Because of the relatively simple composition of most biologic materials as compared, for example, to geologic specimens, it is probable that multiple trace element determinations can be performed on the same sample, provided the characteristic wavelengths used for transmittance measurements are of sufficient energy t o traverse the sample. I t is possible that preparations of lyophilized materials and filtered precipitates can be treated in a similar fashion with the increased advantage of concentration. ACKNOWLEDGMENT
The authors acknowledge the advice and suggestions of P. R. Stout relating to the subject matter of this paper.
The authors thank R. J. Della Rosa for the d a t a in Table I1 and C. K. Hui for technical assistance. LITERATURE CITED
Bowen, H. J. M., Cawse, P. A,, U . K . t . Energy Res. Estab. Re 1.4309 (1963). Campbell, J. T., Shargosky, H. I., Nature 183, 1481 (1959). (3) Compton, A. H., Allison, S.K., “Xrays in Theory and Experiment,” p. 513. Van Nostrand. New York. 1935. (4) -David, D. J., Analyst 8 7 , 576; (1962). ’ (5) Della Rosa, R. J., Pool, R., O’Sullivan, J., U . S. At. Energy Comm. R e p t . UCD 108, p. 66, (1963-); (6) . , Elfers. L. A.. Hallback. P. F.. Velten. R. J., ANAL.CHEM.36, ’540 (1964). ( 7 ) Goldman, M., Anderson, R. P., Gee, W., U . S. At. Energy Comm. Rept. UCD 108, p. 75 (1963). (8) Johnson, C. M., Stout, P. R., ANAL. CHEM.30, 1921 (1958).
(9) Leroux, J., Lennox, D. H., Kay, K., Ibid., 25, 740 (1953). (10) Liebhafsky, H. A., Pfeiffer, H. G., Winslow, E. H., Zemany, P.D., “X-ray Absorption and Emission in Analytical Chemistry,” Wiley, New York, 1960. (11) Mills, A. A., Can. J . Chem. 4 2 , 73 (1964). (12) Mitchell, B. J. “Encyclopedia of Spectroscopy,’’ G. L. Clark, ed., p. 736, Reinhold, New York, 1960. (13) Reynolds, R. C . , A m . Mineralogist 48, 1133 (1963). (14) Roberts, W. Lf. B., Nature 183, 887 (1959). (15) Salmon, M.L., “Advances in X-ray Analysis,” W. hl. hIueller, ed., Vol. 2, p. 303, Plenum Press, New York, 1960. (16) Wade, AI. A., Seim, H. J., ANAL. CHEM.33, 793 (1961). RECEIVEDfor review July 13, 1964. Accepted March 15, 1965. Based on work performed under the auspices of the U. S.Atomic Energy Commission.
Completely Automated Three-phase Countercurrent Apparatus HERBERT L. MELTZER Departments of Biochemistry, New York State Psychiatric Institutes and College of Physicians and Surgeons, Columbia University, New York, N . Y .
JOSEPH BUCHLER Buchler Instruments, Fort lee,
N. 1.
ZACHARY FRANK Warner Chilcott, laboratory Instruments Division, Richmond, Calif. Pertinent details of construction of an automated three-phase countercurrent apparatus are described. The apparatus is capable of running unattended from the time solute is introduced into the first tube until a measured portion of every output sample has been delivered to an analytical station. Its mechanical performance i s so reliable that an operational test of its fail-safe features has not been possible.
T
countercurrent distribution is a technique for the fractionation of complex mixtures. It differs from the familiar (1, 2 ) two-phase distribution in that two phases move at right angles to each other past the third phase stationed within a two-dimensional array. I n contrast, the twophase method requires that one phase move past a linear assembly of another stationary phase. The theory of the three-phase method has been described ( 4 ) . With either method, when the operation is continued for a number of transfers that exceeds the number of stations in the apparatus, the moving phases, containing fractionated solute, may be run into fraction collectors. The automation of the sequence inHREE-PHASE
volving addition of moving phase, distribution, and collection of the output fractions offers obvious advantages in freedom from error and economy of time. The automated apparatus to be described below can perform a variety of useful functions. Foremost among these is its ability to separate complex mixtures. Although the three-phase method was initially devised to deal with mixtures composed of relatively dissimilar components (S), i t has also been applied to the analysis of mixtures of closely related substances (4). A realistic appraisal of the resolving power of the present apparatus will not be possible until the results of many distributions have been studied. However, a n idea of its theoretical limits may be conveyed to the reader by the following. If the apparatus is run without reloading the fraction collector, so that only 59 sets of fractions are collected, and the most favorable distribution coefficients are assumed for the components of the mixture, then nine completely resolved fractions will be obtained in some of the 1770 vials of the fraction collector and another four completely resolved fractions will be found within some of t h e 120 distribution tubes of the apparatus. If reloading of the fraction collector is allowed,
the number of separable components will, of course, be increased. If 20% overlap of component zones is allowed (a condition that still permits quantitative analysis as well as useful isolation of pure material), then the maximum number of components that can be found in 59 sets of fractions, plus the distribution tubes of the apparatus, is increased to 24. The reader should be cautioned that these are theoretical figures only; the probability of obtaining a solvent system with such favorable distribution coefficients for all of the components seems low. I n addition to the separations and analyses discussed above, the apparatus described below has already proved useful for the detection and isolation of useful quantities of trace components. For example, after loading the apparatus with 1 gram of a substance whose purity was in question, an 0.3y0 impurity was detected and isolated. The third principal use of the apparatus is concerned with the proof of purity of a substance. Since the distribution of a single substance produces several sets of output curves, each of which is obtained under conditions of varying peak concentrations, the presence of a poorly resolved impurity will appear as a distortion of a t least one of VOL. 37, NO. 6, MAY 1965
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these curves. Thus, if a set of theoretical curves can he matched to the empirical curves, it can be inferred with reasonable certainty that the substance is pure. Such a requirement is more than equivalent to the frequent practice of rerunning a chromatographic peak to test for purity. The apparatus described below has been designed to test a variant of the three-phase procedure, suggested previously (4). Where one of the phases is substantially aqueous it is possible to make small alterations in its composition (for example, with respect to pH) without producing phase composition alterations upon subsequent equilihrs, tion with the other phases. Under such conditions a progressive variation in solvent composition can he applied to one of the input sides of the apparatus. The effect of such a procedure should he analogous to gradient elution. Previous experience with the threephase technique had indicated that the required analysis of the output fractions was the most time-consuming part of the entire procedure. I t was therefore decided to include provision for automatic analysis in the plans for the automated three-phase apparatus. This purpose was accomplished by transferring a measured portion of each output fraction to a location designated as the analytical station. The particular choice of the aualytical system depends upon the problem at hand; consequently, the description of such systems is beyond the scope of this paper. However, for most purposes the weight of nonvolatile residues will he the most generally useful procedure. An automatic halance to accomplish this purpose is under development and will he described in a subsequent publication. The apparatus is shown in Figure 1. The sdvent dispensing pipets (Figure 2) are visible at the top. Solvent is delivered to the apparatus via the input funnels which appear as a horizontal row just below the first cross brace of the metal outer frame. Each funnel is connected to a n input via an off-angle ball and socket joint; in the photograph these are shown in the closed position. They rotate automatically to the open position just prior to delivery of solvent. The triangular array of distribution tubes (Figure 3) mounted on the rotating frame appears to he somewhat distorted owing to the perspective of the photograph. A t the bottom of this array there is a row of collecting funnels, the vertical stems of which are positioned over a row of tubes in the fraction collector (Figure 6 ) . The three rows of tubes and the top pusher in front of them are the only visible members of the fraction collector. The two panels at the bottom are the pneumatic controls for the sampler 722
ANALYTICAL CHEMISTRY
(Figure 10) and fraction collector (Figure 7). The analytical sampler and carriage drive (Figure 9) mechanism are visible just behind the collecting funnels. The large box at the right center region of the photograph houses the hydraulic drive (Figure 4) and the electrical controls (Figure 5). The operation of the apparatus may he summarized briefly aa follows:
Figure 1 .
Top and middle phases are added to the appropriate input tubes of a 14transfer three-phase countercurrent fractionator. A timed cycle consisting of equilihration, settling, decantation, and transfer then occurs. At each transfer, 30 output fractions (15 each of top and middle phase) are poured into a row of vials In the fraction collector. The fraction collector advances one row. The
Left-front photogrophic view of the apporatus
Figure 3.
T R A N S F E R ROTATION Single stage of the distribution train
" S
See text for description
Figure 2. Solvent reservoir and dispensing pipet Pressure is delivered a t the location marked by the arrow. R, reservoir; P, pipet; M, magnet; S, solenoid; S f M, solenoid valve
analytical sampler then transfers a measured portion from each vial in the row to the analytical station. During this time another sequence of equilibration, settling, and decantation occurs. When the analytical sampler has sampled the last vial in a row, the next transfer operation is allowed to occur. EXPERIMENTAL
Solvent Reservoirs. The reservoirs (Figure 2 ) dispense reproducible portions of solvent in response to two signals during each cycle. At the first signal air pressure is applied for a short time to the reservoir, and solvent is forced into t h e dispensing pipet. Upon release of the pressure, excess solvent siphons back to t h e reservoir and leaves a volume in the pipet determined by the length of ths pipet below the siphon tube. Thie length is adjustable. At the second signal the glass-enclosed solenoid valve opens and discharges solvent via a funnel into the appropriate input tube. Distribution Tube. This tube (Figure 3) consists of a n equilibration section, in which the three phases a r e contained during distribution of the solute, and two decantation sections. K h e n rotated in the direction shown, top a n d middle phase flow into the center sect on. Middle phase is retained in this section while top phase flows into t h e right-hand section. Ot the bottom phase, 10 ml. is retained in the calibrated equilibration section. The center section is similarly calibrated to retain 10 ml. Transfer occurs upon counterrotation to the starting position. Tube Assembly. The tubes are assembled on a metal frame (Figure 1) with individual clamps holding each tube to t h e frame a n d spring clamps securing the ball and socket joints which connect tubes. Although each interior tube has connections to four
neighboring tubes, a n y t u b e can be removed without disturbing the remainder of the assembly. The contents of each tube are available through t h e end ball-and-socket ioint (Figure 3 ) . Driving Mechanism. The drive was reauired t o oscillate the frame i10' around the horizontal position for equilibration, return to -10" for settling, rotate +loo' from this position for decantation, a n d return t o -10" for the transfer of moving phases. I t was also desired to regulate the rate of each of these rotations. All of these motions were accomplished with a n hydraulic drive mechanism. The frame was coupled directly to a Roto-Cy1 of 1700 inch-pound torque (Graham Engineering Co., Palo Alto, Calif.). This is a device which converts a linear motion to a rotary motion, substantially without backlash. The linear motion is obtained by driving the piston of an hydraulic cylinder. A chain moving with this cylinder drives a gear to produce the required rotary motion. The problem of producing the desired oscillations can thus be reduced to a scheme for driving the piston back and forth a t the required times and rates. A schematic of the hydraulic control system is shown in Figure 4. The pistons in the chambers of the air-oil accumulators ( A ) and the Roto-Cy1 ( R ) are each designated by a bar crossed by a two-directional arrow and are shown at positions corresponding to a point midway between the extremes of the linear motion. (A more complete diagram of the Roto-Cyl, showing the way in which the piston is coupled to the rotating members, is available from the manufacturer.) The entire system ir powered by an 80-pound filtered, regulated air pressure supply. Air is admitted to the system through the input port of the four-way valve shown schematically as V in Figure 4. I t is expelled from the system into a pressure regulator (PR) which serves to place a balancing load on the whole apparatus, so that rotation is primarily a function of driving force, rather than free fall. I n the schematic the direction of motion of the pistons in A in response to the application of air pressure is shown by solid arrows. When the four-way valve is switched by solenoid action, as discussed below, to the input and output connections indicated by the broken arrous in the box labeled V,the motion of the pistons
Figure 4. Schematic of hydraulic drive S, solenoid valves; C-1, 2, 3, 4, flow control valves; V, 4-way air valve; P.R., exhaust pressure regulator; R, Roto-Cy1
is reversed. The reverse motion is shown by broken arrows. The flow of hydraulic fluid into and out of the RotoCy1 occurs through parallel circuits. One of these, which can be blocked with solenoid valves, is designated as the fast circuit and serves to control the equilibration oscillation. The rate of this rotation in either direction is preset by two flow control valves. When the fast circuit is shut off, rotation for the decantation and transfer functions depends upon movement of fluid through the slow circuit. The rate of each of these motions is adjustable with flow control valves. Electrical Control. Timed control of the motions described above and integration of these motions with the functioning of t h e solvent reservoirs were accomplished with t h e arrangement of components shown in Figure 5 . T h e system is a n elaboration of a simple electrically timed switch. Each of the four modes of operation, namely, transfer, equilibration, settling, and decantation is governed by its own timer (Type 305-13 Atcotrol Timer, Automatic Timing and Controls, Inc., King of Prussia, Pa.). The timers are arranged in sequence so that the end of a timed interval in one timer closes a switch which starts the next timer. The end of the timed control in the fourth timer closes a switch which recycles the first timer. Each timer is shown schematically in Figure 5 as an assembly of a motor (M), clutch (C), and switches (designated by numbered contact points). The broken lines extending downward from ,If and C indicate mechanical relations with the witches. Timer S o . 1 is shown during operation. At the end of its cycle contact is made between terminals 11 and 13 as well as 4 and 3. Power to operate the motor is thus removed from timer KO. 1 and applied to timer No. 2 . I n the same way timers No. 3 and 4 are activated. When timer No. 4 has reached the end of its timed interval, power is momentarily applied VOL. 37, NO. 6, MAY 1965
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723
.-I
I-
Le, M.S.- 3
Figure 5. Schematic of electrical control for hydraulic drive
'
Timers are indicated within the solid lines. M . 5 - 3 i s a switch operated by the A/E-2 switch of Figure 10
to each clutch through the contacts of R-8, thereby reset'ting all timers. (A manual reset switch is shown at the top left of the schematic.) Power is delivered to the controlled circuits through contacts 4 , 5 , 6, and 7 . The circuit operates as follows: When timer No. 1 is energized, the timed interval for the transfer operation begins. The frame moves from the decant to the transfer position. When it reaches the transfer position it activates a microswitch (MS-1) in series with the controlled output of timer KO. 1. This microswitch activates a 15-second Amperite time delay relay (R-7) through one set of contacts and energizes the primary of an isolation transformer through another set of contacts in series with the contacts of R-7. The secondary of this transformer supplies the input to a bridge rectifier. The filtered d.c. output then energizes the solenoid valves of the reservoir pipets. They remain energized for 15 seconds, during which moving phases are added to the apparatus. When the transfer timer reaches the end of its t'imed period it energizes timer Yo. 2 . The solenoid valves (Ti-3, -4, -5, -6) of the fast circuit (Figure 4) are opened and equilibration begins. The direct'ion of rotation is determined by the four-way air valve (Figure 4) whose solenoids (V-1 and V-2) are activated by microswitches (MS-2) operated by a cam on the drive shaft. During the equilibration period, current is supplied to JfS-2 by the controlled circuit of timer S o . 2 in series with the contacts of R-2. At the end of the equilibration period, timer No. 3, 724
ANALYTICAL CHEMISTRY
which determines the settling period, is activated. Current through the controlled circuit of timer No. 3, in series with the contacts of R-3, energizes Ti-1, thereby maintaining the frame in the transfer position. This is followed by timer S o . 4, which allocates a preselected time for the decantation. During the timing interval of timer No. 4, current through contacts 4, 3 of timer No. 3, in series with the contacts of R-4, activates 1'-2, driving the frame toward the decant position. The timed signal from timer S o . 4 reactivates timer No. 1. d t this point the reservoir pipets are filled. The controlled current from timer KO. 1 drives a rotating valve ( R . V . ) whose electrical components are shown schematically a t the upper right of Figure 5. This valve supplies the brief period of air pressure required to operate the reservoirs (Figure 2), as described above. When the frame reaches the transfer position, the pipet solenoid valves open and dispense solvent, and another cycle begins. All timers are of the nonreset on power failure type. Each timer has maximal setting of 15 minutes, so that the threephase apparatus, operating alone, has a maximal cycle time of 1 hour. However, when it is interlocked with the sampler and fraction collector as described below, the cycle time may be extended to any interval required for sampling a complete set of output fractions before delivery of another output set to the fraction collector. Fraction Collector Mechanism. K i t h each transfer, thirty output samples are poured into a row of receiving vials. This constitutes a sample set. The fraction collector will receive 59 sets. To conserve space they are arranged in two layers. Racks of vials are moved within layers and transferred in a closed loop between layers by a n elevator and pusher mechanism (Figure 6). The motive force is derived from a 30-pound air supply. The problem of guarding against fire in the event of an accidental spill of the volatile inflammable solvents is thereby greatly simplified. The operation is as follows: Upon receipt of an external signal, elevators a t the front and rear of the fraction collector withdraw to the lower level. The front elevator is empty; the one a t the rear carries a vial rack with it. After the upper level pusher retracts, the lower level pusher extends. This moves all the racks on the lower level forward one space and thus empties the rear elevator and loads the front elevator. The lower level pusher then retracts, and after both elevators rise, the upper level pusher extends, moves all racks on the upper level forward one space, and thereby places an empty row of vials under the three-phase apparatus. The rear elevator is filled by this motion and the cycle is completed. Fraction Collector Pneumatic Control. The control system employs the following components: Mead KO.420 style poppit valve; Mead M V series microvalve; Mead square end cylinder (Mead Specialties Co., Chi-
Figure 6. Diagram of fraction collector Two layers of vial racks are shown in the process of cycling. The bottom pusher is retracted, both elevators are raised, and the top pusher is about to push a viol rack off the front elevator on to the upper level
cago, Ill.); Wilkerson S o . 2019-l WH regulator modified to accept a pneumatic control signal (n'ilkerson Compressed Air Products, Englewood, Colo.). The Mead poppit valve is designated as a pneumatic relay in Figures 7 , 8, and 10, and is shown schematically a t the bottom left of Figure 7 . I t operates as follows: Each line within and without the rectangle represents a channel for air flow. The circle a t the top center represents an exhaust port. In the position shown, when air pressure is applied through the channel beginning a t the upper left above the rectangle, it is transmitted through the channel represented by the right-hand diagonal line inside the rectangle to the channel a t the right below the rectangle. At the same time there is a connection between the exhaust port and the channel a t the bottom left, via the left diagonal channel. Assume now that the diagonal channels pivot on centers represented by the junctions with the bottom channels, so that after such a movement the right diagonal channel is connected to the exhaust port and the left diagonal channel is connected to the left branch of the pressure line. The pressure and exhaust relations have thus been switched in a manner analogous to the operation of an electrical switch. The motion described above can be brought about by applying control pressure to the channel at the right center of the rectangle. This control pressure is referred to in the following description as a signal. Reversal of the motion back to the state shown in the diagram can be brought about by applying control pressure to the channel a t the left center of the diagram, provided the pressure a t the opposite end is removed. Thus, the total action of operating a pneumatic switch by the application of control pressure is analogous to the operation of an electric switch by control current, and the unit is therefore designated as a pneumatic relay. Similarly, the pneumatic microswitch shown next to the relay is analogous to an electric microswitch, in that two ports are connected as a result of mechanical operation of a lever. The motions described in the preced-
Io
I__.__ .............................
Figure 8. Figure 7. collector
UD-1
Simplified schematic of fraction collector
-.
0, pneumatic relay; 0, pneumotic microswitch; 0, elevator or pusher;
Schematic of pneumatic control for fraction
..
.Ign.i.,
The condition of a11 components is shown prlor to a start signal. EIevotors and pushers ore represented by vertical rectangle% Ail except B are normally extended. External sipnois are marked with arrows. St-2 ond Re-2 ore applied lo the schematic of Figure i 0. SI-i i s derived from, V-7 of Figure 5. pmswre in; 0,exhaust port; V.A., Y O I U ~omplifiarj C Re, reset; St, s t ~ r t ; B , bottom puiher; 1, top; U, up elevotor; D, down; OD. up-down; C, complete; LO, lockout of start rignol; BR, bottom return
>-,
ing section are sequentially controlled. At each step a pneumatic signal indicates completion and permits the succeeding step to occur. The operation begins upon receipt of an external pneumatic signal and ends with the transmission of a pneumatic completion signal to another part of the apparatus. The schematic for these operations (Figure 7) may appear to he complex. A simplified schematic (Figure 8) is presented &4 a guide to the interrelations among the various functions. The following description is intended a8 a further aid to following Figures 7 and 8. The operational sequence is a8 follows: (1) Pressure through the start relay moves C'D-l to the left. (2) Pressure through UD-l then moves UD-2, LO, and T . (3a) Pressure through UD-2, via the volume amplifier, retracts U and D elevators. (36) C moves to the exhaust position, (3c) Pressure through LO signals reset-2 (which sends a signal t o the sampler) and shifts the start relay to exhaust. ( 3 4 Pressure through relay T retracts pusher T . (4) Microvalves D-I, U-I, and T-I, which serve as position sensors for the moving parts of the fraction collector, are activated. This sends a pressure signal to relay B via relay BR. Pressure through relay B then extends pusher R. (5a) Activation of microvalve R-1 signals relay BR, which in turn signals relay B to reset. Pusher R is then retracted. (5b) A parallel signal from BR resets IrD-1. (6) Pressure through UD-1 resets UD-2. (7a) The volume amplifier is actuated by B-0 and pressure through UD-2 extends the U and D elevators. (7b) Parallel pressure through UD-1 via microvalves U - 0 and D-0 resets relay T. (X) Pressure through relay T extends pusher T. (9) Pressure through microvalve 7'-0 resets BR, C , and LO. (10) The completion signal, from UD-1. through U-0, D - 0 , and C, starts the sampler. Sampler Mechanism. The sampling system employs the WarnerChilcott (Richmond, Calif.) samplerdiluter, connected to a movable probe.
The probe is dipped into a sample solution and a predetermined volume of solution is withdrawn. T h e probe then moves to the analytical station where the sample portion, followed by a measured volume of diluent, is discharged. The sample probe mechanism (Figure 9) is mounted on a carriage driven by a pneumatic dfive mechanism from a "home" station to the first sample position, then to the analytical station, and finally t o the home position. It then moves to the second sample position and repeats the operation. Each position is located by one of 30
Figure 9.
....,sewnd of
first of a pair of signair operating o relay; -.retraction mOtl0"; -, axten.ion motion
(I
pair of
retractable stops activated in the proper sequence by a 30-position stepping valve. All of the drive and control mechanism is pneumatic, with one exception. The samplerdiluter valves are solenoid operated. I t was therefore necessary to convert one pneumatic to an electric signal. The following symbols apply to the schematic of Figure 10 for relays and microvalves: H.S., home station; R , rinse timer; A , cam A of selector valve; S , sample station; S . Pr., sample probe; A.S., analytical station; B, cam B of selector valve; H , hold; C.D., carriage drive; S.D., sampler-diluter; P , pump. For extendable cylinders: R.T., rinse timer; H.D., home detent (spring return); S.V., selector valve actuator (resets when driven to the top,
Photograph of carriage drive and sample probe
The probe, lined iniide ond out with Tefion [Du Pontl. may be seen just to the right of (1 collecting vial. The locotion ihown 1% the "home" station. It is mounted on 0 corrioge that will move on an oppropriote signal until it reaches o top. The stop for the flrst sampling position is shown extended just l o the left of the carriage. At this point the probe will make a vertical e x u n i o n down ond up. meanwhile s~mpling the ont ten ti of the vim1
VOL 37. NO. 6. MAY 1 9 6 5
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steps when driven to the bottom); C.D., carriage drive (forward when driven to the top); S.Pr., sample probe (spring return); S.P., sample pump (spring return); D.P., diluent pump. Other symbols: 0, normally closed port; Z.R., isolation relay (provides oil-less air for the rinse spray); P.R., a secondary pressure regulator; 8-1, solenoid valve, normally open, between sampler and diluter; S-2, solenoid valve, normally closed, between diluter and diluent reservoir; dlE-1, a U-tube containing mercury, to provide a sparkless air to electric signal conversion; AjE-2, an air to electric conversion; M.S.-3, microswitch (see also Figure 5 ) . The pump relay is operated by pneumatic proximity sensors on the sample and diluent pumps. The entire schematic (Figure 10) can be described as was done above for the fraction collector schematic. Although this will not be done in the interests of conserving space, it is advisable to point out several features. Operation is begun upon receipt of the completion signal from the fraction collector. This signal passes through the analytical station, so that a sample portion is not withdrawn unless the analytical system is prepared to receive it. After transfer of each sample portion, the probe rests a t the “home” station until the analytical system calls for a portion of another sample. This is repeated until 30 sample portions have been transferred. At this point the switch activated by cam A (Figure 10) of the stepping valve redirects the cycling signal through the start relay. The signal cannot be transmitted until the start relay is reset. Cam B of the selector switch sends a completion signal to the three-phase apparatus and thus allows movement from the decant to the transfer position. Interconnections among Control Systems. I n the overall operation of the apparatus there are three operating cycles t h a t must function as a master cycle. First, there is the three-phase apparatus itself, whose cycle of equilibration, settling, decantation, and transfer operation produces one set of o u t p u t samples. This must be linked with one cycle of the fraction collector, the net result of which is to present a row of empty vials to the three-phase apparatus. Each operation of the fraction collector is linked to a cycle of operation of the analytical sampler whereby measured portions of the samples in each of the 30 vials of the newly collected sample set are removed and transferred to the analytical stations. (Each transfer operation of the sampler is made dependent upon the state of readiness of the analytical station.) When the sampler has completed this task, its completion signal constitutes a permission signal for the threephase apparatus to move from the decant to the transfer position. Since this motion is also dependent upon the setting of the decant time clock, two conditions are required to begin a new cycle. The particular signals required to link each of these cycles have been indicated on the control schematics described 726
ANALYTICAL CHEMISTRY
TO S.V.
Figure 10.
Schematic of pneumatic control for sampler mechanism
The condition of the components is shown prior to a start signal.
above. When the three-phase frame moves from the settle to the decant position, a pneumatic microswitch, Re-1 (Figure 7) is activated. When the sampler has completed its cycle, C-2 operates on A/E-2 (Figure 10) to close a microswitch in series with the signal from the decant timer to the transfer timer (M.S.-3, Figures 5 and 10). This permits the start of a new cycle. When the three-phase frame begins its equilibration motion, moving away from the transfer position, pneumatic microswitch St-0 (Figure 7 ) in series with a solenoid valve (V-7, Figure 5) in the three-phase controller provides signal St-1 (Figure 7 ) to the start relay of the fraction collector controller. These signals satisfy all of the requirements outlined above. MECHANICAL PERFORMANCE
The apparatus was tested for mechanical reliability by running it continuously for six weeks. No breakdowns occurred during this time under “dry run” conditions or subsequently, during intermittent runs with solutes of interest. However, an alteration of performance that was observed during the ninth week of operation was corrected by replacing a rubber membrane in the four-way air valve. The whole apparatus has been designed to be fail-safe with respect to the fractionation procedure. It is intended that in the event of a failure in any part of a cycle the apparatus will not proceed to the next cycle. To date there has been substantially no experience with failures, so that this concept has not received an operational test. When failures were deliberately invoked by interfering with the operation at several points, the fail-safe effect did occur. The performance of the sampling system was tested in the following
Symbols are as indicated for Fig w e 7
manner. X solution of stearic acid in a 1 : l mixture of ethanol and 1,1,1trichloroethane was placed in selected vials in one row of the fraction collector. The sampler was allowed to transfer eight portions of 0.49 ml. each, followed by a 1.0-ml. ethanol wash, to separate weighing vials. The variation in weights of the residues was .t0.5’%. The performance of the solvent transport section of the apparatus was evaluated as follows. After each tube was filled with bottom phase, moving phases were added automatically. During the decantation stage of the 20th cycle each tube in the output row was inspected for phase separation. The various phases were found to be cleanly separated from each other. Thus the apparatus has been found to be capable of carrying out all of the mechanical requirements of the automated three-phase process. The effectiveness of the separations attainable with this apparatus may be expected to be a function of the stability of the temperature control system as well as of the choice of solvent systems. Distributions of known substances will be discussed in subsequent publications. LITERATURE CITED
(1) Craig, L. C., J . Biol. Chem. 155
519 (1944). (2) Craig, L. C., Hausman, W., Ahrens, E. H., Jr., Harfenist, E. J., A N A L . CHEM.23, 1326 (1951). 13) . , Rleltzer. H. L., Federation Proc. 15, 128 11956’). (4) Meltzer,’ H. L., J . Biol. Chem. 233, 1327 (1958).
RECEIVED for review October 30, 1964. Accepted LIarch 8, 1965. This work was supported by Public Health Service Research Grant B-03191 from the Xational Institutes of Keurological Diseases and Blindness.
