In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC×LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC×LC chromatograms, contours plots, and fcoverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP×RP.
Bibliographical noteFunding Information:
This work was supported by grants from the NIH ( GM 054585 ) and the NSF ( CHE-0911516 ). The authors would like to thank Agilent Technologies, Supelco, and ZirChrom Separations for their generous donation of columns. In addition, the authors acknowledge Prof. Jerry Cohen's research group, Prof. Devin G. Peterson's research group, and Dr. Fred Schendel all at the University of Minnesota for providing several samples used in this study.
- Carbon stationary phases
- Hydrophobic subtraction method
- LC×LC method development
- Reversed phase
- Two dimensional liquid chromatography