New technologies for ultra-high performance liquid chromatography
Brice, Richard W.
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The synthesis of a hybrid organo-silica material containing an octyl moiety has been the focus of our laboratory. Studies of the ammonia saturation effects showed that final volumes of solution above 200 mL would produce particles of roughly equal size and reproducibility. Studies of the stir bar speed indicated that using higher stir speeds improved the batch to batch reproducibility (down to 6% RSD). Studies of the final bath temperature indicated that careful control over the temperature profile throughout the synthesis allowed for size tunability as well as improved batch to batch reproducibility (5% RSD). Previous studies by our laboratory have shown that our in-house synthesized hybrid organo-silica particles containing an octyl moiety offered chromatographically useful columns possessing high pH stability and long retention. A comparison of the chromatographic performance of the C8 hybrid organo-silica material with commercially available Kovasil C8 and C18 materials was performed. For chromatographic testing the C8 hybrid organo-silica material needed to be packed into microbore columns. Studies of various slurry solvents indicated that the best solvents for packing this material will be either dichloromethane or a mixture of 70% hexane/30% acetone. Studies of the effect of the hit pressure indicated that the hit pressure will be dictated not by the optimal value but the pressure limitations of the packing adapter currently being employed. In an attempt to equalize the particle velocity, packing studies using constant flow rate were performed and compared with columns packed using constant pressure methodology. These studies revealed little improvement when packing with the constant flow rate mode. Comparison of the columns performance showed a 5 fold increase in retention as well as a 4 fold increase in the mass loadability for our hybrid material over the commercial materials tested. Three different HPLC column technologies (i.e., monolith, fused core particles, and sub-2 μm particles) were evaluated, comparing vanDeemter plots, speed of analysis, column back pressure, and mobile phase consumption. The columns were also used to separate model neutral and basic compounds. A monolithic column offered very high linear velocities (∼12 mm/sec) with modest pressure requirements (∼1,600 psi or 110bar) at the expense of higher mobile phase consumption, when compared with the particle packed columns. The monolithic column exhibited similar plate heights to those obtained with a 3μm-particle packed column (i.e., ∼8 μm) when operated at optimal linear velocities. The monolithic column showed a substantially lower mass transfer term dependence. The 2.7 μm fused-core packing material yielded only slightly lower efficiency than that observed with the sub-2 μm material, while maintaining a working back pressure under 9,000 psi (620 bar) instead of the up to 15,000 psi (1,030 bar) required for the sub-2 μm material. The fused-core material was able to achieve linear velocities as high as the maximum attained on the sub-2 μm materials (∼5 mm/sec) while staying within the 6,000 psi typically available on most HPLC systems. All of the columns were able to separate a series of basic analytes under various pH conditions with the fused-core yielding the highest efficiencies and best peak shape followed by the 1.7 μm BEH material and then finally the monolithic material. The limited pH stability range on the monolithic material prohibited it from being able to baseline separate a mixture of tricyclic antidepressants under the tested condition. We have evaluated columns packed with two new materials; one is a 2.5 μm totally porous C18 silica (Luna®) and the other is a 2.7 μm superficially porous C18 silica (Halo(TM)). Performance testing of these materials indicated efficiencies of ∼218,000 Plates/Meter on the superficially porous material and ∼180,000 Plates/Meter on the totally porous material. Studies of the crush pressures for these materials showed that the fused-core material could be operated at pressure up to 16,000 p.s.i. (1100 bar) creating theoretically attainable efficiencies of ∼120,000 plates when columns are coupled together verses 8,000 p.s.i. (550 bar) and ∼51,000 plates on the totally porous material. Chromatographic selectivity studies show similar performance on these materials for both the methylene selectivity and silanol activity. The mass loabability of a superficially porous material relative to a totally porous material is often a concern to the chromatographer. This study found similar mass load tolerances (∼11 ug) between these two materials when packed in columns in the 2 mm i.d. regime. (Abstract shortened by UMI.)