APPLICATION OF THE GRADIENT ELUTION TECHNIQUE - DEMONSTRATION WITH A SPECIAL TEST MIXTURE AND THE DRYLAB-G/PLUS METHOD DEVELOPMENT SOFTWARE

A mixture of ten compounds with an overlapping peak pair was analysed with a 90-min elution gradient. To improve the separation, two reversed-phase gradients differing by a factor of 3 in their run times, were applied. Contrary to expectation, two peak pairs were less well separated in the gradient run with the lower slope. The relative resolution map provided a rapid solution to the problem: a gradient with 16-min run time gave the best separation of the mixture. The simulated chromatogram was verified experimentally. The differences between the predicted and experimental retention times averaged 0.03 min. Further improvement was obtained using a segmented gradient, which adequately separated all peaks in only 9 min.