Ruthenium (II) 1,10-Phenanthroline Salts as Mobile Phase Additives for the Separation and Indirect Detection of Free Amino Acids

Abstract

Ruthenium (II) 1,10-phenanthroline, Ru(phen)²⁺ ₃, salts are used as ion interaction reagents in a basic mobile phase for the retention, resolution, and indirect photometric detection (IPD) of free amino acids on a polystyrene divinylbenzene (Hamilton PRP-1) column. Mobile phase Ru(phen)²⁺ ₃ concentration and pH and type and concentration of organic modifier and counteranion affect retention and IPD. Underivatized amino acid elution order is influenced by side chain structure typical of ion exchange processes. Detection limits for the separation and detection of free amino acids using an isocratic elution condition are about 0.1 nmole for lower retained amino acids and 0.25 nmole for higher retained amino acids for a 3:1 signal:noise ratio. Gradient elution is possible but at higher detection limits.     

Enantiomeric Separation of N-Protected Non-Protein Amino Acid Esters by Chiral High-Performance Liquid Chromatography

Abstract

We have found that high-performance liquid chromatographic analysis of enantiomeric N-protected amino acid esters on a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase column (Daicel Chiralcel OD) can be utilized as one of the procedures for determining the optical purities of non-protein amino acids. The methyl esters of the N-benzyloxycarbonyl (Z) derivatives of a number of non-protein amino acids showed excellent to good enantiomeric separations using hexane - 2-propanol as a mobile phase. There was a regularity in the elution order of enantiomers: the L-isomer had a shorter retention time than the D-isomer. We have also investigated the effect of the N-protecting groups and the ester groups on the enantiomeric separation. The Z, 4-methoxybenzyloxycarbonyl (Z(OMe)), and 9-fluorenylmethoxycarbonyl (Fmoc) derivatives gave exceptionally good resolutions. By contrast, the formyl and t-butoxycarbonyl (Boc) groups impaired the enantiomeric separation. Almost all the alkyl esters examined and the benzyl ester gave resolutions better than or of the same order as the methyl ester. The resolution of β-amino acids was worse than that of the corresponding α-amino acids.     

Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

Abstract

An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.     

HPLC Analysis of Nonionic Sufactants. V. Ethoxylated Fatty Acids

Abstract

High performance liquid chromatography technique was used in order to achieve separation and identification of product composition of nonionic surfactants of ethoxylated fatty acids.

Lichrosorb SI-60 (10μm) column, under gradient elution of mixture of isopropanol, methanol and n-hexane (50°C) and UV detector at 220 nm, were used for best separation of ethylene oxide (EO) adducts of fatty acids consisting of up to 20 EO units.

No derivatization of the compounds was needed. An improved baseline, in spite of gradient elution, was achieved by adding negligible amounts of anthracene to the eluents.

Brominated ethoxylated fatty acids resulting from addition of bromine to the double bond of the hydrophobic chain were also separated without a need for change in elution conditions or derivatization.     

Reversed Phase Thin Layer Chromatography of Amino Acids

Abstract

The use of reversed phase layers for the thin layer chromatography of amino acids is described. Only when a modifier was added to the mobile phase was clear separation of the amino acids achieved. Ion paring with trifluoroacetic acid overcame problems with streaking and poor separation on C₂ or C₁₈ reversed phase layers. All amino acids could not be separated with a single mobile phase. Thus, three different combination of acetonitrile-0.4% trifluoroacetic acid were used to separate eighteen amino acids with derivatization. No derivative was required.     

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.     

Direct high-performance liquid chromatographic enantioseparation of free α-, β- and γ-aminophosphonic acids employing cinchona-based chiral zwitterionic ion exchangers

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

Determination of Glutamine and Asparagine by Isocratic Elution Reverse Phase Liquid Chromatography with Fluorescent Detection

Abstract

A method was developed specifically for the determination of glutamine and asparagine in the presence or absence of other amino acids. The amino acids were derivatized by o-phthalaldehyde/ 2-mercaptoethanol and separated by isocratic elution with a mobile phase consisting of acetonitrile and sodium acetate buffer. An application of the method for the analysis of glutamine and asparagine in the enzymatic hydrolysate of cottonseed protein is described.     

Gas Chromatographic Determination of Trimethylsilyl Derivatives of Free Amino Acids from a Botanical Source

Abstract

The application of gas chromatography for the separation of TMS-amino acids from a botanical source was demonstrated. The trimethyl-silyl derivatives of the extracts from germ free tobacco tissue cultures were prepared by reacting amino acid extracts with bis(trimethylsilyl)-trifluoroacetamide (BSTFA) using acetonitrile as a reaction solvent following preliminary separation of the free acids by ion exchange chromatography. Gas chromatographic separation was accomplished with a 10% OV-11 glass column and temperature programming. The findings compare favorably with other chromatographic methods. Structures of the TMS-amino acids were confirmed by gas chromatography-mass spectrometry combination. Mass spectral data for each derivative is presented for the principal protein amino acids observed as well as γ-aminobutyric acid, asparagine, α-aminobutyric acid and β-alanine.     

Ion-Interaction Chromatographic Separation of Free Amino Acids

Abstract

An extensive study of the HPLC separation of 20 free amino acids by the addition of alkanesulfonate salts to the mobile phase was previously reported (1). This paper describes modifications in the procedure that improves the separation and resolution of the 20 free amino acids. Mobile phase variables (type and concentration of alkanesulfonate salt, organic modifier concentration, mobile phase pH, and mobile phase ionic strength), and stationary phase variables (particle size, type of packing) which can affect amino acid separation, resolution and selectivity were studied. Two stationary phases were compared, the 5 μm Hamilton PRP-1 and Phase Separations 3 μm, ODS-2. Longer chain alkanesulfonate salts (octane and decanesulfonate salts) were evaluated as mobile phase additives. A mobile phase gradient of increasing per cent organic modifier was necessary for separating complex mixtures of polar and nonpolar-basic amino acids. It is now possible to separate 19 of 20 free amino acids with this ion-interaction chromatographic procedure.     

Seperation of protein amino acids as their N(O)-Acyl alkyl ester derivatives on glass capillary columns

A comparison of the elution properties of the major protein amino acids as their N(O)-acyl alkyl ester derivatives (O-n-propyl, -n-butyl, -isopentyl; N(O)-trifluoroacetyl, -heptafluorobutyryl) on open-tubular glass capillary columns coated with SE-30, OV-17, OV-210 and EGA is described. A single-column separation to the baseline of the protein amino acids as their N(O)-heptafluorobutyryl n-butyl ester derivatives in less than 35 min was obtained on the SE-30 column. OV-210 columns have properties complementary to those of SE-30 columns and can be used as an aid to compound identification from retention time data. Separations of the amino acids from beer and dialysate from uremic patients are used to illustrate the practical posibilities of the method.     

Direct determination of enantiomeric purity of FMOC amino acids with high performance liquid chromatography

Summary Analytical methods are described which allow a direct determination of enantiomeric purity of seventeen FMOC amino acids commonly used in peptide synthesis. The corresponding ester derivatives can be separated directly on cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel-OD). The methods are suitable for primary as well as secondary FMOC amino acids. The presence of a highly sensitive fluorescence moiety within the molecule, in combination with large separation factors (-values between 1.5–2.2) allowed for a general detection limit below 0.05%. In several cases the antipode has been determined in the ppm-range. An interesting result has been observed with respect to the elution order of the FMOC amino acid esters. The elution order of the Trp enantiomers is opposite to that obtained with the other amino acids. This is contrary to the generally held belief that elution order is identical within a homologous series of racemates when chromatographed under identical conditions on the same chiral stationary phase. In addition, the inversion of elution of the Pro enantiomers depending on the estertype indicates a competition of different separation mechanisms.     

Enantiomeric Separation of Non-Protein Amino Acids by Chiral Ligand-Exchange High-Performance Liquid Chromatography

Abstract

The enantiomeric separation by high-performance liquid chromatography of underivatized non-protein amino acids was investigated by using a column packed with octadecylsilanized silica coated with N,S-dioctyl-D-penicillamine as a chiral ligand-exchange phase (Sumichiral OA-5000). Excellent to good separations of enantiomers were achieved with a variety of nonprotein amino acids carrying aliphatic or aromatic side-chains by optimizing the amount (0-20%, v/v) of the organic component (2-propanol) and the concentration (1-5 mM) of the complexing metal ion (Cu²⁺) in the hydro-organic eluent.     

Monitoring of Protease-Catalyzed Peptide Synthesis by High Performance Liquid Chromatography

Abstract

High performance liquid chromatography (HPLC) on prepacked silica gel columns has been applied to the separation of closely related protected peptides and amino acids. In the course of a protease-catalyzed synthesis of Leu-enkephalin this chromatogra-phic technique was found to be a valuable tool to rapidly and reliably characterize the outcome of enzymatic reactions, the nature of which was often difficult to be predicted. Stepwise gradient elution was employed to enable fractionation of mixture components, which covered only a short polarity range. The solvent systems composed of dichloromethane, anhydrous ethanol and acetic acid were mixed in such ratios so as to provide completely resolved peaks for the sample problems studied so far. Enzymatically prepared compounds and their chemically synthesized, authentic analogues were cochromatographed to enable the assignment of the eluted peaks. The provisional identification thus obtained could finally be established for all compounds under study by standard methods for chemical analysis.     

Retention prediction of a set of amino acids under gradient elution conditions in hydrophilic interaction liquid chromatography

The analysis of amino acids presents significant challenges to contemporary analytical separations. The present paper investigates the possibility of retention prediction in hydrophilic interaction chromatography (HILIC) gradient elution based on the analytical solution of the fundamental equation of the multilinear gradient elution derived for reversed‐phase systems. A simple linear dependence of the logarithm of the solute retention (ln k) upon the volume fraction of organic modifier (φ) in a binary aqueous‐organic mobile is adopted. Utility of the developed methodology was tested on the separation of a mixture of 21 amino acids carried out with 14 different gradient elution programs (from simple linear to multilinear and curved shaped) using ternary eluents in which a mixture of methanol and water (1:1, v/v) was the strong eluting member and acetonitrile was the weak solvent. Starting from at least two gradient runs, the prediction of solute retention obtained under all the rest gradients was excellent, even when curved gradient profiles were used. Development of such methodologies can be of great interest for a wide range of applications.     

HPLC Determination of α-Amino Acids in Phytoplanktonic Marine Cells

Abstract

A procedure for the quantitative determination of 17 amino acids in a marine matrix using HPLC is reported. Pre-column derivatization with o-phthalaldehyde, separation on C₁₈-bonded silica with phosphate buffer (pH 7.2)-acetonitrile as eluent and fluorescence detection have been used. The good variation coefficient (average 2% with working curves in real matrix) and the low detection limit (1-5 fmoles) make the procedure suitable for the determination of total or free amino acids in matrix cultures.     

TLC Separations of Amino Acids on Silica Gel Impregnated Layers

Abstract

Copper sulphate and polyamide were tried as impregnants for improving the separation of twenty amino acids on silica gel ‘G’ layers using a new solvent system MeOH-BuOAc-AcOH-Pyridine(20:20:10:5). Tables are presented to illustrate the improvement in resolution of amino acids on silica gel plates.     

阴离子交换-积分脉冲安培检测法分离测定氨基酸注射液中的氨基酸和葡萄糖

 用阴离子交换 积分脉冲安培检测法测定了氨基酸注射液中 1 7种氨基酸和葡萄糖。研究了氨基酸和葡萄糖在阴离子交换中的保留行为。采用了优化的水、NaOH和NaAc三元梯度淋洗条件。在优化的梯度淋洗条件和积分脉冲安培检测条件下 ,氨基酸和葡萄糖的检出限为 0 3pmol～ 1 0 3pmol,线性范围约为 2个数量级。样品加标回收率为 88 3 %～ 1 0 4 6 %。方法简单、灵敏、准确。     

An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra‐cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at −80°C. Filtered medium samples were derivatized with ortho ‐phthalaldehyde (naphthalene‐2,3‐dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse‐phase columns (LichroCART, Purospher STAR RP_18e or Ascentis Express C₁₈) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra‐assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.     

HPLC Analysis of the Isomeric Thioether Metabolites of Styrene Oxide

Abstract

An efficient separation of the isomeric thioether metabolites of styrene oxide was achieved under reversed-phase conditions. The column was eluted isocratically with 15% methanol in buffered solutions of phosphoric acid-tris-hydroxymethylaminomethane. The thioether conjugates were separated by class, and the order of elution was cysteine, cysteinylglycine, glutathione, and N-acetylcysteine. The effect of pH and buffer salt concentration on the HPLC separation was examined. Optimal conditions for a separation were either found at low pH (pH 3 or 4) or neutral pH, both at a high buffer salt concentration (75mM). The positional isomers and stereoisomers comprising each amino acid conjugate sample were separated into two peaks. The variations in k¹ and α observed with changes in pH were interpreted as reflecting the degree of interaction of the ionizable groups in the amino acid residue and the hydrophobic portion of the molecule. This interaction was found to be strongly influenced by the relative stereochemistry of the benzylic carbon center, thus allowing the separation of diastereoisomeric thioethers.