Applications of monolithic silica capillary columns in proteomics

The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing very good resolution and reproducibility in addition to the known advantages of a low pressure drop over a time period of 6 months. Monoliths were subsequently tested for their suitability to separate proteins and peptides from samples typically encountered in proteomic research such as in-gel digested tryptic peptide mixtures or fractions of proteolytically digested human serum. The monolithic capillaries also proved useful in the analysis of phospholipid species in bronchoalveolar lavage fluid. Compared to particle-filled conventional capillary columns, rapid and highly efficient separation of peptides and proteins was achieved using these bimodal pore size distribution columns, and good quality collision induced dissociation (CID) mass spectra were obtained on an ion trap mass spectrometer. These novel monolithic separation media are thus a promising addition to the methodological toolbox of proteomics research

Fast, high-efficiency peptide separations on a 50-mu m reversed-phase silica monolith in a nanoLC-MS set-up

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-mu m reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETPmin = 5-10 mu m at 2.4 mm/s). Flow rates up to 1.95 mu L/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-mu m monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min. (c) 2006 Elsevier B.V. All rights reserved

CHIRAL CAPILLARY LIQUID CHROMATOGRAPHY BASED ON PENICILLIN G ACYLASE IMMOBILIZED ON MONOLITHIC EPOXY SILICA COLUMN

An epoxy derivatized monolithic silica capillary column (100 micron i.d.) was used as a support for immobilization of penicillin G acylase (PGA), an enzyme used in the production of semisynthetic antibiotics. In order to allow for sensitive UV detection, the PGA-based monolithic capillary column was coupled to an open fused-silica capillary via a TFE (Teflon\uae) shrink tube sleeve (1 cm long, 300 micron i.d.), which proved to be a robust, dead-volume free and easily replaceable connector. This configuration resulted in a duplex fritless column for capillary liquid chromatography (CLC) and electrically assisted CLC (eCLC). In particular, using the driving pressure (2\u201312 bar) supplied by the commercial CE instruments, CLC separations could be obtained in short time due to the low column backpressure of the monolith. In particular, the developed stationary phase characterized by the chiral recognition ability of PGA, was successfully applied in enantioseparation of arylpropionic acids of pharmaceutical interest (i.e., profens). As an example, by using a 7 cm long monolith capillary column, the enantioresolution (Rs > 3.0) of rac-ketoprofen was achieved in less than 2 min (pressure 12 bar) with a minimum plate height in the order of 20 micron and using as a mobile phase a 50 mM phosphate buffer pH 7.0. Validation data such as repeatability of retention time (intraday < 0.62, n = 6; interday < 1.62, n = 9; and column-to-column < 10.5, n = 2), linearity (r2 = 0.999), and sensitivity (LOQ 0.25% (w/w) of (R)-ketoprofen with respect to (S)-ketoprofen) showed good method performance. The method was successfully applied to the determination of (S)-ketoprofen in pharmaceutical samples (tablets)