High-performance precipitation liquid chromatography

High-performance precipitation liquid chromatography is based upon solubility differences and thus especially appropriate for polymers which have a narrow solubility window. The technique offers new possibilities for the analysis of copolymers according to their chemical composition distribution and can be used in combination with size-exclusion separation for chromatographic cross-fractionation.     

High-performance liquid chromatography of alkaloids

Literature information on the HPLC of alkaloids, including adsorption, reversed-phase, ion-exchange, ion-pair, and other variants is generalized. Results are given of the chromatographic analysis of various classes of alkaloids with an indication of the conditions of separation, and the columns used, and also of the methods of detection. The advantages and disadvantages of the HPLC variants are discussed. The review includes the literature over the last 14 years.Scientific Center for Chromatography, Institute of Chemistry and Physics of Polymers, Uzbek Academy of Sciences, Tashkent. Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 595–613, September–October, 1991.     

High-performance liquid chromatography of alkaloids

Literature information on the HPLC of alkaloids, including adsorption, reversed-phase, ion-exchange, ion-pair, and other variants is generalized. Results are given of the chromatographic analysis of various classes of alkaloids with an indication of the conditions of separation, and the columns used, and also of the methods of detection. The advantages and disadvantages of the HPLC variants are discussed. The review includes the literature over the last 14 years. Scientific Center for Chromatography, Institute of Chemistry and Physics of Polymers, Uzbek Academy of Sciences, Tashkent. Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 595–613, September–October, 1991.     

High-performance liquid chromatography of cardiolipin

Resolution of freshly prepared and of commercially available (degraded) samples of cardiolipin into 15-30 components has been accomplished by reversed-phase high-performance liquid chromatography using a 3-micron particulate Microsorb C18 column irrigated with linear gradients of acetonitrile--methanol--10 mM phosphate buffer pH 7.4. Selected resolved components were crystallized and characterized by infrared absorption spectra. Saponification of other components and identification of component fatty acids by reversed-phase high-performance liquid chromatography demonstrated the presence of ten fatty acids (14:0, 14:1, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3, 20:0, 20:4), with linoleic acid (18:2) identified in all resolved components. From fatty acid composition data it appears that several resolved fractions consist of single cardiolipin molecular species.     

High-performance liquid chromatography in microsequencing

The tremendous success of protein structural determinations on a microscale would not have been possible without the introduction of high-performance liquid chromatography (HPLC) into the various steps of this procedure, i.e. separation of peptide mixtures, amino acid analysis and protein sequence analysis. In this review the advantages, limitations and current developments in the application of HPLC to protein structural analysis are discussed.     

High-performance liquid chromatography of sialooligosaccharides and gangliosides

Glycans were cleaved from gangliosides and separated by high-performance liquid chromatography (HPLC). The columns were packed with bonded stationary phases made of microparticulate, macroporous silica with serotonin, phenylpropanolamine or tryptamine as the biogenic amine ligate. The ganglioside oligosaccharides were eluted in the order of increasing number of sialic acid residues in the molecule and their retention decreased with the ionic strength of the mobile phase. Best selectivity was obtained in the pH range from 3.0 to 4.0. The two major sialic acids, N-acetylneuraminic and N-glycolylneuraminic acids, were separated by lectin affinity chromatography using an HPLC column packed with silica-bound wheat germ agglutinin and 10 mM phosphate buffer, pH 4.0, as the eluent. Throughout this study, isocratic elution was used and the column effluent was monitored at 195 nm.     

High-performance liquid chromatography of biotin and analogues

Biotin, analogues, and chemical intermediates were separated by high-performance liquid chromatography (HPLC) using reversed-phase and anion-exchange chromatographic conditions. Reversed-phase separations provided a wide range of retention times and resolution of nearly all the biotin compounds from mixtures of the analogues. Anion-exchange separations gave generally shorter retention times as compared to reversed-phase separations and greater resolution between biotin l- and d-sulfoxide. However, fewer analogues were resolved from mixtures of the compounds with anion-exchange HPLC.     

High-performance liquid chromatography of membrane proteins

Various modes of high-performance liquid chromatography, gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and metal chelate affinity chromatography, were investigated for the separation of membrane proteins. All were found applicable to membrane proteins, although the usefulness of each mode differed. For satisfactory results it was important to select appropriate elution conditions. The type and concentration of detergent was of special importance. The effects of other conditions, flow-rate, gradient steepness, type of buffer and salt, eluent pH, etc., were similar to those observed for soluble proteins.     

High-performance liquid chromatography of glutathione conjugates

Summary An ion-exchange high-performance liquid chromatographic method is described for the quantitative assay of glutathione (GSH) conjugates derived from endogenous electrophilic substances as well as xenobiotics. GSH (reduced and oxidized forms) and GSH conjugates were condensated with o-phthaldialdehyde to highly fluorescent derivatives and monitored at 338 nm excitation and 450 nm emission wavelengths after separation by ion-exchange HPLC on a 60-5NH₂ Polygosil analytical column. The detection limit was 2 pmol for the GSH conjugate of cholesterol epoxide and 6 pmol for the GSH conjugate of oleic acid epoxide. This method allows sensitive determination of all GSH conjugates independent of the chromatographic and spectrophotometric properties of the electrophilic substrates. Using this method we could show for the first time that the endogenous compound oleic acid epoxide is a specific substrate for the cytosolic rat liver GSH S-transferase. The method is applied to the determination of GSH S-transferase activity towards oleic acid epoxide and cholesterol epoxide.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

High-performance liquid chromatography of microbial acid metabolites

The use of high-performance liquid chromatography with a cation-exchange column and effluent monitoring at 210 nm has been evaluated for the profiling of selected microbial metabolites including aliphatic, dicarboxylic, and phenolic acids, as an adjunct to the identification of selected bacteria, detection of bacterial metabolites in foods, and the monitoring of industrial microbial fermentations. Advantages of the technique include the simultaneous profiling of different classes of organic acids without derivatization. Most applications require only qualitative or semi-quantitative data. For others, data are given on the day-to-day reproducibility for several acids.