Analyte solvent and injection volume as variables affecting method development in semipreparative reversed-phase liquid chromatography

Analyte solvent and injection volume were examined as parameters that affect peak elution during method development for semipreparative RP HPLC purification. Analytical and semipreparative scale HPLC with gradient elution were used to analyze a mixture of three standard compounds with significantly different retention factors (k). This mixture was analyzed after (i) dissolution in solvents of varied compositions, and (ii) with progressively larger injection volumes. As analyte solvent composition and injection volume were changed, the most notable effects on peak shape were observed for the compounds with the smallest k values. Overall changes in peak shape were less pronounced when analyte solvent composition was similar to the starting mobile phase regardless of injection volume. Scale-up to semipreparative conditions yielded results consistent with those observed at the analytical scale. These data show that peak shape is greatly affected by analyte solvent composition and injection volume, and that these effects can be ameliorated by the dissolution of analytes in solvent that closely resembles that of the mobile phase used for initial run conditions. The following study addresses the concepts of peak elution in RP HPLC and how they factor into semipreparative purification.