Comprehensive two-dimensional liquid chromatography (2DLC) offers a number of practical advantages over optimized one-dimensional LC in peak capacity and thus in resolving power. The traditional " product rule" for overall peak capacity for a 2DLC system significantly overestimates peak capacity because it neglects under-sampling of the first dimension separation. Here we expand on previous work by more closely examining the effects of the first dimension peak capacity and gradient time, and the second dimension cycle times on the overall peak capacity of the 2DLC system. We also examine the effects of re-equilibration time on under-sampling as measured by the under-sampling factor and the influence of molecular type (peptide vs. small molecule) on peak capacity. We show that in fast 2D separations (less than 1. h), the second dimension is more important than the first dimension in determining overall peak capacity and conclude that extreme measures to enhance the first dimension peak capacity are usually unwarranted. We also examine the influence of sample types (small molecules vs. peptides) on second dimension peak capacity and peak capacity production rates, and how the sample type influences optimum second dimension gradient and re-equilibration times.
Bibliographical noteFunding Information:
This work was supported by a grant from the National Institutes of Health (Grant GM54585 ), Fellowship from the U.S. Pharmacopeia for X.L., Fellowship from the American Chemical Society Division of Analytical Chemistry and Faculty Start-Up Award from the Camille and Henry Dreyfus Foundation to D.R.S., and gifts from MacMod Analytical and the Agilent Foundation.
- 2D on-line comprehensive chromatography
- Cycle times
- Gradient elution
- Isocratic elution
- Multidimensional separations
- Peak capacity
- Sampling times