V O L U M E 2 2 , N O . 1, J A N U A R Y 1 9 5 0 Singh, B., and Rehmann, H., -1. Indian Chem. SOC., 19, 349 (1942).
Smith, G. F., J . Am. Ceram. SOC., 29, 143 (1946). Spacu, G., and Spacu, P., Bull. sect. sci. acad. roumaine, 26, 295 (1944).
Spacu, G., and Spacu, P., Z . anal. Chem., 128, 226 (1948). Zbid., p. 229. Zhid., p. 233.
Stefanovskii, V. F., and Svireiiko, V. D.. Zavodskaya Lah., 9, 1151 (1940).
Stock. J. T., Analyst, 73, 669 (1948). Stock, J. T., Metallurgia, 37, 220 (1948). Stott, J. G.. J . Soc. Motion Picture Engrs.. 39, 37 (1942). Strouts, C. R. N., and MacInnes, C. A., Analyst, 73, 669 (1948).
Swain, C. G . , and Ross, 8. D., .I. '4m. Chem. SOC.,68, 658 (1946).
Talipov, S. T., and Teodorovich, I. L.. Zavodskaya Lab., 15. 529 (1949). Tannaev, I. V., and Lerina, M.I., Zhur. anal. Khim., 1, 236 (1946). Tarayan, V.>I., Zavodskaya Lab., 12,543 (1946). Ibid., 13, 554 (1947). Tomicek, O., Collection Ciechosloe. Chem. Commun., 13, 116 (1948). Tourky, A . R., and El Shamy, H. K., AnaZyst, 68, 2, 40 (1943). Tourky, A. R., Farah, M. Y . , and El Shamy, H. K., Ihid., 73, 258 (1948). Ibid., p. 262. Ihid., p. 268.
41 (215) Treadwell, W. L, , aiid Nieriker, R., Helt. Chim. Acta, 24, 1067 (1941). (216) Zhid., p. 1098. (217) Vasilev, D. V., J . Applied Chem. 1i.S.S R., 14, 689 (1941). (218) Vasil'ev, K. 4.,and Faktorovirh, B. A,, K h i m . RPferat Zhur., 1940, No. 7 , 5 6 . (219) Virasoro, E., Anales inst. invest. cient. y t ~ c n o l .(('nir r i d . litoral Santa Fe, Arg.), 14/15, No. 23, 65 (1946). (220) Volkov, 1'. I., Zavodskaya Lab., 10, 202 (1941). (221) Watters, J. I., and Kolthoff. I. M., J . Am. Chem. Soc., 70, 2455 (1948). (222) Weiner, R.,Z . anal. C'hem., 123, 385 (1942). , Z. Elektrochem , 48, 604 (1942)). (223) Weiner, R., and M a h ~L., (224) Weiss, G., and Blum, P., Bull. soc. chim., 1947. 735. (225) Wernimont, G., and Hopkinson, F. J.. IXD.Eun. ( ' H E M , . 4 N i L . ED., 15, 272 (1943). (226) Wittman. G., Angew. C'hem.. A 60, 330 (1948). (227) Wolff. J. P.. BmZ. C h m Acta. 1 . 9 0 11947). (228) Yadaia, B. P., and Chatterji,'A. b., Indian C'ht'm. Soc.: 20, 115, 123 (1943). (229) Yao, Y . I., Trans. Elecfrochem. Snc.. 85, 213 (1944). (230) Yeck, R. P., and KiHsin, G. H.. IWD. ENG.CHEY.,A s \ i . . J s h . . 17, 692 (1945). (231) Zanko, ,4.M . , Khim.Referat Zhur., 1940, No. 2, 64. (232) Zhidkikh, N. P., Zaaodskaya Lab., 9, 635 (1940). (233) Zhivanovich, D., J . Applied Chem. l'.S.S.R., 19, 1225 (1Mfi). (234) ZUrcher, M., and Hoepe, G., Helv. C'him. Acta, 21, 1272 (1938). (235) Zvenigorodskaya. V. M., Zawdskaya Lab., 11, 1010 (1945). (236) Zvenigorodskaya, V. M.,and Gotsdiner, R. (2.. I h i d . . 12, 142 (1946).
i.
RECEIVED December I!?, 1949.
CHROMATOGRAPHY HAROLD H. STRAIN, Argonne Nutional Laboratory, Chicago, I l l .
ITH the development of the natural sciences, man has based his understanding of the universe upon the properties oi inorganic and organic matter. Among all material substance ranging from the several forms of energy to the intricate associations of living organisms, there are various degrees or levels of complexity. These organizational levels may noiy serve for the comparison and classification of matter and for evaluation of the advancemrnt of knowledge. R l t h some oversimplification and with certain arbitrary assumptions, the relative complexity uf most substance may be summarized in tabular form aq illustrated by Table I. The relationships among matter at the different organizational levels are more complex than the relationships among matter at each lrvel. .Uthough there are many opportunities for progress in th(1 understanding of highly organized material substance in term5 of less complex matter, most investigations and interpretations have been confined to substance a t a single organizational level. This limitation to the prc~grrssof h n o ~ l e d g rcan t w Httiibuted to varioug conditions such a' the extrcme sptxciali-
Table 1. Increasing Complexit?.
j
Relative Complexity of Inorganic and Organir Substance Substaiice Various forms of energy: t h e basic unit of radiant energy, tht, photon: t h e carrier of kinetir r n e w y , t h e neutrino (niasslehparticles) Basic units of electricity and of m a t t r r : t h e electron and t h r positron (lightest particles) Intermediate particles: the m e w w Basic components of atomic nurlei: the proton and the neiitron Chemical elements: hydrogen t o rurium Chemical compounds: inorganir to organir: minerals iucludinr coal a n d petroleum Rather constant associations of r~lirnricalcompounds: soil, a i r . sea water Particular, improbable a-sociatiom of labile conipoitnds: livine cells .Associations of living cells: ~ n ~ l t i c r l l u l aorganisms, r reyrodiictive groups ('oniplex associations of ntlllticeliular organisms: roloniw. hives, flocks, herds, trihes, ritirs. a n d nations
zation of scientific investigators (IO), the limitations of man's sensitivity and perception, and the limitations of scientific methods. Rmognition of most of the degrees of complexity in nature has resulted from the use of sensitive instrumcnts and of specialized analvtical techniques (27, 98). With a few notable exceptions, these methods of investigation have proved most effectivc M it11 matter at b u t one level of coniplc\itv. iPPLICABILITY OF CIIRO\l9TO(;R.~I'HIC ANALYSIS
Of the methods devised for the examination of matter, few have found such wide application under puch a variety of conditions as chromatographic analysis. Invented in different modifications b y Schonbein, by Goppelsroeder, and by Tswett, and improved and applied by many other workers, this method has continued t,o yield significant new w x d t s in many diversib fields ( 1 4 , 28, SO, 69, 118, 119). It has proved useful with SUI)stance varying enormously in degree of complexity-as for example, the chemical elements, various chemical compounds, microscopic parts of wlls, and c w n thc cells themselves (10.7, 118).
Chromatographic analysis is tqeeially effective for t I ic resolution of mistures and for the isolation and identification of the components. As the resolved materials may be isolatrd ivithout chemical changt:, chromatography has become a basic. tool for all scientists whose advance into unexplored regions depends upon knowlrtlpr of the propt'rties of particular substances (11s). Because of the discovery and improvement of chromatographicmethods in remotely related fields, evaluation of the techniquc rvith respect to applicability to new niakrials has been difficult. and timc-consuming. Progress in this direction has been facilifated hy reviews t h a t h a w appeared from time to time (28, 30, 118). .in extensive summary with 688 literature citations cover'ing applications of chromatographic analysis in organic chemixtry for the prars 19:%-194!~has I)ccn prcpared by Lcderer ( 6 9 ) .
ANALYTICAL CHEMISTRY
42 Another review devoted to the properties of ion exchangers, which have been utilized for many chromatographic separations of inorganic ions, has been prepared by Xachod and fifteen collnhorators (87). h general summary of chromatographic techniques, first presented several years ago as part of the Ircture series, “Frontiers in Chemistry,” has also been brought to publication (119). Advances in the use of chromatographic methods for following the chemical and metabolic transformations of carbon compounds labeled with isotopic carbon have been reviewed by Calvin and co-workers (6, 18, 19). Xumerous symposia in Europe and in the United States have also helped to disseminate knowledge about this useful technique (3, 186). The indexes of Chemical Abstracts, of Annual Rei~2’Cv.sof Biochemistry, and of many scientific journals have been expanded to include various applications of the chromatographic technique. In view of the availability of most of this material, no attempt has been made to report here all the recent research or intiustrial applications (8, 11.9). Instead, emphasis has been placed upon the nature, the merhanism, and the modifications of rhromatographic analysis.
varied in many aays, HS tj?, u+v 01 variuur at)sorptive phases and by use of various solvents, the principles involved in the chromatographic resolution of mixtures are few indeed (69, 87, 119). PARTITIOh MECI14NISM IN CHROMATOGRAPIJIC SEPARATIONS
The dynamic partition of solutes betneen the two phases in chroniatographic separations may be represented by equilibrium equations. Stationary phase
e Mobile phase Solutes
Solutes
J/‘
Solvent
\\
Adsorbent
5=
lt
Solvent
These equilibria ma)- involve solution or dispersion of the solute.
IVATURL.: OF CHROMATOGRAPHY
There has been a gradual clarification of the m o t h o d a and procedures which are now recognized as chroniatographir analpsis. This technique depends upon n dynamic partition 01’ distribution of dissolved or dispersed substances kJetwwi two immiscible phases one of which is moving past the other (87, 11.9).
1
S 0 L V E NT
I
Figure 3. Solution in Paper Washed with Fresh Solvent as in Formation of Chromatogram SOLUTION
c
They may iiivolve ionization, solvation, 01’ association of the solute. Thew may be complex formation \vith the phase itself or Kith added su1)stances. There may be a variety of reversible chemiral reactions, and in the stationary phase, there may be various surface-adsorption phenomena. Meinhard (88) has shown that scveml ronihinations of reversible systems remain to be examined as a hasis for chromatographic separations. I n his sixteen arbitrary combinations, adsorption of the solute and solution of the solute in the stationary phase are placed in the although for practical reasons they are usually considered separately (118). Even though ccrtain of these reversible phenomena may predominate in a single absorption system (119), most, of the attractive forces are usually present in varying degree. Recent data on thc absorption of organic acids by ion exchangers suggests that the principal reaction betvieen the acid and the insoluble aminc! resin may vary v i t h the solvent, ion exchange predominating in thr, presence of water, addition reactions predominating in the presrnce of nonaqueous solvents ( 1 0 7 ) . I n absorptive filter paper, the resolution of mixtures of solutes may depend upon surface adsorption. upon ion exchange, or upon partition betn-ren solvents (69, 118, 119). The predominant force varies with the paper, with the solvents, and with the solutes, Both paper and siliceous filter aids may be impregnated with various adsorbents, ion exchangers, and chemical reagents (51, 133) so that the attractive forces are varied enorniously, whereas the desirable physical properties of the support are retained. With absorptive solids, the predominant attractive forces may be chemical reactions, such as ion exchange and complex formation; surface attractions, such as the adsorptive forces of surfaceactive solids; and solution, such as the dissolution of solutes in the liquid retained by a gel or moist solid (107, 119). The amount of solutt. rrtainrtl by the solid phase in relation to the ~
SOLVENT
1
Figure 1 ( L e f t ) . Filtration of Solution through an Adsorption Column Figure 2 (Right). Ascent of Solution in Strip of Filter Paper Distance moved by solution. d , and by solvent, d .
In practice, chromatographie analysis involves a pai?iculai discontinuous procedure. The mixture t o be resolved is absorbed in an absorptive column (Figure l), in one end of a strip of paper (Figure 2), a t the side of a sheet of paper (Figure 3), or in wherein it is washed with fresh solvent a stack of filter paper (S), or with mixtures of solvents so that the solutes migrate through the porous, nonmobile phase a t different rates and separate from one another. Although these physical arrangements may be ~
V O L U M E 22, NO. 1, J A N U A R Y 1950 c
43
the ~oiiiiii011chroniatography based 011 the use of surface-active adsorbents. :9 For the initial passage of solution into a column or strip of ,ne paper, sur11 terms as adsorption or absorption of the mixture. a 7 filtration of the solution, the initial a.clsorption, and zone forniation have been widely employed. Passage of fresh solvent, through the absorptive layer to separate the absorption zonw, though coininonly called development of the chromatogram, is now widely called washing the absorbed substances, irrigation with fresh solvent (10, 73), tre;it,merit iTith complesing agnits (&Y), and formation of the chromatogram (11'). The separate regions in the abmrptive material t h a t coril:iin t h c i individual, resolved solutes liavc been called absorption or CONCENTRATION IN M I L L I M O L E S PER M I L L I L I T E R :tclsorption bands or zones. Because of the possible confusion Figure 4. Isotherms Representing Absorption of Sil, er n-ith spectral absorption bands, these regions i n the coluii~ns Ion by Ion Exchanger ( I I ) , ..idsorption of Acetic .Acid might preferentially be called zones rather than bands. h> Hone Charcoal, and Partition of Solute between Two Immiscible Liquids Irrespectivc of the partition mechanism, the resolut~io~iof iiiixturw in paper is coninionly called paper chromatography, Dakhrd portion. of curves reprewnt extrapolations :ilialogous to columnar chromatography. Resolution of mixtures i n it sheet of paper by irrigation in one direction with one solvent coiicentration of the solute when these different forces 1 ~ - (Figure 3) f o l l o ~ e dby transverse irrigation with another solvcAnt dominate is illustrated by the curves in Figure 4. is no\\- called two-way chromatography (11), a contraction of tlic: t\\(i-dimetisioiial chromatography suggested by con st let^, V A K I WI.E COWITIONS IN C I I H O ~ . ~ , ~ ' ~ O ( : K , ~ P H I ( : ( k~rdot\,wnd XIaitiu, :tiid an improvement on the ci'oss i y d l w y SEPARATIONS :rti:ilysis, first ~ ~ r o p o s eby d Liesegang (118, 119). Sunierous niotlificatioiis of Ihe chroniatographir techniqur Ci~lorlrsu substances stpiratrti by chromatographic adsorpwhich have been reported in the past year pertain to the UPP of tion A ~ Poften rendered visible by addition of reagents to t,lie ahsorptivc paper; hence they are considered in a subseyurnt rhiornatogrmii (69, 119), a procedure ividely utilized iii paper section devot,ed t o paper chromatography. Similarly, variations (69). Colorless substnnces may also be made c~lirorn;2togi;tpl1~~ d c-hroniatopof the absorbents are treated under sections t l ~ w i t to visihle by the addition of re:igents to the absorptive phase (16) raphy by adsorption, by ion exchange, and b y partit,ion. and to the \vas11 liquid (89). Because of the parallelism between .1variety of solvents and solvent mixtures has tieeii eniployetl this production of color and the development of a photograph, for washing the absorbed solutes. These solvents range from forniatioii of colored derivates of solutes separated by adsorption the nonpolar organic hydrocarbons to very polar aqueous soluis widely called the development of the chromatogram, c o n t w y tions. As indicated by the equilibrium equations and as illusro the older meaning of this term. Hy the sheer weight of usztge, trated by many new examples, the solvent must be selected iii Pspecially in the field of paper chromatography, this new meaning relation to the properties of the absorbent and the solute mixture promises to displace the older one. I t may be better, t,herefore, (69, 118). For uniform, re~)roducihlr separations, even filttato call the washing of the absorbed solutes the formation of the tion must lie obtained ( 3 5 ) . vhromatogram, and the production of color with reagents t,lre & i nincreasing number of examples s h o w that many conibiiiaricvelopment or revelation or ripening ( 8 3 ) of the chromatogr:ini. tions of solvents aud absorbents may be employed for resolution The term chromatography, which evolved from Tswett 's of one kind of solute--for example) mixtures o-f organic acids r.h,.o~nutoy,.nl-'hisc/ie ddsorptio,zsunaZyse, no longer applies s o l ~ l y may bc resolved by absorption in columns of ion exchangers to the separation of colored substances, nor does the method dewith watw or with organic liquids as solvents, a variation that' pend entirely upon adsorption phenomena. Moreover, this may lead to inversion of the adsorption sequencc (107). They terminology leads to the anomalous colorless chromatogram may be separated iri columns of silica gel with chloroform or ( 6 9 , 83). I n vimv of the q-idespread use of all chromatographic iso-octanrs as solvent (do, IOY), iri columns of Celite plus strong inethods for the resolution of mixtures, a name indicative of sulfuric arid and with benzene or butanol as solvent (65, 94),in resolution into the ,miallest components would be less amrolumns of charcoal with various solvents (66), in columns of t,iguous than a name indicative of color. From this point of alumina with various solvents (87, 69, 136), and in paper viit,h virw. rwgr:iphy niight substitute for chromatography. water, aqueous phrnol, or butanol as solvent ( 2 1 , 79). R u m OF MIGRATION 51aiiy aiialogous examples of separations of aniino acids performed with various combinatioiis of absorbents and solvents (hit. of the most useful constants in chromatographic iiiv(:htihave been reported in the current literature and summarized in gations is the K value, the rate of migration of a solute through A ttnirul Rcrieios qf Biochemistry. Even the solutes themselves the absorptive phase relative t o the flow of solvent. Ever since may be altercd as by ronversion into various derivatives rlnrl ( ;opprlsroc.tler's extensive invest,igat,ions of the capillary ascent c ~ ~ n i p l w(9, r s 21, .?2). of organic and inorganic solutrs iiit,o strips of filter paper ( 1 l C Y ) , this migration constant h i s p r o ~ e duwful for the comparison TERMIhOLO(;\ ant1 description of the :idsoi~b:il)ilit y of various solutes. Everitu:illy, the quantit,ative considerations of the mechanism of chroI he depentlence of most chromatographic scparat ions upon a niatogi,aphy led to the deduction that the R value was the r:ttio few distribution phenomena has led to widespread acceptance of unabsorbed solute in the interstitial spaces t o the total sol~ilc of such terms as chromatography by ion exchange, l ~ ypartition! in the adsorption zoiie (11!9, 119). and by adsorption (69, 11Y). Sewer terms are electrorhroIn practice, migration rates are determined by filtration of n niatographj-, indicating the use of electromot'ive potential in solution through the absorptive medium as indicated by Figures columns or in paper ( 6 9 ) )and salting-out chromatography (125), 1 and 2, or they are estimated from the movement of an adindicating the increased adsorbability of a proteinaceous solute sorption zone iri a column or in paper, as indicated by Figuro 3 . due to the addition of salt to the solution. Surface chromatogIn these three figures 12 = d l / d z , or R = (amount of solute in raphy, recently suggested as a name for separations of solutes solution in the adsorption Z{JIW) + (the amount of solute in in thin Inyrrs of absorbents (83), is likely Lo be confused with W E
? 7
44
solution plus the amount adsorbed). II-ith an elastic scale, R ran be read directly from the chromatograms (95). As R is determined for a given adsorbent and solvent, the principal variable in the measurements is the concentration of the solute. I n order to compare R with concentration, R values werr calculated from the sorption isotherms illustrated in Figure 4. In this calculation, the interstitial volume was assumed to I)e 0.1 ml. per gram of adsorbent. The results, summarized in Figure 5, show that R values depend upon the concentration of the solute and upon the shape of the sorption isotherm. With surface active adsorbents and with ion exchangers, the R value increases gradually with concentration of solute, but with partition absorbents the R value remains constant over a great range of concenlration as has so often been pointed out (119). Oning to the small rate of change of R with concentration, the R value, determined at a few concentrations of solute, may not be a ciitical measure of the kinds of forces that bind a solute to an :tbsorptive phase. Indeed, the R value must be regarded as the summation of all the absorptive or distribution phenomena that occur during the migration of the solute. At the trailing houndary of an adsorption zone, the variation of R with the decreasing Concentration of solute may be correlated with a rapid and trouhlrsome widening of the zones (59, 119).
3
I ml.
,
~
,
1
INTERSTITIAL
,
,
SPACE
1
PER
,
,
,
,
1
,
1
,
GRAM
-
PARTITION
4
,
lszLLJ ADSORPTION
.8
ION
5
1.0 .90
CONCENTRATION
Figure 6.
EXCHANGE
IS
IO
IN
MILLIMOLES
PER
20
MILLILITER
R Values Calculated from Isotherms of Figure 4
Assuming 1 ml. interstitial space per gram of ahsorhent
widely used as a sensitive descriptive and analytical test. 111 principle, these sequences represent. qualitative comparisons of the R values. In practice they are extremely useful. because they are usually altered less than R values by the presence of impurities. Owing to their extreme sensitivity and because they can be seen during the preparation of many substances by chromatographic methods, adsorption sequences are certain to find further application in many fields. hs the adsorptioii sequence may vary with the solvent and the adsorbent (107, 118), specification of the adsorption conditions is always essentiltl (121).
R
0.1 ml. I N T E R S T I T I A L
1.0:''
"
I
'
CONCENTRATION
Figure 5 .
SPACE
"
IN
'
PER
'
10
I
GRAM
"
MILLIMOLES
" '
PER
'
'
15 MILLILITER
'
3 J
20
R Values, R = dl/d2, Calculated from Isotherms of Figure 4
Assuming 0.1 ml. interstitial space per gram of absorbent
I T an absorptive phase is diluted with a nonabsorptive porous filtrr aid, the proportion of the solute retained in solution in thc interstices will be increased relative to the total amount of solute present in the adsorption zone. With the adsorbent diluted until t,he interstitial volume is 1 ml. per gram of active solid, the R values calculated from Figure 4 are increased as illustrated hy Figure 6. In comparison with Figure 5, these R values of the diluted surface-active and ion exchange absorbents iri,ise more rapidly with concrntration than the R values of thr undiluted absorbent. Moreover, the R value of the diluted pal.titiori absorbent is increased by a constant amount. From these examples, the variation of the R value with tht, cuiicentration of solute provides a measure of the sorption capacity of the stationary phase. This variation also provides clues to the principal kinds of attractive forces in the normobile phase, it illustrates a convenient method for relating adsorption isotherms to chromatographic separations, and it s h o w the importance of the porosity of the absorptive phasr in rhromntographic separations. ADSORPTION SEQUEYCE
Lung before the recent quantitative considerations of chromatographic methods (69),the sequence of substances adsorbed in columns proved useful for the description and identification of many labile compounds obtained from biological materials. Likewise, the mixed chromatogram, the chromatographic comparison of substances suspected of being identical, has heen
Adsorption sequences have been reported in several ways whirh reflect the analytical procedure but which are exceedingly confusing to the reader. \\'hen the flow of solvent, is doanwarti, as in columns (Figure 1 ) arid in some uses of paper, the soluttls are usually tabulated in the direction of decreasing adsorbability, that is, from the top to the bottom of the page (69, 119). But ~rheiithe flow of liquid is upward, as in vertical sheets or strips of paper (Figures 2 and 3 ) , the direction but not the order of the decreasing adsorbability is reversed. Tabulation of t'hr results in this upward direction is convenient for the analyst, but the order of the adsorbability may not be clear (48). In such cases, ambiguity can he avoided only by indication of thi, dirrction of increasing or drweasing adsorbahility. L i . \ i r r i w o y s O Y CHROMATOGRAPHIC SEPARATIO\S
.-llthough much has been written about the great sensitivity aiid wide applicability of chromatographic methods, little ronsider,ation has been given to possible limitations of the technique. h s information has accumulated, however, mixtures of certain kinds of substances have proved difficultly separablr or inseparable in adsorption columns;. An examination of thtl properties of these substances in relation to the mechanism of chroniatographic analysis points to possitile improvements of thc separations. In all chromatographic separations, there are two requisites: a dvnamic partition or equilihrium of the solutes between the t w o phases, and a small difference among the relative amounts of each solute in t h r b t,\vo phases. The dynamic equilibrium is easily achieved for nearly all kinds of solutes, but the difference iri the partition coefficients of certain solutes may be too small for effective chromatographic separations. 1Iost information about the srparabi1it.j- of various kinds of organic (62, 69, 118, 1 2 8 ) and inorganic (59, 66, 87, 110, 111) dubstances has been summarized in qualitative fashion in Tables I1 and 111. From thew tables. chromatographic separability is oi)viously relatrd I O ciiflerences among the polar or reactive groups of the solute molecules. These reactive groups are usually the most absorbed parts of the molecules; consequently, thr separability of pimilar molvculrs is most effective when the immiscible phases attract those parts of the molrculrs in n-hirh the: greatest structural differences reside (118).
V O L U M E 2 2 , N O . 1, J A N U A R Y 1 9 5 0 Table 11. Chromatographic Separability of Organic Compounds in Relation to Variations in Structure Structural Variation lfolecular size Structural isomers Chains us. rings Branched chains Position of double bonds Spatial isomers Cis-trans a t double bond Cis-trans a t ring Optical isomers S u m b e r of double bonds Conjugation of double bonds .Lumber of nonpolar substituents Number of polar aubstituents Polarity of the substituents
Separability Fair t o poor Fair t o poor Fair t o poor Fair t o poor Fair t o poor Good t o poor Poor Good Good t o fair Fair t o poor Good Good
Table 111. Chromatographic Separability of Inorganic Elements in Relation to Variations in Properties Kinds of Variations Ionic charge Different elements One element (oxidation-redurtion i t a t r s ) Ionir radii Different elements Similar elements (rare earths) Tonic mass Different elements One element (i5otopes)
Separability Good Good Good t o fair Fair Good Poor
\\'hew niolwules or ions differ but slightly in the parts or groups that are weakly attracted by solvent or absorbent, mixtures of the solute molecules are difficultly resolvahl(~1)y means of chromatographic methods. This is true of largc, organic molecules Lvhich differ with rcspect to the number or the position of small nonpolar groups. It is characteristic of thc optical antipodes in which identical groups are exposed t o thc absorptive phases. I t is typical of the isotopes wherein the difference in size, mass, and charge of the atomic nucleus is small relative to the solvatioii of the ion and relative to the high polarity and binding force of the urishared electron. \\.hiit cwnditions must be found in order to facilitate the srparat,ioii ( i f optic81 isomers and of isot'opes by chromatographic techiiiclue? Obviously a solvent and a n absoi,bent that will c3xhil)it preferential affinities for the components of the mixturc are rctcluired. In the case of optical isomers, man?- selective systems occur in nature as indicated by the specificity of enzj-mes and t)!. the selectivity of the metabolic systems of living organisms. Siniilarlj-, organic reactions and the exchange systems encouritcmd in the phot,osynthetic apparatus of living cells appear to utilize one isotope of carbon in preference to another (18, I n ) . l l a n y plant and animal cells accumulate certain inorganic ions and exclude others of t h e same periodic group against tremendous concentration gradients. Perhaps if more were kiiown about 6he mechanism of these selective processes, some of t'he partition mechanisms might be adapted to use in columns.. or analogous partition mechanisms might be devised. CHRO%I4TOGR4PHY BY 4I)SORPTIOU (
hi oniatographic analysis based upon the use of surface-active
adsorbents is the most selective procedure for resolution of a great varietj of organic compounds. It has been applied to the detertion of the smallest structural differences among organic molecules such as those involving molecular size, molecular or structural isomers, number of double bonds, conjugation of double bonds, pobition of double bonds, and number and position of various polar and nonpolar substituents (47, 62, 69, 98, 142). This adaptation of the chromatographic technique continues to yield significant results when utilized for the examination of a variety of natural products, especially those t h a t are soluble in organic solvents. Selected recent examples are the investigations of the chloroplast carotenoids (33, 62, 63, 122) and related compound? (88. 98, 142). of vitamin A contained in lipidcs ( 2 3 ) and
45 prepared synthetically (114, 135), of retinene and similar conipounds of the visual-purple complex (IS,%?),and of various unsaturated fatty acids (75, 136). The method has been utilized x i t h vitamin Bi:b ( 9 7 ) , the abalone-shell pigment (26), a pterinlike pigment from the tubercle bacillus ( S I ) , chlorophyll from blackstrap molasses ( 9 ) , and various common indicators (60). It has been employed with a paralytic poison that accumulates in shellfish (115), with a polymeric-homologous series of sugar acetates (Sd), with various amino acids ( I @ ) , and with variow inorganic ions (110, 111). The detection of small molecular differences by the use of surface-active adsorbents usually depends upon the specificity of the active surfaces. T h u s far, this specificity has been discovered largely hy chance. S o w t,Iiat the significance of the specificity is recognized, a systematic investigation of thir property may yield results of great practical value. T h c suggestion by Pauling, that the presence of a solutc during the preparation of a n adsorptive agent may influence the adsorptivc propeitieq, points to one course of investigation. The rclationship betn-een the dimensions of a solute molecule and its abilitb to fit within the interplanar spacings of the adsorbent ( 7 8 ) points to another profitable course of research. In spitc of many investigations of adsorption phenomena, a thorough analysis of the nature of the att,ractive forces b c t w r n adsorbents and various parts of organic molecules (69, 120) and bet\vecn adsorbents and inorganic ions (110, 111) remains a challenging problem (110, 111, 129). CHRO3IATOGRAPHY BY PARTITION
The resolution of mixtures h y partition betn-ecn water bound in a stationary phase and an immiscil)le solvent as chloroform, toluene, or butanol cont,inues to find wide application (69). The water may be fixed to a hydrophilic absorlwit, as a n aqueous gel, diatomaceous filter aid, starch (117), or iiltrr paper (scc section on paper chromatography). Ihaniplcs of separations achieved hy this method are the resolution of mixtures of rihonuclcosides (104), of amino acids in a column of moist starch ( I I ? ) , of sugai'a and methylated sugars in a column of cellulose (58), of tropic , of nnthocyaniand atropic acids in a column of silica gel ( 5 @ )and dines in a column of silica gel containing phosphoric acid (116). llixtures of various lower fatty acids have been resolvccl in columns of siliceous earth impregnated with methanol (1033, with st,rong sulfuric acid ( 9 4 ) , and with strong sulfuric acid plus phosphoric acid (55). Silica columns have also bern eniploycd for isolation of radioactive citric acid (101 ). CIII~O.MM-ITOGR~PHY BY ION EXCHANGE
JVith few exceptions, ion exchange chroniatography is most useful for the resolut,ion of mixtures of ionizable or dissociable solutes. Through preparation of strongly acidic and strongly basic organic resins, the usefulness of ion exchangers has becii extended t o very weakly basic and acidic solutes. For the most part, the preparation of these organic ion exchangers has been in the hands of industrial organizations that have been interested in products of great stability, of high exchange rate, arid of grcat exchange capacity. The exchangers t h a t have been placed on the market represent an extremely small fraction of the grcat number that might be synthesized. With the organic, resinous ion exchangers, the stability, permeability, exchange rate, and exchange capacity are related to two principal structural features, the degree of polymerization (usually called the degree of cross linkage) and the nature of t h e reactive groups. The degree of polymerization determines the permeability of the resin to solutes and to solvent so that i t affeet,s the absorption capacity and rate of exchange. This, in turn, affects the rate of migration of the solutes in columns as well as the distribution of the solutes in the absorption zones, especially in the diffuse trailing boundaries of the zones (59, 87, 119).
ANALYTICAL CHEMISTRY
46 Thus far, only a few kinds of reactive groups have been incorporated in the organic resins. hfost of these are sulfonic acids, carboxylic acids, and tertiary or quaternary amines (87). Although many investigations indicate t h a t these groups react by ion exchange, recent studies of a basic resin (Duolite A-2) manufactured by the Chemical Process Company indicate that addition reactions with fatty acids as well as ion exchange may be involved with a single kind of reactive group (107). Sources of commercial resins and many of the chromatographic applications in organic and inorganic fields have been reviewed by Nachod and his collaborators (87) and will not be repeated here. It is significant, however, that ion exchangers are finding extensive use in the concentration and purification of particular substances from various natural products as, for example, traces of radioactive elements in urine (112), a paralytic basic poison from shellfish that feed on the marine dinoflagellate Gonyaulax (115), the separation of various mononucleotides (S4, 26), the estimation of riboflavin (&), and the separation of various acidic and basic amino acids (87). After the extensive wartime separations of rare earths and fission products with ion exchangers (59), additional attention has been directed to the separation of zirconium and hafnium with acidic and with basic resins (6.5, 123). Radioactive isotopes of cerium and yttrium have also been utilized as an aid in the investigation of column variables such as rate of flo~v,exchange rate, and pH of the solution (128). On a small scale, resins may serve for the preparation of potable water from sea mater (87). For commeicial installationr, however, distillation with efficient heat exchangers seems to bc more efficient (46). Studies of the physical and chemical properties of the synthetic exchange resins and of the natural exchangers, such as alginic acid ( S ) , are continuing to yield results of value in the field of chromatography (61, 54, 66,81). In place of the acidir and basic groups, various oxidation and reduction groups may be combined in the resinous structure (60, 130). These modified resins then become specific reagents, analogoub to the specific t eagents of Feigl (S9), for chromatographic investigation of a variety of solutes. I n this way, the use of resins in chromatogI aphy may be made as widrlr applicable as the use of rhrmical rwigents. (:IIERIICI1RORlATOGRAPHY
Many combinations of chemical reagents and solveiits have been utilized for the separation of various solutes by chromatographic methods as pointed out by hIeinhard (86). This use of reagents promises much wider application in the future because specific reactants may be utilized individually in columns, or in successive layers in columns, or combined with other absorptive or reactive solids ( 3 2 , 5 7 ) . From the author’s own experience, an illustrative example is the separation of ferrous and ferric ions in a column of Celite moistened with a solution of urea plus ammonium acid phosphate. When nashed with a solution of these reagents, the ferrous ions migrate rapidly a m y from the ferric phosphate which precipitates in the Celite. The ferrous ions are readily detected 15 ith o-phenanthroline, the ferric ions with o-phenanthroline plus hydroxylamine or with sulfide. I n many respects, these resultb may be obtained more quickly and moie conveniently in strips of filter paper impregnated with urea plus ammonium phosphate
simultaneously 111 a single mixture, from the minute quantities of solutes that may be separated, from the ease with which R values are determined, and from the convenience with which colorless solutes may be located in the paper after formation of the chromatogram. For chromatographic separations in paper, only about 0.01 ml. of solution containing from a few micrograms to a milligram or t 4 o of solute may be absorbed. If a larger volume of dilute solution is necessary to provide sufficient solute, the solution should be concentrated before addition t o the paper or as it is added to the papel ( 1 3 1 ) . I n paper chromatography, as in columnar chruniatography, the initial w idth of the absorption zone should be small ielative t o the distance moved hy the band (Figure 3) (41, 119). Chromatograms may he formed in paper with water or with various organic liquids as solvent. Many of the organic liquids, such as butanol, furfuryl alcohol, collidine, lutidine, piperidine, and phenol are treated with witer so that partition effects (Figure 4 ) predominate in the resolution of the solute mixtures, Evaporation of solvent fiom solutions in paper exposed to air results in an increase of the R value; hence for quantitative results, the paper must be supported in a closed space saturated mith tlie vapor of the solvent. Sarron paper strips may be placed in a stoppered tube ( l o g ) , but large filtcr sheets, such as those required for tn o-n ity chromatograms, require a large jar, or a glass aquaiium tank (Figure 3). cabinet, a large batt When the solvent is peimitted to flow upil-ard in the paper by capillarity, the paper sheets may be bent into a cylinder (140) or spiral ( 7 7 ) that will fit into a covered glass cylinder (134). When sti ongly absorbed solutes require extensive n ashing with fieRh qolvent, a pad of absorptive paper may be clamped to the end of a short sheet of paper (84)or a large portion of the paper sheet itself may be rolled up like a windon curtain. For downxvard flow of solvent in paper, the liquid must be placed in an elevated trough ( 4 ) from n hich the papei is allox-ed to hang in a closed vessel. Formation of a chromatogram in paper may require a few minutes when the solvent is permitted to penetrate only about 5 cm., but with a penetration of some 15 to 30 cm., about 10 to 24 hours may be necessai y. Paper chromatography has been modified for the separation of acidic, basic, and neutral amino acids by passage of an electiical current along a moist paper strip (137), and across a sheet of filter paper while solution was being drawn upward by capillarity (53). I t has also been utilized for separation of inorganic ions by passage of current through a stack of moist filter paper disks, one of n hich contained the mixture to be resolved (4.5). Flon- of solvent through the paper has been observed to yield small potentials that may affect the separation of adsorbed solutes (109).
Filter paper disks in the form of a column have also been utilized to increase the amount of solutes separable in paper (85). A thick pad of paper may likeaise be suhftituted for a thin sheet of paper. In paper chromatography, salts often separate into ions ivhich mav interfere with the isolation and identification of other hubstances (29, 127). From the reviewer’s experience, ferric chloride yields hydrochloric acid which migrates ax a y from the ten ic ion, leaving ferric hydroxide precipitated in the paper. T,ikemise, sodium sulfide yields hydrogen sulfide which volatilizes, leaving sodium hydrovide in the paper. Impurities in the solvent ( 3 6 ) or in the paper, as copper salts (90) and hydrolyzable PAPER CHROMATOGRAPHY peptides (141 ), sometimes yield spurious results. In the past w a r or two there hai: been a trcniendou9 i ~ i ( w i t ~ t ~ I n view of the small amount of solutes separated in the paper chromatograms, extremely sensitive methods are required for the in tlie number of reports concerning paper chromatography, the detection of the iesolved compounds. Illustrative examples of resolution of mixtures by differential absorption in strips (Figure the kinds of materials investigated by paper chromatography 2) or in sheets of paper (Figure 3 ) (28, 30, 69, 1 1 8 ) This new and the methods utilized for their detection are summarized interest stems from the ease with which many separations may herewith. be made, from the large number of solutes that m a r be detected
V O L U M E 2 2 , N O . 1, J A N U A R Y 1 9 5 0 Radioactive products of photosynthesis were located by photography (6,18,19); nucleic acids were located by absorption of ultraviolet light determined photographically (80); penicillins (61) 64, 7 4 ) and streptomycins (258)were detcrrnined by bact’eric-idaltests; ascorbic acid and related compounds were deterniinctl u.ith 2,6-di1:hlorophenolindophenol (70); r d u c i n g and nonnrducirig sugars wer,; determined with weakly alkaline permangitnate ( 9 1 ) and with aniline phthalate (92); and amino acids were located with ninhydrin (1, 7 , 15, 68,75, 89, 99, 102, 106, 1%) or with copper salts plus ninhydrin (32). Lysine has been detected wit,h ferrocyanide ( 2 ) and glycine with o-pht,halaldehyde (93). Other materials studied by paper chromatography were proteins and enzymes, located with benzidine, hemin, and peroxide (45); nucleotides of ribonucleic acid ( 2 2 ) ; iodoamino acids of the thyroid located by radioactivity (127); amino acids of frog larvae (75), of royal jelly (low),and of diphtheria toxin and toxoid (1%); and a toxic substance formed by treatment of proteins with nitrogen trichloride (6). Still other substances investigated by adsorption in paper were 2,4dinitrophenylhydrazones (21), dinitrophenyl derivatives of amino acids (-96), decomposition products of riboflavin (62), pterins (49), epinephrine aiid norepinephrine (48),and various nucleic acids (80). Various amino compounds have been located with iotiiiie ( 1 2 ) . number of the chemical elements have been separated in paper where they are readily identified by the standard analytical reagents. Examples are the chlorine group of anions (72), heavy met,al ions ( l ? ) ,aluminum and beryllium (go), various cations wit,h R values ( l o o ) , and numerous cations arid complexes (67, 70-72, 76, 110, I l l ) . OpJical scanning niet,hods have been devised for the detection and for the quant,itative estimation of the solutes separated in the paper (15, 40-42, 86). The shape and the area of the absorption zone also provide clues to the nature of t$r ahsorptive forces and t,o the aniount of solute present (IS’, 40). In spite of ita limitations as a preparative method, paper chromatography shons promise of much wider applicatioii in the future. It is especially useful for investigation of substances that) are readily soluble in water and alcohols but that are insoluble in the hydrocarbon solvents. In this respect, paper chromatography c.strnds t h e uwfulnes- of rbhromatogrnphic techniques. LITERATURE CITED
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ANALYTICAL CHEMISTRY
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_--
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PAPER CHROMATOGRAPHY DORIS L. CLEGG Washington Square College, New York University, ,Vew York,N . Y. 66 A good technique sometimes renders more service to science than the elaboration of highly theoretical speculation.”-Claude Bernard
T
HI- stored in a small space, ready for instant use; can be mounted as permanrnt records ( a spray of artist’s “fixative” preserves colors against fading too rapidly); photographed wet, if drying causes change or loss in colors of unknowns (148) or interferrnce due to incarease in hackground intensity (77, 148); or photographed dry (143, 14.7) for preparation of slides or simply to store for future referwce. Purity of substance or the presence of unsuspected const,ituents may he easily determined (104). It is also inexpensive; one 9-em. circle n-ill produce one complete separation using the Rutter technique ( 1 7 3 ) or six if the ascending or descending paper strip is employed; or 450 individual chromatograms per day can be accompliPhed using a series of concentric cylinders as described by Williams and Kirby (207). Paper chromatography is useful as a control of synthesis in industrial processes, because rapid q d i t a t i v e analyses frequently can be macle employing this simple but extremely effective method. T h e extensive literature devoted to paper chromatography ( ~ V P I Ito
