ANALYTICAL CHEMISTRY Roe, H. R., and Mitchell, J., Jr., QNAL. CHEX., 23, 1758 (1851). Sack, W., 2. anal. Chem., 131, 191 (1950). Saeki, T., J . Pharm. SOC.Japan, 70, 644, 853, 680 (1950). Sanderson, P. H., Biochem. J . , 52,502 (1952). Scholten, H. G., and Stone, K. G., ANAL. CHEM.. 24, 749 (1952). Schulek, E., and Punger, E., Anal. Chim. Acta, 5, 422 (1951). Schulek, E., and Punger. E., Magyar Kem. Folyoirat, 57, 11 (1951). van Schuylenbergh, J . , and S‘ervelde, G. J., Rec. hav. rhim.. 71, 609 (1952). Schwarsenbach, G., Anal. Chim. Acta, 7, 141 (1952). Shkodin, -4. M., Iemailov, N. A., and Deyuba, N. P., Zhur. anal. Khim., 6, 273 (1951). Sierra, F., and Carpena, O., Anales real SOC. espafi. fis. y quim., 46B, 547, 627 (1950); 47B, 215 (1951); Pubs. i n s t . quim. “Alonso Barba” (Mad&$, 4, 291 (1950). Sierra, F., and Carpena, 0.. Anales real soc. espafi. f i s . y quim., 47B, 345 (1951). Ibid., p. 527. Simon, V., Sekerka, I., and Dolezal, J., Chem. Listy, 46, 613 (1952). Singh, B., Singh, A., and Singh. R., J . I n d i a n Chem. SOC, 30, 147 (1953) ; (nonpotentiometric studies) 30, 143 (1953). Spicer, G. S., and Stricklmd, J. D. H., Anal. Chim. Acta, 6, 493 (1952). Steele, M. C., and Hall, F. A l . , Ibid., 9,384 (1953). Stock, 5. T., Metallzrrgia, 46, 209 (1952). Stone, K. G., and Scholten, H. G., ANAL. CHEM.,24, 671 (1952). Swinehart, D. F., Ibid., 23, 380 (1951). Takagi, K., and Shimizu, Y., J . Electrochem. SOC.J a p a n , 18, 150 (1950). Takahashi, T.,Kinioto, K., and Minami, S., J . Chem. SOC. Japan,I n d . Chem. Secf., 55, 115, 188 (1952).
Takaki, S., and Maekawa, Y.,Japan. Analyst, 1, 10 (1952). Tanabe, H., and Hasegawa, F., Ann. Repts. Takeda Research Lab., 9,63 (1950). Tanabe, H., and Hidaka, K., Ibid., 9, 682 (1950). Tanner, H., and Rentschler. H., Mitt. Lebensm. Hug., 42,514 (1951). Taryan, V. M., Izvest. Akad. Nauk Armyan S.S.R. Fiz-Mat. Estestven. Tekh. N a u k i , 3, 509 (1950). Taryan, V. M., and Dusepyan, E. N., Ibid., 3, 499 (1950). Tendeloo, H. J. C., Rec. trav. chim., 70, 191 (1951). Teodorovich, I. L., Zhur. Anal. Khim., 7, 175 (1952). Tomicek, O., Blaeek A , and Roubal, Z., Chem. Zvesti, 4, 479 (1950). Tomicek, O., Doleeal. J. and Simon, V., Chem. Listy, 44, 198 (1950). Tomicek, O., and Heyrovskg L , Ibid., 44, 169 (1950). Ibid.. p. 245. Tomicek, O., and Heyrovskg, J., Collection Czech. Chem. Commum., 15, 984 (1951). Tomicek, O., and Valcha, J., Chem. Listy, 44, 283 (1950). Tomicek, O., and Zukriegelova, M., Ibid., 46, 283 (1952). Tsukamoto, T., Kamhara, T., and Tarhi. I., J . Electrochem. SOC.Japan, 19,311 (1951). Wade, P., Analyst, 76, 808 (1951). Wakkad, S. E. S. el, and Riee, H. A. M., Ibid.. 77, 161 (1952). Watt, G. W., Hall, J. L., and Choppin, G. R., J . Phys. Chem’., 57, 567 (1953). Watt, G. W., and Otto, J. B., Jr.. J . Electrochem. Soc.. 98. 1 (1951). Wells, I. C., ANAL.CHEM.,23, 511 (1951). Wenger, P., Monnier, D., and Epars, L., Helv. Chim. Acta, 35, 396 (1952). Wise, E. N., ANAL.CHEM.,23, 1479 (1951). Yundt, A. P., T a p p i , 34, 85 (1951).
chromatsgraphy And Analogous Differential M i g r a t i o n M e t h o d s HAROLD H. STRAIN, T. R. SATO, and JOHN ENGELKE Argonne National Laboratory, Lemont, 111.
T””
A early reviews in this series were devoted solely to the subject of chromatography. Subsequently chromatographic techniques were recognized as prototypes of numerous analytical methods based upon the phenomenon of differential migration (165, 171 ). Consequently, the scope of this revlew has been extended to include a number of these related, widely applicahle, differential migration technique.. I n the 2 years following the last review in this series (166), there has been an enormous acceleration in the development, the improvement, and the application of differential migration methods of analysis. In many productive research laboratories. several of these methods now serve as adaptable tools for exploration, for control, and for corroboration (34, 66, 73, 120, 128, 170). This increase in the number and applications of these basic analytical tools and a concomitant multiplication of the workers versed in their use have stimulated progress in all aspects of science concerned with chemical substances and their reactions. The resultant expansion of knowledge has been so rapid, so great, and so diverse t h a t i t cannot be cited here Even the specialized literature pertaining to the development of the tools themselves ran scarcely be summarized in the space allotted t o this revien,. The examination and correlation of current reports have been complicated tremendously by the description of many minor modifications of earlier methods. Moreover, many of these reports do not include references to the earlier investigations. A note on the separation of chloroplast pigments by paper chromatography (11), for example, gives no reference t o the early reports on the formation of chromatogram with these pigments,
observations which formed the foundation of columnar and paper chromatography (165, 164). For economy, the citations in this review have been restricted to books ( 2 , 6, 19, 26, 42, 4 9 . Y5, 88, 130, 141, 156), surveys (10,16, 27, 51, 32, 35, 41, 64, 74,82, ‘10, 91, 102, 105-107, 109, 118, 127, 129, 131, 158, 160, 164, 165, 171, 174, 182, 184), bibliographies (69, 104, 125), and current reports on basic procedures. This selected material should provide a key to the rapidly expanding literature. Many of the modifications and applications of the techniques can be found only by perusal of the literature of specialized fields. DIFFERENTIAL MIGRATION ANALYSIS
Basic Conditions. For the examination and correlation of various separatory techniques, the concepts of differential migration analysis have now been restudied and extended. I n all these methods of analysis, the migration itself is produced by the application of one or more driving forces, and it is usually opposed by resistive forces. For effective separations, either or both the driving force and the resistive forces must act selectively upon the migrating substances. T h e effects of a nonselective driving force (gravity) acting upon two different kinds of particles with and without a selective resistive force (viscosity) are illustrated by Figure 1. I n differential migration analysis, the migration conditions must be selected so t h a t the components of each mixture migrate a t different rates. The migration rates are a property of the migration system. They depend upon the properties of the
V O L U M E 2 6 , NO. 1, J A N U A R Y 1 9 5 4 migrating substances, upon the properties of the migration medium, and upon the nature and intensity of the driving force. The degree of resolution depends upon the dimensions of the initial zone of the mixture, upon the arrangement of this zone in the medium, and often upon the concentration or the amount of the mixture. It also depends upon the differences among the migration rates of the constituents and upon the distance of the migration. Substances separated by differential migration are located, caompared, identified, and estimated by their physical, nuclear, chemical, or biological properties. Dependent upon the circumhtances, the components of mixtures may be located in the medium either during the migration or after the migration. They may also be examined after removal from the respective parts of the medium.
DF
+
A
DF
c
4
RF
Feather
pynthetic substances of organic or inorganic nat,ure, and they may even be living organisms. Thc components of the mixture should not be bound irrevcrsibly with one another. Ionized or dissociable substances :ire usually separated into the several ionic or dissociated species. Migration Media. M i g r a t i p media may be vacua, gaws, liquids, or solids. With the same driving force, substances migrate fastest in vacua, slower in gases, and much slower in liquids, in gels, and in hydrated resins (92, 100, 159, 172). The migration rate in most solids is so slow that these materials are not widely employed as migration media (8, 9 ) . Zones of gases and liquids have been stabilized in porous media (19, 21, 26, 951, including permeable membranes (46). Liquids have also been stabilized in tubes, capillaries (4,12, 167), annular spaces (79),gels (88,89,125), porous rods (18),and fibrous mcdia such as paper, chamois, and felt (1.9, 26, 95, 100). They h i v e likewise been stabilized in tubes containing many close b:iffleu ( 8 6 ) arid in packed centrifuge beds ( 1 1 6 ) . Driving Forces. Driving forces are any combination of corlditions that cause particles tlo migrate. These forces may be classified from many points of view. With respect t,o their effcut upon the distribution of t,he particles in the migration medium, driving forces fall into two principal groups-those that cause all the particles of each species to migrate, and those that cause the particles of each species to redistribute themselves in the migration system. Forces that can cause all the particles of each species to migrate (usually unidirectionally) and the kinds of particles with which they are effective are: llechanical forces Gravity Centrifugal forces IJltracentrifugal forces Magnetic forces
f.
Leaf
VACUUM
91
CARBON DIOXIDE
Figure 1.
Uniform Migration (left) and Differential Migration (right) Migration with a nonselective driving force (DF,
grayity),unopposed b y a resistive force {left), a n d differential migration with a nonselective driving force opposed by a selective resistive force ( R F ,viscosity of gas) ( r i g h t )
Because separations are freyuent1:- most effective when the concentration or amount of the mixture is small, and because minor constituents must be located, many novel and sensitive detection and assay methods have been devised. These detection methods are without influence upon the differential migr& tions. They depend upon various properties of the separated substances, as, for example, refractive index, optical interference, fluorescence, spectral emission. spectral absorption ( 2 8 ) , dielectric properties with high frequency current (67, 117). radioactivity (10, 21, 144, 175), nuclear activation (94,112, 175), formation of colored products by staining procedures (88, 89, 183) and by chemical reactions (15, 52, 73, 100, 101), bleaching of colored reagents (loo), reactions with hormones or enzymes (1701, the inhibition and the stimulation of the growth of living organisms (the so-called bioautographic methods) (89, 158), and many others (19, 95, 130). Materials to Be Separated. With respect to their physical condition, the materials to be examined by differential migration may be gases, liquids (including solutions), solids, and various combinations of these. With respect to size, these materials may be atomic particles, ions, isotopes, molecules, colloids, and microscopic and macroscopic bodies. They may be natural or
Electrical potential Oscillating electromagnetic fields Flow of liquids Flow of gas
A h c i oscopic to macroscopic bodies Microscopic to macroscopic bodies Colloids t o macroscopic bodies Molecules to microscopic bodies Magnetic particles, moving ions, particles with a dielectric different from that of the surrounding medium and exposed to an electrical field (83) Ions, colloids
Ions
Ions, molecules, colloids, microscopic and macroscopic bodies Ions, molecules, colloids, and microscopic bodies
Forces that produce redistribution of particles in the migration system, and the kinds of particles with which they are effective are: Translational energy Chemical potential gradients Thermal gradients
Ions, molecules, colloids (self-diffusion) Ions, molecules, colloids (diffusion) ( 3 4 , 80, 160,161)
Ions, molecules (thermal diffusion) (12, 87, 79,81,'86, 147,148,167),(thermal osmosis) (46),(distillation) (4, 87,137)
Driving forces differ with respect to their selective action upon the migrating particles. Some are nonselective; others are highly selective. The selectivity, however, depends upon the properties of the particles themselves and upon the properties of the migration system. When two or more driving forces :we utilized, the combined effect is the vector resultant force. Resistive Forces. Resistive forces are the conditions in the migration medium that resist the migration of the particles. These conditions include viscosity, density, density gradients, hydrostatic gradients, sorption, and permeability of the medium, including the permeability of molecular and mechanical barriers. The sorption of solutes or gases by the migration medium is one of the most selective and adaptable resistive forces. This sorption, which is the selective, resistive force in chromatography, depends upon a variety of distribution phenomena in which the sorbed substances are distributed dynamically between a fixed nonmobile phase and a mobile gaseous or liquid phase.
92
ANALYTICAL CHEMISTRY
It serve8 for the separation of all kinds of chemical substances, but i t is not very effective Kith isotopes or with optically active, enantiomorphic substances ( 1 4 , 22, 99, 140). B:rrriers also provide a selective and adaptable resistive force. Barrier membranes permit the selective filtration of polysacvharides (120). They facilitatq the separation of solutes from colloids bv dialysis and by c,lectrodialysis (66, 134, 168, 1 8 2 ) . Screens and the like provide the selective barriers for many me(-hanical separations. Porous barriers provide the stabilizing niedium for the fractionation of isotopes by gaseous diffusion.
A
ir
Figure 2. Differential Migration from a Reservoir Start (left) and partial separation of leading none 0.1 g h t )
hlariy i.c.sistive forces a r t uniformly, so t h a t each kind of particle migratrs through the medium :It a constant rate. -1few resist’ive forces, particularly clcnsity and hydrostatic gradients, provide increasing reaistanre to migration, so that the 1nigrat)ing particles come to :t standstill v h e n t h r resktive force equ:ils the driving force. .in example is the sedimentation of virus 1):irticles in a medium with :I suitable den.sity gradient (53, 261). An andogous efect is the behavior of thc Cartcsim diver in n medium with a density gradient (66 I. Arrangement of Zone of Mixture. The arrangement of the init’ial zon(2 of a mixture determinrs, in large measure, the aepnratioris t h a t may be c+fectetl by particular coml)inations of driving and resistive forces. With a single driving forve, there a r e four principal arrangements for the migration: uniform distribution of the p:irticles in the niigrat,ion syPtem followed by differential rediutribution, differential migration from a reservoir of the mixture. migration from :I zone P O wide that the mixture is b u t partially resolved in the mt.dium. and migration from a zone so narrow that all or most of the compnnmt.: of the niisture are separated in the migration medium. With two driving forces acting t,ransversely to each other. the ixirticles nxiy he distributed throughout the medium, or they may f o r m a witit. initial zone. a narrow spot, a wide stream, or :I.n:trrow Rtrcwn. Differential Redistribution in the Migration System. In a closed or limited migration tem, and with a driving force t h a t produces redistribution of the particles. the migration reaches a steady state in which overlapping zones or concentrat,ion gradients are established for each kind of particle. The degree of separation depends upon the diinensions of the system, the locution in the system, the properties of the components, and the nature of the driving force.
This differential tlistribut,iori in a closed system is the basis for the thermal difusion of gases, and for the thermal osmosis of gases through a membrane ( 4 5 ) . I n closed systems, differential distribution in one direction is frequently combined with migration in a transverse direction. Thermal diffusion transverse to gravity is especially effective for the fractionation of isotopic gases (12, 36, 37. 8 1 , 86, 87! 147, 148), and it has be431 utilized for the fractionation of liquids such as tall oil (7,Y‘j. This combination of forces resembles that in the distillation column (ET)>also adapted to the separation of isotopes ( 4 ) , but here the separation of the vapors of the components is enhanced by the intervening zones of the condensed refluxing phase ( 4 , 12) 87, 137). .in analogous effect is obtained in the thermal diffusion column by the addition of “Hilfsgase,” gases that form zones between those to be separated ( 3 7 , 147, 148). In solutions, e1ectric:d migration h n s v e r s e to gravity provides electrical migration-roni.ection methods (ionophorcsis-convec-convection) that are widely :uhj)t+ble t o the separation of various ions and proteins (15’4, 168, 170). These separations may he made more vffective with suit:i,l)lemc*nihranes as barriers (168). Migration From a Reservoir. Analysis by tlifierential iiiigration from a reservoir is illustrated scheinatically This arrmgement is utilized with both kinds of d t,hose that produce migration of all the particles ai prodwe redistribution of the particles. It provides but it psrtial resolution of the mixture. Only a portion of the fastwt migrating constituent is separated from the others. The proportions of the constit’uents in the overlapping, migrating zones are altered, however, so that repeated recovery and remigration produce a gradual fractionation of the mixture. With this migration from a reservoir, the number of the advancing boundaries provides :in indication of the number of the components in the niisturc.. I-nder st;iiidardized conditions, a bask for idmtificnt,ion and the rate of t,he migration sen e*timation of the migrating s u b h n c e s . The migration nxty bc linear. a~ illustrated in Figure 2. It inny also be radial (126, 1 7 4 ) as from i t narrow source into :I sheet or flat in:iss of the medium; or it may be segnient:tl as from a point source into B three-dimensional mass of the medium in the form of :i sphere, a solid angle, or a cone (see dixussion on paper chromat ography). This migration from a reservoir is employed for the differential diffusion of gases and of solut,es (80, 96, I.%), for the electrical
A+B DF .)
B A+B A
Figure 3. Differential Migration from a Wide Zone Start ( k f ~ and ) partial separation of leading and trailing zone ( r i g h t )
93
V O L U M E 26, N O . 1, J A N U A R Y 1 9 5 4 moving boundary method, for decolorization of Golution with sorptioncolumns, for fractional extraction, and for some spot trsts in paper. It is t h r h i + airangement for hrrakf through analysis ( 6 4 , 80. 161 )) for frontal imilvsis (19, 26, 0.7).and for the capillary adsorption analysis in paper strips so widelyapplied by Gopprlsioeder (164). It is analogous t o the coninion a r r a n g e m e n t f o r aedimentation and centrifugation procedures in d i c h the trailing boundaries of the zones reveal the comF i g u r e 4. Differential 3Iigraposition of the mixture tion f r o m a Narrow Z o n e (23, 34, 63). Start (left) and resolution of t h e mixT h e absorption and the ture ( r i g h t ) e x c r e t i o n of c h e m i c a l substances bv living organisnts are analogous to the differential migration of solutes from a reservoir, Indeed, the selective or differential permeability of the kidney is duplicat’ed by the membranes of the “:wtificial kidney” (182). These selective processes of living org:inisma often yield a partial separation of isotopes (3,IO, 4 7 ) . Differential migration from a reservoir coupled with :t tr:insv c ~ s rniigration permits the fraction:ition of large quantities of mixi urw. .4s a rule, thr srpar:itiona arc iiic*omplete nnd nrc siniihr t o those illustratetl by Figure 2. .In esaniplt. is lhr rption of solutes :It :I gas-liquid interface followrd hy their removal asfoam(f7, 128i. other example is the diffusion of rolutcs through a mt~m1)r:inr tem with transverscl flow of solvcnt, I n practice, this ar1’:ingrment for separation? is usually more efieetive when oper:ttetl. as a countercurrent process a n d esaniplce :ire reported in the srrtion on migration from 3 \ride stream. Migration from a Wide Zone. Differential migration from a \rid(>initial zone, as illustrated in Figure 3, is effective with those driving forces that cnuse all the particles of each species to migrate. With driving forccs that produce redistribution of the particles. the migration from a wide zone yields wparations i,eaenihling those of Figure 2. 3Iigrntion from a wide zone provides but partial rrsolution of the mixture, only a portion of the fastest and slowest migrating substances being separated. This migration arrangement niay be utilized in much the same way as migration from a reservoir. 1301 h the trailing boundaries and the leading boundaries provide infornintion about thc number, the nature. and the proportions of the components. Differential niigration from i i wide zone is utilized in sedimptitntion analysis (34, 65, 162), with sorption columns ( 9 , 181), and with strips or sheets of sorptive paprr. It ia the basic nrrangrmerit for certain niodificat ions of the electrical moving hountlar>method (38) and for the relatrd diffrrential electrophoresis of proteins (2, 12, 88. 8,9. 106, 107). Migration from a Narrow Zone. Differential migration from a n:irrow initial zone of the mixture provides the most effective arr:ingenirnt for the resolution of mixtures. With t,his :irrangrment, modifications of which are illustrated by Figures 4 to 7 . the driving force should cause all the particles of each species t o migrate. Procedures based upon this arrangement represent batch processes for the resolution of mixtures. They make possible the complete resolution of multicomponent mixtures of soluteg, of vapors, and of gases. They are, therefore, of the
A+B DF
B
A
I
greatest usefulness, particularly to chemists whose methods for the determination of physical and chemical properties depend upon the resolution of mixtures and upon the isolation of the components in a s t a t e of high purity. Differential migr:ttion from :t narrow zone produced with a siiigle driving force is illwtr:tted schematically iri Figure 4. The migrat,ion rnsy be linear as illustrated, radial, or segmental (see Dependent upon their section on paper chromatograph!-). properties, the partirles may migrate in one direction orily (Figure 4) or they may migrate in opposit,e directions, :is with positive and negative ions during elec.trolysis (Figwe 5 ) ( I $3,f44, 162). This linr:ir migration from a narrow zone i,?a critical feature of some of the most important differential migration methods as, for exaniplc. chromatography, electrochromatography, sedimentation, :ind centrifugation. It is also a critical feature of t’he time-of-flight mnss spectrometer ( 1 3 , 5 5 , 5 7 ) . I n this inrtrument, ions from a narrolv source are accelerated intermittently by a pulsating electrical field in vacuum, so that only those of R particular mass and velocity pms through a ing of another synchronous, pulsating, clcctrical field (56). This differential migration f r o m a narrow zone niay also be produced with two driving forces in opposition, or it may be caused by two unique driving f o r c e s a p p l i e d transversely. These two transverse forces may be applied intermittently or in succession. They may also be applied simultane+ ously. Two oppowl driving forces, flow of solution and electrical migration, have been utilized in the separation of i o n s . W i t h moist paper i ~ 3themedium for differential clectrical migration, flow of the F i g u r e J . Differential Migrasolution opposed to the t i o n f r o m a Narrow Zone ionic nligration niay be Coinponcnts migrating linearly and produced by electroin opposite d i r e c t i o n s a s i n electroohroinato~raphg. Start (left) and osmosis (106, foe), by resolution of t h e mixtiire ( r i g h t ) hydrostatic pressure, or by evaporation from the paper (elec.trorhrophoresis) (108). Gravity opposed by density gradients provides the h:& of various sink-and-float separations of microscopic particles and macroscopic bodies (161 ). It is of various ore flotation processes. ’(5Tith C:trtesi:in diverp, this combination of force^ 1)rovides a convenivnt :in:iIytiral method for follo\viny vhniigrd in the density of various rr:tc.tion iJ-steni.c ( 2 3 ) . lligration from a narrow zone w i t h t\vo unique driving forces applied t ransvcrsely and in sucrcwion is illustrated by Figure 6. This is thc~h:tsic. procttdure of the t,\vo-w:Ly ptper chromatography with flow of two tliffrrrnt aolvents i n w I t is also the basic. procetlure for two-way and three-\v:iy cblectrochromatography with calrctricd migrittion in different solvents (46. 162)! and of comhimttions of flow of solvent with electrical migration (50, 133). As intlir:itetll)y Figures 6 and i ,the position of the sep:trated Pubstances relative to the p:tper(lcpendti upon the direction in which each force is applied (44). The degree of resolution does not (101). The paths followed by the solutes (lo depend, however, upon the sequence in which the forccs are
A 3 I
C
.
ANALYTICAL CHEMISTRY
94 applied. I n reports concerning the sequences of substances separated by forces applied transversely, the direction of each force should be specified. If there are anomalous trailing effects (40, 108, 154) or if the solvents alter the medium (124), the sequence of the application of the forces should also be reported. Migration from a narrow zone produced by tvio forces applied transversely and simultaneously is illustrated by Figure 8. Here the net result is the same as that obtained by application of the forces in succession. But the paths followed by the solutes are different from those indicated by Figures 6 and 7. A patent pertaining to the separation of mixtures in a narrow spot by the simultaneous flow of solvent transverse to electrical potential indicates that “the novel combined action , . , accomplishes results not obtainable by chromatographic or electrostatic separation severally” (68). From the views set forth here, the separations obtained by the simultaneous application of the forces may also be obtained by application of the forces in succession.
B+C
A
A+C
L-;I +I
,-\
DF;’
I I DF‘ $.
c
B+ C
e
A
6
B
@
Figure 7. DifferentialMigration from a Narrow Zone Two unique forces applied transversely and in succession, as in Figure 6. b u t with the direction of the forces interchanged. Compared t o Figure 6, position of the separated zones relative to t h e medium is changed, but degree of separation is not.
DF” $.
A
@ B
c3 Figure 6. Differential Migration from a Narrow Zone Two unique forces applied transversely and in succession. Force DF’ ( t o p ) and force DF“ ( l e f t ) yield b u t partial separation. Forces applied transversely and in succession (center) produce complete, two-way separation.
I n these differential migration procedures, the narrow zones often become diffuse and enlarge as the migration proceeds. This effect may be attributed t o diffusion (62, 96) and to the heterogeneity of the migration medium. With similar solutes, such as rare earths, or isotopes, or proteins, the rate of enlargement of the zones often exceeds the difference between the rates of migration, so that the zones overlap and the components are 143] 154, 16%). not separated completely from one another (44) Differential migration from a narrow zone is the basis of the batchwise, countercurrent, partition procedure introduced by Craig (41, 127, cf. 38, 103, 114, 122). I n this method, mechanical force produces intermittent counterfloiv of the two immiscible liquids. As in chromatography, differential distribution of the solutes between the tiyo liquid phases is the selective factor. Because the migration is carried out as a series of batch distributions, the separations per unit length of the system are not so effective as those performed with one of the liquids fixed in a partition column. Migration from a Wide Stream. When a wide stream of a mixture is exposed to two transverse forces, arranged as in Figure 9, a partial resolution occurs at opposite sides of the stream.
With this arrangement the fractionation process may be operated continuously. A4nanalogous, continuous, partial fractionation is achieved by many countercurrent procedures (80, 151) in which the components of the mixture migrate transversely and differentially from one stream t o another. With flow of two gas streams as in “free double diffusion” (150), the streams are separated by a porous barrier. With solutions, the streams may be separated by ion exchange membranes (158) or by semipermeable membranes. The intervening, porous, migration barrier may even be a complex differential migration unit, as with electromigration-convection operated continuously. Migration from a Narrow Stream. With two unique, transverse forces applied to a narrow stream of the mixture, each component follows a separate path through the migration medium and emerges a t a different position, as shown by Figure 9. This procedure may not only be operated continuously, but it also serves for the complete resolution of multicomponent mixtures as in continuous electrochromatography (24,65,58, 72, 99, 146). The two forces may be applied simultaneously as in continuous electrochromatography, or in succession as in the early modifications of the mass spectrometer. They may also be applied intermittently as the electrostatic field in the cyclotron and in the ion resonance mass spectrometer (119, f57). CHROMATOGRAPHY
Evolution and Definition. The number of recent publications concerning the modifications and applications of chromatography has been phenomenal. The gradual adoption of Tswett’s “chromatographische adsorptions Ainalyse’’by chemists first led to an entry for “chromatographic” under “adsorption” in the Chemical Abstracts Decennial Index for 1927-36. Not until 1943 was “chromatographic adsorption” made a major entry with 38 citations. In 1950 the citations mere entered under “chromatography and adsorption analysis,” and in 1952 there were some 1000 citations under this heading. Most of the recent investigations have concerned diverse applications of the chromatographic technique. bIany have been devoted to modifications .of the apparatus and procedure. Much has been written about the definition (164, 179) and the
95
V O L U M E 26, NO. 1, J A N U A R Y 1 9 5 4 evolution of chromatography (61, 164, 174, 176, 178, 179). Ten books, several bibliographies, and numerous reviews have summarized various aspects of the technique and its applications (see reviews). All this progress has been so specialized and so rapid that the analytical meaning of chromatography is just now finding its wav into the encyclopedias and the dictionaries which heretofore have included only the older definition-a treatise on colors. K i t h respect to procedure, there is a clear distinction between the flow of solutions through sorptive systems (Figure 2) and the formation of a chromatogram with fresh solvent or wash liquid (Figure 4). Contrary to recent contentions ( b f ,17'6, 178, 179)) the formation of a chromatogram, as reported by Tswett in 1903 and in 1906, must be regarded as the pinnacle in the evolution of the chromatographic technique (164). Chromatography is a unique procedure among a number of closely related techniques. It is no longer restricted to use with pigments, nor is it dependent solely upon the use of surface active adsorbents. I n time the meaning of chromatographv or sorptography may be extended to include all the similar methods of which chromatography is the prototype (Figure 4)) or another collective name for these methods may be devised, leaving chromatography with its present meaning. The folloi&g definition, which differs from some recent proposals (178, 1 7 9 ) , is based upon the principlrs outlined in this reviev. Chromatography is the study and the utilization of widely applicable, analytical procedures for the resolution of mixtures of solutes by differential migration from a narrow zone in porous media. the migration being produced by electrical potential or by flow of liquid or gas. Subdivisions of Chromatography. From the practical viewpoint and in harmony with the definition, there are three principal subdivisions of chromatography: chromatography by flox of solvent or gaq, chromatography by electrical migration, and chromatography by various combinations of flom- of solvent and electrical potential. Techniques iii each of these principal divisions are commonly classified with rebpect to the arrangement and the nature of the migration medium as with porous columns (columnar chromatography, columnar electrochromatography), with fibrous sheets, particularly paper (paper chromatography, paper electrochromatography), or with gels or fixed liquids (electrochromatography in gels). Combinations of flox of solvent with electrical potential provide the two-way method of
B+C
; C
B
I 1
1 1
I
I
chromatography plus electrochromatography (Figures 6 to 8) (40, 133) and the continuous electrochromatography (Figure 9) (58, 145, 160). Electrochromatography in different solvents provides the two-way and three-way electrochroniatographT (46, 162). T h e principal groups of chromatographic methods are frequently subdivided with respect to the mechanism of the sorption process-for example, adsorption a t a solid-liquid interfacr (adsorption chromatography), adsorption a t a solid-gas interfacr (gas chromatography) (9, 64,7 7 , 110, 111, 181, 1851, adsorption a t a liquid-gas interface (foam analysis) (17 , 128), adsorption at a liquid-liquid interface (163)) distribution between two immiscible liquids (partition chromatography) ( 7 4 , 118, 163), distribution betneen a gas and a liquid (gas-liquid partition chromatography) (76). distribution between a chemically reactive substance and a solution (particularly, ion exchange chromatography) (90, 91, 118). and partition between a liquid and a resin containing the same liquid (ion exclusion chromatography) (177). This classification of the chromatographic methods is subject to great variation, not only because the mechanism of the sorption piocess often varics with the experimental conditions, but also becausc several mechanisms may be effective rimultaneously. Dependent upon the circumstances, paper, for example, may be an ineit support. a surface active sorbent, or an ion exchange substance ( 1 , 30, 1 $ 9 , 152). I t may also fix a. liquid \yhich then serves as a partition solvent (74, 163) or.as a surface active sorbent (163).
+
B+C
A
I 1
I I I I
+ A +
C
A B Figure 9
Differential Migration from a Karrow Stream
Two unique transverse forces applied simultaneously or Intermittently. Cdrnponents of the mixture follow separate paths a t different rates. Separation is continuous.
\ \
\
\
\\B
63
Figure 8. Differential Migration from a Narrow Zone Two unique forces DF' a n d DF" as in Figure 6, applied simultaneously or intermittently. Course of migration is vector resultant (dotted lines) of the two transverse forces. Degree of separation IS comparable to t h a t of Figure 6.
The various groups of chromatographic methods are often classified with reFpect t o the field of application. The principal applications are analytical and preparative procedures in inorganic chemistry (98, 130, 1 4 , 1561, organic chemistry, biochemistry (118), clinical chemistry (a,105, 106)) and industrial chemistry (174, 178). All these chromatographic methods depend upon analogous principles. The several techniques yield similar separations, and they can be modified and applied in similar ways. Their range of application varies greatly with the chemical propertie. of the materials to be resolved. Reviews. The general subject of chromatography has been reviewed in a book by Lederer and Lederer (95). Practical techniques in many branches of chromatography have been
ANALYTICAL CHEMISTRY
96
presented by Brimley and Barrett ( 2 6 ) . -4review similar t o the are not so widely applicable. The selective partition between last one in this series (166) has been prepared by Tiselius (171 ). immiscible solvents, so widely employed in columnar chromaThe field of paper chromatography has been surveyed in three tography and in countercurrent extraction, is limited by the books, by Cramer ( 4 2 ) , by Block, LeStrange, and Zweig ( f g ) , miscibility of solvents that have suitable affinit,ies for the nonand by Balston and Talbot ( 6 ) . The latter publication also inpolar solutes (163) or for the strongly polar solutes. Many of cludes results obtained with columns of powdered cellulose. these partition systems are, however, finding wider application for Astonishingly, none of these publications have included Brown’s the separation of fatty, nonpolar .substances (38, 103, 114, early observations on the separation of chloroplast pigments by 222). circular paper adsorption chromatography or Liesegang’s cross From a theoretical viewpoint, each solute s!i 1ul.I forin n single capillary analysis (two-way paper chromatography) (164). zone in a chromatographic system. In prwtice. oil tile c.ontr,try. alteration of the solute (70, 1.54) or mriati,)n of the saq)t,ivr Electrochromatography, particularly as applied to protein separations, has been reviewed by Antweiler (2) and by Mcsystem (explored with chloroplast piginpnts i n 1950) m:iv yicl,l ttvo or more zones (20, 43, 61, 12.6, 15.5, I.?,?) :in 1 may :LIT(,.! : ‘ I ( ’ Donald and his coworkers (106, 106). Conditions requisite for the separation of inorganic ions have been presented by Sato e l aZ. migration rate or the migration distanw of proteins wiic,ii \Y with phosphate buffers (20, 61). (144). Electrochemical separations in ion exchange resins have Experience has now shown that phosliliute ion i p not s,ji,:been reviewed by Spiegler (168). Bibliographies of electrochromatography have been collected paper (144). Cations are strongly sorl>eI. K h t ~ np . 1 ~ buffer flows through paper, a pH gradient is i‘ j m i d . Tiii by McDonald (104) and by Henley and Schuettler (69). The dient determines the location and the migratioii r.itc or h’ v . i l ~ iI ~ J ~~ ‘ latter bibliography contains much material relating to the applisorbed solutes such as proteins. This effect resembles tlie cations of electrochromatography and to the detection of zone boundaries in chromatographic systems. action of sorbed solvents and impurities upon the migration rates Comprehensive books on inorganic chromatography have been of chloroplast pigments. prepared by Lederer (98) and by Pollard and McOmie (130). Columnar Chromatography. Columnar chromatography continues to be widely employed with various powdered sorbents Inorganic chromatography has been cursorily reviewed by Smith (156), who has placed major emphasis upon his own tests of (96, 118, 130, 156). These sorbents include vulcanized rubber (121 ), polysaccharides such as sucrose, cellulose, and starch ag analytical procedures. Practical identification procedures based well as the activated sorbents, such as activated magnesium upon flow of solutions into sorption columns (Figure 2) have been presented by Fillinger for cations of the coinmon qualitative silicate, charcoal, lime, magnesia, and alumina (66). They also include the ion exchange resins (90, 1267, hydrated filter aide, groups (49, cf. 50). Applications of ion exchange resins in analytical chemistry powdered gels, and rods of calcium sulfate (18). Columnar chromatography serves for preparative purposes have been summarized by Samuelson (141). An extensive bibliography pertaining to the analvtical applications of ion exchange and on an industrial scale (17 8 ) as well as on a micro scale. It is often employed as a preliminary or intermediate step in the bv Ochorn (123). The electrochemical resins has been prepared . properties of resins (1.58)and of reqin membranes (180) have also been summarized. Mrtnv aspect9 of the important field of partition chromatography ( 7 4 ) are cited in the bookc dealing v i t h chromatogranhv, paper chromatart tography, and inorgan’c chromatography. Itoutine applications of chromatography, which number about 2000 for 1953, mav be found in Cheinicnl Ahsfmcts. in variouq reviews includine; those published by hnnunl Review. Inc. ( 1181, and in the literature pertaining to particular euhjects and material- (10, ,531. Theory and Distribution Mechanism. Most theoretical treatments of chromatograDhp are concerned with the kinetic aspects of the distribution mechanism Thev relate to zone formation. zone boundaries, and zone migration (92, 136, 158, 172, 173). Thus far no comprehensive theory ha9 been proposed for estimation of the distribution of a IL solute betxeen the two phases of various chromatographic systems. Solubility provides a clue to the distribution of solutes between two immiscible solvents, and “solvent theorv” and the solvent properties of various liquids provide a clue to their effect on the sorption of various solutes (71). The sorption of solutes on a surface active sorbent ip one of the most widelv applicable distribution mechanisms emplored for chromatoFigure 10. Arrangements for Linear Paper Chromatography graphic separations. These sorbents attract all Upward linear flow ( Z J t ) and downward flow (center) (rerformed in closed vessels) ,. kinds of solutes. They sorb hydrocarbons from linear flow a t various angles (right) (between glass plates) S. Solute the least polar, saturated hydrocarbon solvents, WL. K a s h liquid P. Paper and thry sorb polar solutes from polar solvents d. Distance of migration such a? water. hlany other distribution systems For these conditions, Rf value is designated as lin. RI = d s / d w L .
1
[.
t
1
7’
V O L U M E 26, N O . 1, J A N U A R Y 1 9 5 4
97 filter paper is illustrated by Figure 12. This arrangement is subject to variation in many ways. The tab, for example, may extend t o the center of a thick pad of paper and may be insulated so that the chromatogram is formed as a series of concentric spherical zones.
Figure 11. Arrangements for Radial (Sector or Circular) Paper Chromatography Radial Bow in a planar mass, as a narrow sector ( l e f t ' ) rand as a wide eector ( r i g h f ) rad. Rf = d s / d w L
isolation of natural products, partic,ularly on a large scale with fractional elution or extraction of the sorbed substances. The location of t,he resolved substances in columns by the use of reagents is not so convenient as the location of substances resolved in paper (30). With sensitive physical, optical, and tracer methods, the separated substances may be detected as t>hey are washed through the column. So far as has been reported, the resolving power of columns of cellulose is comparable t o that of paper ( 6 ) . When various combinations of sorbents and solvents are employed, the resolving power and the applicability of columns are much greater than those of paper. This resolving power may be increased even further by submitting the fractions obtained from one column to further fractionation in columns of other sorbents and solvents (53, 1a6). Paper Chromatography. Paper chromatography is a remarkably adaptable and sensitive analytical procedure ( 6 , 19, @, 176. 184). I t is effective with very small quantities of mixtures of inorganic. ( i s , 48,132, 166) and organic substances. It i u readily a t l a p t d to two-way (101, 133) and polydimensional (44j migrations (Figures 6 to 9). The paper may be employed as a surface active sorbent (16.9) or as an ion exchange sorbent (1, 78. 14:?). I t is widely employed with a fixed solvent (101), as in paper partition chromatography (74) and in reversed phase partition chromatography (5, 7 , 28, 85, 113, 116, 163). The paper may he altered so that it becomes chemically reactive (48),as an anion or a cation exchange substance ( 1 4 , 6 0 , 7 8 , 9 3 )or so that it exhibits hydrophobic rather than hydrophilic properties (5, 85, 1I S , 116). Paper strips and sheets have usually served for linear migration (132). For short distances of migration. the flow of the solution may be upward, downward, or a t various angles. For long distances of migration, the flow should be downward (Figure 10). With radial migration as introduced by Brown in 19.19 ( 1 6 4 ) , the flow is through the sector of a circle (Figure 11). This technique, which is frequently called sector, radial, ring, or vircular paper chromatography ( 16, 54, 13.9, 16.9), has been modified in many ways. a4 h y the addition of wash liquid through wicks, tabs, capillaries, and dropping funnels (16, 54, 169). As with paper strips, it serves for the comparism of suhstances which are added as small ppots about the apex of the sector (f,?, 5.4). It usually provides narrower, more clearly defined zones \yith less trailing than those formed by linear paper chromatography (15, 54). Paper strips tapered near the starting end are reported t o exhibit some of these desirable features of radial florv in sectors (13,5). Diffusion gradients from the wash liquid improve certain separations (16.9). ;In arrangement for the unexplored segmental flow in a pad of
Figure 12. I..ntested \rrarigement for Segmental (Spherical) Paper Chromatograph?One of n n n v possible ari:xr.;t.icent= for spherical !?ow in a see:nr:nr o i thrcc-di .,.,risteni relative to the rate or distance of migration of t h P solvent provides the R value (Figure 10) which is widely employed for the description of substances and aq a measure of sorbability. This value depends upon many conditions which should be controlled and described. These inc l u d ~ :the Concentration of the mixture; the time and thr distance of migration; the nature, composition, and purity of the wash liquid; the temperature; the porosity of the medium, the nature and activity of the sorbent; the dimensions of the initial zone of the mixture; the dimensions of the migration system; arid the distribution of the solvent in the migration system (15, 31 33, 84, 96, 139, 166, 183). From the theoretical viewpoint, the migration of a solute zone should be determined relative to the region of maximum concentration. As this region of maximum concentration is frequently difficult to detect, the frontal boundary of the zone is regarded as the reference point, and the corresponding R value is indicated by R,. With many chromatographic systems, this boundary is diffuse. The location of the boundary, therefore, depends upon the sensitivity of the methods for the detection of the solute
98
ANALYTICAL CHEMISTRY
In similar chromatographic systems. the R values for particular solutes are subject to less variation if related to the R values of a similar reference substance, for example,
Rb of a = R of a / R of b This ratio of R values is a precise application of the chromatographic sequences so widely employed in qualitative, exploratory investigations. When the R values of two preparations arc, equal, when Rb of a = 1, a and b may be identical. This is a precise application of the mixed chromatogram or eo-rhomatogram. R values determined with linear flow (Figure 10) and with rridial flow from a narrow stream into a planar mass (Figure 11)have been indicated by the same symbol, R ( 1 5 ) . As shown by Figurr 12, R values may also be determined by segmental flow from :t narron- stream into a three-dimensional mass. For careful work. all these R values should be distinguished by distinctive symbols for example, lin. R, rad. R, and seg. R. If the migration systems h a r e the same porosity and the same sorpt,ive capacity, and if the distribution of the liquid in the three systems is uniform, ~~
lin. R = (rad.
= (seg.
R)3
For sugars in filter paper lin. R was found equal to (rad. R)’ ( 1 5 ) . With migration systems of soft paper, however, the distribution of the solvent in the paper is not uniform (183); hence the above relationship is an approximation. Electrochromatography. Differential electrical migration froin a narrow zone of the mixture i n electrolytic solutions was carrictl out in columns and in gels about 15 years ago and subsequently called electrochroniatography. Adapted to use in moist paper about 5 years ago, this method has n o ~ vbeen widely utilized in various fields, not only for the resolution of mistures but also for the determination of varioue prolierties such as isoelectric points and complex formation (e, 69, 10.1-106, 14.i). There :we many reasons for the widespread adoption of this technique-the earlier, estensive experience with thc related paper rhromatography; the numerous, sensitive nir,thods for the detection, elution, recovery, and estimation of the separated substances already perfected with columnar and paper chromatography; the slight decomposition of labile substances such as proteins when migrating in a porous system with sorption at a minimum (88, 8.9, 18.9); the simplicity and economy of the apparatus (154); and the high resolving power for many mixtures that are difficultly separable by other methods. IYrrtrochromatography has been applied to the examination of two principal groups of substances-namely, small ions. both organic and inorganic (36, 46.75, ,97, 125, 143, 144, 168, 162).and large colloids, such as proteins and polysaccharides (21, 52, 7.9, 88) 89,108: 133, 154). This diverse application has led to a complicated nomenclature. As applied to colloidal proteins. differential electrical migration in paper is called paper electrophoresis, zone ionophoresis, zone electrophoresis, elect,ropherography, paper electropherography, and ionography (106, 106). If a distinction is to be made betLveen ionophoresis and electrophoresis, based upon the size of the migrating particles, a collective term applicable to both ions and colloids is desirable. Because a narrow initial zone is employed, as in conventional chromatography, because sorption occurs in many of the migration media, and because sorption is the selective factor in many separations by electrical migration (14.9,1F Z ) , electrochromatography is the most apt. of the terms now in use. Unfortunately, the adverb electrochromatographically contrtins 26 letters! Several forms of electromigration apparatus are available commercially (105, 106). For research purposes, simplified forms of the apparatus may be constructed t o suit each particular investigation (40, 78, 144, 154 180). As in chromatography, the electrolytic solution and the stabilizing medium may be varied in numberless ways (40,78. 205, 106, 144, 158). The common media are gels, porous beds. paper, and ion exchange resins.
With moist paper, two principal arrangements of the clectrodes have been employed. Electrodes have been placed in separate reservoirs of the electrolytic solution in contact with the moist paper (105, 106), and they have been clamped directly to the moist paper (144, 162). With the former arrangement, both electrical inigration and electro-osmotic flow of the solution transport the ionized or chaiged particles. As in the moving boundarp method, the electro-osmotic flow of the solution (d,) may be ascertained by the migration of a zone of an easily detectable, nonionized, nonsorbed substance added to the migration system ( I d $ -of the particles differs from the mobility in free or unstabilized solutions This variation may be attributed to electro-osmosis, to sorption by the stabilization support. t o the action of the support as a barrier which increases the dictance that a particle must migrate, and to orientation of the POIvent and electrolyte molecules at the interfaces between the support and the solution, therebv increaying the heterogeneity of the migration medium (205, 106, 144, 154). -4s in chromatography, the mobilities may be reported in relation to the mobility of some standard or reference substance (62’1 Two-way electrochromatography in different solvents has been employed with inorganic and organic ions (46, 139, 162). Crossed or transverse electrical current in one medium has been reported to provide a more selective migration system than oneway migration (106,106) From the view that the resultant effect is the vector sum of two 1 ieiitical forces, this arrangement is similar to one-nx] migration i n a potential gradient. Centrifugal or magnetic fields transverse to an electrical field has been proposed for the separation of racemic mixtures ( 9 9 ) and for the migration of neutral particle (83). Migration in dry paper has been ohserved (75, 146,, and migration in living organisms (iontophoresis) opens many new fields for exploration ( 6 5 ) . Continuous electrochromatography with flow of solvent transverse to electrical potential has been improved and applied to the separation of ions (145, 2601, dyes (58), and proteins (24. ZC5, 7 2 ) . The theoretical boundary conditions have also been considered (51, 173). ION EXCHANGE RESINS
Continued research on ion exchange substances has increased the number of these materials that are finding increased use in differential migration techniques (123). Resins with oxidationreduction properties ( f 4 2 ) , with chelating properties ( 5 9 ) , with ion-exclusion properties ( 1 7 7 ) , and in the form of membranes (180) offer many possibilities for applications in analysis and in industry (123, I r e ) . The electrochemical properties of these resins have recently become a subject of intensive investigation (158, 169, 180).
.
V O L U M E 2 6 , N O . 1, J A N U A R Y 1 9 5 4
99
LITERATURE CITED
(1) -*llOuf, R.9 and AIachebocuf, 31.)Bull.
SQC.
chim. biol.7 34, 215
(1952). Mediein,” Berlin, Julius Springer, 1952. Baertschi, P., Heli.. Chim. Acta, 36, 773 (1953). Baertschi, P., Kuhn, TT., and Kuhn, H., S a t u r e , 171, 1018
( 2 Antweiler, H. J., “Die Quantitative Elektrophorese in der
,
