Determination of amino acids by ion-pair liquid chromatography with post-column derivatization using...

A new chromatographic method for the determination of amino acids is proposed. The method is based on the separation of amino acids by means of ion-pair liquid chromatography and post-column derivatization using 1,2-naphthoquinone-4-sulfonate. The analytical column was a Spherisorb 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 aqueous 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 100 microl and the total flow-rate for the mobile phase was 0.8 ml/min. The chromatographic outlet was coupled on-line to the two-channel derivatization system which delivered reagent and buffer solutions. The reaction 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 separation of common amino acids was done in 90 min, although an additional period of 15 min was required to stabilize the column. The repeatability of the method for lysine is 2.1% and the reproducibility is 2.6%. The detection limit for lysine is 0.09 nmol. The linear range for lysine 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 powdered milks. The results of the method are in good agreement with those obtained with the standard amino acid autoanalyzer method.