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The characterization and evaluation of three novel 5-microm HPLC column packings, prepared using ethyl-bridged hybrid organic/inorganic materials, is described. These highly spherical hybrid particles, which vary in specific surface area (140, 187, and 270 m(2)/g) and average pore diameter (185, 148, and 108 A), were characterized by elemental analysis, SEM, and nitrogen sorption analysis and were chemically modified in a two-step process using octadecyltrichlorosilane and trimethylchlorosilane. The resultant bonded materials had an octadecyl surface concentration of 3.17-3.35 micromol/m(2), which is comparable to the coverage obtained for an identically bonded silica particle (3.44 micromol/m(2)) that had a surface area of 344 m(2)/g. These hybrid materials were shown to have sufficient mechanical strength under conditions normally employed for traditional reversed-phase HPLC applications, using a high-pressure column flow test. The chromatographic properties of the C(18) bonded hybrid phases were compared to a C(18) bonded silica using a variety of neutral and basic analytes under the same mobile-phase conditions. The hybrid phases exhibited similar selectivity to the silica-based column, yet had improved peak tailing factors for the basic analytes. Column retentivity increased with increasing particle surface area. Elevated pH aging studies of these hybrid materials showed dramatic improvement in chemical stability for both bonded and unbonded hybrid materials compared to the C(18) bonded silica phase, as determined by monitoring the loss in column efficiency through 140-h exposure to a pH 10 triethylamine mobile phase at 50 degrees C.

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Pepsin was immobilized on ethyl-bridged hybrid (BEH) particles and digestion performance was evaluated in a completely online format, with the specific intent of using the particles for hydrogen deuterium exchange mass spectrometry (HDX MS) experiments. Because the BEH particles are mechanically strong, they could withstand prolonged, continuous high-pressure at 10,000 psi. Online digestion was performed under isobaric conditions with continuous solvent flow, in contrast to other approaches where the pressure or flow is cycled. As expected, digestion efficiency at 10,000 psi was increased and reproducibly produced more peptic peptides versus digestion at 1,000 psi. Prototype columns made with the BEH pepsin particles exhibited robust performance and deuterium back-exchange was similar to that of other immobilized pepsin particles. These particles can be easily incorporated in existing HDX MS workflows to provide more peptide coverage in experiments where fast, efficient, and reproducible online pepsin digestion is desired.