Precolumn for High Performance Liquid Chromatography Lawrence A. Pachla and Peter T. Kissinger' Department of Chemistry, Purdue University, West La fayetre, lnd. 47907
Samples for high performance liquid chromatography (HPLC) are frequently derived directly from biological material, pharmaceutical preparations, or TLC spots. I n such cases it is highly desirable to protect the analytical column (10-100 cm) with a small easily replaceable precolumn (1-2 cm). The precolumn is usually packed with the same material as the main column and serves to trap particulate matter (including septum material) and high molecular weight sample constituents (e.g., protein). In common practice, it is difficult to utilize a short precolumn without introducing an unacceptable amount of dead volume. Faced with this problem, we devised a simple precolumn for use with popular commercial HPLC systems based on Teflon tubing and glass columns. Two manufacturers, Altex Scientific and Laboratory Data Control, offer convenient on-column syringe injection ports (Altex Model 251-12, LDC Model 183A8). A very simple precolumn (Figure 1) machined from 5/,-in. Plexiglas or Kel-F roundstock can be directly adapted to these injection ports. The bed in the precolumn is supported by a porous Teflon filter disk (2-7 fim) cut from commercially available material (Chemplast Inc., 150 Dey Road, Wayne, N.J., 07470, Cat. No. 75F Filter Membrane) and held in place by a Teflon tubing (l/l~ in.) flare in the downstream fitting. Suitable filter material is also available from Bel-Art Products (Pequannock, N.J. 07440) and Dilectrix Corp. (61 Allen Boulevard, Farmingdale, L.I., N.Y. 11735). The precolumns are dry packed with the lower end-fitting and filter disk in place and the packing material is added through the top with the aid of another end-fitting (Altex 200-00). If a number of columns are kept on hand, they can be prepacked for immediate replacement use. In constant flow systems, a rise in pressure before the injection port can be used to indicate when replacement is desirable. We have used the above precolumn design for many months and found it to be very helpful in assays in-
G
Figure 1. Simple replaceable precolumn
for Teflon-glass liquid chro-
matography systems (A) y&. dia. round stock
(Kel-F). (B, E) Inlet and outlet ports, 318 in., %-28 thread. (C, D) Filter disk. (F)2-mm dia. X 1-cm bed. (G)bin. tube end fitting
volving direct injection of diluted urine ( I ) , blood (21, and pharmaceuticals ( 3 ) .When high pressure (>600 psi) stainless steel systems are used, an adequate precolumn is available from Altex Scientific, Inc. (Model 250-25).
LITERATURE CITED (1) P. T. Kissinper, L. J. Felice, R . M. Rigpin, ._L. A. Pachla. and D. C. Wenke, Clin. Chem, 20, 992 (1974). (2) L. A. Pachla and P. T. Kissinger, Clin. Chim. Acta, 59, 309 (1975). (3) R. M. Riggin, A. L. Schmidt, and P. T. Kissinger, J. Pharm. Sci., 64, 680 (1975).
RECEIVEDfor review August 1, 1975. Accepted September 15, 1975.
Time Proportioning for Ratio and Gradient Elution for Liquid Chromatography Paul A. Bristow Imperial Chemical Industries Limited, Pharmaceuticals Division. Hurdsfield Industrial Estate, Macclesfield, Cheshire, England
The principle of forming a ranging composition of eluent in liquid chromatography by proportioning the time that two different liquids are being pumped has been widely used by many workers and some commercial instruments ( I , 2 ) . The two (or more) liquids can either be mixed before the pump, which has the advantage that valves and mixer need only operate a t ambient pressure; or alternatively, the valves and mixer can operate on the high pressure output from two pumps or, in the case of the Dupont system ( 3 ) , from one pump and a storage coil. Systems like this using the cheap, simple and reliable gas-pressure coil pump do not seem to have been described. This paper discusses the design considerations of one particular configuration and the results obtained, highlighting especially the increasingly stringent requirements of gradient system when using very small particle packing in short columns which are so fast and efficient.
Indeed, to some extent, very high efficiency columns do reduce the need for gradient elution and lead us to suggest that, for chromatographic method development, the ability to select a particular eluent composition simply by turning a knob is extremely useful.
DESIGN AND PERFORMANCE The basic configuration is shown in Figure 1. Essential characteristics are that pumps A and B (delivering liquids A and B) should be constant pressure types delivering exactly the same pressure. We have used direct gas pressure operated pumps with a holding coil, but pressure intensifier piston pumps driven by compressed air could also be used. The two coil pumps are driven by a common pressure regulator attached to a nitrogen cylinder. The two flow control valves must open or close within a few milliseconds of being actuated. This seems to demand ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
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