The development of high speed separation systems for the miniaturization of liquid chromatography.

摘要

Miniaturization of chromatographic columns offers several advantages over large bore conventional analytical (2.1--4.6 mm ID) columns, typically used today. One such advantage is the increase in mass sensitivity attributed to the reduced column I.D., providing reduced dilution of the chromatographic band. In our lab we have constructed a triskelion instrument that can act as an HPLC, CEC or e-HPLC. The advantage of this configuration is that depending on the column architecture and sample complexity "tailor-made" separation schemes can be designed taking advantage of the either high temperature and/or the applied voltage. Mathematical models have been introduced to describe the effect of temperature on viscosity and analyte retention. In addition, models have been developed describing the quantitative relationships between the e-HPLC retention factor (k″) of charged analytes and the applied pressure and voltage. In the presences of an applied voltage intraparticle flow can become significant and reductions in the C-term of the Knox equations was accounted both experimentally and theoretically. Finally, the instrument was combined to a MALDI mass spectrometer by fraction collection, which allowed a protein digest to be separated and identified in approximately one minute.; In addition to the design of new instruments novel monolithic column designs were developed to eliminate unwanted electrostatic interactions and/or maintain high electroosmotic flow (EOF). To maintain high EOF a monolithic column was constructed that incorporated a charged monomer into the polymer backbone. This produced a column that maintained a high EOF at a broad pH range. The second type of column discussed was a duplex column that eliminated unwanted electrostatic interactions by separation of the charged EOF generating section of the column from the neutral separation section.; To characterize these newly developed monolithic a device was constructed to measure streaming current/potential, which enables measurement of column characteristics such as zeta potential, permeability, porosity, relative-hydrophobicity, and column efficiency. The above characteristics can be assessed using columns as short as 3cm allowing investigation of axial homogeneity. Surface conduction and the electroviscous effect were examined and corrections were made to assure the accuracy of the results.