1 mm ID poly(styrene-co-divinylbenzene) monolithic columns for high-peak capacity one- and two-dimensional liquid chromatographic separations of intact proteins.
The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.
Eeltink
Sebastiaan
S
Dionex, Amsterdam, The Netherlands. sebastiaan.eeltink@dionex.com
Dolman
Sebastiaan
S
Detobel
Frederik
F
Desmet
Gert
G
Swart
Remco
R
Ursem
Mario
M
D002256:Carbonic Anhydrases / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D029968:Escherichia coli Proteins / Q000302:isolation & purification*; D009211:Myoglobin / Q000302:isolation & purification; D011137:Polystyrenes / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000302:isolation & purification
D029968:Escherichia coli Proteins; D009211:Myoglobin; D011137:Polystyrenes; C432006:divinylbenzene-polystyrene copolymer; D012259:Ribonuclease, Pancreatic; D002256:Carbonic Anhydrases