Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1289, Issue -, Pages 58-68Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.chroma.2013.03.008
Keywords
Comprehensive two-dimensional liquid chromatography (LC x LC); Hydrophilic interaction chromatography (HILIC); On-line; Off-line; Procyanidins; Stop-flow
Funding
- National Research Foundation (NRF, South Africa) [70995]
- International Foundation of Science (IFS, Sweden) [F/4904-1]
- SASOL
- Harry Crossley Foundation
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Comprehensive two-dimensional liquid chromatography (LC x LC) provides significantly improved separation for complex real-life samples. LC x 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) x 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 x LC methodologies is presented using HILIC x 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 x 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 x LC system was advantageous for required practical peak capacities up to similar 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 mm. In a separate contribution, the experimental verification of the findings of this study will be reported. (c) 2013 Elsevier B.V. All rights reserved.
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