Abstract
This study has investigated the production of poly(styrene/divinylbenzene) monolithic columns for use in micro LC for protein separations. The 180 µm I.D. x 6 cm poly(styrene/divinylbenzene) monolithic columns were tested for porosity measured as backpressure before a selection of the columns were used for chromatographic separation of proteins utilizing a mobile phase consisting of water and acetonitrile, with trifluoroacetic acid. The best preparation method for the monolithic columns were as follows: A solution consisting of 20 % (v/v) styrene, 20 % (v/v) divinylbenzene, 52 % (v/v) 1-decanol, 8 % (v/v) tetrahydrofuran and 8 mg/ml α,ά-azo-isobutyronitril were injected into a pre-treated and silanized capillary. The capillary ends were then sealed and the capillary was placed in an oven with 70 C for 24h. Finally the capillary ends were cut of and the capillary was cut into segments of 7 cm to be flushed with acetonitrile.
The columns were shown to be suitable for the separation of the model proteins utilized in this study (12.327 -66.000 Da). In addition, sol-gel bonded 7 µm (4000Å) endcapped C18, Nucleosil 5 µm (300Å) diol, YMC 5 µm (300Å) C4, YMC 5 µm (300Å) C8, and microparticles based on melamine resin were tested for their suitability to separate proteins utilizing mobile phases consisting varying amounts of water and acetonitrile, with trifluoroacetic acid. Among these columns the YMC C8 and C4 columns were found to be suitable for use in protein separation. The protein chromatography of the poly(styrene/divinylbenzene) monolithic columns and the C8 and C4 columns were found to be, with some selectivity differences, similar in respect of peak shapes