Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations

The separation of samples that contain more than 15 to 20 analytes (n>15-20 ) is typically difficult and usually requires gradient elution. We have exa mined the reversed-phase liquid chromatographic separation of 24 samples wi th 8 less than or equal to n less than or equal to 48 as a function of temp erature T and gradient time t(G). The required peak capacity was determined for each sample, after selecting T and t(G) for optimum selectivity and ma ximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (whe n T and t(G) have been optimized). These results were also compared with li terature studies of similar isocratic separations as a function of ternary- solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn p rovides information on the relative effectiveness of these two different me thod development procedures (optimization of T and t(G) vs. % MeOH and % TH F) for changing selectivity and achieving maximum resolution. (C) 1999 Else vier Science B.V. All rights reserved.