V O L U M E 28, N O . 4, A P R I L 1 9 5 6 llelpolder, F. IT., Washall, T. A .. Alexander, J. A . , -1s.t~. CHEM.27, 974-7 (1955). llilaeeo, G., Chimica e industria ( M i l a n ) 37, 115-16 (1955). IIuller, R. H., ANAL.CHEM.27, 33.1 (June 1955). IIyers, H. S.,Kiguchi, S.T., A S T M Bull. 195, 39-44 (1954). Sokay, Remei, Lamb, G. G.. Div. Petroleum Chem., ACS, Sew York, 1954, pp. 13-20. Oehler, H. A,, Van Winkle, Matthew, Petroleum Engr. 27, T o . 1, C10-12 (1955). Orchinnikov, B. X., Zhiryakova, S . I., N&yanoe Ichm. 32, SO. 12, 51-3 (1954). Pasrhke, R. F.. Kerns, J. R., Wheeler, D. H., J . A m . Oil Chemists’ Soc. 31, 5-7 (1954). Patterson. G. D., ANAL.CHEM.27, 574-89 (1955). Patton, H. W.,Lewis. J . S.,Kaye, W.I., I h i d . , 27, 170-4 (1955). Petroleum Refiner 33, S o . 4, 223 (1954). Pinkava. J., Chem. Listy 48, 455-7 (1954). Podbielniak, W.J., U. S. Patent 2,676,914 (.ipril 27, 1954). Preston, S. T., Jr., Podbielniak, W. J . , P e t r o l e u ~ nR e f i ~ i e r33, SO. 4, 132-4 (1954). Preston, S. T., Jr., Smith, D. E., Ibid., 33, S o . 2. 137-9 (1954). Reilly, Joseph, Rae, 11‘. N.. “Physico-Chemical Xethods,” Vol. 11, 5th ed., Van Nostrand. New York, 1954. Riley. F. T., Chemistry d I n d u s t r y 1955, 940. Rose, .h‘thur, A N A L . C H E l f . 26, 1 0 1 4 (1954); 24, 60-4 (1952); 23, 38-41 (1951); 22, 59-61 (1950); 21, 8 1 4 (1949). Rose, Arthur, Biles, W.R.. Chem. Eng. Progr. 51, 138-40 (1955). Rose. Arthur. Rose. E. G.. “Distillation Literature. Index and rlbstracts, ’1953-’54,” Applied Science Laboratories, State College, Pa., 1955. Rose, Arthur, Sanders, W , W...\x.%r..CHEU. 27, 331-2 (1955).
723 (84) Roqsini, F. D., AIair, B. J.. Strelff, Ai.J., “Hydrocarbons from
Petroleum,” Reinhold, Sew York, 1953. (85) Kushman, D. F., Simpson, E. 11. G . J . Oil and Colors Chemisfs’ ASSOC. 37. 319-22 (1954). ~, (8G) Serwinski, M., Seapiro, S., Zeszuty S a & . Politech. Lodz., S o . 6 , Chem. No. 2, 15-26 (1955). (87) Sorcina, 11. D., C h i m . A n a l . 37, 172-4 (1955). (88) Stanley, hI. E., Pingrey, G. D.. I n d . Eng. Chem. 46, 2182-5 (1954). (89) Srapiro, Salomon, Zeszyty N a u k . Politeck. Lodz., S o . 6 , Chem., No. 2, 33-7 (1955). (90) Todd, Floyd, “Modern Fract’ional Dihllation Equipment for Your Laboratory,” Todd Scientific Co., Springfield, Pa., 1953. (91) \-an Winkle, Matthew, Petroleum Refirier 33, S o . 11, 171-3 (1954). (92) Vignes, Roger, Compt. rend. 69th congr. i n d . gaz 1952, 903-8. 41, 117-18. (93) Voigt, K. D., Nase, Erhard, ’~ra‘atiLr~issenschalten (1954). (94) T’olk, RZ. E., . ~ N A L . CHEM. 27, 1207 (1955). , S.Patent 2,701,789 (Feb. 8 , (95) White, J. L. (to Cpjohn ‘20.1U. 1955). (96) Wiberley, J. S., Siegfriedt, R. K.. Petroleum Engr. 25, KO.8, C12-14 (1953). (97) Winters, J. C., Dinerstein, R. .A,, ANAL. CHERI.27. 546-50 (1955). (98) Wust, Heinz, Klin. Wochschr. 32, 660-1 (1954). (99) Wustrow, Werner, Erddl u. A-ohle 6 , 321-2 (1953). (100) Yoshida, Fumiktake, Koyaiiagi, Tetsushi, I n d . Eng. Chem. 47, 711-14 (1955). (101) Zaugg, H. E., Shavel, John, < ~ N A L .CHEM. 26, 1999-2001 (1954). (102) Zdichynec, T., Klimerek. 13.. C‘heilf. P r u m y s l 3 (28). 197 (1953). ~~~~
I
REVIEW OF FUNDAMENTAL DEVELOPMENTS IN ANALYSIS
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Extraction LYMAN C. CRAIG The Rockefeller institute for M e d i c a l Research, N e w York 27, N. Y.
HE writing of this review has been undertaken with frank nikgivings about its ultimate worth, partly in view of recent excellent reviews (7S,105,106,147,148,150,152)and books (4, 74, 152, 166) on the general subject of extraction, and partly because the field has beconie so diffuse. That great strides in the use of extraction for separating and identifying compounds have been made during the past 10 years, no one will deny (74). But great strides aleo have been made with other separating techniques useful in analysis, such as chromatography, paper chromatography, ion exchange chromatography, gas chromatography, and zone electrophoresis. The advances made in all these fields taken together have greatly extended the range of the experimental chemist. Thus it is now possible with substances of molecular weight in the order of a few thousand to separate individual conipounds, prove purity, carry out degradations, isolate the degradation products, and determine structure according to the rules of classical orga,nic chemistry with as much or greater reliability than as previously possible with substances in the molecular weight range of a few hundred. Moreover, the published work in several fields, notably in the polypeptide antibiotics and the hormones of the pituitary, has demonstrated beyond doubt that it is now possible to isolate a n unknown natural product, prove purity, and determine its structure without depending on the crystallizability of the substance or its derivative,s. Only those who have spent months in previous
J ears patiently trying t o induce substances to crystallize, often with complete failure, will fully appreciate the advance that has been made. Extraction has played a very important role in this advance. It is certain that extraction as a precise separation method would be a much more highly developed and widely used technique than it now is, had not the majority of the research effort roncerned n ith analytical separations during this recent period been devoted to the various forms of chromatography. Only a relatively few laboratories have been concerned rvith the development of extraction designed for analytical purposes, b u t hundreds of laboratories throughout the world have devoted part of all of their time to research on some phase of chromatography. In spite of this imbalance, a surprising number of structural studies rightly regarded as epoch-making, such as those irith the pituitary hormones, have depended heavily or even been made possible by use of the most highly developed forms of analytical extraction.
FACTORS DETERMINING CHOICE OF SEPARATIOh- TOOL
The major factors which usually determine the choice of a separation tool which is t o be used in conjunction u i t h analytical method are: 1. The ease and speed of accomplishment 2. The amount of sample required
724
3. The general applicability 4. The ultimate over-all selectivity which can be obtained 5. T h e reproducibility regardless of different solute proportions or presence of extraneous material 6 . The number of solutes to be isolated or determined simultaneously
AS the over-all use of some form of extraction in analysis has greatly increased during the past fely years, it must be assumed that improvements in several or all of these points have been made. T h e key to improvement of the first factor is mainly the selectivity of the system. If a single system or a few systems with high selectivity can be found, only a single stage or a few stages will be required. Certainly, a simple extraction is about the easiest analytical separation one could imagine. There has been considerable interest in the development of complexing agents in inorganic analysis (86, 102) for this purpose. Another approach is through the choice of a spectroscopic method for analysis of the phases, which in itself has considerable selectivity. An intriguing example of this is t o be found in a paper by Dean and Lady ( 4 1 ) . I n the determination of iron in nonferrous alloys, the iron was selectively extracted from aqueous acid solution by acetylacetone. The acetylacetone extract was aspirated directly into an oxyacetvlene flame and the iron determined by flame spectrophotometry. An increase in over-all selectivity could easily be reached in such an approach without much additional labor by use of countercurrent distribution, employing only a few stages. I n considering the second factor, it should be remembered that extraction is not really an analytical method in itself. It is only capable of bringing about a separation or presenting an environment from which an analytical determination puch as a weight determination, a titration, or spectroscopic measurement can be made. Thus the amount of material needed for the ovep all process is not necessarilv limited by the extraction procedure, but by the sensitivity of the analytical method used in conjunction with it. Certainly the general applicability of extraction has been greatly improved in the past gear or two by the easy availability of hitherto expensive solvents, various combinations of these, and the discovery of new complexing agents such as the fluoro fatty acids (107). Many more improvements along this line can be expected in the near future. One scarcely need mention the utility of extraction for separation of the fragile solutes such as the hormones and vitamins found in living tissue. For highest over-all selectivity in an extraction process, the countercurrent principle must be employed. Here the over-all selectivity will depend on the inherent selectivity of the system, the selectivity of the analytical method used in conjunction with the extraction, and the number of transfers or plates ryhich can be applied. Obviously the highest possible over-all selectivity cannot be reached without some sacrifice of factor 1. Such separations generally require time and labor. Konetheless, factor 4 is extremely important and well worth making the sacrifice in many cases, such as in purity studies with biologically active principles of relatively high molecular weight. Thus in the separation of pure 8-corticotropin and its enzymatic digestion products for structural studies, Bell and collaborators (8) permitted a 200-tube countercurrent distribution apparatus to run by the recycling procedure for long periods of time; in one case until 10,000 transfers had been applied. However, this gave sufficient pure starting material for elucidation of the entire amino acid sequence of a peptide chain containing 37 amino acids. Such a secure foundation for the Jyork is often the deciding point in the success or failure of a n expensive research undertaking. T h e fractionation studies with insulins form the basis for a concept of ultimate selectivity by correlating it with known cheniical structures as mentioned in the last review of this series (33). Since that time the complete chemical structures of beef, sheep,
ANALYTICAL CHEMISTRY and pork insulins have been established by the work of Sanger, Smith, and Kitai (124, 126) An artificial mixture of beef insulin (molecular weight 5733) and pork insulin (molecular weight 5777) (66) has recently been separated by countercurrent distribution (34) in a system made from 1-butanol, 0.1% acetic acid, and pyridine in the volume proportions of 5, 11, and 3 . This system gives a p H near 6.7 but is not far from the point of mutual miscibility of the phases. Systems of this type with various proportions of pyridine and acetic acid have shown high selectivity with a number of peptides. They offer a range of p H betneen about 4 to 6.7, which is often optimum from the standpoint of the stability of biological materials. Moreover, the solute can easily be recovered by direct freeze drying. Bell and coworkers (8)in their ACTH studies found an interesting difference in the selectivity of 1-butanol-water mixtures as compared to 2-butanol-~vater. A similar result was also obtained in the author’s laboratory with the bacitracin polypeptides. With separation by countercurrent distribution involving thousands of transfers, a shift in beta value of from 1.0 to 1.1may mean the difference betn een success and failure of a difficult separation. Yet such a shift may be caused by a rather small change in the solvent content of the system. An interesting approach to the problem of selectivity stems from a deliberate attempt to overload the system so that there will he marked solute interaction. iln interesting example of this is to be found in the work of Ollev (110). Tn-o different types of phospholipins could be psrtially separated in a single run better a t high concentration than a t lower concentration. T h e interplay of effects in such an experiment would appear to be too romplex to be entirely understandable. This, hoxever, should not p:event practical exploitation. -1similar use oi the overloading principles has been made in separating tannins (90). .A more extreme example of this approach for single-stage extraction is to be found in the study of Tavel (146). Serum proteins iyere fractionated by distribution in pairs of partially miscible phases just below the critical point. The improved separation mav result from a peculiar type of isotherm which changes from a positive deviation to a negative deviation as the concentration increases. .An isotherm of this geneial type is shown by nicotine (60). Such deviations may be the cause for occasional experimental countercurrent distribution curves i s hich are nearly symmetrical but yet distinctly more narrow than those calculated from the binominal expansion. I n the author’s experience this type of distribution curve is found only n i t h larger solutes which show strong tendencies to associate. A too narrow curve may give a separation considerably more selective than nould be expected on the basis of relative partition ratios. When such deviations are encountered, reproducibility from experiment to experiment may depend on duplication of the same initial solute concentration and number of transfers. Otherwise reproducibility from run to run, and with different solute proportions, is one of the favorable features of separation by analytical extraction. KO truly objective research worker Rauld expect to solve the more difficult separation problems by use of only one technique. Successive application of different techniques which depend on widely different properties of the molecule is usually much more effective than attempting to wring the highest possible separation from a single approach. Indeed, the outstanding achievements of the past few years have emphasized the need for all possible techniques. As each technique becomes a more highly developed and effective tool, more of a burden is placed on the research worker, who finds he must be thoroughly familiar not only with one approach but also with the many technical details concerned with a number of different fractionation methods. Even the proper integration of the various techniques becomes a problem in itself. Very good examples of the effective integration of the newer forms of extraction with other techniques are
725
V O L U M E 2 8 , NO. 4, A P R I L 1 9 5 6 t o be found in a number of publications (8, 71, 90, 102, 113, 114). This, in itself, can be considered as part of the advance in the field of extraction as well as in other fields. Certainly the use of extraction for a preliminary separation before application of other separation techniques is not new, It was extensively used this way long before it was recognized in itself as being capable of selectivities of the highest order. I n attempting t o attain the most effective integration of the various techniques, it is recognized that in many cases several of them could be equally effective, the choice depending on personal preferences and the equipment available in the laboratory. Thus ion exchange chromatography is the most effective tool for the quantitative determination of the knon-n amino acids. But for actual isolation work on new fragments or peptides M here ultimate analysis and optical rotations are required, it is not necessarily more effective than countercurrent distribution or zone electrophoresis. Countercurrent distribution has shown itself thus far t o be the most effective separation tool for many naturally occurring peptides-e.g., o q t o c i n , vasopressin, a-corticotropin, @-corticotropin,the tyrocidineF, the bacitracins, and the polymyxins. Although clear generalizations concerning when to use extraction rather than other separation techniques would he highly desirable, the literature a t this time does not justify an attempt t o form them except on a rather vague basis. I n the author’s opinion, extraction is the most promising pieliminary approach for empirical work in a completelv neiv field. Where applicable, it should be used for the final studies of purity. However, other methods may lead to easier separations once something is known specifically of the properties of the solute of interest. is. good example of this is t o be found in the isolation of rererpine, one of the active principles of Indian snake root, which has recently stimulated so much interest in the treatment of mental disease. I n the Ciba laboratories ( 4 6 )the active sedative was found in an intractable oleoresin fraction but it caused trouble when further separation n as tried. However, countercurrent distribution involving a relatively small number of transfers in a chloroform-methanol-water system separated the resin sharply into two groups of substances, which then could be resolved easily by chromatography. Other examples (79, 93) have also appeared of the use of countercurrent distribution in the isolation of this type of alkaloid. Since the last review of this series (33) was written no single major advance in the field can be reported as regards theory, apparatus, or the compounding of systems. However, the work covered in this review shows many improvements for different types of compounds along the lines already suggested in the literature. An attempt t o revise the nomenclature and classification of the different types of extraction procedures has been published by Hecker and Allemann (75). Many authors may prefer the revised terminology they have proposed; others will not care t o change. It seems doubtful if the name “partition chromatography” or “countercurrent distribution” will be changed in the near future in this country. Irrespective of personal preferences, an excellent literature survey of nomenclature has been given. SYSTEMS
I n spite of a much wider use of countercurrent distribution throughout biochemistry and organic chemistry, general rules for the compounding of suitable systems are still badly needed, Some attempts along this line can be mentioned (72, 105), but actual determination of partition ratios with a variety of combinations of solvents still remains the most useful approach. Engel and coworkers (66) have proposed a nomographic approach t o the problem of compounding systems with four components. Rules for compounding three-component systems have been given by Hollingsworth and Taber (80).
Several studies can be cited (29, 53, 74, 105, 115) which list the determination of partition ratios for a considerable number of organic solutes in various systems with the objective of using these data for identification of the solute. Certain speculations concerning the basis for the ratio, such as hydrogen bonding and structure, have also been made (5, 116) and the advantages pointed out of using mixed solvents t o improve selectivity. If these data are t o be used for countercurrent distribution the partition isotherm must also be considered. This makes a much more complicated state of affairs, as the effect of hydrogen bonding is also a function of concentration. Generalizations are much easier if only gross effects are considered, but for countercurrent distribution suitable factors Tvhich shift the K value only slightly -e.g., from 1 t o 1.1-are extremely important. The last review of this series (35) contained a table with many systems listed for different types of compounds. Such a table could be compiled again in this review, but as additional tables can also be found in two other reviews (74, 105), an attempt t o treat the literature again in this way is not made. Perhaps a ehort discussion of research fields where analytical extraction has played an important role nil1 prove of greater intereqt. Complete coverage of the literature will not be attempted. I n most of these fields countercurrent distribution was used only after other fractionation methods had been extensively applied. Perhaps the most spectacular use of countercurrent distribution has been in the field of the pituitary hormones, both those from the anterior ( 2 , 8, 9, 28, 82, 39-101, 120) and posterior (50, 61, 162) lobes of the gland. -411 of these hormones have been found t o be polypeptides or proteins of relatively small molecular size. T h e systems used were of the general type first found useful for insulin (67), in which a complexing agent like trichloroacetic acid or p-toluenesulfonic acid was used t o shift the partition in the direction of the alcohol phase in a butanolwater system. Lysozyme (Sd) has been successfully fractionated this way. An interesting point about these systems is the striking effect of varying concentrations of the complexing agent. Thus with insulin, the system 2-butanol-O.O62% aqueous trichloroacetic acid gave a partition ratio in the right range (0.9), and one which did not vary much with the change of insulin concentration. However, the concentration of trichloroacetic acid was critical. Double the concentration of trichloroacetic acid gave a K of 2.6 but half gave a K of 0.6. The system did not have much capacity because of solubility considerations. A 1yo solution of dichloroacetic acid was found t o give nearly the same K as 0.062% of trichloroacetic acid but the solubility limit was not reached until considerably more insulin was dissolved. The compounding of systems which have a n organic phase with sufficient polarity to dissolve polar solute such as proteins is one of the problems of applying countercurrent distribution in biochemistry. Perhaps one of the most polar systems yet used is one containing phenol and 2,4,6-trimethylpyridine used in isolating cephalosporin (1), a new penicillin more polar than the better known ones. LIPIDES
A valuable review on the fractionation of lipides by countercurrent extraction has been written by Dutton (49). The data obtained by countercurrent distribution have shed new light on the structure of naturally occurring triglycerides. Probably a reasonably “pure” naturally occurring triglyceride has been isolated for the first time in work on the fractionation of the fats of linseed oil (48). Discrete bands having 9, 8, 7, etc., double bonds were obtained. Solvent systems have been worked out for the separation of mono-, di-, and triglycerides ( 10). Diglycerides Rere identified for the first time as components of the human intestine. Other lipide studies have been concerned with those from fish (111)and
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ANALYTICAL CHEMISTRY
from serum (47). The possibility of separating triglycerides by countercurrent distribution has been discussed by Taber (143). T h e highest possible selectivity is needed for this difficult field. A short review on the application of countercurrent distribution methods to the phospholipides has been given hy Baer (6). Precise characterization of many of the phospholipides is very difficult because of their strong tendency to associate and their poor stability. The phosphatidic acids readily undergo hydrolysis even in 85% ethanol. Olley (110) followed this hydidysis by countercurrent distribution. Studies with soybean phospholipides have included a separation of the color in the “lecithin” fraction (151), and further separation of inositoside fractions (130). Countercurrent distribution is an effective way of separating substances that differ in the number of double bonds. This was shown by the separations mentioned above with linseed oil, and was also shown by Klenk and Lindler (91) in the separationfrom brain tissue of CZZfatty acids containing one, two, three, and four double bonds. POLYPEPTIDES
3Iuch work has been done in the field of polypeptides, including isohtion of new naturally occurring ones, isolation of fragments following degradation, and purity studies with synthetic material. T h e most outstanding examples are in the field of the pituitary hormones. These include isolation of probably the first pure ACTH hormone, p-ACTH from beef pituitaries ( 8 ) ) a-ACTH from sheep (100, 101), the lactogenic hormone (28), the melanophore expanding hormone (9, 99), and growth hormone (120). Countercurrent distribution has been used throughout the structural work on oxytocin and vasopressin and synthetic intermediates for isolation and proof of purity (51, 56, 126). The lactogenic hormone probably is the largest molecule (molecular weight 33,000) which has thus far been distributed in a clear-cut way. The hypertensive polypeptides found in the blood of patients with hypertension is another field nThich is being rapidlv advanced, and countercurrent distribution is playing an important role. Skeggs and coworkers (153,154) have isolated tu o polypeptides from horse serum after incubation with hog rennin Bumpus and Page ( 3 1 ) have isolated a similar peptide and Kuether and Haney (95) have isolated a less stable substance with higher activity. The studies on the structure of the bacitracin polypeptides (2‘7, 70, 71, 156, 167) have revealed some unique structural features on very fragile molecules. These results would have been very difficult t o obtain without the extraction techniques. Part of this work involved the separation of pure peptides or derivatives from very complex partial hydrolyzates. Improved selectivity was obtained by distribution of derivatives obtained after distribution of uneubstituted peptide mixtures. Another very complicated peptide field which has been opened up by countercurrent distribution is that of the streptomi cei antibiotics (15-17, 19, 20, 121) A new and interesting member of the penicillin group, cephalosporin, has been isolated ( 1 ) . Countercurrent distribution was found supeiior t o other techniques for isolation of a radioactive penicillin (31). Polymyvin B (69) has been separated into two peptides which differ in the fatty acid conjugated to the peptide part. Some of the amino acid sequences in salmine have been determined by partial hydrolysis by enzymes and fractionation by countercurrent distribution (108). Other peptide separations include one from rye grass (I.&?), an antibiotic, comirin (59), an antibiotic, gramicidin J (112), an antibiotic, cinnamgcin (52)) and one from Fusarium bost ( 2 4 ) . Synthetic higher polypeptides have been separated (18). Countercurrent distribution has been found very effective for
separating D N P derivatives ( 7 1 , 156) and azobenzene sulfonyl derivatives (87)of amino acids and peptides. 4NTIBIOTICS
Separations in this group have included further purification of known types and isolation of new types. Examples are: fummagillin (62), neomycin types ( I % ) , elaiomycin (68),aureomycin and Terramycin (77), magnamycin (78, 154), hygromycin (log), methymycin (is), levomycin ( 2 6 ) , and two erythromycins (119) STEROLS
Urinary metabolites of administered corticosterone (57) have been isolated. A countercurrent distribution procedure for quantitative-qualitative analysis of urinary corticosteroids ( 1 4 4 ) has been suggested. Hormones of the adrenal cortex have been separated (25, 139, 153). ALKALOIDS
When the development of countercurrent distribution was first begun (55) it was with the field of alkaloids in mind because of the possibility of exploiting the high selectivities possible in buffered systems (36). In spite of the fact that this was well demonstrated in the synthetic quinoline antimalarial field (38)) it is only recently that wide use is being made of the method for alkaloids. Recent papers include alkaloids from Rauwolfia (46, 79, 9.3)) veratrine (@), ergot ( 7 6 ) , tropane alkaloids ( I d ) , heliotropium alkaloids (39), pilocereine and anhalonidine (44), cumchichicine (46), and laurifoline (43). MISCELLANEOUS SEPARATIONS
These include sulfonamides (14), bitter principles from hops (81, 135), biotin sulfoxide (160), pyrrolidine carboxylic acid ( 5 6 ) , dermatitic compounds from molds (13), tromesan ( 2 2 ) , methyl homologs of 1,2-~yclopentenophenantbrene( 2 3 ) , tannins (89, 90)) colchicine and derivatives ( 7 ) , degradation product of vitamin BIZ(94), azaserine ( 6 1 ) , pentachloronaphthnlene (11), and radioactive metabolic products (146). INORGANIC SEPARATIONS
The use of extraction in the field of inorganic separations has come t o be a large and very involved subject which cannot be covered adequately in this review. Only a few recent references which give a general idea of the things being emphasized will be cited. It is a field in which much more improvement can be expected in the near future. In the main the extractions are single stage with emphasis on some type of complexing agent used under carefully controlled conditions in order t o achieve selectivity. But when selectivity is not sufficient, attention is being given to some type of countercurrent process such as with the separation of the rare earths ( 5 , 159). However, the inherent possibilities have not as yet been exploited t o the degree cited in any of the organic or bicchemical fields covered in this review. A very good review of systems and complexing agents to be used in analysis of the precious metals has been given by PIcBryde (102). A number of separations are also listed in other reviews (79, 85). Japanese workers have been very active in this field. They have studied the determination of cobalt (159), iron (141), and mercury and bismuth (140) by extraction of an antipyrine complex with ethyl acetate and by extraction of a thiocyanate complex with 2-butanol. The solutions \%-ereanalyzed s p e c t m photometrically. Lead, silver, and mercury (I%), cadmium and arsenic (137), tellurium ( 6 4 ) , and copper and bismuth (138) were determined in a similar manner by extraction of the diethyldithiocarbamate complex with benzene or chloroform. Cobalt was also determined as the thiocyanate (84). Extraction of the iodides of bismuth and mercury was studied (65). An extraction method for separating arsenic and phosphorus (65)was given.
V O L U M E 28, N O . 4, A P R I L 1 9 5 6 The use of extraction for the purification of germanium as the tetrachloride (109) and as the tetrabromide (96) has been studied. Beta-diketones are very useful complexing agents. Acetylacetone (135) has been studied for extraction of aluminum, gallium, and indium. Fluoro-p-diketones (83) are excellent for separating zirconium and hafnium. Separation factors on the order of 13 t o 25 were obtained. Systems made from a n aqueous solution of a strong acid and a solution of methyldioctylamine or tribenzylamine are very selective for separating cobalt or zinc (103) or niobium and tantalum (54). These authors believe a n analogy exists between such long-chain or aromatic amines in liquid-liquid extraction and an ion exchange resin. The system made from tributyl phosphate and hydrochloric acid ( 5 3 ) is selective for scandium. B l STHEMATICS
The mathematics for calculating theoretical curves for countervurrent distribution have been treated in a number of places (30, 74, 155). A nomograph relating number of plates, per cent of impurity, and values for binary mixtures has been given ( $ 7 ) . The mathematics involved ( 7 4 ) in relating these factors have also been discussed elsewhere. -1series of papers (30, 117 , 127-1 29) has given a very thorough treatment of the type of mathematics used in the interpretation and operation of stage continuous columns of the Scheibel type. While this work as it is presented holds considerable more intere.;t for those interested in production than for analysis, it is of great interept for the latter. Compere and Ryland (SO: have applied the probability concept of “random walk” to double withdram-al. Mathematics for double withdrawal calculations are also given in Hecker’s book ( 7 4 ) . The calculation of deviation from the steady state has been treated by Scheibel (129), m-hile holdup and approach t o the steady state have been treated by Peppard and Peppard ( 1 1 7 ) . .Ilthough no good purpose would be served by pointing out specific papers, there has been a certain amount of abuse of the method of countercurrent distribution for the proof of purity. An experimental curve only slightly broader than a calculated cuive cannot be taken in itself as evidence t h a t the preparation is “nearly pure,” unless the reason for the spread has been shoirn bv determination of K’s across the band to be due t o some other cause. A run giving a curve which almost agrees with the theoretical a t 100 transfers may clearly reveal two major components a t 1000 transfers. This is a matter of the ultimate selectivity of the method. APPARATUS
In reading the literature of organic chemistry one frequently finds a paragraph in the experimental part in which some small variation of the Kutscher-Steudel or Soxhlet extractor is dew i b e d . A number of these can be found in fairly recent papers, tiut those seen by this reviewer were not sufficiently different from variations already published and mentioned in earlier reviews t o warrant treatment. There has been much more interest in trying to improve existing apparatus for bringing about some form of countercurrent extraction. Little has appeared concerning the application of the truly continuous type of extractor in the analytical or small qcale preparative field. KO doubt this is due t o the fact that only a few stages a t best are reliably accomplished. An interesting application of this type of extractor deals with the separation of acids and bases (158). The column is so run that there is a p H gradient from the top of the column t o the bottom. This sets up a rather complicated state of affairs which exploits the selectivity of buffered systems. The high selectivity of such systems in countercurrent distribution (36) tvas demonstrated more than 10 years ago. I n contrast to the strictly continuous columns, there has been
727 a moderate amount of interest iii semicontinuous equipment of the “mixer-settler” type. No doubt t’his stems from the desire t o fractionate larger quantities of material. While there is distinct merit in this as contrasted to countercurrent distribution where kilogram or larger amounts are involved, it should not be assumed that preparative amounts cannot be separated by countercurrent distribution. .Uthough exact details of the apparatus were not described, Patterson and others (114) used a 40-tube countercurrent distribution apparatus in the isolation of protogen from 4 tons of liver. Each tube of the apparatus required 1 liter of each phase. An installment in the Squibb Co. contains 21 tubes also accommodating 1 liter in each phase. With volumes of this size the fundamental pattern could b e accomplished with amounts in the order of 1 kg., and by using. the alternate withdrawal procedure much more could be proc-essed. 1111-glass trains with units varying in size from 2 to 1000 ml. are available commercially in this country (H. Post, Maspeth: K.Y.). Although there has been more interest in the development of extractors of the miser-settler types, nearly all the actual separations reported in the literature of organic and biochemistry in the past few years have been made by countercurrent distribution, a. strictly discontinuous type of extraction. A good example of 8: separation by a column of the miser-settler type is t o be found in the work of Karr and Scheibel (86). An interesting and new type of mixer-settler is that of Kepes (88) in which the heavier phase remains stationary as in countercurrent distribution. A series of partitions in a long glass tube keeps the heavier phases from migrating. The lighter phase is caused t o pass up through a small opening in each partition by pressure in such a way as to cause mixing with the heavier phase. .I series of such columns can be connected by a tube leading from the top of one t o the bottom of another. T h e effluent phase is collected from the top of the last tube by a fraction collector. The column operates in a way analogous t o the “single withdrail-al” procedure (37) of countercurrent diatribution, but as the column is continuous in operation, a “countercurrent distribution” is not achieved. Countercurrent distribution is a strictly discontinuous idealized extraction process operating in such a manner that each step coincides with a stage of the binomial expansion. The simplicity of the Kepes column is attractive. Other mixer-settlers include one made from separate units cut from a clear plastic ( 5 ) and clamped together. A stacked series of metal trays clamped together (58) on another offers a compact design, Other designs (40) for accomplishing mixing-settling have been investigated. An interesting column of the pulsating Van Dijck type has been described by Werning and others (159). It is made from polyethylene in order t o permit the use of hydrofluoric acid in the system. The organic phase is methyl isobutyl ketone. This system is favorable for separating t,antalum and niobium. Countercurrent distribution equipment is now made cominercially in the United States (H. Post, 6822 60th Road, Maspeth, N. Y.), England (Quickfit & Quartz, Heart of Stone, Staffs), and Germany (Hermann Iiuhn, Hospital Strasse 4C, Gottengen). All use a design of tube operating by decantation similar to that first made in this country (37). Verzele (149, 151) has modified the tube slightly to permit the heavier phase to be moved as well as the lighter phase which would be moved in the opposite direction. This was also possible in the design of Lathe and Ruthven (98). The advantage of being able to move either or both phases is in accomplishing alternate or double withdrawal for preparative work. There is no difficulty of accomplishing this in handoperated trains by the design of tube ( 3 7 ) most commonly used. However, a fully automatic train in which center feed could be employed would offer greater advantages, if one were available. Complete details of none of the three countercurrent distribution separators commercially available are t o be found in t h e
A N A L Y T I C A L CHEMISTRY
128 literature. The design and arrangement of the tubes are patterned rather closely after that first published by Craig and coworkers ( J T ) , b u t the robots and filling devices are different. T h e one available from Post has a greatly improved filling device and robot compared t o the original. This reviewer has not seen the equipment made in England and Germany. An ingenious new design for the robot has been published by Perry and Weber (118). It uses a three-phase motor to withstand the repeated starting, stopping, and reversing required. T h e excellent step-up switches now commercially available are used t o control most of the motions and intervals. This makes for simplicity of design. BIBLIOGRAPHY
(1) Abraham, E. P., Newton, G. G. F., Hale, C. W., Biochem. J .
58, 94 (1954). (2) Bcher, R., Rlanoussos, G., Olivry, G., Biochem. et Bioghys. Acta. 16, 155 (1955). (3) Alders, L., A p p l . Sci. Researrh A4, 171 (1954). (4) Alders, L., “Liquid-Liquid Extraction,” Elsevier Xew York, 1955. (5) iilter, H. W., Codding, J. W., Jennings, A. S., ANAL.CHEM. 26, 1357 (1954). (6) Baer, E., Ann. Reu. Biochem. 24, 136 (1955). (7) Bailey, S. D., Horowits, R. SI., Dinsmore, H. L., J . .4m. Pharm. Assoc., Sci. E d . 42, 548 (1953). (8) Bell, P. H.. J . Am. Chem. S O C 76.5565 . (1954). (9) Benfey, B. J., Purvis, J. L , Ibid., 77, 5167 (1955). (10) Blankenhorn, D. H., Ahrens, E. H., Jr., J . Biol. Chem. 212, 69 (1955). (11) Blickenstaff,R. T., Callen, J. E., ANAL.CHEM.26, 1586 (1954). (12) Bottomley, W.,Nortimer. P. I., L4ustralian J . Chenb. 7, 189 (1954). (13) Bowden, J. P., Schantz, E. J., J . Biol. Chem. 214, 365 (1955). (14) Braeuniger, H., Spangenberg, K., Pharmazie 9, 343 (1954). (15) Brockmann, H., Angew Chem. 66, 1 (1954). (16) Brockmann, H., Grone, H , Chem. Ber. 87, 1036 (1954). f t e n65 (1954). (17) Brockmann, H., Grone, H., ~ ~ r a t u r ~ i s s e n s c h a 41, (18) Brockmann, H., Musso, H., Ibid., 40, 553 (1953). (19) Brockmann, H., Schmidt-Kastner, G., Chem. Ber. 87, 1460 (1954). (20) Ibid., 88, 57 (1955). (21) Bumpus, F. SI.,Page, I. H.. Science 119, 849 (1954) (22) Burns, J. J., Wexler, S., Brodie, B. B., J . Am. Chem. Soc. 75, 2345 (1953). (23) Butenandt, A., Dannenberg, H., Steidle, R., Z . Satztrforsch. 9b, 288 (1954). (24) Cajori, F. A,, Otani, T. T., Hamilton, 11. A., J . B i d . Chem. 208, 107 (1954). (25) Carstensen, H., Acta Chem. Scand. 9, 1026 (1955). (26) Carter, H. E., Schaffner, C. P., Gottlieb, D., Arch. Biochem. Biophys. 52,282 (1954). (27) Codington, J. F., Antibiotics Ann. 1954-55, 1118. (28) Cole, R. D., Li, C. H., J . B i d . Chem. 213, 197 (1955). (29) Collander, R., Acta Chem. Scand. 3, 717 (1949); 4, 1085 (1950); 5, 774 (1951). (30) Compere, E. L., Ryland, A. L., I n d . Eng. Chem. 46, 24 (1954). (31) Cooper, P. D., Clowes, R. C., Rowley, D., J . Gen. Microbiol. 10,246 (1954). (32) Craenhals, E., Leonis, J., Bull. soc. c h i m , Belg. 64, 58 (1955). (33) Craig, L. C., ANAL.CHEM.26, 110 (1954). (34) Craig, L. C., Ciba Foundation, Symposium on Internal Secretions of the Pancreas; J. and A. Churchill, London, 1956. (35) Craig, L. C., J . Biol. Chem. 155, 519 (1944). (36) Craig, L. C., Golumbic, C., Slighton, H., Titus, E., J . Biol. Chem. 161, 321 (1945). (37) Craig, L. C., Hausmann, W , Ahrens, E. H., Jr., Harfenist, E. J., ANALCHEW.23, 1326 (1951). (38) Craig, L. C., Mighton, H., Titus, E., Golumbic, C., Ibid., 20, 134 (1948). (39) Culvenor, C. C. J., Australian J . Chem. 7, 287 (1954). (40) Davis, M. W., Jr., Hicks, T. E., Vermeulen, T., Chem. Eng. Progr. 50, 188 (1954). (41) Dean, J. A., Lady J. H., ANAL.CHEM.27, 1533 (1955). (42) Djerassi, C., Smith, C. R., Figdor, S.K., Herran, J., Romo, J., J . Am. Chem SOC.76,5889 (1954). (43) Djerassi, C., Smith, C. R., Lippman, A. E., Figdor, S. K., Herran, J., Ihid.. 77,4801 (1955). (44) Djerassi, C., Smith, C. R., Marfey, S. P., McDonald, R. N., Lemin, A. J., Figdor, S. K., Estrada, H., Ibid., 76, 3215 (1954). (45) Donin, M. N., Pagano, J., Dutcher, J. D., McKee, C. M . , Antibiotics Ann. I, 179 (1953).
Dorfman, V. L., Furlenmeier, A, Huebner, C. F., Lucas, R., MacPhillamy, H. B., hfueller, J. M., Schlittler, E., Schwyser, R., St. Andre, A. F., Helu. Chim. Acta 37, 66 (1954). Douste-Blasy, L., Compt. rend. 239,460 (1954). Dutton, H. J., J . Am. Oil Chemists SOC.32, 652 (1955). Dutton, H. J., Progr. Chem. of Fats and Other L i p i d s 2, 292 (1954). DU Vigneaud, V., Gish, D. T., Katsoyannis, P. G., J . Am. Chem. SOC.76,4751 (1954). Du Vigneaud, V., Ressler, C., Swan, J. -M., Roberts, C. W., Katsoyannis, P. G., Ibid., 76, 3115 (1954). Dvonch, W., Shotwell, 0. L., Benedict. R. G. Pridham. T . G.. Lindenfelser, L. A,, Antibiotics and Chemotherapy IV, 1135 (1954). ANAL.CHEM.27, 1551 (1955). (53) Eberle, 9. R., Lerner, M. W., (54) Ellenburg, J. Y., Leddicotte, G. W., RIoore, F. L., Ibid., 26, 1045 (1954). (55) Ellfolk, N.,Synge, R. L. M., Biochem. J . 59, 523 (1955). (56) Engel, L. L., Alexander, J., Carter, P., Elliott, J., Webster, XI., ANAL.CHEM.26,639 (1954). Engel, L. L., Carter, P., Fielding, L. L., J . Biol. Chem. 213, 99 (1955). Fenske, AI. R., Long, R. B., Chem. Eng. Progr. 51, 194 (1955). Forsyth, W.G. C., Biochem. J . 59, 500 (1955). Fowler, R. T . , Koble, R. A. S., J . A p p l . Chem. (London) 4, 546 (1954). Fusari, S. A, Frohardt, R. P., Ryder, A., Haskell, T. H., Johannessen, D. W., Elder, C. C., Barts, Q. R., J . Am. Chem. SOC.76, 2878 (1954). Goodall, R. R., Landquist, J. K., Analyst80,499 (1955). Goto, H., Kakita, Y., Sci. Repts. Tohoku C’nia. A 7, 294 (1955). Ibid., A 7, 365 (1955). Ibid., A 6, 130 (1954). Goto. H., Susuki, S., Harfenist, E. J., J . Am. Chem. SOC.75, 5528 (1953). Harfenist, E. J.. Craig, L. C. Ibid., 74, 3083 (1952). Haskell, T. H., Ryder, A, Barte, Q. R , Antibiotics & Chemotherapy 4, 141 (1954). Hausmann, W., Craig, L. C., J . Am Chem. SOC.76,4892 (1954). Hausmann, W.,Weisiger. J. It., Craig, L. C., Ibid., 77, 721 (1955). Ibid.; p.’723. Hecker, E., Chimia 8,229 (1954). Hecker, E. V., Osterr. Chem. 2. 56,3 (1955). Hecker, E. V., “Verteilungsverfahren im Laboratorium,” Verlag Chemie Weinheim/Bergstr., 1955. Hecker, E., Allemann, K.. Angew. Chem. 66, 557 (1954). Hellberg, H., Farm. Reuy. 53, 605 (1954). Hickey, R.J., Phillips, W.F., ANAL.CHEM.26, 1640 (1954). Murai, K., J . Am. Chem. SOC.76,5080 (1954). Hochstein, F. -4., Hocbstein, F A , Murai, K., Boegemann, W. H., Ibid., 77,3551 (1955). Hollingsworth, C. A., Taber, J. J.. Daubert, B. F., Science 120, 306 (1954). Howard, G. A., Tatchell, A. R., Chemistry and Industry 1954, 514. Howard, K. S., Shepherd, R. G., Eigner, E. A , Davies, D. S., Bell, P. H., J . Am. Chem Soc. 77, 3419 (1955). Huffman, E. H., Iddings, G. SI., Osborne, R. N., Shalimoff, G V., Ibid., 77, 881 (1955). Ikeda, S., Sci. Repts. Tohoku Univ. A 6, 417 (1954). Irving, H. M., Quart. Revs. (London) 5, 200 (1951). i86j Karr, A. E., Scheibel, E. G., I n d . Eng. Chem. 46, 1583 (1954). (87) Keil, B., Knesslova, V., Sorm, F., Collection Czech. Chem. Communs. 18, 131 (1953). Kepes, A . , Bull. soc chim. biol. 35, 1243 (1953). Kirby, K. S.,Knowles, E., White, T., J . SOC.Leather Trades Chemists 36, 45 (1952). Kirby, K. S.,Knowles, E., White, T., Ibid., 37, 283 (1953). Klenk, E., Lindlar, F., 2. physiol. Chem. 301, 156 (1955). Klohs, R1. W., Draper, AI., Keller, F., Koster, S , Malesh, W., Petracek, F. J., J . Am. Chem. SOC.76, 1152 (1954). Klohs, SI. W.,Draper, bl. D., Keller, F., hlalesh, W., Chemistry and Industry 1954, 1264. Kuehl, F. A., Jr., Shunk, C. H., Moore, M., Folkers, K., J . Am. Chem. SOC.77, 4418 (1955). Kuether, C. A., Haney, M.E., Jr., Science 121, 65 (1955). Ladenbauer, I. XI., Saama, O., Hecht, F., Mikrochim. Acta 1955, 118. Lancaster, C. R., Lancaster, E. B., Dutton, H. J., J . Am. Oil Chemists’ SOC.27, 386 (1950). Lathe, G. H., Ruthven, C. R. J., Biochem. J.49, 540 (1951). Lerner, A. B., Lee, T. H., J . Am. Chem. Soc. 77, 1066 (1955). Li, C. H., Geschwind, I. I , Dixon, J. S.,Levy, A. L., Harris, J. I.,J . B b l . Chem. 213,171 (1955). Li, C. H., Geschwind, I. I., Levy, A. L., Harris, J. I., Dixon, J. S.,Pon, N. G., Porath, J. O., Nature 173,251 (1954).
V O L U M E 28, NO. 4, A P R I L 1 9 5 6 (102) McBryde, W. A. E., A n a l y s t 8 0 , 5 0 3 (1955). (103) Mahlman, H. A , , Leddicotte, G. W., hloore, F. L., ANAL. CHEM.26, 1939 (1954). (104) iCIann, R . L., Gale, R. M., Van Abeele, F. R., Antibiotics Ann. I, 167 (1953). (105) hletzsch, F. A. V., A n g e w . Chem. 65, 586 (1953). (106) hletzsch, E’. A. V., Chem. 2 . 78, 391, 423 (1954). (107) Mills, G. F., Whetsel, H. B., J . Am. Chem. S O C . 77, 4690 (1955). (108) Nonier, R., Jutisa, M., Biochim. B i o p h y s . Acta 15, 62 (1954). (109) Morrison, G. H., Dorfman, E. G., Cosgrove, J. F.. J . ,417~. Chem. SOC. 76,4236 (1954). (110) Olley, J., Biochem. et Biophys. Acta 10, 493 (1953). (111) Olley, J., Lavern, J. rl.,Biochem. J . 57, 610 (1954). (112) Otani, S., Saito, Y., Proc. J a p a n A c a d . 30, 991 (1954) (113) Paladini, A . , Craig, L. C., J . Am. Chem. Soc. 76, 688 (1954). (114) Patterson, E. L., Pierce, J. V., Stokstad, E. L. R., Hoffmann, C. E., Brockman, J. A , , Jr., Day, F. P., Macchi, 11. E., Jukes, T. H., Ibid., 76, 1823 (1954). (115) Pearson, D. E., Levine, hI., J . Org. Chem. 17, 1351 (1952). (116) Ibid., p. 1356. (117) Peppard, D. F., Peppard, JI. A., I n d . Eng. Chem. 46, 34 (1954). (118) Perry, E. S., Weber, W.H., ANAL.CHmr. 2 6 , 4 9 8 (1954). (119) Pettinga, C. TV., Stark, IT’.hl., Van Abeele, F. R., J . Am. Chem. SOC. 76, 569 (1954). (120) Pierce, J. G., Biochem. J . 57, 16 (1954). (121) Rao, K. V., Peterson, W. H., J . Am. Chem. S O C . 76, 1335 (1954). (122) Ressler, C., Du Vigneaud, V., Ibid., 76, 3107 (1964). (123) Rigby, F. L., Bethune, J. L., Ibid., 77, 2828 (1955). (124) Sanger, F., Bull. SOC. chim. b i d . 37, 23 (1955). (125) Sanger, F., Smith, L.F., Kitai, R., Biochem. J . 58,Proc. vi (1954). (126) Schaffner, C. P., Antibiotics Ann. 2 , 153 (1954). (127) Scheibel, E. G., Chem. Ing. Tech. 27, 341 (1955). (128) Scheibel, E. G., I n d . Eng. Chem. 46, 16 (1954). (129) Ibid., p. 42. (130) Schofield, C. R., Dutton, H. J., J . Biol. Chem. 208,461 (1964). (131) Schofield, C. R., Dutton, H. J., J . Am. 0 2 1 Chemists’ SOC.31, 258 (1954).
729 (132) Schroeder, W., Voigt, K. D., Van der Werth, H., Beckmann, I., Acta Endocrinol. 14, 14 (1953). (133) Skeggs, L. T., Jr., Marsh, W. H., Kahn, J. R., Shumway, K.P., J . Ezptl. M e d . 99, 275 (1954). (134) Ibid., 100, 363 (1954). (135) Steinbach, J. F., Freiser, H., AXAL.CHEM.26, 375 (1954). (136) Sudo, E., Sci. liepts. Tohoku C‘niu. A 6 , 137 (1954). (137) Ibid., p. 142. (138) Ibid., p. 253. (139) Ibid., p. 324. (140) Ibid., A 7 , 306. (141) Ibid., p. 312. (142) Synge, R.L. RI., Wood, J. C., Biochem. J., 56, Proc. xix (1954). (143) Taber, J. J., Dissertation Abstr. 15, 727 (1955). (144) Talbot, N. B., Ulick, S.,Koupreianow, A, Zygrnuntowics, A., J . Clin. Endocrinol. and Metabolism 15, 301 (1955). (145) Tavel, P. V., Helu. C h i m . Acta 38, 520 (1955). (146) Titus, E., Weiss, H., J . Biol. Chem. 214, 807 (1955). (147) Treybal, R. E., I n d . Eng. Chem. 46, 91 (1954). (148) Ibid., 47, 536 (1955). (149) Versele, M., Bull. soc. chim. Belg. 62, 619 (1953). (150) Verzele, M., I n d . chim. Belge. 19, 135 (1954). (151) Versele, LI., Alderweireldt, F., Nature 174, 702 (1954). “Fractionnernents par Solvents,” Vigot Frhres, (152) Vigneron, M,, Paris, 1954. (153) Voigt, K . D., Schroeder, W., Beckmann, I., Van der Werth, H., Acta Endocrinol. 14, 1 (1953). (154) Wagner, R. L., Hochstein, F. A,, hlurai, K., Messina, N., Regna, P., J . Am. Chem. SOC.75,4684 (1953). (155) Weisiger, J. R., in ”Organic Analysis,” vol. 11, pp. 277-326, Interscience, Ken- York, 1954. (156) Weisiger, J. R., Hausmann, W., Craig, L. C., J . Am. Chem. S O C 77, . 731 (1955). (157) Ibid., p. 3123. (158) Reiss, D. E., I n d . Chemist 31, 230 (1955). (159) Werning, J. R., Higbie, K. B., Grace, J. T., Speece, B. F., Gilbert, H. L., I n d . Eng. Chem. 46, 644 (1954). (160) Wright, L. D., Cresson, E. L., Valiant, J., Wolf, D. E., Folkers, K., J . Am. Chem. Soc. 76, 4163 (1954).
I
REVIEW OF FUNDAMENTAL DEVELOPMENTS IN ANALYSIS
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Ion Exchange ROBERT KUNIN, FRANCES X. MCGARVEY, and ANN FARREN Rohm & Haas Co., Philadelphia, Pa.
F ONE judges the value of ion exchange techniques to the
analytical chemist b y the number of published articles and books on the use of ion exchange in analytical chemistry, as well as b y the number of commercially available ion exchange resins tailored specifically for the analyst, one must concede that this analytical principle or technique is well accepted b y the majority of analytical chemists. This status has been achieved only recently, as it has been only during the past few years t h a t specific procedures and quantitative principles for ion exchange techniques were available to the analyst. T h e continued development of ion exchange techniques in analytical chemistry must be accompanied b y a better understanding on the part of the analytical chemist of t h e physical chemistry involved and of the nature and properties of the ion exchange resins. Steps in this direction are now evident, judging by the fact t h a t many undergraduate and graduate analytical chemistry courses in many universities and colleges include the subject of ion exchange. A review of the developments in this field of activity during the past 2 years reveals several important facts. T h e number of detailed quantitative procedures involving the use of ion exchange resins is gradually increasing. In addition, the availability of
ion exchange resins tailored for the analyst has improved considerably and the resins can now be obtained readily from several sources of supply. Simplification of the chromatographic theory as applied to ion exchange columns has resulted in a set of equations less cumbersome, although less rigorous, t h a n those available previously; and these are readily employed by the analytical chemist in developing and modifying procedures. It is obvious to those familiar with ion exchange in analytical processes t h a t ion exchange techniques function primarily a8 a means for separating and concentrating various ionic substances and are used where conventional qualitative or quantitative measurements cannot be directly applied t o the system in question. REVIEWS
During the past 2 years, many developments in ion exchange of interest t o the analytical chemist have appeared. Several reviews summarize these advances. Deuel ( / A) reviewed rather extensively the properties of ion exchange resins from a structural standpoint. Kunin ( 1 1 A ) outlined progress in the use of ion exchange in a highly condensed but comprehensive article. A
