Optimization of separation in two-dimensional high-performance liquid chromatography by adjusting phase system selectivity and using programmed elution techniques.

The overall peak capacity in comprehensive two-dimensional liquid chromatographic (LC x LC) separation can be considerably increased using efficient columns and carefully optimized mobile phases providing large differences in the retention mechanisms and separation selectivity between the first and the second dimension. Gradient-elution operation and fraction-transfer modulation by matching the retention and the elution strength of the mobile phases in the two dimensions are useful means to suppress the band broadening in the second dimension and to increase the number of sample compounds separated in LC x LC. Matching parallel gradients in the first and second dimension eliminate the necessity of second-dimension column re-equilibration after the independent gradient runs for each fraction, increase the use of the available second-dimension separation time and can significantly improve the regularity of the coverage of the available retention space in LC x LC separations, especially with the first- and second-dimension systems showing partial selectivity correlations.