We report on a new approach to obtain highly homogeneous silica-monolithic columns, applying a sol-gel fabrication process inside a rectangular pillar-array column (1 mm in width, 29 microm in height and 33.75 mm in length) having a cross-sectional area comparable to that of a 200 microm diameter circular capillary. Starting from a silicon-based pillar array and working under high phase-separation-tendency conditions (low poly(ethylene glycol) (PEG)-concentration), highly regular silica-based chromatographic systems with an external porosity in the order of 66-68% were obtained. The pillars, 2.4 microm in diameter, were typically clad with a 0.5 microm shell layer of silica, thus creating a 3.4 microm total outer pillar diameter and leaving a minimal through-pore size of 2.2 microm. After mesopore creation by hydrothermal treatment and column derivatization with octyldimethylchlorosilane, the monolithic column was used for chip-based liquid-chromatographic separations of coumarin dyes. Minimal plate heights ranging between 3.9 microm (nonretaining conditions) and 6 mum (for a retention factor of 6.5) were obtained, corresponding to domain-size-reduced plate heights ranging between 0.7 and 1.2. The column permeability was in the order of 1.3 x 10(13) m(2), lower than theoretically expected, but this is probably due to obstructions induced by the sol-gel process in the supply channels.
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.
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