Recent progress in online, comprehensive two-dimensional high-performance liquid chromatography for non-proteomic applications

AbstractComprehensive two-dimensional HPLC (2DLC) has been used very successfully in proteomics applications for over a decade. Increasingly we are seeing online, comprehensive 2DLC used for non-proteomic applications. This article gives an overview of the state of the art of this technique, with emphasis on current trends in theory and practice that are exciting and hold the most promise for advancing the performance of 2DLC. Specifically, the recently commercialized small, superficially porous packing materials are very well suited to use in the second dimension of online 2DLC where analysis speed is critical and largely dictates the performance of the 2DLC system. Further, the recent development of optimization schemes and associated software that support the use of different second-dimension elution modes in a single 2DLC analysis will improve the flexibility and effectiveness of 2DLC separations. Excluding separations of peptides, proteins, and polymers, 2DLC systems utilizing reversed-phase separation in one dimension, and either normal phase or reversed-phase in the other dimension continue to be the most popular. Although these systems have been applied mostly to complex biological materials, we are beginning to see applications in the analysis of pharmaceutical materials for ingredient purity and degradation profiling. The general lack of robust, easy-to-use commercially available software is arguably the greatest impediment to wider application of 2DLC methods. This situation is improving slowly, however, and at least two commercially available software packages have been described in the peer-reviewed literature. This is an exciting time in the development of online 2DLC. Online abstract figureTwo-dimensional HPLC separation of the low molecular weight constituents of a human urine sample, yielding several hundred chromatographic peaks. Reversed-phase HPLC columns and conditions are used in both dimensions, and the entire analysis is complete in 30 minutes.