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
237
130 n
Cas pressure
Mixer
supply
Detector 1
Relay contact
1
j
1 Hhi DC
Figure 3. Coil activation circuit
Figure 1. Configuration : v ,
De - Energized
Figure 2. Soionoid valve electromagnetic operation, which unfortunately means that the corrosion resistance of commercially available types is limited, some very severely. The best grade of stainless steel that is magnetic and used commercially is 17-4 PH, regrettably inferior to 316 or 321 grades normally used in liquid chromatography equipment. (A more common magnetic grade is 430F which is hardly stainless at all.) The impact operation system shown in Figure 2 gives an approximately 20-msec opening time with a precision of f 2 msec. This information comes partly from details provided by the manufacturers, and from the observed system performance, but was also measured directly by displaying on an oscilloscope the waveform from a microphone placed very close to the valve. The electric actuation signal was used to trigger the scope. The time before the slug hits the pin was measured for successive actuations. The valve opens when the slug impacts the pin, lifting it off the seat. The stored inertial energy of the slug gives a fast and positive opening. When the actuating current is removed, the spring returns the pin to the seat and the valve closes. It was difficult to see clearly the signal resulting from closing because the pin now hits a soft seat. In any case, the opening impact is much larger because it must overcome both the spring force and the system differential across the closed valve. The time to close was estimated to be about 10 msec and precise to a few msec. Different valves took different times to open, presumably a result of the inevitable manufacturing tolerances. Since timing is the essence of eluent composition control, the precision and accuracy of the valves are of paramount importance. Note that quite long but constant delays are involved with these valves which will affect the absolute ratio of eluents but still allow the equally important high precision of ratio control. A number of electronic control systems are easily conceived. The simplest dialable ratio was used in this investigation to give a “eluent ratio controller”. This was a percent timer which has a fixed time cycle (in this case 10 sec), and produces a microswitch closure for an adjustable percentage of the cycle. The coil current required to open the valve was found to be about double that for holding the 238
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1, JANUARY 1976
valve open. It was also observed both on the oscilloscope studies above and in operation, that the delay was reduced and the timing precision improved by operating at a current about twice the nominal rating given by the manufacturers. The difference in operating speed between the two valves was also much reduced by a higher current. Unfortunately, a continuous high current can lead to overheating. A simple circuit (which is duplicated for the other coil) to overcome this problem is shown in Figure 3. The large capacitor is charged through the continuous current limiting resistor to the full supply voltage (50 V) when the valve is off and then the stored energy is available to open the valve quickly when it is switched on (0.9 A, 50 V). The resistors values are chosen to pass only the required holding current (0.2 A, 12 V). This system gives a slightly higher timing precision and will help overcome differences due to valve manufacturing tolerances. A final point to note about selection and operation of the valves is that it is difficult (even with capacitor discharge actuation) to operate against a very high differential pressure that will build up across the valves if the pump pressure is applied before either of the valves is open. After the first valve opens, the pressure drop will all be across the column and almost none as differential pressure across the valve although it may be at 2000 psi above ambient pressure. The practical implications are that at least one valve must be open before the pump pressure is applied, and that the system will not work unless the column is connected and has an impedance much larger than that of the valve orifices and pipework. The accuracy of the system is highly dependent on this last condition. Note that this implies that some large diameter preparative size columns will have insufficient impedance for use with valves chosen for analytical columns. For the eluent composition to reflect accurately the time proportion allocation, the variations in orifice diameter must have a negligible effect on the flow rate. The mixer can consist of a simple tee but this gives poor mixing in a stream that consists of slugs of alternating liquids (in contrast to continuous streams from two differential speed piston pumps where different amounts of the two eluents emerge at the same time). A simple static mixer, a short inefficient column of glass beads, has been used but a direct experimental comparison of this with a magnetically stirred chamber (Figure 4) showed positive stirring to be much superior (Figure 5) especially for liquids of widely different density andlor optical density. Short (100 mm) and very efficient columns (10000 plates) packed with 5-pm silica provide very little additional mixing and eluent composition variations appear very little attenuated at the final detector leading to random or cyclic noise. The positive mixer used was similar to one commercially available. The stator of an ac motor produces a rotating magnetic field which is followed by an encapsulated magnet with N and S poles across its diameter (instead of the usual end to end magnetization). It thus rotates a t 50 Hz
t"NPT - t"tube Swagelok ref. S 5-200-1-2
From
Small bore 316. Stainless tube &''O.dia. 0.049" wall thickness.
i" NPT - h ' l tube Swagelok ref. s s -100-1-2
;"O.D.
i " NPT - I \ ' ' tube Swagelok ref. -
mn
Ref. SS-200-3
Polythene encapsulated magnet 6 mm. diameter. LKB part no. 949 203655 (for 11300 Ultrograd)
$' AlFlats
Mixer consists of a stator from Edgecumbe Peebles heavy duty type C motor. Mixer must be verticle so that magnet spins freely and to allow bubbles to rise and escape. Volume of chamber=volume of magnet t0.3 ml. For flow l m l l m i n . Figure 4. Mixer
(or line frequency). The theory of this type of mixer has already been worked out in detail. The performance of the system was assessed by placing a UV detector first in position 1 to monitor the output direct from the mixer and then in position 2 after the column to assess the addition smoothing by the column. Pump A delivered pure solvent (denatured alcohol) and pump B, a solution of dye (2,4-dinitrophenyl hydrazine) in industrial alcohol. One hundred percent transmission (zero absorbance) was taken with pure solvent flowing. A suitable wavelength and dye concentration was chosen to give 10% transmission (1 absorbance unit) with only eluent B flowing. Over the range 10-90%, the precision of the composition ratio was as great as the control and measuring system (about f0.5% concentration). The mixing efficiency (or lack of it) and the lag on changing the ratio setting were clearly visible (Figure 6). The ratio could be reset to within f 2 % accuracy, reset with a precision of fl%, and maintained ratio with a change of
