Calculating Optimal Modulation Periods to Maximize the Peak Capacity in Two-Dimensional HPLC

Abstract

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