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 ((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.
Eeltink
Sebastiaan
S
Department of Chemical Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. seeltink@vub.ac.be
Dolman
Sebastiaan
S
Vivo-Truyols
Gabriel
G
Schoenmakers
Peter
P
Swart
Remco
R
Ursem
Mario
M
Desmet
Gert
G
D002853:Chromatography, Liquid / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D011137:Polystyrenes / Q000737:chemistry; D018892:Proton-Motive Force
D010455:Peptides; D011137:Polystyrenes; C432006:divinylbenzene-polystyrene copolymer