Table 2. Gradient program employed for the separation of PITC derivatized amino acids^a.

Run timeb (min)	Flow rate (ml/min)	% Buffer Ac	% Buffer B (60% acetonitrile in water)
0	1	100	0
0.1	1	95	5
5	1	90	10
14	1	90	10
25	1	60	40
30	1	50	50
35	1	40	60
40	1	10	90
52	1	10	90
62	1	95	5
65	1	100	0

^a Column temperature was maintained at 39°C.

^b Run time was 62 min with 3 min column regeneration time.

^c Sodium acetate buffer [19 g of sodium acetate trihydrate was dissolved in 1 l of HPLC grade water. To this 0.5 ml of TEA was added and the contents were mixed properly. The pH of the solution was adjusted to 6.4 with glacial acetic acid and filtered. To the filtrate (940 ml) acetonitrile (60 ml) was added, mixed and filtered through a 0.22 µm Millipore membrane].