An Automatic Programmer for Chromatographic Eluents 5. R. Lerner, Radioisotope Service, Veterans Administration Hospital, ond Biochemistry Department, Baylor University College of Medicine, Houston, Texas
HE VARYINQ INFLUENT buffer reTqnirements for ion exchange chromatography lead to the development of the automatic apparatus described to set up elution schemes free of delays or human error in the butrer program. The flexibility of the appartus allows its use for simple huffer changes at predetermined times or the setting up of buffer gradients with changes in the inflowing buffer to provide for shaping the elution gradient. In contrast to other apparatus (1, 3-6), this programmer allows flexibility in both the total volume and shape of the gradient sequence by simply controlling the time of inflow of each huffer into the stirred gradient vessel. As each asymptote of pH or salt concentration is approached in the gradient vessel, the timing card programs in a new buffer to increase the gradient slope. To prolong a gradient requires only the substitution of a fresh program card, notched a t differenttimes t o delay the start of the new buffer. With adequate reservoirs of each buffer,
there is no possibility of emptying the gradient vessel inadvertently, with consequent introduction of air into the column. The apparatus consists essentially of a multi-inlet Teflon plug stopcock with a common outlet similar to the apparatus described by Hamilton and Anderson (e). The inletroutlet pathway is selected by a timing mechanism which actuates a motor to rotate the stopcock to the next position in sequence; a most useful apparatus for liquid chromatography has four inlet
CONSTRUCTION DETAILS
The apparatus, which is available from Adelco Labs., P.O. Box 592, Bellaire, Texas, may be constructed for self-contained mounting or for rack mounting as a component of a larger apparatus. All of the components are mounted on the front panel (Figure la). As shown in Figure l b , the motor-
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driven stopcock arrangement is mounted on standoffs with a clevis (Figure Id) to fit the motor shaft to the stopcock handle. A cam (Figure IC), notched at appropriate points for the stopcock to be used, is mounted on the motor shaft, with a roller leaf-actuated, snapaction switch (Hetherington S2B6F1) mounted to bear on the cam. When the time clock actuates the pulse generator, the stopcock motor (W. VI. Grainger, Inc., Chicago, Ill., Catalog No. 5K208, 7 r.p.m.) is energized for a period of about 0.5 second, which rotates the motor shaft enough to raise the switch arm from the cam notch and energizes he motor directly, allowing it to run inti1 the next notch stops the motor by eturning the switch to the first position. The motor magnetic brake stops the motor shaft promptly; coasting may lead to erratic results. A block diagram of the apparatus is shown in Figure 2. The stopcock is a modified, four-vay Teflon plug stopcock (similar to Scientific Glass Apparatus Co., Bloomficld, h’, J., Catalog No. JS-4llO/lmm.) with four 12/1 ball joints on the sidearms and s solid Teflon plug which is drilled
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Figure 1. Automatic programmer
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ANALYTICAL CHEMISTRY
view
of
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ovtomatic
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programmer
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vffer ot column Motor-stopcock assembly, 01 mounted on anel, showing switch, cam, and Teflon-glaar
lopcock Motor shaft cam Motor shaft clevis e Stopcock plug detail f. Outlet ball ioint detail
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Ball joint
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Front
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0.001-0.002 mailerthan 0.0. of 7/1 ball joint tubing, 1/2”deep
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115 V. 60 cycles Stopcock Motor
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Pulse
Generator
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'Ill 4-
Inlet5
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Teflon plug
Outlet Figure: 2.
Time Clock and Program
Block diagram of automatic programmer
as in(lieated in Figure l e . A 7/1 ball joint iis bent as shown in Figure 1f and inserted into the axial hole of the Teflon plug. The low coefficient of fr iction of the T with held i tation Tht quire( closure of a switch for a prolonged period. This is achieved by the circuit shown in Figure 3. The alternating current input is rectified by diode D and furnished through a limiting resistor R to charge the ele'etrolytic eondenser C, and to maintain it charged. When the actuating switch S1 is closed, the charge is dumped through the relay coil, closing the re1ay:momentarily; the time constant is governed by the product of C and the resistance of the coil, as long as R is large enough to provide negligible recharging of C during the discharge period. The timing clock consists of a standard one-revolution-per-day clock motor lC:mmnr Cont,rol Corn.. Centerbrook.
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toS2-N.C.
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externalorswitch { to51 Figure 3.
Di
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Circuit diagram for long pulse generator
Diode, Sarkei-Tmrim M-500or equal Relay, 10,000-ohm coil, Potter & Bwmfleld RSSD-1OK or equal R: Resistor, loOK, 1 1 2 W C: Capacitor.50 mf., 1 5 0 volts SI: Switch, actuating, Switchcroft 20031 SZ: Switch, motor, Hetherington S2A6F1 01 equal
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hours, a 0.2N sodium phosphate, pH 7.5, 30 mg. per liter methyl orange solution is automatically applied to the column. At 16 hours, the stopcock rotates back to the first position and the 0.1N acetic acid regenerates the simulated column. It is readily apparent that alteration of the pH, ionic strength, gradient vessel size, and time of each take place during normal working hours. period can produce changes in the The small amount of eluent trapped in shape of the pH and/or ionic strength the 1-mm. capillary of the stopcock curves. This can be done with little can be flushed out with new eluent, if maninulation bevond chaneine the time desired. Rubber or olastic washers are not& positions; buffers, -or- gradient desirsihle for all the'baii joint connections. vessel size. Once the optimal conditions have been selected, they can be In the ease of columns which are ilrai.erl an4 _-_*_.., ron.04 II fnnrih "doh regenL.-.-.. _...__"_.. repeated automatically. on the program card will return the stopcock to the first eluent at the end DISCUSSION of the elution seauence. and the column This apparatus is a versatile controller of the flow of liquids or gases through any stopcock that can be mounted on the panel face. Three-way stopcocks may he used by changing the cam to one notched at 120" intervals. Flow may be directed from one pair of arms to another pair, as in the usual three or four-way stopcock. A gradient vessel may be used through a
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example, a column started at 11:OO A.M. would elute with the starting buffer until 1:00 P.M., when the second buffer would be started, either to ~recorder mark time oi CUIUIIII,. --'.~-~ ~ inpntto-ulue into a gradient vessel or into the $ contacts (in change), to ueri with aetuat.in6 s&h eration of cLanging mechani closure of e:xternal switch by collector, et1:,), and timer motor A rimnla, .-_..___.Ledion exchange r' made as shown in Figure 5. of the exit solution and the ligh mission were recorded a t a flow 25 mi. ner hour. The lieht trans of methyl orange a t itsisosbest (470 mp) is independent of the was chosen to simulate a chang concentration. Initially, the 0.1N acetic acid (28.0 ml. per recorded. At 2 hours, the first starts a gradient (100 ml.) magnm stirred flask toward 0.1N sodii tate, pH 5.0, 7.5 mg. per liter of orange. At 8 hours, the buffer-methyl orange is applied directly to the column. bypassing the gradient vessel, and a t 11 Figure 4. Program card
rarbance of simulated eluent system vs. time VOL. 35, NO. 8, JULY 1 9 6 3
1 109
for later portions of the elution scheme. The programming of several buffers into a gradient vessel offers a finer control of the shme of the gradient than the use of a single concenk&ed, remote pH inflow. The flow of gases or samples for gas chromatography could be automatically Programmed, for example, to repeatedly analyze a flowing stream by
injecting into the chromatograph the constant volume sample trapped in the cross-drilled hole in the Teflon plug of a four-arm stomock.
and T. R.Kressman~ed8.j PP. 16C-2, lg5'. R. A., ( 2 InterscienceY ) Hamilton, New P. B., Anderson,
A ~ ~cHEM. ~ , 31, , 1504 (1959). (3) Kellie. A. E.. Wade. A. P.. Biochern. J . 66, igg'(i957j. (4) Peterson, E. A., Sober, H. A., ANAL. CHEM.31, 857 (1959). (5) Smith, Ivor, "Chromatogrraphic Techniques," pp. 252-4, Interscience, New York, 1958. ~
LITERATURE CITED
(1) Hale, D. K., "Ion Exchangers in
Organic and Biochemistry,'J C. Calmon
Multiple Hydrogenation insert R. A. Eisenhauer and R. E. Beal, Northern Regional Research Laboratory, Peoria, 111.
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HYDROGENATE
threads, except for the bottom of the sixth section. All internal threads have a l/lsinch relief at the base of the threads. The bottom of one section forms the top of the next, except for the first and sixth sections. The first section has a 6/16-in~hthreaded cap and the sixth, a flat bottom and no threads. Teflon gaskets, cut from a lj16-inch sheet, were used between each section. The gaskets fit between faced surfaces giving an excellent seal. A '/,-inch hole was drilled in the center of each section, and a piece of l/d-inch pressure tubing, 3/4 inch long, was welded in each hole projecting into the section thus giving an opening for hydrogen. The inside diameter of the pressure tubing is a/92 inch. Without this tubing, leakage occurs and samples intermix. Procedure. Materials t o be hydrogenated are placed in each of the six sections of the insert and a suitable catalyst is added. The sections are fitted together with fingertip pressure. Knurled surfaces on each section prevent the fingers from slipping over the machined stainless steel. This pres-
samples on a semi-
T micro scale, a stainless-steel insert, which has a capacity of 10 ml., is avail-
able for use with an Aminco, 300-ml. reaction vessel. However, when many samples must be hydrogenated for analytical purposes, time becomes a n important factor because only one sample can be hydrogenated at a time with the 10-ml. insert. Methods for hydrogenating several samples simultaneously were explored, and a new insert was designed that allows the hydrogenation of six samples concurrently. EXPERIMENTAL
Fabrication. Individuai sections of this insert were cut from solid stainless-steel rods of approximately l 6 / 8 inch diameter. The inside of each section was bored to a depth of Is/, inches, and the section was cut to approximate dimensions (Figure 1). The top of each section n-as threaded with 24 internal threads, the bottoms with 24 external
sure on the Teflon gaskets is sufficient to obtain an adequate seal to prevent leakage. Hydrogen inlet holes need not be aligned because the pressure tubing extends to the center of each section, and with approximately 1 to 5 grams in each section, no leakage occurs. When the insert is completely loaded and assembled, it is inserted into a n Aminco 29/16-in~h0.d. micro-series reactor. This reactor is bored from a solid forging and has an over-all length of l13/a inches. The reactor containing the insert is then placed in a rocker assembly, which includes a heating jacket and means for introducing hydrogen. The pressure vessel tilts through an angle of 30' at 36 cycles per minute. This movement gives the material in each section ample agitation and catalyst contact. The Northern Laboratory is a laboratory of the Northern Utllization Research and Development Division, Agricultural Service, U. S. De artment of Agriculture. Mention of tra& names is for identification only and does not imply endorsement by the Department.
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Teflon gasket.
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Figure 1. Insert for hydrogenating six samples concurrently in an Aminco reaction vessel
24 Intwnal
ONE OF I TYPICAL SECTIONS
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ANALYTICAL CHEMISTRY
