Optimum Combination of Reversed-Phase Column Type and Mobile-Phase Composition for Gradient Elution Ion-Pair Chromatography of Amino Acids

Abstract

An optimum combination of reversed-phase column type and eluent composition was selected for the practical separation of amino acids by gradient elution ion-pair chromatography.A butyl-silica (C4) column with a concentration gradient elution between 5 mM SDS-10 mM HC104 solution and acetonitrile provided the best resolution for amino acids. The C4 column was most adequate for reducing the chromatographic cycle time including the column regeneration. The use of 5 mM SDS as ion-pairing agent in relatively low concentration is economical and practical for replicate and routine analyses. The developed HPLC system is applicable to the analysis of organic cations at low cost. The analysis of amino acids using high-performance liquid chromatography (HPLC) techniques is very important in biological and biomedical laboratories. The widely accepted methods, so-called amino acid analyzers, are mainly based on cation-exchange separation followed by postcolumn derivatization such as o-phthalaldehyde (OPA) fluorescence derivatizationl-3 and reversed-phase separation of precolumn derivatization products such as OPA derivatives.4-6 Such instruments designed and specialized for routine use are of course commercially available, but they are expensive and unavailable for general purposes.As a relatively fast and simple procedure for amino acid analysis, gradient elution ion-pair chromatography techniques have also been proposed'-9, but these have been unpopular so far owing to their poor reproducibility in replicate chromatography.To overcome the poor reproducibility, auto-samplers are sometimes employed for timed sample injections between the gradient runs. In any case, however, it is difficult to obtain reproducible retention times unless we know the real column reequilibration. It is known that the gradient ion-pair HPLC needs a long time for regenerating the reversed-phase column to the initial ionic equilibrium between the stationary and mobile phases, requiring more than 15 -20 column volumes of initial solvent.1 Patthyl1 has experimentally estimated the reequilibration of a reversed-phase column between gradient runs, by finding the plateau of retention times of standard analytes after several different regeneration times. Even t To whom correspondence should be addressed .by this method, however, we can not know the real reequilibration finish.We recently have developed a time and cost efficient HPLC method for amino acid analysis using dual-mode gradient ion-pair chromatography.12 A butyl-silica (C4) reversed-phase column and a sodium dodecylsulfate (SDS)/perchloric acid/acetonitrile eluent system with a simultaneous concentration and flow-rate gradient elution technique has been efficiently applied. In the process of developing the above method, we have discovered that the ghost peak appearing on the UV baseline is due to the completion of reequilibration of the separation column after the acetonitrile gradient elutions. In the previous paper, however, the optimum combination of reversed-phase column and mobile phase com...

Ion-pair reversed phase liquid chromatography-indirect conductivity detection and response deviations of underivatised amino acids

“…Anion-exchange chromatography (2 mM of Ba(OH) 2 as mobile phase) has been used for the separation of Lys, Met, Asn, and Gly and indirect conductivity for their detection. However, conductivity detection has never been investigated after separation of underivatised amino acids by ion-pair reversed-phase liquid chromatography (IP-RPLC) as current applications of IP-RPLC for these molecules contain high concentrations of ionised species > 50 mM [3,19] and/or a high concentration of organic modifiers (> 15%) [20] which causes a high background signal (low sensitivity) and several system peaks [21]. System peaks appear in chromatographic systems containing more than one eluent component after injection of a solution with a composition different from the bulk eluent.…”