Micellar liquid chromatographic separation of amino acids using pre- and post-column o-phthalaldehyde/N-acetylcysteine derivatization

The isocratic analysis of primary proteic amino acids using a C-18 column, micellar mobile phases of sodium dodecyl sulphate (SDS) and 1-propanol at v arying pH, and pre- and po st-column formation of the o-phthalaldehyde (OPA )/N-acetyl-L-cysteine (NAC) isoindoles, is examined. An interpretive optimi zation strategy was applied to find the best separation conditions. For thi s purpose, empirical polynomial models were used to describe the elution be havior of the isoindoles and free amino acids, using as separation factors the concentrations of surfactant and modifier (two-factor problem), or thes e two factors and the pH of the mobile phase (three-factor problem). The se paration of the OPA/NAC amino acid isoindoles was very poor: only eight com pounds could be separated satisfactorily. In contrast, 14 free amino acids (ordered according to their elution times: aspartic acid, glutamine, threon ine, cysteine, alanine, tyrosine, valine, methionine, phenylalanine, leucin e, lysine, histidine, tryptophan and arginine) could be well resolved with 0.05 M SDS-1.2% propanol at pH 3 in 42 min, partially due to the higher eff iciencies frequently five- to 10-fold those achieved for the isoindoles at this pH. A mobile phase of 0.05 M SDS without alcohol allowed the resolutio n of a mixture of aspartic acid, glutamine, asparagine, glutamic acid, thre onine, cysteine and alanine, which eluted at the lower retention times. In these conditions, only serine and glycine overlapped with glutamine and thr eonine, respectively. (C) 2000 Elsevier Science B.V. All rights reserved.