Comparison of various second‐dimension gradient types in comprehensive two‐dimensional liquid chromatography

Abstract

Gradient elution provides significant improvement in peak capacity with respect to isocratic conditions. In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC x LC are compared: (i) "full in fraction", (ii) "segment in fraction" and (iii) "continuously shifting" gradients, applied in orthogonal LC x LC separations of phenolic acids and flavones on a polyethylene glycol column in the first dimension and two types of porous shell fused-core C18 columns in the second dimension (Ascentis Express and Kinetex). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated. The type of gradient program controls the range of lipophilicity of sample compounds that can be separated in the second-dimension reversed-phase time period. This range can be calibrated using alkylbenzene standards, to design the separation conditions for complete sample separation, avoiding harmful wrap around of non-eluted compounds to the subsequent second-dimension fractions.

Comprehensive two-dimensional liquid chromatography–tandem mass spectrometry for the simultaneous determination of wine polyphenols and target contaminants

“…The LC×LC separation of wine polyphenols was optimized using the standard mixture, at first, in There are some examples in the literature demonstrating how a tailored 2 D gradient program leads to a greater coverage of the available separation space [18,33]; the total analysis time may also be reduced [34] and the modulation time reduced accordingly, resulting in a better fractionation of the a narrower organic solvent range was adopted, spanning from 0% to 20%, so that retention was stronger on the 2 D column, and the separation improved.…”

Strategies in the Analysis of Plant Flavonoids

“…In these systems, it is preferable to use the more polar of the two columns in the first dimension and the more retentive (usually a bonded alkylsilica) in the second dimension, so as to suppress band broadening connected with the sample transfer (Jandera, Hajek, and Cesla, 2010). Hajek et al (2008) combined a polyethylene glycol (PEG) column in the first dimension with different alkylsilica stationary phases in the second dimension for the separation of 27 antioxidant polyphenols including various flavonoids, concluding that monoliths and SPP columns provided lower hold-up time and improved resolution in the second dimension than totally porous columns of similar length.…”

Determination of Pesticides in Complex Samples by One Dimensional (1D-), Two-Dimensional (2D-) and Multidimensional Chromatography

“…In the second dimension, gradients are limited to a short-time period available for separation. Various types of second-dimension gradients in comprehensive LC x LC are compared: (i) ''full in fraction'', (ii) ''segment in fraction'' and (iii) ''continuously shifting'' gradients, applied in orthogonal LC x LC separations was described by Jandera et al (Jandera et al, 2010). The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations.…”

“…The porous shell columns provide narrow bandwidths and fast second-dimension separations at moderate operating pressure that allows important savings of the overall separation time in comprehensive LC x LC separations. The effects of the gradient type on the bandwidths, theoretical peak capacity, separation time and column pressure in the second dimension were investigated by these authors (Jandera et al, 2010). The coupled-column (LC-LC) configuration consisting of a 3 µm C column (50x4.6 mm I.D.)…”