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/×/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A first-dimension (1D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the 1D 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/×/RP was performed at undersampling conditions using a short 10 min 1D gradient. The peak-production rate was highest using a 50 mm long 2D column applying an 8−10 min 2D 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/×/LC proved to be an increase in the number of 1D fractions collected. Increasing the 2D gradient time was less effective. The potential of the optimized RP/×/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/×/RP) approach. At the conditions applied, the RP/×/RP approach provided a higher peak-production rate than the 1D-LC approach when collecting three 1D fractions, which corresponds to a total analysis time of 60 min.