ANALYTICAL CHEMISTRY
1858
Automatic Fraction Collector Dean Frarer, Virvi Labarotory, University of Califwnio, Berkeley, Calif.
great rise in popularity of chromatographic separations T.involving . the collection and assay of many effluent fractions HE
which momentarily removes the bias, causing the tube to provide a direct current impulse to the "units" stepping relay. The "Int" and "Home" contacts me built into the stepping relays. The Int contact orovides for return of the ratchet uawl of the relay a t each s t e i o f the reset cycle. The Home c o n h t is oper-
-e.g. [Moore, S.,and Stein, VI. H., J . Bid. Chem., 192, 663 (1951)l-has led to a number of designs far automatic collectors [ANAL. CHEM., 25, 1423-9 (1953)l. The collector described below is of a new type which offers a number of advantages. The design is that of a conveyor chain whose individual links are themselves the collection tube holders and which is motor driven, actuated by an impulse from a positive, direct-contact, dropcounter accumulator (Pedersen Electronics Co., Lafayette, Calif.). I
The tube conveyor chain consists of a double row of brass washers punched to hold 18 X 150 mm. test tubes and linked and spaced by riveted brass posts (Figured 1 and 2). Each tenth post, instead of being riveted, is held by a nut on the top and a nylonbutton-tipped skid post on the bottom. One such post is shown in the center foreground of Figure 2. The chain passes around brass sprockets, each made of two duplicate die-punched plates rigidly assembled and spaced by three riveted hrsss posts (Figure 2). Of these sprockets only one is driven; the rest are idlers (Figure 1). The idlers are on steel axles rigidly mounted on the base of a rectangular box consisting of z stainless steelhovered plywood base with wooden sides and a close-fitting Lueite top. The drive sprocket axle and its drive motor are mounted on a brass plate bolted through slats in the plywood base and held in position by a long machine screw which serves to adjust the conveyor chain tension. The drive consists of a small gear-head motor which turns two cams bolted one on tap of the other, ooncentrically, with the bottom cam mounted on the motor shaft (Figure 2). The top (drive) cam, shown removed in Figure 2, engages the drive sprocket, moving it one position per impulse, three positions per revolution. The bottom cam, shown in position in Figure 2, actuates a mierosait.ch xs-hich controls this cycle. The ratchet tally-counter mounted on the cover plate is driven by a spider gear fastened to the shaft of the drive cam (shown above the cam in Figure 2) and counts the number of tubes which have received fractions, a great convenience. The collector, as shown in Figure 1, also has a brass tube column SuDDort mounted on 8. steel past, so that the whole column asdimbly may be swiveled from the chain to the stationary tube holder while the collector is being loaded. A rack far blank tubw i s mounted along one side.
Figure 2.
Exploded Detail of Drive Mechanism
"tend' stepping relay and immediately resets itself to Eem through Home-l and I n e l . When the meset, number (here shown as 5 5 )
1g ho
or and of constructing the driven sprocket, a t lea!rt, of a harder metal than hrnss-perhaps chmnne plating. The layout can be custom made to fit available space and tlhe number of tubes is easily varied-the chains have been assembll lin ks in units of ten riveted links, The author has units of (cabinet 29 X 44 inches), as shown in Figure 1, and of 100 lin ks (cabinet 10 X 67 inches) in a single loop between two sproekelts,
Figure 1. Over-all View of Fraction Collector and Counting Unit
The actuating unit (Figure 3) divides the oolumn effluent by direct drop counting. The solution passes through a calibrated funnel tip and each drop falls between a pair of platinum electrodes. The plate of the 2050 thyratron is supplied with isolated 60-cycle alternating current, and the tube is held from firing by a s m d direct ourrent bias. Each falling drop closes a contact
:a
1859
V O L U M E 2 6 , N O . 11, N O V E M B E R 1 9 5 4 an estremely simple arrangement. The apparatus could readily he adapted to further automatic operations on the tubes-the chain could, for example, pass under a pipetting machine or through a colorimeter. The drive is self-indexing, in that no chance for cumulative error exists and no individual error barring failure of the drive unit.
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Figure 3.
R x E m SET JP AND AT 55
ZEC
Counting Circuit
Loadiiig is particularly simple, as the drive can lie switched to continuous operation (Figure 3) a t about 14 r.p.ni. or about 1.5 seconds per link. Three t'ube displacements per cam revolution rombirie the advantage of high gear reduction with reasonable chain speed during loading. Counting continues during continuous drive operation. The indicator lights on the counter show thrs riuniber of drops ahead!. in the tube and prevent missing a tulle change. Loading of a reel-tj-pe collector with several rows of tuhes, while the essence of simplicity in theory, actually leads to appreciable likelihood of mistakes and often requires an inconvenient change-over from the last row to the first to permit operation overnight or over a week end. The present collector (*:in be started at an)- point. The automatic loading is more than a superfluous conveniencp, as it means that the collector need not l i e accessible except at the front and it allow loading and unloading wit,h a minimum exposure of the collector tubes to the outside air. The present collect'or is completely closed except for a small opening in the plastic cover for the drop?. Onlj- a small wction of the cover is removed for loading. COUNTING UNIT
It is necessary to quantitative chromatograph)- that the fractions be of identical, or at least known, size. Many methods have lieen employed to assure constant fract,ion size. -4most practical and flexible one for small samples (of the order of 1 ml.) srcm to be drop counting. (Timed flow operation is not satisf:ictory.) The author has had a great deal of trouble with vquipment which featured phot,oelectric counting of the drops.
Because the method depends on exact optical and electrical balance, the difficulties seem rather inherent. The many sources of mechanical, optical, and electrical instability plus occasional unavoidable failures of parts meant that a considerable proportion of the week-long runs, frequently involving valuable material, failed a t some point. inherent weakness of the chromatographic technique is the necessity ,for repeating the entire run if even only a part of one peak is deranged through accident. For these reasons direct counting v a s employed based on the momentary contact of the falling drop between a pair of wires asymptotically disposed to the line of fall. This is satisfactory onlj with conducting solutions but is essentiallj- an all or nothing contact which does not require the careful balancing of a method dependent on maintenance of a sensitive threshold. The limitation to conducting solutions does not appear to be serious, since, in general, the trend of quantitative chromatography is toward aqueous or part-aqueous solvents. The direct counting method has proved completely reliable; only minor difficulties have been encountered in over a year of operation of several collectors. The possilile objection that the flow of current might electro1)-zethe passing material has been obviated by designing the relay t 3 require only extremely tiny currents-the design shown passes less than lpa. through the drop-13 hich pass for only a Fraction of a second as the drop falls (as distinguished from passing the curlent through the forming drop). Calculation shows that even contact of 0.1 second-several times the theoretical contact y of time-would result in a maximum loss of only some an amino acid. Holdup on the electrodes is not a problem, as the residue from each drop is rinsed by the succeeding drop, hut it is minimized by making the electrodes from small ( S o . 22) platinum wire and filing the ends to sharp points of long taper. T\ith this design the standard deviation of individual drops (at some 40 per ml.) is f 2 9 / , and, hence, in a 1-ml. sample is approximately kO.370. Symmetrical disposition of the electrodes with respect to the falling drop is necessary t o avoid wildly eccentric falling. The electronic design of the counting unit ha5 been given a great deal of consideration. The primary criteria are simplicity and dependability The circuit shown (Figure 3 ) involves only a single thyratron, two relays in the mot80r circuit, and two counting relays of a type designed for several million operations n ithout service after the initial adjustment (Ahtomatic Electric Co., Chicago, Xodel STT--52 ADXC T j p e 11 rotarr). (This i h simply an adjustment of spring tension requiring only observation of the resetting operation.) Careful tests have shown that the circuit is very stable against external voltage fluctuations and poner surges caused by other equipment in the vicinity. Deliberate variation of the voltage supply, for ruample, causes no misbehavior of the counter in the range 100 to 135 volts and merely cessation of response below 100 volts. Cnder no conditions have false responses been obtained. Over a year of operation of four fraction collectors has revealed no n eaknesses in the units. ACKNOWLEDG\IENT
The author ta,kes pleasure in ncknon-ledging the interest and cooperation of S. R. Pedersen, Pedersen Electronics Co., Lafayette, Calif., T T ~ Odid much more than simply construct the machines t o order and is responsihle for many features of the design of 120th the electronic and mechanical aspects of the collectors. TTORK aided by a grant from the Xational Foundation for Infantile Paralysk. T h e Pedersen Electronics C o . will consider inquiries for complete collectors coiinting unite, or chains in the ten-link units and sprockets.
