Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography×reversed phase liquid chromatographic analysis of procyanidins. Part II: application to cocoa procyanidins.

Procyanidins present a severe analytical challenge due to their structural complexity and diversity. Comprehensive two-dimensional hydrophilic interaction chromatography×reversed-phase liquid chromatography (HILIC×RP-LC) provides a highly efficient separation system for procyanidins. In the first part of this contribution a systematic approach for the optimisation and evaluation of HILIC×RP-LC analyses in on-line, off-line and stop-flow modes was presented. The three systems were compared in terms of peak capacity, the number of peaks produced per unit time as well as the total analysis time required to carry out such analyses by taking under-sampling, degree of orthogonality and stop-flow band-broadening into account. In this paper, the experimental verification of these findings using cocoa procyanidins as an application is presented. The results show that while optimisation procedures based on theoretical considerations remain largely valid in practice, several important experimental considerations should also be taken into account to achieve maximum performance in all three modes of HILIC×RP-LC. On-line analysis provides an effective tool for the screening of procyanidin content within reasonable times, provided that under-sampling of first dimension peaks is minimised. Off-line- and stop-flow HILIC×RP-LC analyses on the other hand are more suited for the detailed analysis of complex procyanidin fractions, with the latter being shown to be a promising automated alternative providing the same performance as the off-line approach. Experimental data presented verify the conclusion that stop-flow operation has a negligible effect on first dimension band broadening under the optimised experimental conditions used.