The history of HPLC has seen a progression in the use of columns packed with particles of decreasing size. Decreasing particle size has led to smaller values of the plate height and faster optimum velocities. Due to pressure limitations of existing HPLC equipment however, this trend has translated, not into columns of increasing separation efficiency, but instead, into columns offering faster analysis times. The 400 bar pressure limit of current HPLC technology is an arbitrary limit. The use of ten to ten-fold higher pressure allows the use of columns 40 cm long, packed with 1 micron particles, delivering 250,000 theoretical plates with column void times of 3 minutes.
Hardware (pumps, valves, injectors, connecting tubing, columns) must be made which can withstand such high pressure while in contact with solvents ranging from aqueous salt solutions to polar and non-polar organic solvents. Significant amounts of heat can be generated in pumping solvents at optimum velocities through such a highly restrictive bed of particles. In a column of conventional diameter (4.6 mm), this heat will result in axial and radial temperature gradients, which will lead to excessive band spreading. Packed capillary columns can be used to reduce this difficulty. Analyte distribution coefficients are also a function of pressure. This might result in inconvenient and/or confusing changes in relative retention times of analytes as a function of operating pressure. The design and performance of a system capable of gradient elution liquid chromatography in packed capillary columns at pressures in excess of 7,000 bar (100,000 PSI) will be described. Results of the separations of small organics, peptides, and proteins using this system, and its coupling to a mass spectrometer will also be described.
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