75
V O L U M E 2 1 , N O . 1, J A N U A R Y 1 9 4 9 (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60) (61)
Popova, M. M., Zaaodskaya Lab., 11, 887-98 (1945). Rabbitts, F. T., ANAL.CHEM.,20, 181-2 (19481. Rogers, D., and Heron, A. E.; Analyst, 71, 414-17 (1946). Rollet, A. P., Ann. chim., [ l o ] 13, 137-252 (1930). Sand, €1. J. S.,Metallurgia, 30, KO. 176, 107-9 (1944). Sand, H. J. S., Nature, 154, 696 (Dec. 2, 1944). Schleicher, A., “Elektroanalytische Schnellmethoden,”3rd neubearbeitete Auflage von Fischer-Schleicher “Elektroanalytische Schnellmethoden,” Stuttgart, F. Enke, 1947. Schleicher, A., 2.anal. Chem., 126, 412-17 (1944). Schleicher,A., and Todoroff, T., Chem. Ztg., 68, 48-9 (1944). Schleicher, A., and Todoroff, T., 2 . Elektrochem., 50, 2-7 (1944). Schucht, 2.anal. Chem. 22, 485-95 (1883). Sease, J. W., Niemann, C . , and Swift, E. H., ANAL.CHEM.,19,
197-200 (1947). (62) Shapiro, IM. M . , Zavodslcaya Lab., 12, 369-72 (1946). (63) Stein, P. von, Chemist-Analyst, 34, 87 (1945).
Buvorovskaya, N. A., Zavodskaya Lab,, 11, 474 (1945). Szebellbdy, S., and Somagyi, Z., 2. anal. Chem., 112, 313-23 (1938).
Tamburrini, V.,“Analisi quantitativa per elletrolisi,” Arpino, Italy, Tip G. Frailoi, 1946. Trishin, F. I., Zhur. A n a l . Khzm. 3, 21-8 (1948). Ibid., 3, 29-30 (1948). Tucker, P. A , , Analyst, 71, 319-21 (1946). Webb, H. R.,Ibid., 70, 301-4 (1945). Weinberg, S.,and Boyd, T. F., IND. ENG.CHEM.,AXAL.ED., 16, 460-1 (1944).
Zakhar’evskil, M. S., Voprosy Pitaniya, 7, No. 415, 180-8 (1938) ; K h i m . Referat. Zhur., 2, No. 4, 84 (1939). Zischkau, C., Proc. Am. Soc Testing Materials, Preprint 39, (1944). RECEIT-ED h-overnber 18,1948.
CHROMATOGRAPHIC SEPARATIONS HAROLD €1. STRAIN, Carnegie Institution of Wushington, Stanford, Calif.
T
HE interpretation of natural phenomena in terms of physics and chemistry depends upon knowledge of the properties of particular chemical substances. Because each substance must be isolated in a high state of purity before many of its physical and chemical properties can be determined, progress in the natural sciences hinges upon the development of efficient analytical methods (10, 61). Of the various techniques that have been devised for the resolution of mixtures, the chromatographic adsorption analysis, first described by Tswett in 1906, is one of the most effective (25). Xow that scientists have become aware of the wide applicability, the extreme sensitivity, and the great rapidity of this adsorption method, hundreds of new uses and numerous modifications of the procedure have been described (147). From pedagogic and developmental points of view, it is significant, as related by Dh6r6 (45),that Tswett, the son of a Russian father and an Italian mother, obtained his doctorate in botany with hlarc Thury a t Geneva. Later, a t the veterinary institute a t Warsaw, this unlicensed chemist of that day observed that pigments in the extracts of green leaves form a series of green and yellow bands when a solution of the mixture is filtered through a glass tube filled with precipitated chalk. Moreover, Tswett found that complete separation of these pigments from one another could be effected only by washing the adsorbed substances with fresh solvent or with mixtures of solvents. illthough adsorption columns had previously been used for the partial resolution of mixtures, this development of the chromatogram with various solvents represented a unique advance in the use of adsorption methods. In the 45 years preceding Tswett’b basic discovery, Schonbein, Goppelsroeder, and many others had observed that solutes concentrate in distinct zones as solutions are drawn into strips of filter paper by capillary action (76, 77, 101). But not untilrecently has it been realized that the principles involved in this socalled “capillary analysis” are identical with those in chromatographic analysi.: (32, 117 , 148). Viewed from the vantage point of current knowledge, many early modihcations of the capillary adsorption method, such as the addition of reagents and indicators to the paper ($7, 63, 77, 98), have been utilized in Tswett’s columnar analysis. Conversely, numetous advances in the use of adsorption columns, eapecially the development of the chromatogram, have now proved applicable to capillary analysis (37, 76). In view of the similarity between these two methods, capillary analysis is frequently called “paper chromatography” and “paper pa1 tition chromatography” (35, 37, 84,86, 86). The efficiencyand convenience of chromatographic separations in relation to those obtainable by other methods vary greatly with
the field of investigation. Applied to some substances, such as the inorganic ions (70, 112, 213, 13.2) and the amino acids from various natural sources (35, 36, 37, 84,90, 115, 116), chromatographic analysij has often contributed little more than a convenient confirmation of the results obtained by the conventional methods of analysis. In other fields, as with the polyene codpounds, each new application of the chromatographic technique has revealed many new compound3 that had escaped detection or isolation by other methods (124, 149, 150). In the past five years, advances in chromatography have followed many courses. There have been significant improvements of the apparatus and procedure. New adsorbents, particularly the ion exchangers and the hydrated gels, have been introduced on a large scale. A variety of solvents, such as the cyclic nitrogen compounds, higher alcohols, and salt solutions, have been employed as solvents and eluants. As in the past, most studies have dealt with the resolution of mixtures, both of inorganic and of organic substances. These applications of the method have been enlarged to an industrial scale, and they have been refined to microgram proportions for qualitative and quantitative analysis. Adsorption in paper has been widely employed for the examination of biological products; a recent review contained as many as 51 references ( 3 5 ) . Xurnerous attempts have been made to standardize the paper and the columnar methods as an aid to reproducibility, as a basis for engineering applications, and as a foundation for physical and mathematical interpretations of the process (;?4,73,74,107,113,114,126,128,129, IS;?). Several reviews of various aspects of thc, paper (51, 77) and columnar chromatography (6, 29, 46, 64, 84,85, 89, 105, 113, 132, 134, 144, 148, 151) have treated many mpects of the subject, that could not he considrrt?d here. METHODS
Apparatus. Certain modifications of the apparatus facilitate particular applications of the chromatographic methods. Glass adsorption tubes, TTith a slight, uniform taper toward the base, and aluminum tubes aid the removal of the intact, cohesive mass of the adsorbent after the chromatogram has been developed (16, 7 5 ) . A transparent plastic adsorption tube with a removable, longitudinal section permits addition of reagents to the adsorbent while it is still in place (14, 42). Rotating columns with a hollow center, akin to the tubular basket of a centrifugal filter partially filled with adsorbent, accelerate the flow of solvent and, therefore, the rate of the separations (62). Columns with removable quartz windows have also been described (SO). Removal of the cohesive mass of the adsorbent from the adsorption tubes, which is desirable for the detection of colorless substances Kith reagents, is most easily accomplished when the columns are formed from a slurry of solvent and adsorbent. When columns are packed with certain dry adsorbents, such‘as precipitated chalk, the slug of adsorbent can often be removed after it has been sucked free of excess solvent. But with long
76 columns formed by pressing dry, resilient, powdered sugar or Celite into the tubes, successive portions of the adsorbent must be dug out one by one with a long spatula (124). Devices for the collection and examination of successive portions oi the percolate facilitate the detection of solutes as they are washed through the columns. Successive portions of the percolate may be collected and recorded automatically by gravimetric (28, 29, 128, l e g ) , volumetric ( g o ) , and spot testing (47) techniques. Variable portions of the percolate may be collected ~ solvent or the vacuum pressure without interrupting the f l o of by use of the all-glass receivers designed for use in vacuum distillation (1). Solutes in the percolate from a column may be detected and estimated by continuous recordings of optical density or refractive index of the effluent (3,28, 29, 48, 90, 128, 18.9).
1
Procedure and Objectives of Columnar Adsorption. Thanks to the studics of Tiselius and his eo-workers, much basic information pertaining to the separation of mixtures has recently been obtained by the percolation of a solution through a column until the solutes appear in the percolate (28, 29, 128, 129). Under these conditions, the distance migrated by the solutes is usually expressed in relation to the distance migrated by the solvent, as has long been done for capillary analysis. This kind of information provides a measure of the adsorption capacity of columns; it reflects the adsorbability of the solutes, and it provides some information about the effect of one solute upon the adsorbability of another (28, 29, 121). With a given solute and solvent, thi, percolation method serves as a basis for the standardization of adsorbents (73, 7 4 ) , and with a given solvent and adsorbent, i t provides a means for the detection and identification of solutes. Because the analyst is concerned chiefly with the advancing boundaries of the migrating solutes, this continued percolation of solution through the adsorption columns is commonly called “frontal analysis” (28, 128, 129). This procedure never results in a complete separation of a single component from a mixture; hence frontal analysis is not equivalent to the complete chromatographic analysis as described by Tswett. I t is more nearly equivalent to capillary analysis in its original form. Adsorption of a small quantity of solution followed by development of the chromatogram remains the common procedure for the resolution of mixtures. Development of the chromatogram until the solutes are carried into the percolate is usually called the “flowing” or “liquid” chromatogram or “elution analysis” (6, 25). The amount and the concentration of the solution drawn into the column before development of the chromatogram influence the degree of separation of the bands and the distribution of the solutes in the bands (121). The smaller the amount of the solution the greater will be the separation of the bands, but the more difficult will be the detection of the bands, particularly those of the minor constituents. For example, in a small column, 0.03 microgram of chlorophyll b was required to form a perceptible band. Yet this amount of chlorophyll b could be deLected in the presence of a t leait 2000 parts of chlorophyll a (123). Development of the chromatogram with a series of solvents or solvent mixtures of increasing polarity, as first described by Tswctt, often facilitates the separation of mixtures of similar substances (118, 124). mhen the solutes are carried into the percolate, this use of polar solvents is sometimes called “displacement development” (28, 128, 189). But variation of the solvent occasionally reverses the sequence or order in Khich the solutes separate in the columns (118); hence separations obtained by one solvent may be enhanced or they may be neutralized by the subsequent use of another (118, 119). When different mixtures of two solvents cause a binary mixture of solutes to separate in two sequences, there should be one mixture of the solvents that will not effect a separation of the solutes (118). Similar considerations should apply to mixtures of adsorbents. All these deductions point to obvious precautions that should be observed in the use of mixtures of solvents and mixtures of adsorbents. There are many objectives in the use of chromatographic adsorption methods in addition to the resolution of mixtures and the isolation of the components. Some of these aims are the com-
ANALYTICAL CHEMISTRY pariaon of substances, especially those suspected of being identical, and the description and identification of substances by means of their adsorbability relative to that of various reference materials (117, 118, 124, 150). The reversal of the adsorbability with variat’ion of the solvents may be utilized in order to remove the last traces of a less adsorbed substance from a more adsorbed substance (82, 118, 124). Additional examples of special objectives in the use of adsorption columns are presented in the sections devoted to adsorbents and to applications of the method. Procedure and Objectives of Paper Chromatography. In the carly applications of capillary analysis, one end of a strip of adsorpt’ivepaper was dipped into a solution and the distances penetrated by the solutes and by the solvent were compared. For the development of a paper chromatogram, a drop of the solution is commonly placed near one end of the paper strip, which is then dipped into the solvent (77). The paper may be held vertically as in the first applications of the capillary method (143) or it may be bent across a horizontal glass rod, so that gravity hastens the flow of the solvent and thereby aids in the development of the chromatogram (35, 36, 116). In long strips of paper, extensive development of a chromatogram may require nearly 24 hours, whereas comparable development in columns may require only an hour. Development of the chromatogram in paper must be performed in a closed vessel or cabinet in order to prevent excessive evaporation of solvent. Chromatography in paper lends itself t’othe aptly called “twodimensional development” or “cross capillary analysis” which was briefly described by Liesegang (?7) and subsequently more extensively studied by Consden, Gordon, and Martin (87). In this method, a drop of the solution is placed near one corner of a square sheet of paper, an adjacent edge of which is then dipped into the developing solvent contained in a long narrow dish or trough (78, 116,14b). After the chromatogram has been formed, the paper is dried, and the edge adjacent to the chromatogram is dipped into another solvent, so that the chromatogram is developed farther in a‘direction a t right angles to that of the first development. Under these conditions, the solutes appear as a series of spots distributed in a specific pattern in the paper (35, 37, 77, 94, 96). In columns, a comparable separation of solutes can be approached only by transference of the adsorbent from various sections of the chromatogram to a fresh column followed by further development with another solvent as has been done in the separation of chlorophylls from xanthophylls (82, 124). In paper, extremely small quantities of complex mixtures may be completely resolved, especially by use of the two-dimensional chromatogram. d few micrograms of some 20 amino acids have been isolated and identified both in relation to their position in the paper and by the addition of reagents, such as ninhydrin, which form colored products with the separated acids (36,37, 44). Similar results have been obtained by adsorption of sugars (20, 44, 94, 95) and other colorless substances. This use of reagents in paper is akin to the sensitive spot testing technique that has been widely employed by Feigl (32, 33, 62, 53). As in columns, radioactive substances in paper chromatograms can be located with photographic film or with counters (44,55,115,131). Polarographic and enzymatic reactions may also be applied t o solutes separated in paper (67). Dissolved salts and impurities in t,he solvents and in the paper often have pronounced effects upon the separability of mixtures of sugars and of amino acids (94, 139). In general, the objectives of paper chromatography are identical with those of columnar chromatography. However, paper chromatography is not readily adaptable to use with very volatile solvents, with various adsorbents, or for large scale preparations (97). But the use of finely divided polysaccharides, such as cellulose pulp (66), cotton ( l a f ) ,starch (go), and cellulose acetate (18) in columns, overcomes some of these limitations of paper chromatography.
17
V O L U M E 21, N O . 1, J A N U A R Y 1 9 4 9 ADSORBENTS
Ion Exchangers. One of the major recent developments in the preparation and use of adsorbents has been in the field of ionexchange compounds. A variety of organic ion exchangers of great combining capacity, of rapid exchange rate, and of highly reversible reaction are available commercially (iimerican Cyanamid and Chemical Company, Dow Chemical Company, The Permutit Company, and Resinous Products and Chemical Company). Many of these ion exchangers, although formed in large particles or beads in order to facilitate rapid filtration, appear to be porous to the reactive ions and do not disintegrate upon repeated reaction and regeneration (17 , 58). Particular properties of many of these products have been reviewed by Myers (92) and by Applezweig ( 7 ) . Surface-active substances such as charcoal or alumina may be converted into ion-exchange adsorbents by preliminary adsorption of acids or bases (70, 138) just as cloth is rendered more attractive to dyes by treatment with mordants. In columns, ion exchangers are especially useful for the separation of anions from cations, of ionic substances, as the alkaloids, from nonionic substances, and of mixtures of ionic substances that differ in valence. They also facilitate determination of the trace elements (102) and the total equivalence of acids and bases in salt solutions (4,79). They have been used extensively in the separation of the rare earths and the products of atomic disintegration; some 113 pages of the Journal of the American Chemical Society have been devoted to a single symposium on this subject (112, 113, 232). In spite of all this effort, development of chromatograms in columns of ion exchangers has not reached a high state of perfection, although promising results have been obt,aint:d bg t,he use of acids and of salt solutions as developers (112, 113, 132). Partition Adsorbents. A solvent, as water or nitromethane, held in structural material, such as silica gel or diatomaceous earth, may sometimes serve as the adsorptive, stationary phase in an adsorptioii column (2,84).In columns of Lhese adsorbents, the solutes are, in effect, partitioned between two immiscible 86). This liquids, hence the term “partition chromatography” (8.4, use of partition adsorbents in columns is analogous to countercurrent extraction, particularly the fractional partition procedure improved so extensively by Craig (21, 38, 39, 40, 88, 11.4). Columns of partition adsorbents are useful for the resolution of mixtures of compounds which are decomposed by adsorption on surface-active substances, a noteworthy example being the separation of the peniciilins (13,26, 56, 127). But even under favorable conditions, amino acids were found to be bound by the silicic acid itself as well as by the water in the gel (36,84,85).With suitable solvents, partition adsorbents may have very great capacity for the adsorption of dissolved solutes. When higher alcohols are used as solvents, water held by filter paper may also serve as the adsorptive phase, a phenomenon that has led to the term “paper partition chromatography” as indicated already (36, 84). With water as solvent, however, inorganic iouic substances may be held in paper by ion-exchange reactions as pointed out by Kolthoff some 30 years ago. Moreover, with water as solvent, the proteins phycocyanin and phycoerythrin are retained by the paper so that a mixture of these pigments can be separated as described by Kylin ( 7 1 ) . Even with aqueous ethanol as solvent, the nonionic, wealtly polar, fatsoluble chloroplij-lis and xant,hophylls are bound by the surfaceactive forces of filter paper arid can be separated from one another. Surface-Active Adsorbents. Kumerous investigations of the surface-active adsorbents confirm the enormous variability of their adsorption capacity and of their specificity or se1ectivit)y. Special attention has been given to the properties of diatomaceous earth (51, clays (80), maguesia (15%),alumina (QI), paper (36), starch (go), and many other substances (43). The specificity of any given adsorbent, as indicated by the
sequence in which substances are adsorbed in columns, may vary greatly with the solvent (118, 120, 124). This effect, which has been attributed, in part, to preferential affinity of the adsorbent for certain structural units or groups of the organic molecules (119), holds considerable promise for further investigation of the complex relationship between molecular structure and adsorbability. Addition of fluorescent organic dyes or inorganic phosphors to nonfluorescent adsorbents facilitates the detection of colorless solutes that absorb ultraviolet light. In columns of these mixtures, the solutes appear as dark, nonfluorescent bands (19,108). Selection of Adsorbents. Mixtures of solutes may be resolved under such a great variety of conditions that the choice of adsorbents remains, to a great degree, a matter of trial and error. Indeed, by variation of the solvent and by adjustment of the concentration and the amount of the solutes adsorbed, mixtures of fatty acids and mixtures of amino acids have been resolved in columns of a variety of adsorbents such as the ion-exchange adsorbents, the surface-active adsorbents, and the partition adsorbents. In many uses of adsorbents, it is impossible to distinguish clearly among partition, ion-exchange, and surfaceactive forces, as all may be effective simultaneously. The number of variables is so great and experience is so limited that selertion of adsorbents is determined to a greater extent by limited personal experience and by the objectives of the worker rather than by comparative results or by theoretical considerations. In spite of all these complexities, the practical chromatographer has developed several procedures that serve as guides to the selection of adsorbents. When the nature of compounds in mixtures is known, selection of adsorbents can be based upon previous experience. With mixtures of unknown substances, a variety of adsorbents can be tested quickly in small columns. As a rule, the solutes should be weakly adsorbed so that they can be forced to migrate rapidly through the columns. In these exploratory experiments, the initial band of the adsorbed substances should not occupy more than about a tenth to a twentieth of the column, so that extensive development of the chromatograms will be possible (119). In these tests, the solvents should also be varied, because different solvents have a pronounced effect upon the adsorption capacity and the specificity of many adsorbents. SOLVENTS 4ND ELUANTS
Selection of Solvents. The use of different kinds of solvents and eluants has scarcely kept pace with the increasing number of adsorbents. As the separability of mixtures varies tremendously with the solvent (15, 37, 118, 119), much more attention might profitably be given to the effect of solvents upon the relative adsorbability of various solutes. I n columns and in paper there must be an interaction between solvent and adsorbent and also an interaction between solvent and solutes in addition to the reaction between solutes and adsorbent. Disproportional variation of these forces may account for alteration of the adsorption sequence when solutions of chloroplast pigments in different solvents are adsorbed in columns of one adsorbent (118, 119). From this point of view, solvents may be selected so that they affect primarily the adsorbents or the solutes. With surface-active adsorbents including filter paper, a variety of solvents ranging from water and the alcohols to the nonpolar hydrocarbons have been employed for the adsorption of various substances. Substituted cyclic nitrogen compounds and aliphatic and aromatic alcohols that are immiscible in water have found extensive use with the partition adsorbents (37, 84, 90, 94, 96, 116, 116). Solutions of acids and of organic ions have been utilized to adjust the equilibrium of inorganic ions between the ion-exchange adsorbents and the solvent so that the cliromatograms are developed more effectively than with water alone (111,113, 131).
ANALYTICAL CHEMISTRY
70
Table I. Approximate Molecular Weights and Kinds of Suhstances Investigated hy c h r o m a t o g r a p h i c Adsorption Met1lads hloleoular Weisht 1-10 10-100
1oo-1,ooo
1ooo-loooo
io:ooo-iob,ooo 1oo,ooo-1,ooo,ooo
Kinds of Substance8 Hydr ogen ion , s,"+est inorgD*nicions-., .... ,". InOW.anie ions an" s m a n r s r LlrFLlrls lllUIYYYLrl ."oh as 'alcohols, aldehydes. ketones, esters. and 60me smino aoids . . H I:avy metal ions Complex inorgmic ions, most iynthrtio m g a d c eomwunds and many natural organic produots such as ~ u c a r ?fats, , +erols.,hlkaloids. ohlorophylb, carotenoids, amino aolds, and antibiotirs Pepti+. polysaeoharides, phosphatidPr. etc. ~ r o t e i n ri n o i u d i n ~enwmes Lamest proteins and
~~..~~.
-
Vnriable proportions of impurities in WIVL-ILIIU UL mi ~ U U Dp.0nounced and unpredictable effect,s upon the adsorhability of various solutes. These effcets, which may result in the alteration of the adsorption sequence of substances in a column (118, l Z 4 ) , complicate the standmdization and interpretation of chromatographic adsorption procedures. On the other hand, when more fully underst,ood,these effects of impurities may point the way to further refinement of t,he chromatographic adsorption techniques (84, 90,118). APPLICATIONS O F CAROMAMGRAPHIC ADSORPTION METHODS
One of the remarkable feat,uresof the chromatogrtLphic adsorptions methods is their wide applicability. All kinds of com.... pounds of molecular weights from one to .2 million can be Studled with adsorption columns provided these substances are soluble or dispersible in the.liquids that are suitable for formation of the chromatograms. An indication of this wide applicability of the adsorption methods is provided by Table I. There have been so inmy recent applications of the chromatographic adsorption method that it is impossible to present all of them, even in tabular form. As a conssquence, selected applications that illustrate generalizations already presented in this review are summarized in Table 11. I n most of the examples, recent referenoeshave been cited 60 that t,heyprovide a key to the earlier literstuw.
.. .
determined in relation to tho sharp leading houndaries of the unsymmetrical bands; hence R ir sometimes ropresented hy Rn (97, 49, 75, 74, 84). An equivalent expression for the rate of migration of ions in paper was formerly developed by Skrsup and by Kolthoff, who recognized that R vas a function of concentration, a complex relationship as shown by subsequent theoretical and experimental invost.igations (24, 28, 29, 79, 74, 84, 95, 121, 157). The variation a i R with concentration depcndv upon t,he properties of the adsorhents. With surfaoe-active adsorhents, the amount of solute not adsorbed relative to that adsorbed increases with increasing conecntration, as is well known from studies of adsorption isotherms (24, 121, 157); hcnee R increases with concentration. With partition adsorhents, R may yemain relatively constant ovor wide variations of concentration just as the partltion coefficient is known to be indopendent of concentration (24,97, 4 4 84? 86). Determmatmn of R a t various concentrations of salute provides anothei method for estimation of adsorpt,ion isot.horms (75, 74, 115, 126, 152, 197). This information provides a clue to the kin& of forces that aremost effective in columns of a given adsorbent (57, 49, 57, 79, / 4 >115,152). The rate of migration of onc solute can also be described in relstion to that of another. For the adsorption of sugars in paper this procedure is reported to yield reproducible values, but it introduces another variable (20). Identification of carotenoid pigments an the basis of their position above or below various reference 'materials (118, 119, !?O, IBG), involves, qualitatively, the same principles as those ut1hEed in the estimation of the R value. The amount of solvent which flows t:hraugh an adsorption column before a salute appears in the percolate serves as a meaeure of the adsorption capscity of the column and IS related t o the R value. It is variously known 8s the "threshold volume" (28, 75, 74,92), the "retention volume" (28,29,128,129), and the "break-through, volume" (S?, 9?LL:he last term being widely ~~
PRINCIPLES OF CHROMATOGRAPHY
Mechanism. Chromatographic separations, either in paper or in columns, depend upon repeated partition of the solute between the adsorbent and the solvent. This repeated partition can take plaoe only when there is a dynamic equilibrium among the solutes, the adsorbent, and the solvent. A single solute molecule migrates in an adsorption column only while it is in solution. When i t is held by tho adsorbent, it remaim stationary as the solvent flow past. The longer it is held by the adsorbent the slowcr it migrates. If two solute molecules differ in the time that they are retained hy the adsorbent, they will migrabe through an adsorption column a t different rates. This dynamic effect may he illustrated by the mechanical n Figure 1. model shown in gration of Solutes. Many molecules of a single Rate of Migration solute migrate t,hroueh through an adsorotion adsorption column at a rate which is determined by the flow of solvent and by the ratio of the molecules in solution to those adsorbed. On this basis, the distance moved by the bands of the adsorbed solutes i s also related to the disbsnce moved hy the solvent. Amount solute not adsorbed = distance moved by solute Amount solute adsorbed distance moved by solvent (28, 57, 75, 74, 84) This constant, R, has been determined in several ways. With pahition adsorbents, R is determined in relation to the region of hiohmt. snlnto mnwntrat,inn in t,he middle of t,he svmmet,rinal
Figure 1. Mechanical Model Illustrating Separation of Two Different Molecules in Adsorption Column
..
~~
pl+
,+,e?de,,
..~.~~
L.moleds,, . and th. vhito _L-l.ll. . . 2-
" . . , I* I."-,"-
.I.._
V O L U M E 21, NO. 1, J A N U A R Y 1 9 4 9 Table 11. Substances Various rare earths Fission products of uranium Ions of salts Inorganic ions Trace elements solutions, Various anions
Substances Separated or Isolated by Adsorption and Adsorbents and Solvents Employed Inorganic Substances Adsorbents A rnberlite 1R- 100
salt
Stearic a n d oleic acids Higher f a t t y acids Tartaric a n d lower f a t t y acids Lower f a t t v acids ( R values) Various f a t t y acids I n relation t o molecular weight I n relation t o chain length Lower f a t t y acids as p phenylphenyacyl esters Nitrogenous lipides. amino acids, sugars F a t t y acids a n d bile acids Phospholipides
Amberlite 1R-100 Alumina
Petroleum ether (2s) Grape juice ( 8 7 )
Silica gel
Chloroform
+
Petroleum ether (48)
(49)
Silica
+ butanol
Charcoal
Heptane (31)
D r y silicic acid
Benzene petroleum ether (69) Phenol water, colwater, etc. lidine
Paper
+ ++
Foam hlg0
Collidine, benzyl alcohol, butyl alcohol, etc. (37, 1 4 s ) Phenol ammonia
+
(67)
Water (effect of p H ) (69) Water phenol (188, 129) Charcoal, silica, AlzOl, Water, acids (106) etc. Amberlite 1R-100 Water (34)
+
Charcoal
Tofatit C Amberlite 1R-4
Water, 1 N HC1 ( 1 4 1 ) 0.01 N HC1 (47)
Amberlite 1R-100
Water a t p H 6 t o 7 (8)
Paper
+
From plants From liver
Duolite resin Paper
Phenol water, ete. (116 116) Water '($2) Phenol water, etc.
From silk hydrolyzate
Paper
Phenol
From urine
Paper
Phenol
ZnCOr
Petroleum ether 10% benzene (68)
Various amino acids aa p phenylazobenzoyl esters Various sugars ( E values)
+
(130) (91)
(44)
Carbohydrates Paper
+ Celite +
+ water, + water,
etc. etc.
+
Various sugars
Florex
Amberlite IR-4B Florex Celitk Amberlite 1R-4B Paper
Water, ethanol
Paper
Phenol water collidine ($0) Phenol water etc. (66)
76)
Cellulose pulp
Streptomycin Penicillin G Various penicillins Various penicillins
Antibiotics AllOr charcoal Super Filtrol Paper Silica gel
(4)
Water (80) Phenol S K I etc. (SA)
Various sugars
+
+ +
Various Isomeric Substance2 Silica gel nitro- Hexane (I) methane d , I forms of Troger's base Lactose Petroleum ether (99) Brucine-d, Z-mandelate Glucose Benzene (61) Z-iMenthyl-d,Z-mandelate Alumina Petroleum ether (66) cis-trans-Carotenoids Lime Petroleum ether 4various solvents ( 1 6 , 149, 160) cis-trans-Carotenoids Sucrose Petroleum ether propanol ( I % $ . 188) Isomeric chlorophylls Sucrose Petroleum ether propanol (82) Benzene hexachlorides
Enzymes HyAo Super-Cel Paper
Glycerophosphatasr
.%bo3
Various Vitamin A , carotenes carotenes. chloVitamin -1. rophylls Vitamin A alcohol and ester Vitamin .%? Vitamin A aldehyde (retinene) Vitamin A aldehyde Carotenes Carotene isomers Carotenoids phylls
and
chloro-
,
.
Ethylene dichloride
AISiOa, 1 1 ~ 0ZnCOt ,
Petroleum ether (109) Petroleum ether (9)
CaCOa All03 Ca(OH)>
Petroleum ether (196) Chloroform ( 1 2 ) Petroleum ether various solvents (16, 149, 160) Petroleum ether propanol (119, 180,
A1203
Sucrose
(100)
+ + +
114)
Petroleum ether benzene ( l a 6 )
+ +
Agent of chicken tumor
I
AlrOa
CaCOa
hyoscya-
+
Carotenoids and Vitamin A A1201 Petroleum ether (91) Celite Petroleum ether (1LI)
Various Organic Substances filter 3,5-Dinitrobenzoates of ali- Silicic acid phatic alcohols aid filter 2,4-DinitrophenylhydraSilicic acid zones aid Purines, pyrimidines, etc. Paper Anthocyanins (R values) Paper Hyogcine and mine Coal bitumens
Water (81) Collidine or butanol acetic acid ( 4 1 ) Water ( 6 0 )
Silicic acid Silicic acid Celite
Hexane
+ ether
(140)
+
Petroleum ether ether ( 1 0 4 ) Butanol ( 6 4 ) Butanol acetic acid water ( 1 1 ) Benzene (193)
+
+
Benzene, benzene 5% ethanol ( 7 % ) Aqueous XaCl(103)
+
mathematical description of chromat,ographic separations regarded as kinet,ic (46, 111, f%), partition (38, 39, 40, 84,86, 88, 114), and adsorption (28, 46, 93, 126, 137) phenomena. The amount of solute adsorbed and the distribution of the solutes at the leading and trailing boundaries of the adsorption bands have been correlated with adsorption isotherms (24, 84, 93, 113, 121, 137). Qualitatively t>hereis excellent agreement betyeen many deductions based upon the theories of adsorption and the phenomena observed in columns. Quantitatively, much additional precise information is needed in order to distinguish other important effects such as the rates of adsorption and desorption in columns (113, 126, I S ) , the effect of particle size, the effect of one solute upon the adsorbability of another (29, 128, 129), and the rc~lationshipbetween heat, of adsorption and adsorbability (911.
++
Phenol water, colli water, etc' dine (80,96) Water, alcohols (16,
Sugats of milk Uronic acid,, sugars Polysaccharide of bacterium dysenteriae Methylated sugars
Penicillinase Flavine nucleotides
Galloxanthin
Heptane (31)
Various amino acids (R Pauer values L ' S . concentration a n d spot size) Various amino acids AgzS
Acidic, neutral, and bwic amino acids Basic amino acids Asuartic a n d elutamic acids Citrulline and allantoin Various amino acids F r o m plants
(69)
0.1 .V salt solutions (102) Water ( 7 0 )
F a t t y Acids Charcoal diatomaceous earth Charcoal .4mberlite 1R-4
Amino .\cids Various amino acids ( R Paper values)
Various amino acids (retardation values) Various amino acids
Solvents .%queous solutions of citric and oxalic acids (112, 113, 192,) Phenol water, collidine x a t e r . etc. (199) Water and aqueous HC1
++
Paper Zeo-Karb
in
79
+
+
+
LITERATURE CITED (1) Ace Glass Inc., Vineland, N. J., Catalog 40 (1940). (2) Aepli, 0. T., Munter, P. A , , and Gall, J. F., Axar,. CHEN., 20, 610 (1948). (3) American Instrument Co., Bull. 2167 (1948). (4) Anantakrishnan, C. P., and Herrington, B. L., Arch. Biochem., 18, 327 (1948).
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ANALYTICAL CHEMISTRY (83) Mantell, C. L.. “Adsorption,” New York, McGraw-Hill Book Co., 1944. (84) Martin, A. J. P , Ann. .V. Y . Acad. Sci., 49, 249 (1948). (85) Martin, A. J. P., Endeavour,6, 21 (1947). (86) Martin, A. J. P., and Synge, R. L. M., Biochem. J., 35, 1358 (1941). (87) Matchett, J. R., Legault, R. R., Nimmo, C. C., and Notter, G. K., I d . Eng. Chem., 36, 851 (1944). (88) Mayer, S. W., and Tompkins, E. R., J . Am. Chem. Soc., 69, 2866 (1947). (89) Meunier, P., and Vinet, A., “Chromatographie et MBsomerie, Adsorption et RBsonance,” Paris, Masson & Cie., 1947. (90) Moore, S.,and Stein, W.H., Ann. N. Y.Acad. Sci., 49, 265 (1948); J. Bid. Chem., 176, 337, 367 (1948). (91) Muller, P. B., Helv. Chim. Acta, 27, 404,443 (1944). (92) Myers, F. J., Ind. Eng. Chem.,35, 858 (1948). (93) Offord, A. C., and Weiss, J., Nature, 155, 725 (1945). (94) Partridge, S.M . , Biochem. J., 42, 251 (1948). (95) Partridge, S. M., and Westall, R. G., Biochem. J . , 42, 238 (1948). (96) Peck, R. L., Ann. S . Y . Acad. Sci., 49, 235 (1948). (97) Polson, 4 . , Mosley, V. M., and Wyckoff, R. W. G., Science, 105, 603 (1947). (98) Pratt, J. J., Jr., and Auclair, J. L., Science, 108,213 (1948). (99) Prelog, V., and Wieland, P., Helv. Chim. acta, 27, 1127 (1944). (100) Reed, G., Wise, E. C., and Frundt, R. J. L., IND.ENG.CHEM., ANAL.ED.. 16, 509 (1944). (101) Rheinboldt, H., in Houben, J., “Die Methoden der organischen Chemie,” 3rd ed., Vol. I, p. 291, Leipsig, Georg Thieme, 1925. (102) Riches, J. P. R., Nature, 158,96 (1946). (103) Riley, V. T., Science, 107, 573 (1948). (104) Roberts, J. D., and Green, C., IKD.ENG.CHEM.,AKAL.ED., 18, 335 (1946). (105) Robinson, F. A . , Pharm. J., 158, 46 (1947). (106) Schramm, G., and Primosigh, J., Ber., 77,417 (1944). (107) Schroeder, W.A., Ann. S.?. Acad. Sci., 49, 204 (1948). (108) Sease, J. W., J . Am. Chem. S o c , 69, 2242 (1947). (109) Shantz, E. M., Science, 108,417 (1948). (110) Shedlovsky, L , Ann. -V.Y . Acad. Sci., 49, 279 (1948). (111) SillBn, L. G., Arkiv KemiXineral. Geol., A22, l(1946). (112) Spedding, F. H., Fulmer, E. I., Ayers, B., Butler, T. A., Powell, J., Tevebaugh, A. D., and Thompson, R., J . Am. Chem. Soc., 70, 1671 (1948). (113) Spedding, F. H., Voigt, A. F., Gladrow, E. M., and Sleight, N. R., Ibid., 69, 2777 (1947). Foi other papers inthisseries. see Ibid., 69, 2771-881 (1947). (114) Stene, S., Arkiv KemiMineral. Geol., A18, 1 (1944). (115) Stepka, W., Benson, A. A., and Calvin, M., Science, 108, 308 (1948). (116) Steward, F. C., Stepka, W., and Thompson, J. F., Ibid., 107, 451 (1948). (117) Strain, H. H., “Chromatographic Adsorption Analysis,” p. 7, New York, Interscience Publishers, 1942. (118) Strain, H. H., IKD.EXG.CHEM.,ANAL.ED.,18, 605 (1946). (119) Strain, H. H., J . Am. Chem. Soc., 70, 588 (1948). (120) Ibid., 1672 (1948). (121) Strain, H. H., J . Phys. Chem., 46, 1151 (1942). (122) Strain, H. H., and Manning, W. M., J . Am. Chem. Soc., 64, 1235 (1942). (123) Strain, H. H., and Manning, W. M., J. Biol. Chem., 144, 625 (1942). (124) Strain, H. H., Manning, W. M.,and Hardin, G., Biol.Bull., 86, 169 (1944). (125) Tauroe. 9.. Entenman. C.. Fries. B. A.. and Chaikoff. I. L.. Ibid,’ 155, 19 (1945): (126) Thomas, H. C., Ann. N . Y. Acad. Sci., 49, 161 (1948). (127) Thorn, J. A., and Johnson, M.J., ANAL.CHEM.,20, 614 (1948). (128) Tiselius, A,, “Advances in Protein Chemistry,” Vol. 111,Academic Press, Sew York, 1947. (129) Tiselius, A , , “The Svedberg,” p. 82 Uppsala, Almquist and Wicksell, 1944. (130) Tishkoff, G. H., Zaffaroni, A , , and Tesluk, H., J . Biol. Chem., 175, 887 (1948). (131) Tomarelli, R. M., and Florey, K., Science, 107, 630 (1948). (132) Tompkins, E. R., Khym, J. X., and Cohn, W. E., J . Am. Chem. Soc., 69, 2769-77(1947). (133) Trautner, E. M . , and Roberts, M.,Analyst, 73, 140 (1948). (134) Vahrman, M., Bull. Brit. Coal Utilisation Research Assoc., 10, 305 (1946). (135) Wald, G., J . Gen. Physiol., 31, 377 (1948). (136) Ibid., 459 (1948). (137) Weil-Malherbe, H.. J . Chem. Soc., 1943, 303. (138) Weiss, D. E., Nature, 162, 372 (1948). (139) Westall, R. G., Biochem. J , , 42, 249 (1948). ~I
V O L U M E 21, NO. 1, J A N U A R Y 1 9 4 9 (140) Khite, J. W., Jr., and Dryden, E. C., ANAL. CHEM.,20, 853 (1948). (141) Wieland, T., Ber., 77, 539 (1944). ENG.CHEM., ANAL.ED.,18, 702 (1946). (142) Wilkes, J. B., IND. (143) Williams, R. J., and Kirby, H., Science, 107,481 (1948). (144) Williams. T. I., “Introduction t o Chromatography,” London, Blackie and Son, 1946. (145) Winsten, W.A,, Science, 107, 605 (1948). (146) Winsten, W. A , , and Spark, A. H., Ibid., 106, 192 (1947).
81 (147) (148) (149) (150) (151) (152)
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RECEIVED November 8 , 1948.
DISTILLATION ARTHUR ROSE The Pennsylvania S t a t e College, S t a t e College, P a .
T
HIS paper is written from the point of vievi of developments during the years 1946 to 1948. A few earlier papers are commented on or listed as references, but in general it is assumed that the earlier items of interest will be obtained by consulting the references in the more recent papers, or found in bibliographies or indexes. Analytical distillation may be defined in various ways, but in this paper it is assumed to include simple laboratory distillation procedures and all the forms of distillation that involve rectification or fractionation regardless of whether quantitative analysis, qualitative separation, or mere detection is the objective. Production operations are naturally excluded, but preparative distillation, even on a pilot plant scale, is often of analytical interest. Significant trends and developments in analytical distillation during the past several years have included the following: Extensive utilization of fractionation in the analysis of complex mixtures of hydrocarbons, fluorocarbons, and many other types of compounds. Efficient fractionation has become a powerful and frequently used tool, so that success in achieving difficult separations or analyses is often a mere incident in solving broader problems. Recognition of the limitatioris of distillation as a method of separation and analysis, and consequent combination of the process with various physical and chemical methods of analysis. Realistic evaluatioq of analytical fractionation apparatus, with emphasis on time required, ease of operation, and actual separation a t finite reflux in addition t o the older use of theoretical plate standards a t total reflux. Improvement of older apparatus, particularly as to details and accessories, as n-ell as t’he introduction of several entirely new types of contacting devices. Development of special distillation apparatus such as rotary columns for vacuum distillation, small molecular stills, and apparatus for semimicro and microdistillation. Further study and improvement of low temperature distillation, particularly by use of automatic controls to gain reproducible operation, and also by the use of simple isothermal distillation a$ low pressures to take advantage of the improved relative volatility, small sample size, and speed of analysis. Limited progress in practical application of theoretical concepts, but considerable interest and activity along these lines. Continued improvement and extension of A.S.T. RI. distillation tests and similar methods involving simple standardized distillation as part of an analysis. The relatively slow progress in catching up on the part of European scientists, except for general use of Podbielniak columns. Publication of basic data and general bibliographies. UTILIZATION OF FRACTIONATION
A classical example of successful analytical fractionation is the work of the National Bureau of Standards and its cooperating groups, dealing with the composition of petroleum (45, 53-56, 119, 120, 13X, 152). This work is particularly notable for the balance between the use of distillation and other separation processes such as selective adsorption. Similar use of fractionation has been reported by the Bureau of Mines and others (83, 126, 147). Various complete fractionating columns have been developed or improved for general laboratory use (40, 52, 61, 66,
65, 85, 90, 98,113, 1S1, 136, 141, 143). Extensive application of precise fractionation has also occurred in the development of fluorine chemistry (11, 50, 104). Almost all the papers in the 1947 A.C.S. Symposium on Fluorine Chemistry ( 4 9 ) describe or mention the use of distillation as a method of analysis or as a separation or purification step related to analysis. Other similar applications are widespread (107). The most recent involve the isolation of the oxygenated organics obtained from the FischerTropsch or related processes. The prevalence of azeotropes in these mixtures complicates the use of distillation. There are no published papers as yet. SPECIAL METHODS
Early enthusiasm for efficient fractionation led to the extreme of even attempting the separation of materials of almost identical volatility. This has been succeeded by a period of the development of a multitude of special tests which can identify components in the fractions resulting from the distillation. Such procedures are not new, but during the past fern years there have been much more attention and activity in the perfecting of the special physical and chemical methods than in the associated distillation itself. Typical publications are cited (30, 72, 78, 89, 119,120). EVALEATION OF APPARATUS
Early evaluation of fractionating colunins ww confined almost entirely to the determination of the number of theoretical plates a t total reflux. It has become increasingly evident that this is but one among many factors that determine the utility of a particular apparatus, and that it is easier to satisfy and measure plate requirements than some others. The American Petroleum Institute Symposium on High Temperature Analytical Distillation in Soveniber 1946 was a major contribution in the direction of overall evaluation. This series of papers dealt in detail with the results obtainable with heligrid, helices, perforated plate, and screen packings, and in columns ranging in size from those for samples of a few milliliters to gallon scale operation (17 , 21, 34, 4O,57, 66, 90, 114, 116, 162). Among the factors considered were the time required to reach equilibrium, throughput rates, holdup, pressure drop, and deterioration, as well as theoretical plates a t total reflux. Some attention has been given to determination of the ]atter at finite reflux (34, 67, 117) and to expression of results in terms of actual distillation curves instead of theoretical plates (21, 57, 90). Hilberath ( 7 1 ) has presented an interesting and thorough paper on column evaluation Willingham and eoworkers (153) have compared various columns and packings through use of the A factor. The evaluation of vacuum columns has progressed to the extent that several additional binary test mixtures have been developed ( 4 7 , 106, 151). IMPROVEMENT OF APPARATUS
A great variety of column heads and other devices has been presented for controlling and determining reflux ratio, for auto-
