6533b823fe1ef96bd127df2e

RESEARCH PRODUCT

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

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Abstract The isocratic analysis of primary proteic amino acids using a C18 column, micellar mobile phases of sodium dodecyl sulphate (SDS) and 1-propanol at varying pH, and pre- and post-column formation of the o-phthalaldehyde (OPA)/N-acetyl- l -cysteine (NAC) isoindoles, is examined. An interpretive optimization strategy was applied to find the best separation conditions. For this purpose, empirical polynomial models were used to describe the elution behavior of the isoindoles and free amino acids, using as separation factors the concentrations of surfactant and modifier (two-factor problem), or these two factors and the pH of the mobile phase (three-factor problem). The separation of the OPA/NAC amino acid isoindoles was very poor: only eight compounds could be separated satisfactorily. In contrast, 14 free amino acids (ordered according to their elution times: aspartic acid, glutamine, threonine, cysteine, alanine, tyrosine, valine, methionine, phenylalanine, leucine, 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 efficiencies 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 resolution of a mixture of aspartic acid, glutamine, asparagine, glutamic acid, threonine, cysteine and alanine, which eluted at the lower retention times. In these conditions, only serine and glycine overlapped with glutamine and threonine, respectively.