Systematic optimisation and evaluation of on-line, off-line and stop-flow comprehensive hydrophilic interaction chromatography × reversed phase liquid chromatographic analysis of procyanidins, Part I: Theoretical considerations
Stellenbosch University, Department of Chemistry and Polymer Science, Private Bag X1, Matieland 7602, South Africa
Kalili, K.M., Stellenbosch University, Department of Chemistry and Polymer Science, Private Bag X1, Matieland 7602, South Africa; De Villiers, A., Stellenbosch University, Department of Chemistry and Polymer Science, Private Bag X1, Matieland 7602, South Africa
Comprehensive two-dimensional liquid chromatography (LC × LC) provides significantly improved separation for complex real-life samples. LC × LC can be performed in one of three different ways, using on-line, off-line, or stop-flow configurations. We have previously shown how off-line comprehensive hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography (RP-LC) provides a powerful separation system for procyanidins (PCs), one of the most complex fractions of natural phenolics. In the current contribution, a systematic approach for the optimisation and evaluation of each of the LC × LC methodologies is presented using HILIC × RP-LC analysis of PCs as application. Optimisation was performed using the peak capacities of individual one-dimensional separations measured for different gradient times and flow rates and their combination in each of the three LC × LC modes by taking into account the effects of first dimension under-sampling, the degree of orthogonality between the two dimensions and additional band broadening associated with stop-flow analysis. The performance of all three methods is compared in terms of practical peak capacities, analysis times and peak production rates. One-dimensional LC provided the best performance for separations requiring relatively low peak capacities, whereas the on-line LC × LC system was advantageous for required practical peak capacities up to ~600. For higher resolution, the off-line or stop-flow systems should be used. Especially noteworthy is the fact that, due to slow diffusion of PCs, the contribution of stop-flow to first dimension band broadening was negligible for stop-flow times of up to 15. min. In a separate contribution, the experimental verification of the findings of this study will be reported. © 2013 Elsevier B.V.
Comprehensive two-dimensional liquid chromatography; Hydrophilic interaction chromatography; Off-line; On-line; Procyanidins; Stop-flow; Flavonoids; Optimization; Two dimensional; Liquid chromatography; catechin; epicatechin; procyanidin derivative; article; cacao; diffusion; flow rate; hydrophilic interaction chromatography; intermethod comparison; mathematical analysis; nonhuman; online system; priority journal; process optimization; reversed phase liquid chromatography; separation technique; Chromatography, High Pressure Liquid; Chromatography, Liquid; Chromatography, Reverse-Phase; Hydrophobic and Hydrophilic Interactions; Models, Theoretical; Proanthocyanidins