OPTIMIZATION OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION FOR PROGESTOGENS

An efficient, rapid HPLC optimization method based on semiempirical model and simplex algorithm was proposed and examplified by the optimization separation of six progestogens within a given time (1≤K≤10). The resultant composition of optimum ternary mobile phase was MeOH—THF—H₂O (41:22:37), MeOH—ACN—H₂O (61.6:8.9:29.5) respectively.The procedure consists of three main steps: 1. Initial estimation of isocratic condition using MeOH—H₂O linear gradient run. 2. Transfer MeOH—H₂O to other iso-eluotropic binary mobile phases. 3. Simplex algorithm. In step three, a small step size simplex was used instead of "large step size simplex". By combining the binary conditions obtained from step 1 and 2, the iso-eluotropic ternary mobile phase composition was made as initial simplex points, which were closer to the optimum regions than that of "large step size" because many failed experiments caused by boundary violation and analysis time excess were avoided.A computer program in BASIC was presented for "off line" optimization of HPLC using the simplex method.