Towards a comprehensive 2-D-LC-MS separation.
A comprehensive 2-D-LC-MS method has been developed by coupling columns of different selectivity. The primary column eluate is alternately trapped and sampled onto the secondary columns through a guard column interface. When one guard column traps the eluate, the other injects the previously trapped components onto a secondary column. This cycle is repeated throughout the chromatogram. The use of dual secondary columns provides the secondary columns with additional time to generate high-speed chromatograms. Each secondary column generates alternate chromatograms which when combined generate the entire chromatogram. The primary column separation is comparable to conventional HPLC, whereas the secondary column separation is fast. With both the columns operating in reverse phase mode, one would expect strong correlation in the two-dimensional retention and hence inefficiency in separation. However, differences in column operation modes, interaction mechanisms, and vendor silica result in a complementary separation. The system was evaluated by comparing it to one-dimensional counterparts and coupled column chromatography. Although some correlations were observed in 2-D-LC-MS, peaks do show two-dimensional distribution with superior UV and MS data as co-elution is minimized. Also, the ease of converting conventional systems to 2-D-LC-MS is discussed.