Analysis of unresolved chromatograms by the absorbance ratio and sequential chromatogram ratio techniques coupled with peak suppression

Abstract

The sequential chromatogram ratio (SCR) technique was successively applied or was coupled with the absorbance ratio (AR) technique to facilitate analysis of a peak of interest that was overlapped with two other peaks. First, the AR or SCR technique was used to suppress one undesired peak. This created or elongated a region of pure-component elution for the peak of interest. Then the appropriate ratio technique was applied to this region for qualitative and quantitative analysis. The AR technique allows suppression and qualitative analysis of unknown components via the absorptivity ratio. For the SCR technique, peak identity can be deduced prior to suppression and quantitative analysis. A statistical peak matching procedure that employs user-selected standards is described for the SCR technique, by which components in sequentially injected samples may be identified based on differences in retention time, t R, or in diffusion coefficient, D m, which controls peak width. For two similarly retained analytes in sequentially injected samples, having a factor-of-two difference in D m, the problem of reduced resolution, R s, with a third peak was investigated by manipulating the data to simulate a reduction in selectivity factor. Below a limit, R s = 0.38, the original two analytes could no longer be qualitatively distinguished. At this same R s limit, the two-times difference in D m, for two analytes having essentially zero t R-based resolution, provided equivalent qualitative discrimination of peaks as a t R-based resolution of 0.12 for two analytes having equal D m. The classical problems of inaccurate baseline correction and run-to-run retention variation were examined, and the latter was more limiting for the SCR technique. Still, small t R shifts were adequately corrected by selecting and aligning a common peak in sequential chromatograms.