Anal. Chem. 2005, 77, 7489-7494
Injection Valve for Ultrahigh-Pressure Liquid Chromatography Jason A. Anspach,† Todd D. Maloney,‡ Richard W. Brice,† and Luis A. Colo´n*,†
Department of Chemistry, University at Buffalo, The State University of New York, Natural Science Complex, Buffalo, New York 14260, and Michigan Laboratories, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105
The increased interest in HPLC at elevated pressures, beyond the conventional 6000 psi (400 bar), has created a demand for injection systems capable of withstanding pressures beyond the 20 000 psi (1380 bar). To achieve high-resolution separations, an appropriate length of columns packed with sub 2-µm packing materials, a 30 000-40 000 psi (2070-2760 bar) pressure range is desirable. A new air-actuated needle valve injection system rated to withstand pressures of up to 40 000 psi (2760 bar) has been evaluated. Under isocratic chromatographic conditions, injecting 200 nL and operated at ∼20 000 psi (1380 bar), the system showed a peak area reproducibility of ∼2.5% RSD, contrasting the 5% RSD of a pressured-balanced injection system operated under similar conditions. Programmed for partial loop injections using injection times of 300-700 ms (injection volumes in the range of 1-2.5 µL) and operated at pressures close to 30 000 psi (2070 bar), the reproducibility in peak area for the amounts injected was ∼1.5% RSD or lower, while an injection time of 100 ms resulted in a reproducibility of 3-4% RSD. The new injection system did not show any significant carryover, and after thousands of injections, the system has not shown sign of wear, loss of pressure during injection, or loss in chromatographic performance. The reduction in size of the chromatographic packing material used in liquid chromatography leads to an increase in separation efficiency, while decreasing analysis time.1,2 However, the column inlet pressure (∆P) that is necessary to drive the mobile phase through the chromatographic column increases as a function of the size of the packing material, according to eq 1,3 where φ is 2
∆P ) φηLu/dp
(1)
the flow resistance factor, η is the mobile phase viscosity, L is the length of the column, u is the linear velocity, and dp is the * To whom correspondence should be addressed: (e-mail) lacolon@ buffalo.edu. † University at Buffalo. ‡ Pfizer Global Research & Development. (1) Kirkland, J. J. J. Chromatogr. Sci. 2000, 38, 535-544. (2) Giddings, J. C. Unified Separation Science, 1st ed.; John Wiley and Sons: New York, 1991. (3) Jerkovich, A. D.; Mellors, J. S.; Jorgenson, J. W. LC-GC 2003, 21, 600, 604, 606, 608, 610. 10.1021/ac051213f CCC: $30.25 Published on Web 09/27/2005
© 2005 American Chemical Society
diameter of the packing material. In addition, the optimum linear velocity is inversely proportional to the size of the packing material; thus, the pressure required to drive the mobile phase through the column is inversely proportional to the particle diameter cubed.3,4 Due to mechanical constraints, most commercial HPLC systems have been limited to a pressure of ∼5000 psi (345 bar). It, therefore, becomes necessary to shorten the length of the column as the size of the packing material is reduced, typically to 5 cm or less for packing materials smaller than 2 µm in diameter. Consequently, there is no net gain in the number of theoretical plates, although there is a substantial reduction in analysis time. To fully realize the true benefits of smaller packing materials, longer columns must be utilized, which will require pressures greater than the conventional 5000 psi (345 bar) to operate. Ultrahigh-pressure liquid chromatography (UHPLC) has emerged as a viable approach to drive the mobile phase through packed columns longer than 5 cm with materials of
