Reversed-Phase Ion-Pair High-Performance Liquid Chromatography of Phosphatidylinositols

Abstract

Reversed-phase ion-pal, high-performance liquid chromatographic (HPLC) separations of molecular species of phosphatidylinositols (PI) were studied. Mobile phases of acetonitrile-methanol-water containing various tetraalkylammonium phosphates (TAAP) were used for optimization. Stationary phases of macroporous polystyrene divinylbenzene (MPD), octylsilica, and octadecylsilica exhibited arying degrees of retentivity toward PI solutes. Without exception capacity factors (k') of PI on either alkylsilica or MPD increased with the alkyl chain length and the concentration of the quaternary ammonium counter ions evaluated. the results can be interpred in terms of an ion-pair retention mechanism. Logarithmic k' values were linearly related to the total number of carbons in TAAP. Perbenzoylation of PI yielded derivatives readily resolvable without the use of any mobile phase additive. Incorporation of TAAP to a mobile phase facilitated component separations of early-eluting perbenzoates. In HPLC with     

Reversed-Phase High-Performance Liquid Chromatography of Unsubstituted Aminobenzoic Acids

Abstract

High-performance liquid chromatographic (HPLC) characteristics of three position isomers of aminobenzoic acids (potential metabolites of important anesthetic drugs), were delineated with respect to their interactions with various mobile phases and stationary phases. HPLC with five hydrocarbonaceous phase, β-cyclodextrin silica (CDS), macrophase MP-1 polymer (MP), macroporous polystyrene/divinylbenzene (MPD), octadecylsilica (ODS), and propylphenylsilica (PPS), yielded results explicable in terms of substituent effects derived from the bifunctional amino– and carboxy groups. For cases where mobile phases contained sulfonates or quaternary ammonium salts both having longer chain alkyls, retention of analytes on all but CDS appeared to proceed predominantly via an ion-pairing mechanism. The extent of the corresponding counter-ion effects decreased in the order: MPD > ODS > PPS > MP, while the analyte retention order paralleled thier pH₂ values. On the other hand, an inverse relationship between the magnitude of capacity factors (k′) and pK₁ values of the title compounds was observed in experiments that produced retention data incompatible with ion-pair interaction rationales. The unique HPLC results obtained with the CDS phase are compared with those obtained with other phases.     

A Novel Approach to Reversed-Phase Preparative High-Performance Liquid Chromatography of Peptides

Abstract

In this study, we describe a novel approach to preparative liquid chromatography which takes advantage of the different relative hydrophobicities of components of a sample mixture, so that when a column is optimally loaded with an aqueous solution of the sample mixture, there is competition among the sample components for the adsorption sites on the hydrophobic stationary phase. The more hydrophobic components compete more successfully for these sites than more hydrophilic components, which are displaced and immediately eluted from the column. Thus, the major separation takes place in water. Subsequent treatment with an aqueous organic modifier is only required to wash retained components off the column and takes no part in the major separation process. This approach was applied to the preparative purification of mixtures of closely-related peptides, representing the crude peptide mixtures typically obtained from solid-phase peptide synthesis. The excellent separation profiles and high yields of pure peptide products on analytical columns reported in this study demonstrate that this methodology has great potential for preparative separation of a major component from hydrophilic and/or hydrophobic impurities.     

Ion-Pair Reversed-Phase Thin-Layer Chromatography and High-Performance Liquid Chromatography of Benzoic Acids

The separation of benzoic acids by ion-pair reversed-phase thin-layer chromatography and high-performance liquid chromatography was studied. In the optimization of separation conditions, the effect of the nature and concentration of the organic solvent, acidity of the mobile phase, concentration of salts of quaternary ammonium bases, and the length of their hydrocarbon radical on the retention of benzoic acids was studied. Conditions of the separation of a mixture of benzoic, hydroxybenzoic, aminobenzoic, and nitrobenzoic acids on Silufol plates impregnated with cetyltrimethylammonium bromide were selected; the mobile phase was isopropanol–ammonium acetate buffer solution with pH 5–dodecyltrimethylammonium bromide.     

反相高效液相色谱技术鉴定小黑麦品种

通过反相高效液相色谱(RP-HPLC)分析小黑麦种子醇溶蛋白,对供试的18个不同品种的小黑麦进行了品种鉴定和倍性水平鉴定,同时也对育种系谱相同的品种进行了系谱证实。     

The Analysis of Insulin-Related Peptides by Reversed-Phase High-Performance Liquid Chromatography

Abstract

This paper describes the use of high performance liquid chromatography (HPLC) for the rapid analysis and purification of insulin-related peptides prepared by solid-phase synthetic procedures. Examples include the bovine insulin C-peptide (34–45), the porcine insulin C-peptide (41–53) and the insulin B-chain fragment (22–27). Chromatographic elution systems containing reducing reagents like B-mercaptoethanol allow the direct analysis of insulin reduction products. Similar systems should allow the rapid analysis of disulphide bond pairing patterns in appropriate polypeptides and proteins either directly or following proteolytic digestion.

Reversed-phase high performance liquid chromatography (RP-HPLC) is a versatile and rapid technique useful for the analysis and purification of biological substances. In a series of recent publication^1–4 we have described methods for the analysis of underivatised amino acids, peptides and proteins on reversed-phase packings using ion-pairing or stationary phase modifying reagents as components of the mobile phase. These studies demonstrated that excellent resolution of closely related peptides can be achieved under a variety of elution conditions. The addition of low levels of phosphoric acid, inorganic or organic phosphates to a mobile phase (generally water-organic solvent mixtures), in particular, allows rapid and reproducible analysis of peptidic compounds with high sensitivity detection at wavelengths down to 190nm^5,6. It is the purpose of this report to show that these chromatographic conditions allow the facile analysis, and purification, of a variety of insulin-related peptides.     

Determination of Catecholamines by Capillary Electrophoresis and Reversed-Phase High-Performance Liquid Chromatography

A method is proposed for the electrophoretic determination of adrenaline, noradrenaline, and dopamine with mass-spectrometric or UV detection. A procedure is proposed for the sample preparation of biological fluids with the use of solid-phase extraction on alumina. A comparative assessment of the determination of catecholamines by capillary electrophoresis and reversed-phase high-performance liquid chromatography was performed.     

Quantitative Determination of Tabersonine and Methoxytabersonine by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A method for the quantitative determination of tabersonine and methoxytabersonine by reversed-phase high-performance liquid chromatography has been developed based on employing amine modifiers of the mobile phase as silanol group masking agents. Thus peak tailing is reduced and peak shapes are improved. The effect of pH of the eluent and the type of the organic modifier upon the overall separation has been examined. A mobile phase optimization procedure has been carried out for selecting the most favorable eluent mixture. The accuracy and precision achieved by the quantitative analysis are satisfactory.     

Determination of Amino Acid Enantiomers in Pharmaceuticals by Reversed-Phase High-Performance Liquid Chromatography

Precolumn derivatization with the reagent o-phthalic aldehyde/N-acetyl-L-cysteine (OPA/NAC) was used for the determination of amino acid enantiomers by reversed-phase high-performance liquid chromatography. The influence of the composition and pH of the eluent on the separation of the resulting derivatives was studied with the example of four amino acids. It was found that the highest selectivity and efficiency of the separation of OPA/NAC derivatives of amino acids is attained with the use of the eluent methanol–0.01 M Na₂HPO₄ (pH 6.0). The optimum composition of the mobile phase and conditions of the gradient elution were selected for the separation of a mixture of 20 amino acid derivatives. A procedure was developed for the determination of amino acid enantiomers in parenteral nutrition preparations. The procedure was used for the determination of D-isomers of arginine, alanine, methionine, phenylalanine, and leucine in the preparation Polyamine.     

Steroidogenesis in Patients with Various Adrenal Cortex Diseases as Studied by Reversed-Phase High-Performance Liquid Chromatography

A method is proposed for the quantitative determination of diagnostically important corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycorticosterone, and 11-deoxycortisol) in blood serum and urine (cortisol and cortisone) in an isocratic mode of reversed-phase high-performance liquid chromatography (RP HPLC) with the use of -cyclodextrin as a component of the mobile phase (CH₃CN : H₂O). Biological fluids (blood serum and urine) from a group of healthy donors and patients with various endocrine diseases (Cushing's syndrome, congenital adrenal hyperplasia, aldosteroma, and adrenal cortex carcinoma) were examined, and characteristic chromatographic steroid profiles were obtained for these disturbances.     

High-Performance Thin-Layer Chromatography and Reversed-Phase High-Performance Liquid Chromatography Methods for Fingerprinting of Salvadora persica Root Powder Extract Using Benzyl Isothiocyanate as Biomarker

JPC – Journal of Planar Chromatography – Modern TLC - Salvadora persica plant having a number of antimicrobial substances and the roots of the S. persica shrub have been demonstrated to...     

Separation of Multicomponent Mixture of Naphthalene Derivatives by Reversed-Phase High-Performance Liquid Chromatography

Separation of a mixture of naphthalene derivatives by reversed-phase high-performance liquid chromatography with UV detection was studied. Chromatographic characteristics were calculated for nine compounds, including naphthalene.     

Cellular Fatty Acid Composition of Vibrio parahaemolyticus by Reversed-Phase High-Performance Liquid Chromatography

Abstract

High-performance liquid chromatography was used to separate and identify cellular fatty acids isolated from Vibrio parahaemolyticus, a gram-negative estuarine microorganism associated with seafood-borne enteritis in man. Fatty acids were isolated from statically grown bacterial cultures, saponified, and derivatized with an ultraviolet tag. Aliquots of derivatized fatty acids were injected onto a reversed-phase column with water:acetonitrile gradient as the mobile phase and ultraviolet detection at 254 nm. The predominant fatty acids found for the V. parahaemolyticus strains studied were C12, C14, C16:1, C16, C18:1, and C18. In addition, previously unreported fatty acids C13, C17, C19, and C21 were identified. Comparison of HPLC with GLC fatty acid separations showed good agreement with the exception that HPLC was able to resolve previously unidentified fatty acid constituents.     

Multiresidue Analysis of Some Insect Growth Regulators by Reversed-Phase High-Performance Liquid Chromatography

Abstract

A general procedure has been developed for the analysis of 8 different insect growth regulators (IGRs) by using reversed-phase high-performance liquid chromatography with gradient solvent systems. The method has been used to identify and separate 8 insect growth regulators from a mixture of the standards. The method has been evaluated with different column conditions and under different solvent systems. Best resolution was obtained by using a double column and methanol/water gradient system.     

Use of Reversed-Phase High-Performance Liquid Chromatography in QSAR Analysis of 2,4-Dihydroxythiobenzanilide Analogues

Abstract

Thiobenzanilides are found to show strong biological activity as antimicrobial, antimycotic, and tuberculostatic agents. In addition, they are relatively weakly toxic to higher organisms.

A large set of new (N-phenyl-)-2,4-dihydroxybenzenecarbothioamide derivatives was obtained. Preliminary studies showed high microbiological action of some of them. In the process of chromatographic analysis, several different chromatographic parameters were obtained. In case of RP-HPLC, these parameters correspond to hydrophobicity of the solute. Obtained chromatographic parameters exhibited moderate correlation with calculated log P parameter.

Linear dependence of bacteriostatic or fungostatic activity on lipophilicity was observed. The degree of correlation of different parameters was compared. The lipophilicity of analysed tioamides was the most important factor responsible for fungostatic and bacteriostatic activity. In comparison to methanol eluent system, chromatographic parameters obtained in acetonitrile system were better correlated with bioactivity. Conversely with the calculated log P values, the experimentally derived parameters exhibited significant higher correlation to fungostatic activity determined on dermatophytes. While in case of other tested microorganisms log P was comparably or sometimes slightly better correlated.     

反相离子对高效液相色谱法检测兔脑中的磷酸腺苷

采用反相离子对高效液相色谱法测定兔脑提取液中的磷酸腺苷。分别考察了流动相中ＴＢＡＨ，磷酸盐和甲醇的浓度对磷酸腺苷保留行为的影响。确认最佳色谱条件为甲醇２０％，２０ｍｍｏｌ／Ｌ ＫＨ２ＰＯ４－２ｍｍｏｌ／ＬＴＢＡＨ８０％，等度洗脱。保留时间和峰高的相对标准偏差分别为０．２９９％－０．４４３％和０．１８０％－０。．８９％；样品标准加入回收率为１０１．７２％－８６．３６％．最低检测限为ＡＭＰ０．２１，ｇ     

Effect of Ionic Liquids as Mobile-Phase Additives on Chromatographic Parameters of Neuroleptic Drugs in Reversed-Phase High-Performance Liquid Chromatography

Abstract

Ionogenic basic compounds belonging to phenothiazine derivatives were analyzed in the reversed-phase system and were modified with the addition of three ionic liquids: 1-ethyl-3-methyl-imidazolium hexafluorophospate (EMIM PF₆), 1-butyl-3-methyl-imidazolium hexafluorophosphate (BMIM PF₆), and 1-butyl-3-methyl-imidazolium chloride (BMIM Cl). The effects of the concentration and the type of ionic liquid on the analytes' retention, peak symmetry, and efficiency were examined. The following trends increase analytes' retention factor and improve system efficiency: BMIM PF₆ > EMIM PF6 > BMIM Cl. With its asymmetric cation enlarged with hydrophobic substituents and a chaotropic anion, BMIM PF₆ appeared to be the most advantageous one. The isotherm of adsorption of this reagent presents a typical Langmuir course. By the application of high-performance liquid chromatography, lipophilicity parameters were established (lnk_w, S, ? ₀) for the investigated compounds. Chromatographic systems modified with ionic liquids were compared to buffered organic–aqueous mobile phase and eluent containing chaotropic salt additive.     

Determination of Iodate in Iodized Salt by Reversed-Phase High-Performance Liquid Chromatography with UV Detection

A method for the determination of iodate was developed by reversed-phase high-performance liquid chromatography with UV detection. Iodate was converted to iodine, which was separated from the matrix using a reversed-phase Ultrasphere C₁₈ column (250 × 4.6 mm, 5 μm) with methanol-1 mmol L^?1 H₃PO₄ (20:80, v/v) as mobile phase at 1.00 mL min^?1 and UV detection at 224 nm. The calibration graph was linear from 0.05 μg mL^?1 to 5.00μg mL^?1 for iodine with a correlation coefficient of 0.9994 (n=7). The detection limit was 0.01 μg mL^?1. The method was successfully applied to the determination of iodate in iodized salt. The recovery was from 96% to 101% and the relative standard deviation was in the range of 1.5% to 2.9%.     

Evaluation of Mobile and Stationary Phases in Reversed-Phase High-Performance Liquid Chromatography of Conjugated Bile Acids

Abstract

The reversed-phase high-performance liquid chromatographic separation of the ten major conjugated bile acids of man using isocratic conditions is described. Each component of the mobile and stationary phases was examined for its ability to influence the separation selectivity. Manipulation of pH, buffer species, organic modifier and different types of packings showed that optimal resolution was obtained with a mobile phase of methanol-0.02M sodium acetate (60:30) adjusted to pH 4.2 with phosphoric acid, on a Supelcosil LC-18-DB column. Advantages of the optimized phase system are the complete baseline separation of compounds within a short period of time, improved peak symmetry and a high rate of reproducibility. This new chromatographic method, coupled with UV detection at 205 nm, is suitable for the simultaneous determination of bile acid conjugates in routine clinical analysis.