Temperature, eluent flow-rate and column effects on the retention and quantitation properties of phenylthiocarbamyl derivatives of amino acids in reversed-phase high-performance liquid chromatography.

The separation and identification possibilities of 27 PTC-amino acids (with particular attention to those present in apples in free forms), are reported on seven RP columns such as, Nucleosil, 3 and 5 microns: 150(+20 guard) x 4.0 mm; Gromsil 3 microns; 150(+10 guard) x 4.0 mm; Hypersil 5 microns: 130(+20 guard) x 4.0 mm, 150(+20 guard) x 4.0 mm and 200(+20 guard) x 4.0 mm, as well as, Hypersil 3 microns: 150(+20 guard) x 4.0 mm: a UV range photodiode array (PDA) detection was employed. Optimization studies carried out in model solutions, as a function of the temperature (30-55 degrees C) and flow-rate (0.8-2.5 ml/min) of eluents proved that optimum resolutions are associated 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 of 30-50 degrees C. Twenty-seven amino acids, characteristic in apples in free forms, have been separated and determined on all seven columns, performing the same gradient program, (the main component asparagine, present in overwhelming excess, and the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, homoarginine and 1-aminocyclopropane-1-carboxylic acid). Optimum conditions, at 2.1 ml/min, at 50 degrees C, with 40 min run time, including equilibration, have been obtained with the Hypersil, 150(+20 guard) x 4 mm column, performing elutions. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range of 15-1500 pmol was < 4.0% R.S.D. (relative standard deviation). Detailed study of the PDA spectra revealed that in addition to the identification/peak purity possibilities further characteristics can be obtained taking advantage of the difference in maximum values and of those of their special ratio values, respectively. The utility of the protocol was shown in the quantitation of the free amino acid content of three apple varieties.