Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

Retention/quantitation properties of the o-phthaldialdehyde-3-mercaptopropionic acid and the o-phthaldialdehyde-N-acetyl-L-cysteine amino acid derivatives in reversed-phase high-performance liquid chromatography

The separation/identification of 25 amino acids as their o-phthaldialdehyde-3-mercaptopropionic acid (OPA/MPA) and o-phthaldialdehyde-N-acetyl-L-cysteine (OPA/NAC) derivatives have been optimized [paying particular attention to those amino acids which elute with more than one derivative (glycine, histidine, gamma-aminobutyric acid, beta-alanine, ornithine, lysine) and that are expected to be present in apples in their free form]. Optimum separation conditions are reported on six reversed-phase columns: Nucleosil 3 and 5 microm, 150(+20 guard)x4.0 mm; Gromsil 3 microm, 150(+10 guard)x4.0 mm; Hypersil 5 microm, 150(+20 guard)x4.0 mm and 200(+20 guard)x4.0 mm; and Hypersil 3 microm, 150(+20 guard)x4.0 mm. Elutions were followed, simultaneously, with photodiode array and fluorescence detectors connected in line. Optimization studies carried out in model solutions as a function of temperature (30-55 degrees C) and eluent flow-rate (0.8-2.5 mL/min) demonstrated that optimum resolutions are obtained with the highest flow-rate applicable (remaining on the safe side with a column pressure of < 3500 p.s.i.; 1 p.s.i.=6894.76 Pa) in the temperature range 30-50 degrees C. Twenty-five amino acids, eluting in 31 separate, characteristic derivatives, were determined on all six columns (the main component, asparagine, present in overwhelming excess, together with the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, and homoarginine). Optimum conditions in the case of both derivatives were obtained on the same type of column (Hypersil, 5 microm), as follows: for the OPA/MPA amino acids with programmed flow-rate [1.3-2.3 ml/min; column, 200(+20 guard)x4 mm], at 50 degrees C, while, for the OPA/NAC amino acids at 2.1 ml/min flow rate, at 30 degrees C [column, 150(+20 guard)x4 mm], with 40 and 37 min run times, including equilibration. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range 6-12,000 pmol, related to the injected amount of amino acids, was <3.4% RSD (average relative standard deviation percentage). The utility of the protocol was demonstrated in the quantitation of the free amino acid content of five apple varieties (Jonagored, Idared, Jonica, Florina, Freedom) on various harvesting dates and after different storage times. Derivatization of the apple pulp was performed with filtered samples, applying any special isolation processes.     

Precolumn diastereomerization and micellar electrokinetic chromatography on a plastic microchip: rapid chiral analysis of amino acids

Precolumn derivatization and chiral separation of DL-amino acids based on diastereomerization have been performed on an integrated poly(dimethylsiloxane) microchip. Diastereomeric derivatives were formed in a microfabricated precolumn reactor by the reaction of amino acid enantiomers with o-phthaldialdehyde/2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose (OPA/TATG), and separated by MEKC in an achiral environment without chiral selectors in the running buffer. Optimized precolumn reactions and chiral separations of amino acids were achieved within 2.5 min. Resolutions of diastereomers of OPA/TATG-amino acids were in the range of 2.5-6.1 at optimized separation conditions. Simultaneous separation of a mixture of five chiral amino acids was successfully performed in a single run in less than 100 s.     

Advances in the evaluation of the stability and characteristics of the amino acid and amine derivatives obtained with the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/N-acetyl-L-cysteine reagents. High-performance liquid chromatography-mass spectrometry study

The composition of the amino acid and amine derivatives obtained with the o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and with the OPA/N-acetyl-L-cysteine (NAC) reagents was investigated by on-line HPLC-electrospray ionization MS. The initially formed derivatives proved to be, as expected, the corresponding isoindoles while their transformed species contained one additional OPA molecule. Based on the MS spectra of all transformed OPA derivatives a reaction pathway is suggested. This reaction mechanism was supported both by the molecular ions of the endproducts and by the presence of several selective fragment ions that served as an explanation to the structure of the believed to be less stable OPA derivatives. It has been shown that more than one OPA derivative forms in all those cases when the compound to be derivatized does contain the NH2-CH2-R moiety. Thus, amino acids like e.g. glycine, histidine, beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid, ornithine, and also several aliphatic mono- and diamines provide more than one OPA derivative. Analytical consequences of this experience were utilized by altering the reagent's composition. Reagents containing mole ratios of [OPA]/[MPA] or [OPA]/[NAC]=1/50 resulted in two benefits, simultaneously: (i) in a decrease of the transformation rate of the initially formed derivative, and, (ii) in an increase of the overall stability of the total of derivatives.     

The solute molecule—Rigid or partially flexible? calculation of benzodiazepineR F values as an example

Summary The liquid-chromatographic separation of the enantiomers of amino acid esters as benzophenone Schiff-base derivatives on polysaccharide-derived chiral stationary phases (CSPs) is described. The performance of Chiralcel OF was superior to that of the other CSPs for resolution of benzophenone imine derivatives of amino acid ethyl and methyl esters. The enantiomers of most of the amino acid esters examined as their benzophenone imine derivatives were resolved to baseline on Chiralcel OF. The L-(−) enantiomers of all the analytes were preferentially retained on Chiralcel OF. The resolution of several imine derivatives of amino acid esters was investigated, as was the effect of eluent composition on the resolution of amino acid ethyl esters as their benzophenone imine derivatives.     

Direct high-performance liquid chromatographic enantioseparation of apolar beta-amino acids on a quinine-derived chiral anion-exchanger stationary phase

A quinine-derived chiral anion-exchanger stationary phase was applied for the direct high-performance liquid chromatographic separation of the enantiomers of N-protected unusual beta-amino acids, i.e. 3-aminobutanoic acid, 3-aminopentanoic acid, 3-amino-4-methylpentanoic acid, 3-amino-4,4-dimethylpentanoic acid, 3-amino-4-methylhexanoic acid, 3-amino-4-ethylhexanoic acid, 3-amino-3-cyclohexylpropanoic acid, 3-amino-3-(3-cyclohexen-1-yl)propanoic acid and 3-amino-3-phenylpropanoic acid. The readily prepared N-2,4-dinitrophenyl derivatives were well separable, with good efficiency and high resolution. The chromatographic conditions (eluent composition, pH and buffer concentration) were varied to achieve optimal separation. In some cases, the elution sequences of the enantiomers of the derivatives were determined.     

OPA柱前衍生反相高效液相色谱法测定氨基酸含量

建立了邻苯二甲醛（ＯＰＡ）手动柱前衍生反相高效液相色谱法测定样品中氨基酸含量的方法。以邻苯二甲醛（ＯＰＡ）／３－巯基丙酸（３－ＭＰＡ）为衍生试剂进行衍生,ＯＤＳ柱分离,３４０ｎｍ检测,在４０ｍｉｎ内１８种氨基酸全部得到基线分离。测定牛血清白蛋白（ＢＳＡ）的氨基酸组成和小鼠血清中的游离氨基酸,取得满意的结果。     

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.     

Automated Optimised High Performance Liquid Chromatographic Analysis of Pre-Column O-Phthaldialdehyde-Amino Acid Derivatives

Abstract

The feasibility of injecting o-phthaldialdehyde/2-mercaptoethanol reagent and amino acid mixture simultaneously, from separate Injector vials in order to allow automation of HPLC pre-column amino acid derivative analysis was investigated. The modified duel injection system described proved to be successful. Reaction of amino acids and reagent takes place during flushing of the injection loop. This is immediately followed by the introduction of the formed derivatives into the main HPLC eluent stream leading to the chromatographic column. The described procedure gave reproducible results with no loss in sensitivity. Further established was the optimum pH of reagent for derivative formation and of eluents for the separation of derivatives.     

On the use of solid phase ion exchangers for isolation of amino acids from liquid samples and their enantioselective gas chromatographic analysis

This work focuses on the development of a suitable working procedure for preconcentration of amino acids enantiomers from water samples using a solid phase extraction. The three types of ion exchangers with various capabilities have been used. The effect of experimental conditions in SPE procedure employing strong anion exchange (SAX), weak (WCX) and strong cation exchange (SCX) cartridges (such as sample volume, pH, origin of elution solvent and its volume) on effective preconcentration of the model set of amino acids has been studied in detail. The enantiomers of isolated and preconcentrated amino acids have been analysed by GC on three capillary columns coated with chiral selectors. The different amino acids derivatives have been investigated in order to achieve optimal resolution of biogenic amino acids and their enantiomers. The best separation of amino acid enantiomers has been obtained on a Chirasil-L-Val column analysing their N-TFA methyl esters. It has been shown that SCX-SPE cartridge with sulfonic groups attached on silicagel support is most suitable for isolation and preconcentration of amino acids from water samples. For this sample treatment procedure, the overall recovery of extraction process has been calculated as an average value from three measurements. It has been found, that recoveries are practically identical for both enantiomers of a particular amino acid and varies in the range 75-99% depending on the type of amino acid. The effectivity of this sample preparation and GC method has been verified by preconcentration of amino acids from orange juice fortified by racemic mixture of some selected amino acids.     

Proline-coated column for the capillary electrochromatographic separation of amino acids by in-column derivatization

With 3-trimethoxysilylpropyl chloride as the spacer, a proline-coated capillary column was prepared for the capillary electrochromatographic (CEC) separation of amino acids by in-column derivatization. Nine standard mixtures, including aspartic acid, glutamic acid, valine, phenylalanine, alanine, isoleucine, leucine, tyrosine, and tryptophan, were injected. o-Phthalaldehyde (OPA), OPA/2-mercaptoethanol (2-ME) and OPA/N-acetylcysteine (NAC) in borate buffer were tested as the derivatizing agent. Among them, OPA (50 mM) in borate buffer (pH 9.5, 50 mM) gave the best performance. The formation of isoindole could be detected by UV detection. The sandwich-type injection was carried out in hydrostatic mode (10 cm) with the program R(10 s)S(10 s) R(10 s)W(10 min) with R, S, and W being the reagent, sample, and waiting times. Mesityl oxide, benzyl alcohol, and acetone showed some interaction with the column. A current monitoring method was used instead of the determination of the electroosmotic flow (EOF). The direction of EOF was from anode to cathode even under acidic condition lower than the pI value (6.31) of the bonded group due to some unreacted silanol groups. Some parameters including pH, nature, and concentration of the mobile phase and the effect of organic modifier with regard to the CEC separation were investigated. With the proline-coated column (75 (50) cm x 75 microm ID) the best separation was performed in phosphate buffer (pH 4.00, 100 mM) with an applied voltage of -15 kV. The established method was also compared with those precolumn derivatized prior to the separation with proline-coated column as well as with in-capillary derivatization and separation with a bare fused-silica column.     

Advances in the o-phthalaldehyde derivatizations. Comeback to the o-phthalaldehyde-ethanethiol reagent

The main aims of this work were (a) to present the characteristics and stability of the o-phthalaldehyde (OPA)-ethanethiol (ET) derivatives of 22 amino acids, including the believed-to-be less stable OPA derivatives providing glycine, gamma-aminobutyric acid, beta-alanine, histidine, ornithine, lysine and the C(1)-C(5) aliphatic amines; (b) to compare the stability properties of the most common amino acids and amines as OPA-ET-fluorenylmethyl chloroformate (FMOC) derivatives to the corresponding ones obtained from OPA reagents containing various (SH)-additives; (c) to show the molar responses of alanine and lysine depending on the OPA reagent's composition; as well as (d) to prove the practical utility of these basic researches, by the simultaneous HPLC separation of 22 amino acids and 15 amines as their OPA-ET-FMOC derivatives. Investigations have been carried out by varying the composition of the reagents, the molar ratios of reactants and the reaction time, applying diode array and fluorescence detections simultaneously. Average reproducibility of quantitations, characterized with the relative standard deviations (RSDs) based on the fluorescence intensities of derivatives, in the order of listing, proved to be 1.2-5.9% for amino acids and 1.1-8.7% for amines. The practical utility of the method is demonstrated by the analysis of the amino acid and amine contents of mouse tissues, with an average reproducibility of 3.5%.     

Dual-gradient capillary electrochromatography by varying the mobile phase composition and applied voltage.

Dual-gradient capillary electrochromatography (DG-CEC) was developed to provide superior performance with regard to the separation of ionized analytes; in this method, both the eluent composition and the applied voltage are varied during the separation procedure. As for the gradient in the eluent composition, a shift in the pH is employed to control not only the electrophoretic mobility, but also the retention factor of the analytes. The dual-gradient method was shown to be effective in increasing the resolution and reducing the chromatographic period of ionized analytes. Fourteen kinds of o-phthalaldehyde labeled amino acids were separated within 8 min using DG-CEC with multistage enlargement in the applied voltage. The separation efficiency increased particularly for highly retained amino acids in the dual-gradient, as compared to those in the ordinary single-gradient for the eluent.     

Separation of enantiomers of <Emphasis Type="Italic">N</Emphasis>-derivatives of amino acids by capillary electrophoresis using macrocyclic antibiotics

The potential for the application of macrocyclic antibiotic eremomycin as a chiral selector for the separation of enantiomers of N-derivatives of amino acids in capillary electrophoresis was estimated. The influence of several factors (the composition and pH of the running electrolyte, the concentration of the chiral selector, capillary geometry, the value of the applied voltage) on the migration of the derivatives of amino acids and enantioselectivity in the presence of eremomycin was studied in order to choose the separation conditions. Using the example of the dansyl derivatives of amino acids the enantioselectivities of vancomycin and eremomycin in capillary electrophoresis were compared.     

Capillary electrophoresis and column chromatography in biomedical chiral amino acid analysis

Free amino acids are typically quantified as the sum of their enantiomers, because in terrestrial organisms they mainly exist in the left-handed form. However, with increasing understanding of the biological significance of right-handed amino acids interest in enantioselective quantification of amino acids has steadily increased. Initially, electrophoretic and chromatographic methods using chiral (pseudo)-stationary phases or chiral eluents were applied to the separation of amino acid enantiomers. Later, derivatization of amino acids prior to chromatography with chiral reagents gained in popularity, because the diastereomers formed can be resolved on conventional reversed-phase columns. Novel multi-interaction chiral columns turned attention back to direct chiral chromatographic methods. Hyphenation to mass spectrometry has increasingly replaced optical detection because of superior selectivity, although this has not obviated the need for baseline resolution of amino acid enantiomers. Despite the progress made, enantioselective separation and quantification of amino acids remains an analytical challenge owing to frequently incomplete resolution of all naturally occurring enantiomers and insufficient sensitivity for the determination of the trace amounts of d-amino acids typically found in biological fluids and tissues. Chiral GC-MS analysis of heptafluorobutanol/pentafluoropropionanhydride amino acid derivatives on an Rt-gDEXsa column     

Performance of throughout in-capillary derivatization capillary electrophoresis employing an on-line sample and run buffer loading device

We report on the effect on performance of varying the length of the capillary during throughout in-capillary derivatization (TICD) capillary electrophoresis (CE). Performance was evaluated by on-line coupling with a sample and CE runbuffer loading device that was newly introduced for this study. The device was assembled with a low cost using two 5 mm inner diameter (ID) disposable polyethylene syringes. First, a sequence was manually formed consisting of a 200 microL run buffer solution plug, a 100 microL sample plug and another 200 microL run buffer solution plug. Each plug was separated from its neighbor by a 100 microL air plug. When each plug reached the injection point where both a platinum-wire anode and the end of the separation capillary tube were located, 340 V/cm separation voltage (electrophoresis voltage) and 34 V/cm injection voltage were applied to the capillary for 3 s. Then the analytes were derivatized during migration in 50 microm ID capillaries filled with 2 mM o-phthalaldehyde (OPA)/N-acetylcysteine (NAC) in a 20 mM phosphate-borate buffer (pH 10), followed by separating and detecting of OPA derivatives by absorbance of 340 nm. Derivatization, separation, and detection were performed systematically using capillaries which varied in length from 5 to 80 cm. In the case of TICD-CE of a mixture containing 1 mM aspartic acid (Asp) and 20 mM m-nitorophenol (MNP) as a test solution, it was determined that peak area and peak width ratios of Asp to MNP did not depend on capillary length. Enantiomeric separations of DL-alanine (Ala) and Asp were examined using a run buffer consisting of a 45 microM beta-cyclodextrin (CD)-2 mM OPA/NAC-20 mM phosphate-borate buffer (pH 10). Even though the resolution of these enantiomeric pairs decreased with decreasing capillary length, as expected, the peaks corresponding to both enantiomeric amino acids were identified even when a 5 cm capillary was used. An 8-component amino acid mixture was also tested with 5 cm and 10 cm capillaries.     

Liquid chromatographic enantiomeric resolution of amino acids with β-cyclodextrin bonded phases and derivatization with o-phthalaldehyde

The reaction of amino acids with o-phthalaldehyde (OPA) and 2-mercaptoethanol is evaluated as a precolumn derivatization technique in order to achieve the chiral separation and final fluorimetric detection of l,d-glutamic acid in β-cylodextrin bonded phases. The pertinent chromatographic parameters were determined and the effect of organic modifiers, pH, ionic strength and temperature on retention and resolution were evaluated. The chromatographic method finally developed allowed the optical resolution of OPA-derivatized l- and d-glutamic acid using methanol—1% triethylammonium acetate (pH 7.4) (30 + 70, v/v) as eluent.     

Method for on-line derivatization and separation of aspartic acid enantiomer in pharmaceuticals application by the coupling of flow injection with micellar electrokinetic chromatography

A novel, easy and accurate capillary electrophoresis (CE) coupled with flow injection (FI) method for the separation and determination of aspartic acid (Asp) enantiomers by on-line derivatization had been developed, and it had been applied to the real sample for the first time. The derivatization reagents were o-phthalaldehyde (OPA) and mercaptoethanol (ME), which were obtained easily, the chiral selector was beta-cyclodextrin (beta-CD), the micellar chemical was sodium dodecyl sulfate (SDS), and the modifier was methanol. By on-line derivatization, aspartic acid enantiomers were automatically and reproducibly converted to the ultraviolet (UV)-absorbing diastereoisomer derivates, which were separated by micellar electrokinetic chromatography (MEKC). According to the factors affecting the separation and sensitivity of aspartic acid enantiomer and other amino acids in the real sample, the pH value and concentration of the buffer, the concentration of beta-CD and SDS, the volume percentage of the methanol (v/v) in the buffer, the applied voltage and the conversion time were selected as the investigating variates. Under the investigated separation conditions, D-aspartic acid (D-Asp), L-aspartic acid (L-Asp) and other four amino acids achieved the baseline separation in not only the standard mixture of amino acids but also the real sample (Compound Amino Acid Injection (6AA)). The repeatability (defined as relative standard deviation (RSD), n = 5) was 4.0% and 4.0% with peak area evaluation, and 4.2% and 3.7% with peak height evaluation for D-Asp and L-Asp in the real sample. Recovery at added standard levels of 1.0, 3.0 and 6.0 mM was 92%, 104% and 109%, respectively.     

Separation and determination of neuraminic acids in nanogram quantities by liquid chromatography — A comparative study with different amino phases

Summary Neuraminic acid derivatives were separated easily by liquid chromatography on amino phases and using acetonitrile/15 mM phosphate buffer, pH 5.2 as mobile phase. Detection at 200 nm allowed determination of substances in the nanomole range. All solvent systems were used isocratically at room temperature. The separation did not depend on ion-exchange but on a partition mechanism, the solutes being separated according to their polarity. Thus, it was possible to optimize the resolution by varying the stationary phase and the composition of the eluent.Part of this work has been presented at the 15th International Symposium on Chromatography, Nürnberg, October 1–5, 1984.     

HPLC-fluorescence detection and MEKC-LIF detection for the study of amino acids and catecholamines labelled with naphthalene-2,3-dicarboxyaldehyde

Naphthalene-2,3-dicarboxyaldehyde (NDA) is commonly used for detection of primary amines in conjunction with their separation with HPLC and CE. The fluorescence of the derivatives can be measured by a conventional fluorometer or via LIF. NDA is a reactive dye, which can replace o-phthaldehyde (OPA) and provides for derivatives which are considerably more stable than OPA derivatives. In addition, NDA can be used to derivatize primary amines at concentrations as low as 100 pM. In this work, HPLC/fluorescence and MEKC/LIF experiments were performed to separate/detect six neuroactive compounds, the amino acids, Gly, Glu, Asp, gamma-aminobutyric acid (GABA) and the catecholamines, dopamine and noradrenaline. The two methods were compared in terms of performance of separation. The amino acids can be separated in HPLC in less than 30 min and an identical separation is obtained in CE using MEKC and lithium salts with greater resolution (the number of theoretical plates was approximately 5000 for HPLC and 200 000 for MEKC). The lowest detected concentration was in the range of 0.1 nM for CE/LIF. The presence of a high salt concentration does not affect the separation of the samples. Examples of the analysis of microdialysate samples as well as amino acids in Ringer's solution are presented.