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  • Title: Selection of column dimensions and gradient conditions to maximize the peak-production rate in comprehensive off-line two-dimensional liquid chromatography using monolithic columns.
    Author: Eeltink S, Dolman S, Vivo-Truyols G, Schoenmakers P, Swart R, Ursem M, Desmet G.
    Journal: Anal Chem; 2010 Aug 15; 82(16):7015-20. PubMed ID: 20666432.
    Abstract:
    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 ((1)D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the (1)D 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 (1)D gradient. The peak-production rate was highest using a 50 mm long (2)D column applying an 8-10 min (2)D 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 (1)D fractions collected. Increasing the (2)D 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 (1)D-LC approach when collecting three (1)D fractions, which corresponds to a total analysis time of 60 min.
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