title: Effects of the gradient profile, sample volume and solvent on the separation in very fast gradients, with special attention to the second-dimension gradient in comprehensive two-dimensional liquid chromatography.
abstract: Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions and therefore should be used in comprehensive two-dimensional LC×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×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.
authors.affiliation: Department of Analytical Chemistry, University of Pardubice, Studentská 573, 53210 Pardubice, Czech Republic. Pavel.Jandera@upce.cz
authors.forename: Pavel
authors.initials: P
authors.lastname: Jandera
authors.forename: Tomáš
authors.initials: T
authors.lastname: Hájek
authors.forename: Petr
authors.initials: P
authors.lastname: Cesla
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D001555:Benzene Derivatives / Q000737:chemistry; D055598:Chemical Phenomena; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008956:Models, Chemical
chemical_list: D000097:Acetonitriles; D001555:Benzene Derivatives; C032159:acetonitrile