External electric field control of electroosmotic flow in non-coated and coated fused-silica capillaries and its application for capillary electrophoretic separations of peptides.

The influence of an external electric field on the electroosmotic flow in the noncoated (bare) fused-silica capillaries and in the fused-silica capillaries with covalent coating of the inner surface by the polymer of a new acrylamido derivative, N-(acryloylaminoethoxy)ethyl-beta-D-glucopyranose, has been tested in the capillary electrophoretic separations of peptide analytes. The effect of magnitude and polarity of the external electric field on the flow-rate of the electroosmotic flow, the migration times of charged analytes and the separation efficiency and resolution of separations of synthetic oligopeptides, diglycine, triglycine, glycyl-proline and prolyl-glycine, by capillary zone electrophoresis has been evaluated. The effect of the external electric field on the velocity of the electroosmotic flow was much higher in the bare fused-silica capillaries than in the coated capillaries. Better separation of the analyzed peptides was achieved in the coated fused-silica capillaries. An external electric field proved to be an effective tool for control of the electroosmotic flow and for optimization of the speed and resolution of capillary electrophoretic separations of synthetic peptides.

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.

Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes. Application to analysis and preparation of fragments of insulin.

A basic theoretical description of the correlation between capillary zone electrophoresis (CZE) and free-flow zone electrophoresis (FFZE) is presented. The theory of the correlation between CZE and FFZE results from the fact that both methods are based on the same separation principle, zone electrophoresis, and both are performed in the carrierless separation medium with the same composition of the background electrolyte. The equations describing the movement of the charged and noncharged particles in the d.c. electric field applied in the capillary and in the flow-through electrophoretic chamber are presented and used for the quantitative description of the correlation between CZE and FFZE. Based on the theory of the correlation between CZE and FFZE a procedure has been developed for conversion of analytical, microscale CZE separations into continuous preparative separation processes realized by FFZE. Practical application of the developed procedure is demonstrated by CZE analysis and FFZE preparation of an octapeptide fragment of human insulin.

Contribution of capillary coiling to zone dispersion in capillary zone electrophoresis.

A mathematical model of the deformation of the analyte zone in capillary zone electrophoresis (CZE) due to capillary coiling is presented and the influence of capillary coiling on the distribution of electric current density inside the capillary is described. The model gives a quantitative description of the potential contribution of capillary coiling to the total zone dispersion in CZE. The zone broadening caused by capillary coiling is calculated as the difference of migration distances of the particles migrating at the inner circumference of the capillary coil and those migrating at the outer circumference. This difference is shown to be directly proportional to the capillary diameter and to the number of capillary coils, i.e. indirectly proportional to the radius of the capillary coils into which the capillary of the given length is coiled. The contribution of capillary coiling to the total zone dispersion is compared with the contribution of the longitudinal diffusion of low and high molecular mass analytes and bioparticles. It is shown that, especially in the case of CZE separation of macromolecules and particles, capillary coiling can significantly decrease the separation efficiency. For that reason the small radius coiling of the capillary column in the CZE apparatuses should be avoided.

Capillary electrophoresis device with double UV detection and its application to the determination of effective mobilities of peptides.

A new experimental device for high-performance capillary electrophoresis (HPCE) with a double UV detection system and with thermostating of the whole separation compartment was developed. UV detection is doubled by producing two apertures placed symmetrically close to the axis of the optical path of the single-beam UV detector and by the adjustment of the capillary loop on these two apertures. The double-detection system allows exact measurements of electrophoretic and electroosmotic flow velocities. A procedure for the determination of effective mobilities from the data obtained by the double UV detection system was developed and applied to determine the effective mobilities of synthetic peptides (diglycine, triglycine, growth hormone releasing peptide and its derivatives and fragments). The measurements are performed at constant temperature (25 degrees C) and low input power at which temperature increase in the capillary can be neglected and temperature corrections of temperature-dependent magnitudes need not be included in the calculations.

Application of capillary and free-flow zone electrophoresis and isotachophoresis to the analysis and preparation of the synthetic tetrapeptide fragment of growth hormone-releasing peptide.

The use of high-performance electromigration separation methods, capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) and continuous free-flow arrangements of these two separation principles, free-flow zone electrophoresis (FFZE) and free-flow isotachophoresis (FFITP), was investigated in the analysis and purification of the synthetic C-terminal tetrapeptide fragment (H2N-Ala-Trp-D-Phe-Lys.NH2) of the growth hormone-releasing peptide. CZE and CITP were used for microanalysis of peptide preparations after different steps of their purification. The homogeneity of the peptide preparations, including fractions of preparative separations, was quantified by relative zone length (CITP) and/or relative peak height (CZE). In addition, the data obtained by CZE and CITP (electrophoretic and electroosmotic flow migration velocities) were utilized for conversion of micro-scale capillary separations (nano- to picomole level) into the preparative separations realized by FFZE and FFITP with a capacity from tens to hundreds of milligrams per hour.

Application of capillary isotachophoresis in peptide analysis.

This paper gives a broad and detailed review of the applications of one of the modern high-performance electromigration separation techniques--capillary isotachophoresis (ITP)--in peptide analysis. Examples are presented of the utilization of capillary ITP for peptide analysis in the fields of chemistry, general and clinical biochemistry, biology, biotechnology, pharmacy and the food industry. The complete composition of all the electrolyte systems used for peptide ITP analyses in both cationic and anionic techniques is given in tabular form. According to the purpose of analysis the applications are divided into several sections: model studies, determination of physico-chemical characteristics, purity control of both intermediate and final peptide preparations, including the determination of low-molecular-mass ionogenic admixtures, and the analysis of peptides in biological fluids and tissue extracts. In addition to the main applications the theoretical and methodological aspects of peptide ITP analysis are discussed. The basic electromigration properties of peptides (their polyampholyte character, effective and absolute mobilities, acid-base equilibria) are explained and the selection of parameters for peptide ITP analysis is described in detail. The advantages and disadvantages of ITP compared with other electrophoretic and chromatographic methods used for peptide analysis are discussed.

Correlation of capillary zone electrophoresis with continuous free-flow zone electrophoresis: application to the analysis and purification of synthetic growth hormone releasing peptide.

Two carrier-free electrophoretic separation methods, capillary zone electrophoresis (CZE) and continuous free-flow zone electrophoresis (FFZE), have been applied to both microanalysis at the nanogram level and preparative fractionation, with a throughput of 30 mg/h, of synthetic growth hormone releasing peptide (GHRP). A crude product of GHRP, a hexapeptide with the sequence His-D-Trp-Ala-Trp-D-Phe-Lys-NH2, synthesized by the solid phase methodology, was desalted and analyzed by CZE. Based on the results of analytical CZE the separation was converted into a preparative purification procedure by continuous FFZE, employing the same separation medium (0.5 mol/L acetic acid, pH 2.6). The purifity of peptide fractions obtained by FFZE was reevaluated by CZE. The combination of these two techniques proved to be a valuable tool for both peptide analysis and peptide purification. A close correlation of CZE and FFZE, resulting from the fact that both methods are based on the same separation principle (zone electrophoresis) and that both are performed in a free solution of the same composition, was confirmed. However, when transforming data from CZE to FFZE, the different electroosmotic flow, temperature and electric field intensity in the capillary and in the flow-through cell, respectively, have to be taken into account and corresponding corrections have to be made.

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.

Micropreparative isotachophoretic electrodesorption of monoclonal antibodies from an affinity adsorbent.

The principle of isotachophoretic desorption of proteins from an affinity sorbent was used for the preparative isolation of monoclonal antibodies against transferrin, under a cationic regimen. Electrodesorption was carried out on an apparatus of our own construction with a photometric detector and a detector of potential gradient. The apparatus was provided with a sorption element filled with an exchangeable affinity active sorbent. The construction of the apparatus and the procedure suitable for the isolation of the antibody of the IgG type are described. The possibility of repeating the sorption-desorption cycle at least sixteen times was demonstrated, with relatively good yields of the protein.

Isotachophoretic electrodesorption of proteins from an affinity adsorbent on a microscale.

Cationic isotachophoresis was used for the desorption of mouse monoclonal antibody to transferrin strongly affinity-bonded to transferrin immobilized on polyethyleneglycol terephthalate powder. The electrodesorption under nondestructive conditions was effected in the capillary isotachophoresis apparatus of our own construction which was equipped with an adsorption element. The electrodesorption is on-line connected with quantitative isotachophoretic analysis of the antibody desorbed. Only a few tens of microliters of the affinity adsorbent and several nanomoles of the antibody are needed for the characterization of the capacity of the affinity adsorbent and the conditions of adsorption and desorption.

Isolation of human haemopexin by bioaffinity chromatography on haeme-sepharose.

A preparative procedure was developed for the isolation of human apohaemopexin from Cohn fraction IV or blood serum, based on bioaffinity chromatography on haeme-Sepharose. The isolation is carried out in the pH range 4-8; hence the possibility of degradation of the carbohydrate moiety of the glycoprotein in the acidic media used in other isolation procedures is decreased. Owing to the conditions of the separation and the good stability of the affinity support, the column can be used repeatedly for long periods without a significant loss of binding capacity. The reversibility of the conformational changes that haemopexin undergoes in acidic media was examined by hydrophobic chromatography. The original hydrophobic characteristics were restored only approximately 48 h after haemopexin had been brought into a neutral medium.

Induction of extrajunctional acetylcholine sensitivity of rat EDL muscle by peptidic component of peripheral nerve.

The induction of extrajunctional ACh-sensitivity was studied electrophysiologically 4 days after implantation of segments of the sciatic nerve, segments of dorsal and ventral spinal roots and silastic plates, containing some components of peripheral nerve onto surface fibres of innervated rat extensor digitorum longus muscle. It has been found that extrajunctional ACh-sensitivity can be induced by a piece of rat sciatic nerve and by peptidic material of about 8,000 and 10,000 mol. wt. No sensitivity was induced by segments of ventral and dorsal roots, by a piece of rabbit sciatic nerve and by rat sciatic nerve sectioned 7 days prior to implantation. The active nerve fractions lost its inducing potency after pepsin pre-treatment and irradiation with UV-light. Peripheral nerve probably contains some specific factor, peptidic in nature, which is able to induce the ACh-sensitivity of muscle fibre membrane.