of CZE and EOF to the ends of the inner capillary compartment dipped into the electrode vessels and filled with background electrolyte. Two power supplies are connected to the ends of the outer low-conductivity coating of the capillary which is formed by the dispersion of copolymer of aniline and p-phenylenediamine in polystyrene matrix. The difference between electric potentials on the outer capillary surface and inside the capillary determines the voltage of radial electric field across the capillary wall and affects the electrokinetic potential at the solid-liquid interface inside the capillary. The effect of magnitude and polarity of external radial electric field on the flow rate of EOF, on the migration times of charged analytes and on the separation efficiency and resolution of CZE separations of synthetic oligopeptides, diglycine, triglycine and octapeptide fragments of human insulin was evaluated. Through the EOF control by external electric field the dynamic effective length of the capillary was obtained and the speed of analysis and resolution of CZE separations of peptide analytes could be optimized.     

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte-based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4-9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline-rich di- to decapeptides, and phosphinic pseudopeptides--tetrapeptide mimetics synthesized as a mixture of four diastereomers having the -P(O)(OH)-CH(2)- moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.     

Transient isotachophoresis in carrier ampholyte-based capillary electrophoresis for protein analysis

Transient ITP (t-ITP) has been used in carrier ampholyte-based CE (CABCE) to enhance the sensitivity of protein analysis. The characteristics of carrier ampholytes (CAs) narrow pH cuts-based buffers, when used as BGEs in CE are compatible with t-ITP requirements. Indeed, being the sole buffering species of such solutions, CAs impose a pH close to their pI. Thus, in these solutions, the CAs possess low electrophoretic mobility. As a consequence, by adding an ionic component with high electrophoretic mobility either in the studied sample or in the BGE, a t-ITP step can be generated. This has first been demonstrated for protein test mixtures. Then, the combination of t-ITP with CABCE has been applied to study a real sample, the bovine milk.     

Analysis and characterization of phosphinic pseudopeptides by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) was applied to analysis and characterization of phosphinic pseudopeptides with the general structure N-Ac-Val-Ala(psi)(PO2(-)-CH(2)) Leu-Xaa-NH(2), where Xaa represents one of 20 proteinogenic amino acid residues. Pseudopeptides containing neutral or acidic amino acid residues in position Xaa were analyzed as anions in weakly alkaline (pH 8.1) Tris-Tricine background electrolyte (BGE), pseudopeptides with basic amino acid residues in position Xaa were analyzed as cations in acid BGEs (Tris-phosphate buffers). Acidity of phosphinic acid moiety in peptides with basic amino acid residues was determined from the dependence of effective mobility of these peptides on pH in the acid pH region (pH 1.4-2.8). Additionally, separation of diastereomers of some peptides was achieved.