Selection of column dimensions and gradient conditions to maximize the peak-production rate in comprehensive off-line two-dimensional liquid chromatography using monolithic columns

The peak-production rate (peak capacity per unit time) in comprehensive off-line two-dimensional liquid chromatography (LC/x/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A first-dimension (D-1) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the D-1 fractions on a monolithic column operating at a pH of 2. To obtain the highest peak-production rate, effects of column length, gradient duration, and sampling time were examined. RP/x/RP was performed at undersampling conditions using a short 10 min D-1 gradient. The peak-production rate was highest using a 50 mm long D-2 column applying an 8-10 min D-2 gradient time and was almost a factor of two higher than when a 250 mm monolithic column was used. The best way to obtain a higher peak-production rate in off-line LC/x/LC proved to be an increase in the number of D-1 fractions collected. Increasing the D-2 gradient time was less effective. The potential of the optimized RP/x/RP method is demonstrated by analyzing proteomics samples of various complexities. Finally, the trade-off between peak capacity and analysis time is discussed in quantitative terms for both one-dimensional RP gradient-elution chromatography and the off-line two-dimensional (RP/x/RP) approach. At the conditions applied, the RP/x/RP approach provided a higher peak-production rate than the D-1-LC approach when collecting three D-1 fractions, which corresponds to a total analysis time of 60 min.