THE OPTIMIZATION OF STEP-GRADIENT ELUTION CONDITIONS IN LIQUID-CHROMATOGRAPHY - APPLICATION TO PESTICIDE-RESIDUE ANALYSIS USING COUPLED-COLUMN REVERSED-PHASE LC

A computer spreadsheet application has been developed for the optimiza tion of step-gradient elution conditions as applied in coupled-column RPLC for online clean-up and separation in the analysis of pesticide r esidues. The procedure is based on the experimentally determined reten tion behaviour of the analytes as a function of mobile phase compositi on. Retention and peak volumes of the analytes eluting under isocratic and one- and/or two-step gradient conditions can be calculated. For a given set of analytes, the spreadsheet systematically calculates the resolution of the least efficiently separated pair of peaks in the spa ce defined by the eluotropic strengths of the mobile phases and the ti me at which the step-gradient takes place. The performance of the prop osed procedure was tested and evaluated for the analysis of nine polar pesticides for which in a previous study adequate conditions had been found by computer-aided chromatogram simulation. The optimum conditio ns predicted by the procedure were in good agreement with those establ ished experimentally and by simulation. The simultaneous production of data on retention, peak volume and resolution for a group of analytes under selected one- and/or two step-gradient elution conditions makes the calculation procedure highly productive and flexible in searching for and/or adjusting appropriate separation conditions.