Sense and nonsense of shifting gradients in on-line comprehensive reversed-phase LC × reversed-phase LC.

In on-line comprehensive two-dimensional liquid chromatography (LC × LC), the combination of similar chromatographic modes such as reversed-phase liquid chromatography × reversed-phase liquid chromatography (RPLC × RPLC) usually leads to the partial occupation of the available separation space. A possible solution to circumvent this issue may be to dynamically adjust the gradient elution in the second dimension (D) throughout the LC × LC analysis. This allows the gradient elution to be tailored to the elution conditions of each fraction instead of using a conventional full gradient program in which the same gradient profile is repeated for each D-fraction. In this study, an online RPLC × RPLC method is optimized with shifting gradients in D. The logic behind implementing such programs in on-line LC × LC is explained. The optimized method consists of a combination of segmented and shifted gradient modes. It is shown that the retention space coverage can be increased by 50 % compared to a conventional full gradient program, leading to a significant increase in peak capacity (about 35 %). However, such an increase comes at the expense of larger peak widths in D and thus lower peak intensities. It is shown here that the use of shifting gradients raises another serious issue related to the repeatability of retention times between two successive D-separations.