Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1218, Issue 15, Pages 1995-2006Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2010.10.095
Keywords
Two-dimensional comprehensive LC x LC; Fast gradients; Porous shell particles; Sample volume; Sample solvent; Bandwidths; Band distortion
Funding
- Grant Agency of the Czech Republic [203/07/0641]
- Ministry of Education, Youth and Sports of the Czech Republic [MSM 0021627502]
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Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions and therefore should be used in comprehensive two-dimensional LC x LC, both in the first and in the second dimension, where, however, gradients are limited to a short time period available for separation, usually 1 min or less. Gradient conditions spanning over a broad mobile phase composition range in each second-dimension fraction analysis are used with generic full in fraction (FIF) gradients. Segment in fraction (SIF) gradients cover a limited gradient range adjusted independently to suit changing lipophilicity range of compounds transferred to the second dimension during the first-dimension gradient run and to provide regular coverage of the two-dimensional retention space. Optimization of the gradient profiles is important tool for achieving high two-dimensional peak capacity and savings of the separation time in comprehensive LC x LC. Calculations based on the well-established gradient-elution theory can be used to predict the elution times and bandwidths in fast gradients, taking into account increased probability of pre-gradient or post-gradient elution. The fraction volumes transferred into the second dimension may significantly affect the second-dimension bandwidths, especially at high elution strength of the fraction solvent, which may cause even band distortion or splitting in combined normal-phase (HILIC)-RP systems, but also in some two-dimensional RP-RP systems. In the present work, the effects of the fast gradient profile, of the sample volume and solvent on the elution time and bandwidths were investigated on a short column packed with fused-core porous-shell particles, providing narrow bandwidths and fast separations at moderate operating pressure. (C) 2010 Elsevier B.V. All rights reserved.
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