Comparison of the practical resolving power of one- and two-dimensional high-performance liquid chromatography...

Two-dimensional liquid chromatography (2DLC) has become a mainstay of proteomics research due to its higher peak capacity compared to one-dimensional LC (1DLC). Because of the long analysis times typically associated with 2DLC (tens of hours) and its use primarily in proteomics applications, 2DLC in the context of general HPLC has been regarded as a niche technique for use in analysis of mixtures containing hundreds to thousands of components compared to the far more common techniques of isocratic and gradient elution 1DLC. A significant next step in the analytical development of 2DLC is to consider using its higher resolving power to reduce the analysis time of rather "simple" mixtures, in the range of only tens to hundreds of chemical constituents. The chief objective of this paper is to provide guidance to practitioners who need to decide whether 1DLC or 2DLC gives the superior separation in a given analysis time. Conditional peak capacities are predicted for fully optimized 1DLC and practical 2DLC separations of the low molecular weight constituents of an extract of corn seed at several analysis times using a model based on the chromatographic properties of compounds that are representative of real mixtures of lower molecular weight species. Two important corrections to the ideal 2DLC peak capacity are made to account for both incomplete usage of the separation space and the serious effect of first-dimension undersampling; this allows, we believe for the first time, a fair comparison of the resolving power of 1D- and 2DLC under realistic conditions. The predicted optimum conditions are then used to carry out experimental separations of low molecular weight corn seed extract, and peaks are counted in each 1D- and 2DLC chromatogram. Based on comparisons of both the predicted peak capacities and number of peaks observed in experimental chromatograms, we believe that practical 2DLC will be superior to fully optimized gradient 1DLC for separations lasting more than only about 10 min. This crossover time is much shorter than intuitively expected, and we believe this finding will inevitably have a major impact on the practice of 2DLC in liquid-phase separations in general.