Tailing and spreading of spots prevented clean-cut resolution of either pair. By resorting to multiple development or downward displacement, a resolution may be possible with the butanolammonia system, particularly if a more delicate detecting device could be found to permit the use of smaller amounts of acids. The 1-butanol-ammonia system is available for better resolution in specific cases, or as a different solvent when two-dimensional development seems desirable. EXPERIMENTAL
The acids were dissolved either in water or in acetone, the latter being preferred when prolonged storage was involved, even with refrigeration. Samples were treated with equal volumes of concentrated ammonium hydroxide and promptly applied to the paper (Whatman KO,1). A sample of 1 pl., containing from 40 to 80 y of each acid present, was used.
The rest of the procedure was essentially that of Reid and Lederer (9). The developer was 1-butanol saturated with aqueous 1.5N ammonium hydroxide. The spray was 0.04% (wt. per vol.) bromocresolpurple in a 1 to 5 (vol. per vol.) dilution of 35 to 40% formalin in ethanol, adjusted finally to pH 5.0. The spots were revealed by intermittent exposure to the vapors above concentrated ammonium hydroxide and, because of their transient nature, were marked as soon as they were established. The usual precautions were taken to maintain a saturated atmosphere in the chromatographic chamber, and the temperature mas maintained a t 25.0" zt 0.1 O c. ACKNOWLEDGMENT
Needed equipment was obtained through grants from the Sigma Xi RESA Research Fund and the William H. Wilson Fund a t The College of TTooster.
LITERATURE CITED
Brown, F., Biochem. J . 47, 598 (1950). Brown, F., Hall, L. P., Nature 166, 66 (1950). Burton, H. S., Ibid., 173, 127 (1954). Hashmi, M. H., Cullis, C. F., Anal. Chim. Acta 14, 336 (1956). Hiscock, E. R., Berridge, N. J., Nature 166,522 (1950). Isherwood, F. h., Hanes, C. S., Biochem. J . 55,824 (1953). Kennedy, E. P., Barker, H. A., ANAL. CHEW23, 1033 (1951). Long, A. G., Quag-le, J. R., Stedman, R. J., J . Chern. SOC.1951, 2197. Reid, R. L., Lederer, M., Biochem. J . 50,60 (1952). Renard, M., Bull. SOC. chim. belges 59,34 (1950). RECEIVEDfor review August 12, 1957. Accepted March 7, 1958. Based on the senior theses of Thomas A. Gustin, Robert L. McGuire, and John T. Sweeney, submitted in partial fulfillment of the requirementa for the BCS Certified B.A. degree and the Inde endent Study program at The College o f Wooster, June 1955, 1956, and 1957, respectively.
Compact Countercurrent Distribution Apparatus SAMUEL RAYMOND' College of Physicians and Surgeons, Columbia University, New York, N. Y.
b Countercurrent distribution apparatus of greater flexibility and compactness is needed, if this valuable analytical tool is to be used to its fullest extent in the average laboratory. The apparatus described includes both automatic drive mechanism and 100tube extraction train on a base 24 inches square. The extraction train includes tubes of special design which permit close mounting in compact racks. These tubes produce significantly less co-current flow than previous designs. The drive mechanism analyzes the necessary motions of the tube into two components, each separately driven b y its individual motor. The mechanism is controlled by electrical switches rather than mechanical devices, so that adjustment of parts of the cycle is rapid. With this apparatus, countercurrent distribution procedures can b e used routinely in the laboratory for separating mixtures quantitatively into their components.
F
optimum use, countercurrent distribution requires automatic apparatus with large numbers of tubes in the extraction train. The apparatus described is more compact than the OR
1 Present address, Graduate Hospital, University of Pennsylvania, Philadelphia, Pa.
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ANALYTICAL CHEMISTRY
apparatus of Craig and Post @), and has been used successfully in several laboratories. ARRANGEMENT
OF APPARATUS
The apparatus with automatic drive mechanism and 100-tube extraction train of 20-ml. capacity per tube is mounted on a base 24 inches square (Figure 1). The extraction train consists of 10 banks of 10 tubes, each mounted in a rack rotating on a main shaft. The shaft is driven by the automatic drive mechanism housed in a cabinet adjacent to the extraction train on the same base and supporting one end of the shaft. Additional racks of tubes can be installed on the same shaft by extending the base and main shaft; 10 inches of extension are required for each additional rack. Larger tubes can be installed, up to 35 tubes of 100-ml. capacity in the rack shown. Rack Mounting. The rack consists of two end plates of aluminum, 18 inches s q u r e , supporting cross bars a t each end of the banks. At each end, the individual tube is positioned in a notch on the cross bar on one side and separated from the cross bar on the other side by a rubber pad. Appropriate choice of dimensions in relation t o the diameter of the tube permits each cross bar (except the outer ones) to support two banks. The main shaft passes through flanges mounted at the center of each end plate
One flange is equipped with a locking device, which when released permits rotation of the rack independently of the shaft. Extraction Train. The extraction train in the standard 100-tube rack consists of a series of 100 tubes arranged in banks of 10. When seen from the front tube 1 is at the left, followed by tubes 2 to 10 from left t o right in the front bank. Directly behind tube 10 and connected to it by a ground joint is tube 11; tubes 12 to 20 follow from right t o left in the second bank, so that tube 20 is directly behind tube 1. The entire series is arranged in this alternating manner, ending with tube 100 as the left-hand member of the last bank. Tubes 1 and 100, respectively, carry socket and ball joint connections for various accessory tubes, providing operation as a single withdrawal or as recycle procedure. Between banks 5 and 6 there is a space for clearance of the main shaft and for mounting a reservoir bottle when automatic feed operation is required. A short tube bridging this space connects banks 5 and 6 and can be removed to divide the train into two trains of 50 tubes each. I n apparatus of more than one rack, the outlet tube of one rack is connected to the inlet tube of another by a special tube, which runs diagonally to connect the two racks. Extraction Tube. The tubes are a new design, shown in Figure 2. Each tube consists of a mixing cham-
her, A , nith inlet, B , and a decanting chamber, C, containing a fixed-volume trap and a drain tube, D. The outlet of the drain tube is connected to the inlet of the folloning tube in the extraction train to transfer upper phase from one tube t o the next. For a tube of 20-ml. capacity (10-ml. upper and 10-ml. lower phases) appropriate dimensions of the tube are 45 cm. long overd l , each chamber 16 mm. in diameter. Access to the interior of the tube 1s provided by a screw cap a t either end. \\-hen the tulles are clowmounted in the rack shorn, it is more convenient to have only one cap per tube, loc'ntetl
I
a t alternate ends of the tubes in the rack, as shown in Figure 1. Automatic Filling Accessories. Provision is made between banks 5 and 6 for mounting a reservoir bottle. Attached t o the reservoir bottle is a volume tube with a syringe adjustment which can be adjusted to deliver a single present volume during each cycle of the extraction train. At one point in the cycle, the volume tube depends on a liquid lock to pFevent complete draining of the reservoir; it cannot be used, therefore, rrith highly volatile solvents such as ether, but it delivers accurately measured volumes of lower
1 ll
/I 1
I
I
24"
I
Figure 1.
Countercurrent distribution apparatus
Figure 2.
7
Extraction tube
L
G
M2
, 9 POS. S/*//lir/ Figure 3.
Operating Cycle. Operation of the extraction tube as an extraction unit in a train consists of a cycle of oscillations and rotations about a n axis perpendicular t o the plane of the two chambers. The position of this axis and the principal direction of rotation are indicated in Figure 2 . The distance from the rotation axis to the tube does not affect its operation. The axis may be between chambers A and C or to either side of them. Therefore, rows of tubes may be mounted in parallel a t different distances from a single main shaft, effecting a saving in space required for the apparatus. The operating cycle of this tube requires the follom-ing motions: 1. Oscillation about the horizontal position with the mixing chamber lowermost for mixing. 2. Rotation approximately 170" in the principal direction of rotation. 3. Rest in the 170" position for settling out the phases. 4. Rotation t o the 2iO" position, again in the principal direction, for decantation. 5 . Draining in the 270' position with the tubes vertical. 6. Rotation in the principal direction t o 0" (starting) position.
9 P O 5 SWiTLH
6
volatility solvents. (Volatility does not affect operation, once the train is filled.) The volumes delivered by the volume tube are conducted to the train through the filling tube, connector tube, and rack inlet tube. The filling tube and rack inlet tube form the equivalent of a zero tube in the train. Fraction Withdrawal. The fraction take-off is attached to the drain tube outlet of tube 100. It is a siphon which drains the outflow from tube 100 a t the point in the cycle where the drain tube is positioned directly over the funnel leading to the fraction collector. Recycle Operation. The recycle operation of the train requires outflow from tube 100 to be returned t o tube 1. This is accomplished by rack outlet, connector tube, and rack inlet tube.
Wiring diagram for distribution apparatus
Automatic Drive. The motions required in the operating cycle described are of two kinds: (1) oscillation of the main shaft and ( 2 ) rotation of this shaft. TKOseparate motors can be used to effect these motions. The fact that all the rotations are progressive and in the same direction makes it possible to use a nonreversing motor and greatly simplifies the drive mechanism. In the drive mechanism shown, a crank arm is suspended from the main shaft by a bearing block in which the main shaft turns freely. Mounted on the crank arm and geared directly to the main shaft is the "rotate motor." VOL. 30, NO. 7,JULY 1958
1215
When this motor is energized, the shaft turns with respect to the crank arm, but, when it is not energized, the shaft is locked to the crank arm by the very high ratio gear train of the rotate motor and an electromechanical brake on the motor armature. The crank arm in turn is linked to the “shake motor,” which when energized causes the shaft to oscillate, but when not energized effectively holds the crank in a fixed position. Thus, these two motors alternately energized and de-energized a t appropriate intervals cause the shaft to oscillate and to rotate as desired. The circuit controlling the two motors is a relay circuit, shown in Figure 3. Switches are provided to control the number of oscillations and the length of the settling period. Power from the line is conducted to the shake motor through positions 1 to 10 of W5. With every five oscillations, W1 delivers an impulse to W 5 , advancing it one position. Xhen the number of oscillations set by switch W6 is reached, W5 automatically advances through the remaining positions to position 11, R-here power is diverted from the shake motor to the rotate motor through switch W 2 . This motor causes the shaft to rotate, carrying a cam which trips 81 when the settling position is reached. S1 causes W 2 to advance to position 1, stopping the rotate motor and activating a timer which delivers pulses a t 1-minute intervals to W2, advancing it one position each minute. Khen the number of minutes set by switch W 3 is reached, m’2 automatically advances through the remaining positions to position 11, re-energizing the rotate motor. At the transfer point in the rotation, switch 86 stops the rotate motor for a 1-minute interval to permit complete draining of the tubes. The shaft rotates into the shaking position where S4 closes, advancing W5 to position 1, stopping the rotate motor, and restarting the shake motor to repeat the cycle. A predetermining counter is inserted in the circuit t o stop the apparatus after a preset number of cycles. A switch is also provided to cut out both shake and settling periods when the apparatus is being filled.
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ANALYTICAL CHEMISTRY
Emptying. Any tube can be emptied a t any time through the screw caps. A number of tubes can be emptied simultaneously by use of special receiving tube racks holding test tubes appropriately spaced to receive the contents. Kith the extraction train in the shaking position (tubes horizontal), the cap ends are tilted slightly upward and the caps are removed from the upper banks. The receiving tubes in their rack are applied over the ends of the open tubes and are rotated downward to drain the train. Filling. Before a run is begun, the extraction train must be filled with a sufficient quantity of the more dense solvent (lower phase) to fill all the fixed-volume traps in the apparatus. This can be done by any of the procedures previously described ( I ) . An expeditious way of filling the apparatus is to use a motor-driven automatic syringe pipet of commercially available type. With such a pipet, both upper and lower phases can be added to every tube in the train in less than 30 minutes, including removal and resetting of the caps. The train is then ready for use either as a single withdrawal or a recycle operation. If a motor-driven or other automatic pipet is not available, the train can be filled with the automatic filling attachment described above. First, every tenth tube is filled directly through its cap with a slight excess over 100 ml. of lower solvent. Kith the caps replaced, the train is rotated about 15 times to distribute the solvent through the train. This operation leaves an excess of lower solvent in some of the tubes. Second, upper phase is placed in the reservoir of the automatic filling accessory. It is now in order t o add the sample to the first tube. The drive is set for 100 cycles (in a 100-cycle train) and the distribution is carried out while upper phase is being added from the reservoir. During the run, the excess lower phase in the tubes runs ahead and is discarded through the fraction take-off. If two or three portions of upper phase are run ahead of the first tube containing the
sample, allowing a gap of a few tubes, and are discarded with the excess lower phase, this preconditions the train and prevents depletion of upper phase in the leading tubes. At the end of the first 100 cycles, the accessory tubes can be readjusted for either single withdrawal or recycle operation as desired. SUMMARY
This apparatus is offered as a new design for carrying out countercurrent distribution studies. It has compactness, ease of adjustment, and flexibility in use. It has been used in several independent laboratories. Some users have reported that it is difficult to visualize the progress of the distribution and to evaluate the settling out of the phases, because only the first 10 and last 10 tubes in the rack are fully visible. This has not been a serious difficulty in the author’s hands, as about 2 inches of each tube are exposed a t the ends, through which it is possible to visualize the contents when the tubes are horizontal. If it is essential to see the entire contents of each tube a t any time, the advantages of compactness will have to be foregone. ACKNOWLEDGMENT
The author acknowledges with gratitude the encouragement of H. T. Clarke of Columbia Cniversity and David Seligson of the University of Pennsylvania in the work leading to this apparatus. Ines Mandl of Columbia University tested the apparatus in actual use and has reported results obtained with it. A custom-built model of this apparatus can be obtained from the E-C Apparatus Co., 538 Walnut Lane, Swarthmore, Pa. LITERATURE CITED
(1) Craig, L. C., Hausmann, Werner,
Ahrens, E. H., Jr., Harfenist, E. J., A X A L . CHEM. 23, 1236 (1951). (2) Craig, L. C., Post, O., Ibid., 21, 500-4 (1919).
RECEIVEDfor review June 17, 1957. Accepted December 16, 1957. Meetingin-Miniature, New York Section, SCS, New York, ?;. Y., February 15, 1957.
