Capillary electrophoresis separation of heterocyclic nitrosoamino acid-conformers at sub-ambient temperatures

The separation of a selected group of naturally occurring, heterocyclic nitrosoamino acids was achieved by capillary zone electrophoresis and the resolution of the syn and anti conformers improved as the temperature was lowered to 5 °C. The double peaks observed for each nitrosoamino acid are probably caused by the slow kinetics of conformational isomerism. The experimental setup consisted of a P/ACE 5510 electrophoresis system, a 57 cm polyacrylamidecoated capillary, and a 10 mM phosphate buffer, pH 7.2, containing 2 mM of 3-(N-N-dimethylmyristylammonio)propanesulfonate (DMMAPS) and 0.1% Tween 20. Our study shows that (a) the lower the temperature, the greater the resolution and the longer the migration times; and (b) different nitrosoamino acid conformers were resolved at different temperatures because of differences in the rate of isomerization. For example, the conformers of N-nitrosothiazolidine-4-carboxylic acid were partially resolved at 30 °C while those of N-nitrosoproline were well resolved at the same temperature.     

p-Alkoxy- and p-alkylbenzoates of α, a'-bi-p-hydroxytoluene. New series of liquid-crystalline compounds

Abstract

Six p-alkoxy and eight p-alkylbenzoates of 1,2-(p-hydroxyphenyl)ethane were synthesized for potential use as stationary phases in gas-liquid chromatography. All the p-alkoxy derivatives were nematic. The first five members of the alkyl series were nematic and the rest exhibited both smectic and nematic character. Most possess the desired combination of a wide liquid-crystalline temperature range and a high mesomorphic-isotropic transition temperature. The nematic-isotropic transition temperatures and entropies show the usual alternations characteristic of a high-melting series.     

Peptide mapping by capillary zone electrophoresis: how close is theoretical simulation to experimental determination

A multi-variable computer model is presented for the prediction of the electrophoretic mobilities of peptides at pH 2.5 from known physico-chemical constants of their amino acid residues. The model is empirical and does not claim any theoretical dependencies; however, the results suggest that, at least at this pH, peptides may be theoretically represented as classical polymers of freely joined amino acid residues of unequal sizes. The model assumes that the electrophoretic mobility can be represented by a product of three functions that return the contributions of peptide charge, length and width, respectively to the mobility. The model relies on accurate experimental determination of the electrophoretic mobilities of a diverse set of peptides, by capillary zone electrophoresis (CZE), at 22 degrees C, with a 50 mM phosphate buffer, at pH 2.5. The electrophoretic mobilities of a basis set of 102 peptides that varied in charge from 0.65 to 16 and in size from two to 42 amino acid residues were accurately measured at these fixed experimental conditions using a stable 10% linear polyacrylamide-coated column. Data from this basis set was used to derive the peptide charge, length, and width functions respectively. The main purpose of this endeavor is to use the model for the prediction of peptide mobilities at pH 2.5, and for simulation of CZE peptide maps of protein digests. Excellent agreement was obtained between predicted and experimental electrophoretic mobilities for all categories of peptides, including the highly charged and the hydrophobic. To illustrate the utility of this model in protein studies it was used to simulate theoretical peptide maps of the digests of glucagon and horse cytochrome c. The resulting maps were compared and contrasted with their experimental counterparts. The potential of this approach and its limitations are discussed.     

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.     

p-Alkoxy- and p-alkylbenzoates of α, a'-bi-p-hydroxytoluene. New series of liquid-crystalline compounds

Six p-alkoxy and eight p-alkylbenzoates of 1,2-(p-hydroxyphenyl)ethane were synthesized for potential use as stationary phases in gas-liquid chromatography. All the p-alkoxy derivatives were nematic. The first five members of the alkyl series were nematic and the rest exhibited both smectic and nematic character. Most possess the desired combination of a wide liquid-crystalline temperature range and a high mesomorphic-isotropic transition temperature. The nematic-isotropic transition temperatures and entropies show the usual alternations characteristic of a high-melting series.     

Synthesis,Thermodynamic Properties,and Gas- Liquid Chromatographic Evaluation of a High-Melting Liquid Crystal Series

The synthesis, phase transition temperatures and thermodynamic data (enthalpies and entropies) are reported for the methoxy through n-octyloxy (and n-decyloxy) homologues of the liquid crystal series represented by N,N′-bis[p-alkoxybenylidene]-α, aα′-bi-p-toluidine. The trends in transition temperatures are more or less analogous to previously-studied liquid crystal series. Nematic-isotropic enthalpy values are appreciably higher than average values for other series. Entropy values show the usual odd-even effect, but in contrast to observed trend for other series, they decrease with increasing terminal substituent chain length. Di-Schiff' s bases with various terminal substituents that showed interesting linear correlation between nematicisotropic transition temperatures and the anisotropy of polarizability of the substituent were also synthesized. A simple gas-liquid chromatographic method for the investigation of the selectivity of nematic stationary phases and its dependence on the degree of molecular order in the nematic state is also presented.     

The effect of electric field strength,buffer type and concentration on separation parameters in capillary zone electrophoresis

Summary Probe solutes were used to investigate the effect of buffer type, concentration and applied voltage on solute mobility, column efficiency and resolution in capillary zone electrophoresis. With low conductivity buffers higher concentrations and/or higher voltages could be used to improve column efficiency and resolution. Doubling the concentration of the buffer doubles the amount of heat generated inside the column while doubling the applied voltage cause a 4-fold increase. Solute migration time is approximately an inverse function of the charge density of the buffer's cation. Analysis time is increased by about 30% if the buffer concentration is doubled while it is cut in half if the applied voltage is doubled. Column efficiency is improved (higher theoretical plate count) with increasing buffer concentration and/or applied voltage as long as the heat generated is efficiently dissipated. The separation factor is directly related to analysis time and, therefore, selectivity improves with increasing buffer concentration but decreases with increasing applied voltage. Hence, resolution is optimized by increasing buffer concentration at a moderate applied voltage.     

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.