Gradation of the mobile phase elution.

Time (min)	Flow rate (mL min−1)	A%	B%
Initial	0.300	98	2
0.50	0.300	98	2
1.50	0.300	81	19
3.00	0.300	81	19
3.50	0.300	20	80
4.50	0.300	20	80
4.60	0.300	98	2
6.00	0.300	98	2