No abstract

A solution of methacryloxypropyltrimethoxysilane in the presence of an acid catalyst, water, toluene, and a photoinitiator was irradiated at 365 nm for 5 min in a 75-microm i.d. capillary to prepare a porous monolithic sol-gel column by a one-step, in situ, process. The photopolymerized sol-gel (PSG) column shows reversed-phase behavior. Using this column, a variety of low-molecular-weight neutral compounds, including polycyclic aromatic hydrocarbons, alkyl benzenes, alkyl phenyl ketones, and steroids are separated from mixtures. Various different operational parameters, such as buffer composition, field strength, and column temperature, were varied to assess their influence on column performance. Use of PSG as a stationary phase for a pressure-driven separation is also demonstrated.

Using sol-gel technology, a porous glass matrix (xerogel) is formed in a capillary column and acts as a support for a stationary phase of chromatographic particles used in capillary electrochromatography. Preparation of the sol-gel matrix and immobilization of the octadecylsilica (ODS) stationary phase occur in a single step. The presence of the particles in the column greatly reduces matrix cracking caused by internal pressure differentials within the pores of the sol-gel matrix. Good electroosmotic flow is achieved in part because of the inherent negative charge of both the particles and the sol-gel matrix. The performance of these sol-gel/ODS capillary columns was evaluated with a mixture of aromatic and nonaromatic organic compounds. Efficiencies of up to 80 000 plates/m were observed in columns with immobilized 3-μm ODS particles. The efficiency and resolution are enhanced when 3-μm ODS particles are used in place of the 5-μm particles.

Macroporous polymer frits have been fabricated in fused-silica capillaries by the UV photopolymerization of a solution of glycidyl methacrylate and trimethylopropane trimethacrylate. This in situ preparation is a simple, rapid, and reproducible process. The frit can be placed at any desired position along the column. Photopolymer frits can withstand the short exposure to a high pressure (over 6000 psi). Bubble formation is no observed to occur with these frits under our experimental conditions. By choice of porogens, it is possible to control the porous properties. The use of such frits in capillaries to retain particles of chromatographic packing has been demonstrated to be stable and robust with continuous operation over 3 days.

Trypsin is covalently linked to a photopolymerized sol-gel monolith modified by incorporating poly(ethylene glycol) (PSG-PEG) for on-column digestion of N(alpha)-benzoyl-l-arginine ethyl ester (BAEE) and two peptides, neurotensin and insulin chain B. The coupling of the enzyme to the monolith is via room-temperature Schiff chemistry in which an alkoxysilane reagent (linker) with an aldehyde functional group links to an inactive amine on trypsin to form an imine bond. The proteolytic activity of the immobilized trypsin was measured by monitoring the formation of N alpha-benzoyl-L-arginine (BA), the digestion product of BAEE. The BA is separated from BAEE by capillary electrophoresis and detected downstream (18.5 cm from the microreactor) by absorption (254 nm). Using the Bradford assay, we determined that 97 ng of trypsin is bound to the 1-cm microreactor located at the entrance of capillary column. The bioactivity of the trypsin-PSG-PEG microreactor at 20 degrees C for the digestion of BAEE was found to be 2270 units/mg of immobilized trypsin. The bioactivity of trypsin bound to the capillary wall in the open segment upstream from the monolith was 332 units/mg of immobilized trypsin under the same conditions. In contrast, the activity of free trypsin could not be observed for the digestion of BAEE at 20 degrees C after 16 h of incubation time.