Extra c t,ion LYMAN CRAIG The Rockefeller Institute for M e d i c a l Research, N e w York 21,
T
N. Y.
1. Systems Stability Partition ratio Partition isotherms Selectivity of system Rate of break of dispersions 2. Apparatus Single stage Discontinuous Continuous Multiple stage Discontinuous Countercurrent Discontinuous Continuous 3. Analysis Direct weight Spectroscopic 4. Purpose Removal
HIS review covers primarily the papers dealing with analytical extraction which have appeared since the 1952 review of this series. Much of the work covered has already been mentioned in other general or specialized reviews (36,90, 165,148, 150, 165, 17i,177) on extraction or some phase of it b u t from a different standpoint. No claim is made here of complete coverage of all aspects of the subject. Extraction in some form has long been a part of many analytical procedures and accordingly is often given little emphasis in the description of the work. I t is obviously impossible to learn of all such procedures, as they are not commonly indexed in abstract journals under the heading of extraction or even under a related subject. T h e particular phase of extraction covered perhaps can best be defined in terms of the purpose for which the extraction is to be made. If i t is to be made and used as a method to learn something, either qualitatively or quantitatively, of the nature of an unknown, i t properly comes within the scope of this review. Processes designed for large scale purification are not usually covered, as they are so well covered in the review in Industrial and Engineering Chemistry (171). Only in cases where the reasoning OT theory has a bearing on procedures which might he used in analytical extraction are such papers mentioned. However, when the process is designed to permit purification of sufficient material for structural study or further analysis, it properly falls within the scope here intended. Xo attempt is made to cover the field called “Partition Chromatography,” although this certainly is a countercurrent extraction process, irrespective of whether or not solid-liquid or liquid-liquid extraction is responsible for the effect. However, this type of extraction is a specialized field and has been covered in a separate review of this series. il survey of the literature since the review of 1952 ( 3 5 ) was written does not indicate major progress in different types of apparatus, although a number of papers describing new apparatus have been published. It would appear that extension of the usefulness of extraction for analysis does not now depend so much on apparatus as on a better understanding of systems and the choice of solvents or discovery of complexing agents, so that much higher selectivities can be reached. Definite advances along this line are to be reported in several fields, most notably in systems for the separation of inorganic substances and for the separation of peptides. It is thought by TYeaver and collaborators (180) that liquid-liquid extraction offers the most promising approach to the problem of the separation of the rare earths. Systems with p values of 1.6 between adjacent members of the atomic series (146) apparently have been developed by the use of tributyl phosphate. Those familiar with countercurrent distribution ill recognize this as a rather easy separation. I n trying to review the recent literature on analytical extraction the writer has been impressed by the many confusing facets of the subject and the interrelation of the various factors which have a bearing on whether or not a given extractor and procedure are the best for the problem. +illtoo often a publication describes a new apparatus and procedure but on the basis of its use for a restricted purpose has gone on to recommpnd its general use without giving sufficient consideration to the interrelation of the various factors. I n attempting to give the proper balance in this review to the many claims which are to be found in the literature, the following flow sheet has been prepared. I n it most of the considerations are listed which contribute to the science of extraction and which necessarily form the basis for the choice of any particular extraction procedure.
Fractionation
Distorting effect of solutes Recovery of solute Speed of interchange Complexity of mixture Molecular size and complexity of solutes Cost of apparatus and availability Selectivity Reproducibility Labor involved Time involved
Titration Time for analysis Amount of pure substance desired Purity required
The most important consideration from almost any point of viev, in any extraction process is the choice of a suitable system. Obviously, the ideal system would be one so selective t h a t a single contact would accomplish the full purpose of the extraction, thus eliminating the need for extensive apparatus. This often is the case but, ivhen not, more extensive apparatus is brought into play in order to increase the owr-all selectivitv of the process. SYSTEMS
The choice of a suitable system is indeed one of the major difficulties in the use of extraction. Unfortunately, the compounding of systems cannot be done very successfully on the basis of theoretical predictions, although some correlations have been made (68, 84, 139). Experience in compounding systems is of the greatest assistance, but unfortunately there is no substitute for a n actual experimental trial. I n general, however, in a n unknown field a good system may be found more quickly than the best solvent for purification by recrystallization or the best adsorbent for resolution of an unknown mixture by chromatography. .4 system as treated here includes not only the liquids but all sorts of additives incorporated to produce a desired effect. These agents include salts for salting out or salting in, complexing agents like the hydrotropic agents such as the sulfonic acids, and coordinating agents or chelating agents such as those so widelv used in inorganic analysis. I n the past fern years many satisfactory systems have been compounded for different separations. In the hope that a list of many of these will be helpful in suggesting other systems, a number of tables of different systems‘which have proved useful in various separations during the past two years are given. Where the separation desired warrants a careful study of systems in order to reach the highest selectivity, an approach such as t h a t suggested by Engel and collaborators (51) will prove helpful if the system involves combinations of three or more solvents. A survey of the tables reveals considerable use of very simple systems. Where these fail to give the required selectivity or behavior, more complex systems have been devised. Buffers have proved especially helpful. I n many cases careful studies of complex formation (55, 62, 6S, 151) have been made. This is a familiar approach to inorganic chemists ( 9 0 ) and now has become very important in separations with the rare earths. I n comparing the relative use for analytical extraction which
110
V O L U M E 2 6 , N O . 1, J A N U A R Y 1 9 5 4 Table 1. Inorganic Compounds Solutes Separated Aletal thiocyanate complexes Subgroup VI1 elements Zirconium, hafnium Pitchblende residues, ionium Ionium. scandium Ionium, uranium Uranyl nitrate Ferric chloride Th 8-Qii inolinol Antimony
N b and T a In Group I11 B metal halides Cn dithizone Ce Afetal bromides Rare earths Rare earths Uranium perchlorate
*
Pyridine, 4 S NaOH Benzene, various 6-diketones, aqueous HC10r soln. Tributylphosphate, aqueous, " 0 3 , Ca(KO8) Dibiitoxytetraethylene glycol diethyl ether, aqueous xHaN03, "01 Ethyl ether, aqueous Al(Sod, NaAc, HAC Dibutyl carhitol, pentaether, Hz0 Ainyl acetate, ?A- H C l Benzene, thenoyl trifluoroacetone, HKOs solns. Aqueous buffer, chloroform E t h y l acetate, Hz0, oxalic citric acids Xylene. S.TT HCl, methyldioctylanine Diisopropyl ketone, aqueous H?90a, €IF E t h y l ether, aqueous H I E t h v l ether, aaueous halog i n acids . CHC13 or CClr and H20 Ethyl ether, 5.V IINOa E t h y l ether, H B r Tributyl phosphate, nitric acid Tributyl phosphate. HCl Ethyl ether, perchloric acid
+
iYb and T a
a
System Tributyl phosphate, H10
Apparatus
. .
..
... ...
APPARATUS
..
During the past two years a number of continuous extractore designed for extraction for removal purposes have been reported. S o n e of them offer new principles but many offer technical improvements of a practical nature. Napoli and Schmall (132) and Kamphausen (102) describe apparatus for extraction of solids. Gage et nl. (56) describe a laboratory wet grinder which can be used also for extraction. 4 microextractor set, up also for steam distillation has been described by Connolly and Oldham (33) and one for ext,racting a t reduced pressures by Bick and Clezy (15). Extractors in which the solid is extracted with the cold condensate have been described b y Parks (142) and Herian and Moignard (81). .ill-
.,.
...
... ... ... ,..
CCa
SC6 CCD
.
Table 11. Peptides
Oxytocin Desulfurized oxytocin Bromine oxidation products of oxytocin Hog vasopressin Oxidized oxytocin Partial hydrolysis products of vasopressin Synthetic oxytocin ACTH ACTH ACTH .1CTH Benzoyl peptides Hydrolyzate of grainipidin Peptide from B. sublilis Sisins Kisins A, B, C , and D Licheniformins llicrococcin Clupein, salmine Pipsyl deriratives from peptides Pipsyl derivatives from peptides Bacitracins Bacitracins Amino acids from bacitracin D N P derivatives of bacitracin Polypeptin Tyrocidines Tyrocidine tives
A deriva-
System N e B u ether. sodium nanh-
(PH 3) 2-Butanol, HAC 2-Butanol %Butanol: 2-Butanol,
Apparatus CCD
0.5A- .iqueous
CCD
0.01M ammonia water 0.5% acetic acid
CCD CCD CCD
1-Butanol, 0.09 p-toluenesulfonic acid 1-Butanol 6% acetic acid 2-Butanol: 0.l.V acetic acid
CCD CCD
2-Butanol, 0 . 1 5 acetic acid 2-Butanol, 0.01'V ammonia 2-Butanol, 0.2% aqueous trichloroarptir arid 2,4,6-Collidine,~wate; 2-Butanol, 0.5% aqueous trichloroacetic acid 2-Butanol, 0.1% aqueous trichloroacetic acidEthanol, Hz0, CHCls, cyclohexane I-Butanol, dilute aretic acid I-Butanol, 0.1M phosphate buffer Methanol, butanol, acetic acid mater YaCl NaAc Methinol, 1-b;itandl. acet a t e buffer Phenol, dilute HC1 Ethanol, acetic acid, CHCla, CClr, H2O I-Butanol, lauric acid, NaAc butfer Various organic solvents with water Various organic solvents with water Pentanol, butanol, phosrihate buffer 2-Butanol 3% acetic acid 2% HCI, bhenol 2-Butanol, 3 % acetic acid, CHCls water 2-Butandl. isopropyl ether, dilute HC1 Nethanol, CHCla, 0.1N HCl Methanol, benzene, CHCla, 0.1.V HC1
has been made of the strictly discontinuous process, with t h a t of the continuous processes, a survey of Tables I to V will be found very instructive. ?;early all the applications of the past two years have been made by countercurrent distribution (CCD), except in the inorganic field, and here simple extraction has been the choice. Most of the papers refer to some application of countercurrent distribution rather than to attempts to improve the procedure. This has not been true for attempts involving the continuous column approach. For a short account of countercurrent distribution reference (36) may be consulted. For a more detailed account, the excellent review of Rauen and S t a n p i (150) will be found very useful.
...
Continuous countercurrent. Stage continuous.
Solutes Separated Actinornvcins
111
CCD
Solutes Separated Lipides from placenta Lipides from brain Phospholipides from ox brain Linseed phosphatides Uridine-5-pyrophosphate derivatives M e esters of higher f a t t y acids Peroxides of methyl linoleate Urinary estrogens Steroid mixtures
CCD CCD CCD CCD
serine Phospholipins
Table 111. Lipides System Mixtures of CClr, CHCla, CHIC11, CHaOH, and HzO CHaOH, CClr, HzO Pet. ether. ethanol, HzO
Apparatus CCD CCD CCD
Hexane, CHaOH, H?O Phenol, sulfate buffer
CCD CCD
Pentane hexane, nitromethane, nitroethane Pentane, hexane, ethanol, Hz0 CHsOH. CClr, Hz0, ethanol, cyclohexane, ethyl acetate, H20 Cyclohexane, ethyl acetate, ethanol, water Pet. ether, water
CCD
Heptane, 90Y0 ethanol Benzene, 5-V HC1, methyl butyl ketone, citrate buffer P e t . ether, acetone, ethanol, 1120
CCD CCD CCD
CCD CCD CCD
CCD CCD CCD CCD CCD
Solutes Separated Alkaloids of veratrine
CCD
Desacetylneoprotoveratrine Alkaloids of veratrine
CCD CCD CCD CCD CCD CCD CCD CCD CCD CCD
Table IV.
Alkaloids Sys t eIn
CHC18, CClr, 2.M acetate buffer Benzene, 2.11 acetate buffer
Apparatus CCD CCD CCD
Zygadenus alkaloids Zygadeniis alkaloids
Cyclohexane, benzene, 2.11 acetate buffer CHCls, 2 M acetate buffer Benzene, phosphate buffer
Neogerinitrine Alkaloids of veratrine Alkaloids of veratrine -4lkaloids of veratrine Alkaloids of veratrine Germbudine, isogermi-
Benzene 2 M acetate buffer Benzene: o h o m h a t e buffer Benzene, i,Zr acetate buffer CIICI?, acetic acid, Hz0 0 5.Y HC1. CHC13 Benzene, 2 1f aretate buffer
CCD CCD CCD CCD CCD CCD
WiliGgine Wilfortrine Wilforgine Ryanodine Corynanthein T a r bases
Benzene. hexane, 2% HCl Benzene, 1.8% IICl Benzene, hexane, 2% HC1 Ethvl ether, water E t h y l ether, citrate buffer Cyclohexane. phosrlhate-citrate buffer: CIIC17 chosphate-citrate buffer ' iert-Amyl alcohol, various buffers C H C h , phosphate-citrate buffer
CCD CCD CCD CCD CCD CCD
dine
CCD
Pyridine alkaloids
CCD
Garraya alkaloids
CCD CCD
CCD
(6)
CCD
(184)
112
A N A L Y T I C A L CHEMISTRY Table V. iMiscellaneous Organic Solutes
Solutes Separated Porphyrins Aromatic amines Cresols
System Apparatus Ether, HC1 CCD Cyclohexane H20 A g + ... Cineole, cyciohedne, HzO, . Ag Butanol, trisodium phosCCD phate soln. E t h y l ether, phosphate bufCCD fer CHCla 0.6M phosphate CCD Or a & solvents, buffer CCD Et& ether, 0.5M phosCCD phate buffer: ethyl ether, H C l ( p H 2) Benzene, 50% acetic acid CCD Benzene, 50% acetic acid CCD 2,2 4-Trimethylpentane CCD phosphate-citrate buffer Iso-octane. 0.5.W phosphate CCD buffer Ethanol, water, CHClr CCD
..
+
hlaple lignin Carbobenzoxygluta mic esters a-Lipoi7 acid or-Lipoic acid Pyruvate oxidation factor Lipoic acid a-Lipoic acid Cohumulone Cohumulone Cardiac glycosides Antibiotics from basidiomycetes Antibiotics from basidiomycetes Antibiotics from basidiomycetes Flavines Rhizopterin Vitamins Bn? Bnb, etc. Transformation products of vitamin BIZ Biocytin Gliotoxin 2,4-D metabolic products Germination factor Antibiotics from ceohalosporium Magnamycin Mycomycin Streptomycin Antimycins Formic acid Various solutes Various organic bases Higher fatty acids
Conjugated bile acids
CaroJenoids from orange juice Various aldehydes from lignin
CHCla, phosphate buffers
CCD
E t h y l ether, hexane, methanol. H r O Benzene, hexane, HIO 1-Butanol, 0.02.V HC1 1-Butanol, 0.02.V HC1 Phenol, CCh, H9O Benzyl alcohol, HzO
CCD
CCD CCD CCD CCD
CHCla. cresol, ddute HC1 CHC13, HzO Ethyl ether, phosphate buf-
CCD CCD CCD
CCD
(0,.
Irl
tert-Butanol, pet. ether, acetone. H9O Hexane isopropyl ether acetone, phosphate buffe; Benzene, acetate buffer CHCls, phosphate buffer Pentasol, aqueous buffered stearate Benzene, pet. ether, 80% ethanol N e isobutyl ketone, water 1-Pentanol, water 2-hIethyl-l-pro~ano1,water Liquid paraffin, phosphate buffer Various combinations of methanol, heptane, isooctane, acetic acid, formamide, and acetonitrile Heptane, isopropyl ether, aretic acid, 2-butanol 3% acetic acid, heptane: 97% acetic acid Pet. ether, 99% methanol Various organic solvents, water
CCD CCD CCD CCD CCD CCD
... .,.
...
( 8 3 ) has described a mechanical robot for operating a countercurrent distribution train. Its design is different from that designed by Craig and collaborators (59) and may well appeal to one who builds his own robot and fraction collector. I t operates by vacuum drive in order to avoid the danger with organic solvents resulting from an electric spark. Short and Twigg (164 j, Short ( 1631, Bock and Meyer (18), and Spence and Streeton (167) have described continuous countercurrent evtractors for analvtical purposes. All have made use of a spinning center pole in the column. Extractors of the stage continuous type have been reported by Fischer and Jdbermann (65), Nagata, Eguchi, and Yokoyama (131), and JVeygand, Wacker, and Dellweg (181’). The latter is an intrresting modification of the countercurrent distribution machine which provides for continuous flow of the lighter solvent. Certain of the advantages of the discontinuous process for liquid-liquid evtraction for fractionation have been mentioned elsep$here in this review. That similar advantages might be found in the liquid-solid type of extraction had also been suggested hv Craig and collaborators (38). Recently Morris (129) has found this approach to be promising and has built a countercurrent distribution apparatus especially for liquid-solid eutraction At each stage the liquid is transferred ljv decantation through a filter. Granick and BogoraJ ( 6 7 ) designed an apparatus to be inserted in the Beckman spectrograph for determining partition ratios rapid) I+ hen analyzing countercurrent distribution runs. In countercurrent distribution the most informative distnhution pattern is one which is based on direct weight analvsis. When properly done, this analysis need not be particularly tinieconwming. Two procedures (@, 7 7 ) for accomplishing this have recently been reported. THEORETIC4L CONSIDERATIONS
CCD
CCD
CCD CCD
puipose extractors have been described by Icratz anti Frank (111), Hemmings (7‘9), and Tfflnnd (89). The latter has an adjustable elbow on the overflow tube which permits adjustment of the level of the liquid in the extraction chamber. Schmall et al. (161) have designed an extractor for the determination of the salts of organic acids. Extractors were designed especially for use in the purificatim of radioactive substances by several workers (69, 98, 124). Liquid-liquid extractors with stirrers in the extraction chamber t o promote interchange Rere described by Holliman (86), Henry and Sorba (801, and Dobson and Randall (47). The latter also has a builtin rapid evaporator for concentrating the extract. An extractor with a diffuser plate and arranged for use under reduced pressure has been given by Heftmann and Johnson (78). Other extractors (46, 792, 88, 106) have been described. Less interest has been shown in the design of new equipment for extraction for fractionation purposes. For fractionation purposes the interest appears largely to have been directed toward the search for suitable systems for countercurrent distribution as Tables I to V show. Netasch (115) has described an automatic all-glass 200-tube countercurent distribution train, which has a number of different features from the one described by Craig and collaborators (39) and mentioned in the last review of this series. Tschesche, Grimmer, and Neuwald (17 4 ) have described a hand-operated train made from glass. It requires individual transfers. Hickey
After reading many papers dealing with the theory of countercurrent extraction, this reviewer has the impression that many research workers are not really aware of the basic differences between the strictly discontinuous process and the continuous process. Merely evaluating a continuous process in terms of theoretical plates calculated on the basis of the changes in concentration produced in an arbitrarily chosen mixture does not satisfactorily explain the basis for the effect the column has produced. The term “countercurrent distribution” has even been applied more than once erroneously to a continuous process. I n countercurrent distribution either phase is caused to move in a strictly stepwise manner. This is not a “cascade” and the difference as far as separation is concerned is more than superficial. In countercurrent distribution each transfer is made only after complete equilibrium (or a known degree of disequilibrium) IS reached. Discrete volumes of a constant amount of the phases are moved on to the next contact each time. The ranges of concentration of the solutes are known and the partition isotherm has a real meaning. These factors are all vital i n permitting a theoretical distribution curve to be drawn. K i t h the continuous process a different state of affairs exists. Equilibrium in the sense of the discontinuous process is never reached. Some operating stage, often far short of equilibrium as far as partition is concerned, determines the separation. It is therefore a rate process, in the sense t h a t its efficiency may largelv depend on the relative rates the different solutes approach true equilibrium. These may or mav not be different and may even permit separation of solutes nith very similar partition ratios, (37, p. 220; 91, 181). All solutes do not approach equilibrium a t the same rate in a given system. Even more serious with continuous columns may be the effect of solute concentrations building up in the column when the solute is fed in continuously. This can contribute toward an easier separation, but usually it has the opposite effect. Phase shifts are usually undesired.
V O L U M E 2 6 , N O . 1, J A N U A R Y 1 9 5 4 These effects are not easy to evaluate in the continuous process, but they are undoubtedly reflected in the well-known variation a given continuous countercurrent extractor will show when its plate efficiency is measured with different solutes, different systems, different concentrations of solutes in the same system, and different flow rates of the solvents. Further examples of such variations can be found in recent studies with continuous ext,ractors (8, 30, 69, 163, 164, 167). Even sound can have a n effect (73). The rate of approach t o equilibrium is a basic phenomenon in extraction which merits much further study, in spite of the fact that, the literature on this subject in chemical engineering is voluminous (34, 1.90, 172). I T ~ Kvariable this is with different solutes and solvents can be $hon-n experimentally ( 7 ) in a very simple manner. Of all the solutes anti systems thus far studied by countercurrent distribution, by non- a considerable number, as Tables I to 1‘ show, among organic solutes the vast majority easily reach essential equilibrium with only the few gentle shakes which the distribution machine provides. Only the penicillins (7’) with buffered solutions seemed t o be slow. This fortunate state of affairs does not, appear to hold for the complexes of certain inorganic ions (90, 91, 9 6 ) . In the extraction of zinc dithizone ( 9 2 ) from aqueous buffers with chloroform, shaking for a matter of minutes was required. \\.hen carbon tetrachloride n-as uscil, less time was required. Irving, Bell, and Williams give a good account of this observation and show the desirability of studying the approach to equilibrium x i t h the solute first in one phase and then in the other, as had previously been done with the penicillins ( 7 ) in the author’s laboratory. The many confusing aspects of the phenomenon of the transport of solute from one phase to the other are still best explained on the basis of the t,w-o-filni theory (37, 172). Simple diffusion cannot explain the resistance to transport. Ward (l7,9) has presented data pertinent to this problem in studies on the kinetics of adsorption a t liquid-licluiii interfaces. With certain systemse.g., hexane, water----anti iritli long-chain fatty acids 3 s solutes a newly formed interface would not reach its maximum interfacial tension until several minutec: had passed. T h e effect n-as attributed to an “entropy :ic.tivation barrier.” From the practical standpoint as applied to columns attempts to accomplish better interchange have been made with pulse extraction columns (SO,62) and with a spinning tube in the column (18, 163, 164, 167). H E T P values as low as 0.6 inch have been obtained but not with very high total number of plates in the column. Perhaps also from the practical standpoint it would be well to point out t h a t the glass countercurrent distribution train can be conveniently used for the same type of extraction as these columns were designed to accomplish. For example, a handoperated unit with twenty tubes of the design given ( 3 9 ) (available commercially from H. 0. Post, 6822-60th Road, hiaspeth, N. Y., in any size desired) and arranged for recycling with ten t’ubes in the upper row ant1 t r n in the lower row is ideal for this purpose. T h e alternate or tlouble withdrawal procedure given (37, p. 202) can be used and frwh portions of solute added to the appropriate tube on each t ranefer t o maintain desirable solute concentrations. For this purpose the excellent formula8 developed by Scheibel (159, 160) can he used for calculating the amount of solute to be added in order to reach and maintain the proper balance. This method of operation gives the well-knoFn triangular pattern which Scheitwl and Klinkenberg (107) have treated. In spite of its discontinuous nature, a favorable system with phases which separate in 30 seconds will permit 20 extract and 30 raffinate phases t o be collected per hour. Even with the smallest distribution apparatus which is built for 10 ml. in each phase, this mould mean a throughput of 200 ml. of each phase per hour. This compares favorably with continuous small laboratory ex-
113 tractors with this number of plates. With the distributors built for 100 ml. in each phase, the throughput would be 2 liters of each phase per hour, If this is not sufficient capacity, there is no mechanical reason against building distributors which hold 1 liter of each phase per tube. The advantage of such a distribution apparatus over the continuous columns would be that it operates on the basis of true equilibrium and a constant number of actual stages. It would also be very easy to increase or decrease the number of stages if required. Moreover, solute concentration effects could be observed and readily studied a t any stage of the process. The Podbielniak centrifugal extractor is a very efficient tool and is widely used in industry for production. One of its major advantages is the speed of extraction Although even the smallest model, the (‘Pup,” is somewhat beyond the range of this review, many readers will find the excellent study by Barson and Beyer (8) of the characteristics of the P u p of great interest. As resards the mathematics of countercurrent distribution, several papers may be cited. Sewton and Abraham (136) give equations for calculating theoretical curves Hecker (76) has given a mathematical treatment and comparisons with the continuous process. Correlations of numbers of stages, p valurs, yield, anti purity Rere made. Bland, Hillis, and Killiams (16) have given a rather extensive mathematical treatment of these same considmations. A general mathematical treatment for continuous countercurrent piocesses of all types has been given by Kuhn (113). When countercurrent distribution is used for a separation, it is usually best to try to operate so t h a t a symmetrical distribution curve is obtained-i.e., a t complete equilibrium and in a concentration range which gives a nearly linear partition isotherm. On the other hand, rn hen it is not practical to operate this way, many useful separations can be made in spite of the deviations. The deviations can even be exploited. Weygand, Wacker, and Dellneg (181) used this approach i’n separating the hydrolysis products of desoxyribonucleic acid. T h e train wan operated a t disequilibrium. I n the separation of the bacitracin polypeptides (43) the system was deliberately overloaded in order to produce a skened pattern and thus obtain a higher yield of bacitracin A of high purity. However, for analytical purposes conformance to ideality is definitely to be preferred. Deviation from the linear partition isotherm seems to be caused by association of solute molecules as given by the equilibrium S e (S)n. Higher concentrations favor the formation of associated molecules, (Sjn. T h e difficulty as far as extraction is concerned is caused by the fact that the equilibrium is not the same in the two phases. With acidic or basic substances a buffer added to the system will usually help to produce a linear isotherm, probably because a high proportion of ionized molecules, S + or S - molecules, exists and these scarcely partition in the organic phase. The net effect amounts t o a lowering of the effective concentration and a more linear isotherm is favored. Any solute added as part of the system which does not interfere otherwise and which associates strongly with the solute may help to give a more linear isotherm. With acidic solutes a good additive for the system is glacial acetic acid. The reasoning here is t h a t the complex S.(HAc), is predominantly formed and (S), or S,.(HAc), repressed. Since there are so many H:Ir molecules available in both phases in equilibrium, the over-all effect amounts t o that of the partition of simple S molecules. Irrespective of the validity of this reasoning, glacial acetic acid has permitted linear partition isotherms n ith the higher fatty acids (a), and both conjugated and unconjugated bile acids (3). A similar type of reasoning has played a role in the development of complexes for partitioning inorganic solutes (90). Here buffers and strong acids are often used, Golumbic ( 6 2 ) and Golumbic and Weller (63) have used complex formation with silver ion in the attempt to develop selective systems for aromatic amines.
114
ANALYTICAL CHEMISTRY
As a practical consideration i t may be well to ask the limits of the present stage of development of analytical extraction and its over-all selectivity. There are bright points and others rather discouraging. For this reason the outlook would not be too bright if extraction were the only tool available. The same, however, would hold true for chromatography or any other separation approach. It is only when extraction is properly integrated a i t h the other approaches that it finds its maximum effectiveness. Examples of this are to be found in the work with lipoic acid (166, 156), oxytocin (49), ACTH (19, 111 ), the carotenoids from orange juice (44),and others. Individual cases of high selectivity are to be found in work on B12(101, 137), tyrocidines (9),bacitracins (43),vasopressin (149)) and actinomycins (63). Here separations a ere obtained, although slight structural changes in molecules of molecular weight 1000 to 1500 were the only differences. With the peptides the differences often resulted from the substitution of one single amino acid residue for another. The insulin studies (70, 7 1 ) are interesting in this connection. The method of partial substitution ( 7 1 ) plainly showed beef insulin to have a molecular weight in the range of 6000, yet it could be fractionated by countercurrent distribution and a component B separated from the major component A. B was found to differ from -4by having only five amide groups, n-hereas A contained six. All other analytical data, including quantitative amino acid analyses, were identical. LITERATURE CITED
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