Technology for ultra-high pressure liquid chromatography
Anspach, Jason A
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The evolution of chromatography has led to the reduction in the size of the materials used as column packing. As one reduces the size of the packing material, there is an increase in the efficiency and a decrease in the analysis time. The inter-particle space, however, is also decreased, leading to an increase in the overall flow resistance. It is therefore necessary to increase the pressure used to drive the mobile phase through the column. Commercial HPLC equipment, until recently, has been limited to operating pressures of about 5000 p.s.i. (350 bar). At the time of this investigation, columns packed with sub 2 μm materials were not commercially available. We therefore built a packing system, and developed packing methods that enabled us to pack columns with both porous and non-porous 1.5 μm materials. While other methods to drive the mobile phase through columns packed with sub 2 μm materials has been proposed, the use of pressure is still the most popular choice. When moving mobile phase through columns at high pressures, there is a generation of frictional heat. Previous investigations have proposed that the use of capillary columns (<100 μm i.d.) were necessary. We investigated using 1--1.5 mm i.d. columns in ultrahigh pressure HPLC (UHPLC) and found that there was no net decrease in the retention factor at pressures up to 20,000 p.s.i. (1380 bar) as would be expected in cases where there was an excess of heat generated in the column. We compared columns packed with 1.5 μm packing materials with columns that were packed with 3 μm packing materials and found that by reducing the particle size we were able to double the separation efficiency while decreasing the overall analysis time by a factor of two. We went on to investigate a new injection system for use in UHPLC which was capable of performing injections at 40,000 p.s.i. (2750 bar). We found that the peak area RSD for the new injection system was lower than that of a conventional pressure balanced injection system. We also found that there was only minimal sample carryover.