Calculating optimal modulation periods to maximize the peak capacity in two-dimensional HPLC

Theoretical calculations are presented to optimize modulation period for maximum total peak capacity in comprehensive two-dimensional HPLC (2D-HPLC) taking into account the effect of modulation on the apparent peak capacity of the first-dimension (D-1) separation. Results indicate that modulation periods are most favorable when they are adjusted to similar to 2.2-4 times the standard deviation of a D-1 peak in order to avoid excessively short run times at the second dimension (D-2). Data are presented that effective peak capacities of several thousand in 60 min can be expected for practical 2D-HPLC conditions, utilizing D-1 gradient elution followed by D-2 isocratic elution, that remain at similar to 50-70% of the theoretical maximum peak capacity. This work suggests that lower modulation frequencies and longer D-2 separation times than previously proposed are favorable under realistic chromatographic conditions, alleviating some practical problems associated with 2D-HPLC.