Apparatus for Countercurrent Distribution LYMAN C. CRAIG AND OTTO POST
Rockefeller Institute f o r Medical Research, New York, N . Y.
4 serious hindranee to the use of multiple extraction or countercurrent distribution for 81eparating mixtures of closely related compounds is the amount of labor involtwd in making the extractions. The apparatus described here permits s e ~ e r1a thousand almost quantitative extractions in a few hours of operation. Tnvo other types of supplementary extractors are also described. The most us&ul prooedures for using the extractors are given.
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HE possibility inherent in entraotion as a tool for the septlmtion and isohtion of chemical oompounds has been recognized almost from the beginning of chemistry. Although the pracedure has been used as an analytical tool for investigating crude mixtures containing components with wide differences in salubility, further extension of its use to the analysis of mixtures with closely related components has heen attempted only more recently. The analogy of fractionation by distillation to fractionation by extraction has been considered (9) and it is only logical on the basis of the analogy to turn to some type of column extraction in order to gain the greatcst efficiency and thus permit separations in spite of closely rela.ted partition ratios. On the other hand, attempts to develop smd-scale continuous laboratory extraction columns for fractionation have met with great teehnieal difficulties and the attempt hits, in general, not been rewarding. For this reason the attention of the aut,hors' laborstory, several years ago, was turned toward some type of discontinuous process with the hope that furthcr information in regard to the requisites of quantitative extraction might be gained. Irrespective of tliis possibility, certain definite advantages of the discontinuous procoss, particularly for analytical purposes, have become apparent. These include an oppvrt,unity for the direct application of the type of calculation already developed in the maihematics o f ' probability (11, 14). These mathematics have permitted a more precise interprotittian of the results than is possible in the continuous case. The name "countercurrent distribution" hay heen niven t,o the t v w of entraotion that permits direct appl sion in order to interpret the rcsul Investigations along this line.ha"" .I "-I_-..that a g m e r d method of considerahle precision and wide applicability has been develapcd. The method has been particularly useful in proving purity and in characterizing substances that are difficult to study by the more established procedures. Types of substanoes studied have iricludod synthetic antimalarials (7), the ponicillins (a, 6), the streptomycins ( I S ) , fatty acids ( l o ) , purines and pyrimidines (le),and polypeptides of the gramicidin type (8). For further development of the method it is the intention of this laboratory to consider mainly two separate lines of rcsearch. One involves the study of systems-i.e., the search for two immiscible phases (one may he a solid, 5) which give the greatest possible differences in the partition ratios of the individual members of a mixture. Thcse may he spoken of BS "solectivc" systems. Nat.urally, such s y s t e m must not intcrfere too greatly with the final isolation of the fractions. Tho other line of research is purely a technical one and involves tho dovelopment of suitable apparatus for the performance of large numbers of quantitative extractions with speed and minimal labor. If a sufficient number of oxtractions could be applied, high selectivity of a, system would not he required for many purposes. In order to achieve such multiple extractions, several types of apparatus offer possibilities. Up to the presmt time the best
devicc tram the analytlch.1slandpoint has provod to bo B modifieittian of the original count.orcuricnt distribution machine (4). The present paper describes t,ho model eurrontly in use and mentions two other types of diatrihution apparatus which an. under investigation. 3IGN OF APPARATUS ,
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The tubes p& krough the steel plates with as perfect a fit as is mechanically possible, and are pressed in with considerable force. No detectable leakage has thus far been observed around the joints. The distance between adjacent tubes is approximately 0.125 inch: the over-all diameter of the part is approximately 6 inches. A' hole slightly larger than the-central ;6d, 3, extends through the center of each end plate. Three aluminum studs ioin t,hn t,wo nlat,es ,~~~~ inst, inside t,he row of tuhes and are held h r three stainless steel Allen head cap screws whose heads &n be seen on the plate of part 1. These makc the part more rigid. The contaoting surfaces of the two plates are ground flat with great accuracy. The tubes arc numbered, 0 t,o 24, in counterclockwise direction an the top end plate. Part 2 is constructed almost, like 1, except that the tubes are only 2.5 inches in length and a heavy piece of aluminum is used I
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rigure I IS a, pnarograpn or an appamrus con-camng za T U I W Figure 2 shows the same apparatus eomplatel:y disassemblrcl. The various parts will be described mostly from Fi gure 2.
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Figure 1.
26Tuhe Distribution Apparatus
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V O L U M E 21, N O . 4, A P R I L I949
Fixure 2.
25-Tube Distribution Apparatus Disassembled
on the plate by three Allen head cap screws. When 1 is placed over 2 8s shown in Figure 1, its tubes can be made to coincide exartly with those of 2 by rotat,ing the part around the central rod, 3. The tubes of 2 are numbered 0 to 24 in clockwise direction. The plate glass cover, 4, then seals each of the lower ends of the t,uhesof 2. The glass is held in position by the aluminum cover, 5, which in turn is held in position bv the circular nut, 6. The steel rod. 7. is used for tightening the circular nuts.
position b$ the U'lig nut, IO. A spring 11, fits beneath the wing nut, so that a slight pressure will be exerted on the cover even when the wing nut is loosened. The glass cover, 8, has two small holes on either side of the central hole and still another hole near itsouteredge. Thelatterpermitsaecesstoanytubeof theappppamtm during operation. Two short round rods extend from the
moved and in this way the opening may b i br&ht
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over any tube
to the upright position' slowly, in order not to cause d i s p e r s h again. As the end of each bube is of glass, the separation can be followed visually. In order to make a transfer, the upright position is first ensured by the plug 22. The wing nut is backed off one quarter iurn, the machine plug is withdrawn, and the upper section is rotated slightly in the clockwise direction. The plug is then allowed to snap back against the steel plate of 1. Upon rotating 1further, 23 snaps into the next hole and the tubes are again superimposed. The wing nut is tightened and the apparatus is ready far a. second equilibration. A system which equilibrates reasonably well will permit from 20 to 25 transfers per hour. Aside from great,er speed in operation as compared to t,he original design (4), the appartratus desoribed here is more flexible. When the volume ratios desired of the two phases are not 1 to 1, a correspondingly longer or shorter section can be substituted for 1. Both longer and shorter soetions have been successfully used. An apparatus with tubes of larger diameter can also be built. An apparatus of the design of Figure 1, containing 54 tubes of 0.5-inch diamder, has been constructed and has been in successful operation for more t,han a year (Figure 3). An interesting modification of this larger apparatus is that part
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.~ 18 is itself mounted on a bearing a t 25 and conveniently drops t o
&ms is pressed firmly theopening by wedge, 13. The small end of the wedge is made from steel wire which fits into a tiny
hole in ?4, wde& it isheld firmly in p&ion by one of then&, 15 The other nut is screwed lightly on the top of 3. The aluminum
round nut. 15. is screwed upward. i t presses against the bar and
ofwaud b i t is oovered with stainless steel shkking. *hitubes of the apparatus can be held in an upright position by virtue of the plug, 22, which extends through one of the bearings. M-hen the wing nut,, 10, is backed off R quarter turn, the upper
the rear when the nut, 15, is released. Thus the tap of the apparatus is completely clear of obstruction far filling, etc. In this apparatus, part of the central shaft is also removable by releasing a wing nut, 26, a t the bottom. A hollow tube forms the lower part of the shaft from the top of part 2 to 26. The upper part of t.he shaft is held in position by a narrower extension which passes through the tube and further extends through 26. The spring, 11, of Figure 2 is not necessary for the larger apparatus.
With the 54-tube apparatus and 20 transfers per hour, some 2.7 hours are required for one complete circuit of the upper section and 1431 separate extractions are thus made. If a second circuit of the upper section is undertaken, the number of separate extractions made per hour remains constant a t 20 X 54 = 1080. If the single withdrawal procedure is used, and the upper phase considered in discontinuous flow, this would give a flow rate,
ANALYTICAL CHEMISTRY
502
An appaiatus ot the type of Figme 4 with l a g c r units serves another uscful purpose as a supplement to the apparatus of Figure 1 or 3. When considerable mateiial t o he fractionated 1s a t hand, a 12- to 20-stage prehminary distribution 1s made m t h the individual tuhos, for many timos the volume of solvent here is possible. The soluto in a selected tube 1s then rccovered and subjoctod to distribution in the apparatus of F~guro3, I n which 200 to 300 transfers can he apphod if desired The combined soparating powcr is thus considerable, although I t 1s done a t tho cxpenrc oi yirld
Figure 3.
54-Tube Distribution Apparatus
a t 20 transfers per hour, of 340 ml. per hour u-hen 17 ml. of the upper phase are used for each cell. Among tho many apparatus designs considered by the authors for multiple extractions, other than that of Figures 1and 3, two deserve mention. One of these, Figure 4, is particularly useful in studying mixtures preliminarily before taking the time for a more prolonged run in the apparatus described above. The contacting units are ground glass stoppered tubes 0.5 inch in inside diameter and 7.5 inches in length, and numberedo, 1 , s r. The tubes are held by spring clips attached to a Flexaframe rod. The stainless steel Gee clips, available from scientific supply houses, may be conveniently adapted. The rads attached to the clips are in turn attached to a longer rod which extends through two hearings on each end of the rad. The bearings are simply Flexaframe clamps in which the screw remains loose. The bearings are supported by two stands. A crank is attached to one end of the rod for tumbling tho tubes. In this design each tube receives its equal portion of stationary phase initially. The sample to be distributed is placed in tube 0. An arbitrary portion of the other phase, previously equilibrated with the first, is then introduced and equilibrium is reached by inverting the tubes with the crank. Twenty-five inversions aro usually sufficient ( 1 ) . The glass stopper will stlek sufficiently not to fall out, if it is given a slight turn when it is placed tightly in position, Small interchangeable stoppers are therefore more reliable than larger ones. After the layers have separated, the phase to be moved is transferred to the adjoining tube, 1, by the small siphon shown in Figure 4,preferably made from stainless steel tubing. Pressing the rubber collar, through uphich the siphon extends, against the mouth of the tube permits act,uatian by either air pressure or vatuum. The glass tube to which the pressure or vacuum line is attached also passes through the rubber collar and has a hole a t the bend. The operator's finger is placed over,this hole, so that instant release of the vacuum or pressure 16 possible. Following the first transfer, fresh mobile phase is added to 0 snd an equilibration made. The mobile phasc of 1 then goes to 2, that of 0 goes to 1 , and fresh phase goes to 0. This Drocedure is pontinued until all the tubes of the series contai n bdtb phases,.e., the fundamental operation is complete. A E.eries of 12 tubes can be filled in this manner in approximately 1ha "1'. The apparatus of Figure 4 is useful whcn stable emulsmis iena to form, hccause the individual tubes can be placed directly in t.he centrifugc. Often only a single unit d l emulsify and this can he withdrawn latcr from the serics. Distributions which givo initial difficulty, oither from the tendency ta emulsify or from volume shift duo to a high concentration of solute, can he carried aut in these tubes until the difficulty is overcome, The contents of oach tuhe can then be transferred to the corresponding tube of the machine of Figure 1 or 3 (after moving the upper section the corresponding number of stages).
Figure
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Extraction Train Permitting Transfers bv a Siohon
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Figure 5 shows a third type of distribution apparatus under
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cinetinatinn in t" h.i. s". lahorutnvu ainalo nnits ,".r"".aw..r I " _ _ i . The ~ . - -.. int,erlork . .
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shown at left, with the exit tube from one unit inscited into the mtisncr tube il IS tho equumsuuli L L I I L I I I U ~wlllbll ~ Vr uLIYrU xu angle, then hack to a. 45" angle in the other direction without a t h e r of the phases splashing out through E or B. Tilting hack and forth gives good dispersion of the two phases and equilibnurn IS reached exactly .as with the other two types of counterourrent dmtnhutian apparatus described here. After the phases have been allowed t o separate, the unit IS tiltcd somewhat more than 90" so that chembor C I S lower thrm tube B. The upper phase wll then decant through B into n x hde the lower phase i-emrms behmd as I t IS of such volume th r t 11 leaches B only when the apparatus'is in the decanting posltlon. \\'hen tho unit is tilted back to the horizontal position, the upp phasem C~nllRowthraughDintotheopening,E',oftheadjolnn unit of the series. Fresh uppei phase 1s then added through, etr The units of a. series can he indimdually suuuorted by stanaar d clamps and rods available in any lahoraiory: 2.
METHODS OF OPERATION
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I n studies with the distribution apparatus thus far, four . . different methods oi opcratmn have pmvecl most usciul: l l i fundamental, (2) single withdramal, (3) alternatc withdrawal, and (4) complet,ion of squsrcs. Another me1thod which would bo of considerable interest from thc theoreticalI standpoint might . .. he some procodure which would introduce the principle or rcnux. However, tllis must he left for a later occasion when a thooretical discussion ii3 also presontcd. Fundame,ntal. In the fundamental procedure the apparatu8 is . . ,.. , , loaded as given m the previous section anu wnm sumcienc c r a m fers have been applied so th%tupper 0 is over the tube adjacent to lower 0 on the right, the operation is complete. A fixed numI
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V O L U M E 21, NO. 4, A P R I L 1 9 4 9
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her of tubes, 12, thus permit n - 1 transfers. The content8 of each tube are withdrawn and analyzed for their solute content by some suitable tLnalytica1 procedure. For purposes of simplification, the total content of a tube, both layers combined, is of primary interest, hut often useful deductions may be dorived by considering the layers separatdy. The amount of solute in each tube is plotted against the consecutive tube number to give tho .distribution pattern ( 2 4 ) . Single Withdrawal. If, when the state of affairs obtaining a t t h e ond of the fundamental procedure is reached, the process should he continued, a fast-moving band would be overtaking a slaw-moving one and remixing of fractionated solute would occur. However, in certain cases where fast-moving bands are known to be absent, a second circuit of the upper section of the apparatus may he accomplished without remixing fractionated solute, because for practical purposes, all the solute will have been extracted from the advancing upper phase. In most cases 8. better procedure for the application of transfers of number greater than the total number of tubes consists of withdrawing the contents of a particular tube and substituting a fresh phase for the one wiihdrawn. In the mast simple of these modificationr, the fundamental procedure is followed until upper 0 has reached the second tube from the rieht of low.er 0. lower 52 in the aooamtus of Figure 3. The conte& of tube 53153 are then withdkvn and disiarded,
503 matography than are m y of the other procedures herein described. Rapid methods are available for oalculating theoretical distributions for this type of operation. These will be treated in a forthcoming contribution. If the upper phase should be tho one that interferes with the analysis or if the solute of interest should favor the upper phase considerably, i t may be desirable to withdraw from the trailing edge of the band. This may bo aecomplishcd just as easily as in t,he above case. When the first transfer into t.he empty tube is made, fresh lower phase is added to tube 0/53 and an equilibration is made. While the phases are separating the lower phase present in tube 53/0 is withdrawn and numbered 1. On the next transfer, fresh lower phase is added to tube 0 / 0 and the lower phase present in tube 5 3 / 1 is withdrawn as number 2 of the series. This process may be continued until a11 the solute has been withdrawn. I n case the proeoss is interrupted while solut,e yet remains in the a p p a r s h , thp distribution pattern would consist. of two separate curves, as described in 8, previous papor of this scries (4). Alternate Withdrawal. In this procedure three separate patterns are obtained on a single run. One pattern represents a withdrawn series of the slowcr moving oomponents-i.e., those of lower partition ratio-the second represents those of intermediate partition ratio, near 1 which remain in the machine, while the third represents the faster moving components of higher partition ratio. It,,like the first, is a withdrawn series. Specific directions for carrvine out this orocedure have been given (6;). Howovor, a modification is possible whii:h gives only lower piI&EO in the first 51-ithdrawnseries and only up]per phase for the t,hir,a. _
emptit"be. "owe;, at'this point tube 53iO will have no upper ohase. The aoproorittte volume of fresh upper phase is accord-
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of a &hon:'It
'constitutes the first member of the wi
