J. Saurina et S. Hernandezcassou, DETERMINATION OF AMINO-ACIDS BY ION-PAIR LIQUID-CHROMATOGRAPHY WITH POSTCOLUMN DERIVATIZATION USING 1,2-NAPHTHOQUINONE-4-SULFONATE, Journal of chromatography, 676(2), 1994, pp. 311-319
A new chromatographic method for the determination of amino acids is p
roposed. The method is based on the separation of amino acids by means
of ion-pair liquid chromatography and post-column derivatization usin
g 1,2-naphthoquinone-4-sulfonate. The analytical column was a Spheriso
rb ODS 2. Amino acids were separated by an elution gradient with four
linear steps based on increasing the concentration of 2-propanol. Two
eluents were used to create the gradient profile: eluent A was an aque
ous solution of 20 mM H3PO4 + 20 mM H2PO4- + 15 mM dodecyl sulfate and
eluent B was a mixture of aqueous (25 mM H3PO4 + 25 mM H2PO4- + 18.5
mM dodecyl sulfate)-2-propanol (1:1, v/v). The injection volume was 10
0 mu l and the total flow-rate for the mobile phase was 0.8 ml/min. Th
e chromatographic outlet was coupled on-line to the two-channel deriva
tization system which delivered reagent and buffer solutions. The reac
tion took place at 65 degrees C in a reaction coil of 4 m x 1.1 mm I.D
. The spectrophotometric detection was performed at 305 nm. The separa
tion of common amino acids was done in 90 min, although an additional
period of 15 min was required to stabilize the column. The repeatabili
ty of the method for lysine is 2.1% and the reproducibility is 2.6%. T
he detection limit for lysine is 0.09 nmol. The linear range for lysin
e is up to 32 nmol with a correlation coefficient of 0.999. The method
was applied to the determination of amino acids in animal feed and po
wdered milks. The results of the method are in good agreement with tho
se obtained with the standard amino acid autoanalyzer method.