No abstract

A low-capacity cation-exchange column was newly developed for the separation of amino acids. A highly cross-linked macro-porous polystyrene-divinylbenzene co-polymer was functionalized by a sulfoacylation reaction. The exchange capacity was controllable at the acylation step. The capacity between 55 and 60 µmol/column was adequate for the practical separations in acceptable retention times. The 5-µm base polymers having average pore diameters smaller than 3 nm gave satisfactory results, and those having 1.5-nm pore was most favorable. Several isocratic elution conditions at different pH values adjusted by phosphate buffer of mM order with or without acetonitrile could provide good separations for individual classes of amino acids, i.e., acidic, neutral, hydrophobic, and basic groups. The results provided fundamental data for constructing gradient elution systems required for the simultaneous separation of protein amino acids.

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...