Affinity chromatography - Analytical Chemistry (ACS Publications)

Affinity chromatography - Analytical Chemistry (ACS Publications) GR Gray - â€...

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Anal. Chem. 1980,52,9R-15R (332) Ibid., 1978, 78,251-254; CA, 89, 156782e (1978). (333) Yoshimori, T.; Tanaka, T . BUN. Chem. SOC. Jpn. 1979,52,1366-1367. (334) Yoshimori, T.; Yamarnoto, K . ; Aizawa, K . ; Sato, H . Bunseki Kagaku 1978, 27, 377-379; CAI 89, 99043p (1978). (335) Zakharov, V. A.; Bekturova, G. B.; Songina, 0. A. Zh.Anal. Khim. 1979, 34, 755-758; CA, 91, 116670b (1979).

(336) Zakharov, V. A.; Gavva, N. F . ; Songina, 0.A . Zh. Anal. Khim. 1979, 3 4 , 174-177; CA, 90, 197052f (1979). (337) Zakharov, V. A.; Songina, 0. A . ; Aitkhozaeva, T. A . Zh. Anal. Khim. 1977,32, 1786-1789; CA, 88,2024616 (1978). (338) Zauls, L.; Veiss, A. Latv. P.S.R. Zinat Akad. Vesfis, Kim. Ser. 1977 (3), 359-360; CA, 88, 15578k (1978).

Affinity Chromatography Gary R. Gray Department of Chemistry, University of Minnesota, 207 Pleasant Street, S.E., Minneapolis, Minnesota 55455

Affinity chromatography has become an extensively utilized technique in a variety of analytical and separatory procedures. A search of article titles in Chemical Abstracts, in fact, reveals that in the period covered by this review (January 1977 through September 1979), the title words received 665 citations! See Table I for nomenclature. It is not the purpose of this review to summarize the basic principles of this technique, practical considerations for its effective use, or the myriad of ways in which it has found application, as excellent recent monographs are readily available (2-6). Rather, it is my purpose to present selected recent advances in immobilization chemistry, as advances in this area have important consequences for all applications of affinity chromatography. In order for affinity chromatography to be effective, the ligand must be attached to a support in such a way that it is physically accessible, that recognition between it and the molecule being isolated occurs, and that the chemical linkage between ligand and support is completely stable to the conditions required for elution of the adsorbed molecule. Despite the apparent simplicity of these requirements, they are difficult to meet because of stringent requirements imposed by biological molecules for expression of their activity. As a consequence, new and varied coupling procedures and supports with altered chemical and physical properties are constantly being sought. The general strategy employed in ligand immobilization is to incorporate chemically reactive and compatible groups into both the support and ligand in such a way that coupling can be accomplished in an efficient and chemically well-defined manner. Given the variety of organic functional groups present in biological molecules and the vast array of modified supports currently available, chemical modification of both the ligand and support is rarely necessary. However, steric requirements usually demand that linkage of the ligand be accomplished through a "spacer arm", and for this reason, further modification of the support or li and is usually necessary. In the discussion that follows, kvelopments in the area of ligand immobilization chemistry are arbitrarily divided into those involving support modification and those involving ligand modification.

SUPPORT MODIFICATION T h e reaction of polysaccharide supports with cyanogen bromide (7) continues to be the most widely used method of activation, despite the difficulty of handling the reagent and the instability of the linkage formed to the support. The activated polysaccharide is subsequently reacted with ligands containing amine functionalities to produce isourea derivatives of structure 1.

Support -0


-C-"-Ligand 1

Cyanogen bromide-activated polysaccharide gels are also 0003-2700/80/0352-9R$Ol.OO/O

Table I. Nomenclature ( I ) affinity chromatography

separation of molecules using biospecific interactions between the molecule being isolated a n d another molecule immobi1izc:d on an insoluble polymer insoluble polymer sites o n support available for ligand binding specific molecule interacting with the biological molecule being isolated

support matrix ligand

frequently used to incorporate chemically reactive spacer arms through which various ligands are subsequently attached. Wilchek and Miron (8),for example, reacted CNBr-activated agarose with various bishydrazides to give hydrazidoagarose (21, u










I1 It II Agarose -0 - C -N H N t l C (CH2),C N H N H 2 2

which was subsequently converted to several other reactive derivatives by standard procedures. O'Brien et al. (9) prepared affinity resins containing immobilized ethanolamine pyrophosphate and thiamine pyrophosphate via the aminohexyl derivative of Sepharose (3), formed by addition of 1,6-diaminohexane to the CNBr-activated polysaccharide gel. The ethanolamine pyrophosphate gel ( 5 ) was formed by successive succinoylation of 3 to give the carboxylate gel 4 and treatment of the latter with the water-soluble carbodiimide l-ethyl-3-(3-dimethylaminopropy1)carbodiimide and ethanolamine pyrophosphate. Preparation of the thiamine pyrophosphate resin (7) was accomplished by coupling thiamine pyrophosphate (TPP) to the diazotized p-aminobenzoyl derivative (6) of Sepharose, the latter prepared as described by Cuatrecasas (10). A similar strategy was employed by Joshi et al. ( 1 1 ) to prepare a regenerable affinity chromatography support (RACS) for protein immobilization (10). Diazotized p aminobenzoyl Sepharose (10) was coupled to wN-acetyl-1,tyrosine to give resin 8. Protein coupling to 8 was accomplished via the active ester 9, and regeneration of the resin was accomplished by dithionite reduction and recycling of the p-aminobenzoyl derivative (11) as described above. All immobilization procedures based on CNBr activation of polysaccharide gels suffer, however, from the lability of the isourea linkage to the support, and other activation procedures have therefore been sought. Schnapp and Shalitin (12) [email protected] 1980 American Chemical Society







mobilized amine ligands on aminoalkylated polyacrylamide and glass by CNBr activation, and the guanido linkage (12) Support



similar procedure was used by Kucera ( 1 7) for the preparation of epoxide-activated cellulose (16),


?\ - Cellulose - 0 C H 2 C H C H 2


-N H -C -N H -L i g a n d ,I




that was formed was found to have much greater chemical stability. Bethell et al. (13),employed 1,l'-carbonyldiimidazole t o activate cross-linked agarose gels. Treatment of agarose with the carbonylating reagent gave the imidazolyl carbamate (13),which, on treatment with amine ligands, gave the nonbasic N-substituted carbamate (14). A

/ N -b. V

N Q \ C

I1 A g a r o s e -OH





which was used to immobilize several aliphatic and aromatic diamines. A variety of s-triazines have also been used to activate polysaccharides (6) or synthetic hydroxylated polymers. Treatment of agarose with dichloro(methoxy)-s-triazinegave the chloro(methoxy)-s-triazinylagarose derivative (17), which was subsequently reacted with amine ligands to give immobilized ligands of structure 18 (18). OMe



9 L





17 /Llganc--"Z





-N H - h g a n d

T h e preparation of epoxide-activated polysaccharide gels has been accomplished by a number of workers. Sundberg and Porath (14) reacted agarose with the bisoxirane, 1,4-butanediol diglycidyl ether, which resulted in simultaneous introduction of the reactive oxirane group (15) and stabilization of the gel by cross-linking. Agarose-OH




C H2CHC H 2 0 ( C H2 )qOC H2C HC H 2




This method of activation, which was later investigated in detail by Uy and Wold ( 1 5 ) ,has been used to immobilize a variety of amine and hydroxyl-containing ligands (14-16). A 10R

A g a r o s e -0



In a similar manner, the hydroxyalkylmethacrylate gel, Spheron P 1000, was activated with 2-amino-4,6-dichloro-striazine, and protein antigens were then coupled to give immunoadsorbents of structure 19 (19).


-0 -CH2CH C H 2 0 ( C H e )4OC H 2 C/"\ H C H2


1; 491, N



sup p o r



+ -0



%-Substituted4,6-dichloro-s-triazines are used in preference to trichloro-s-triazines in the activation step in order t o eliminate the need for removal of the remaining reactive chlorine prior to affinity chromatography (18);however, trichloro-s-triazine is sometimes used (20).


Gary R. Gray is Associate Professor of Chemistry and Biochemistry at the University of Minnesota, Minneapolis. He received his B.S. degree in Chemistry from Ouachita Baptist University in 1964 and his Ph.D. in Biochemistry from the University of Iowa in 1969. After spending two years as an NIH Postdoctoral Fellow and a year as a Visiting Assistant Professor in the Department of Biochemistry, University of California, Berkeley, he joined the staff at Minnesota. Dr. Gray's research is focused on the isolation, structural characterization, and synthesis of mycobacterial immunostimulants and in the development of these components as immunotherapeutic agents for cancer. He is the recipient of Faculty Research Award 143 from the American Cancer Society.

Divinyl sulfone is another reagent that has found considerable use for the activation of polysaccharide gels. The reagent is normally used to bind ligands to agarose gels (21); however, it has recently been used to make cross-linked gels of soluble polysaccharides which already contain the ligand of interest (22). There have been several recent advances in the development of affinity matrices that involve either the synthesis of new support materials or the modification of existing synthetic materials. Schnaar and Lee (23)prepared polyacrylamide gels containing active esters (20a,b) by copolymerization of the N-succinimidyl and N-phthalimidyl esters, respectively, of acrylic acid, with acrylamide and N,N'-methylenebisacrylamide. Displacement of the active esters with ligands containing aliphatic amino groups resulted in the formation of stable amide bonds between the ligand and polyacrylamide gel (21). 0




phenyllactic acid and p-aminobenzyl-l-thio-:[email protected],respectively (25). Polymers prepared by the copolymerization of 22 or 23 and N-[tris(hydroxymethyl)methyl]acrylamide or N- [ tris(hydroxymethy1)methyllmethacrylamide were examined in another study and were found to also be suitable affinity chromatographic supports; the fucose-binding lectin from Ulex europeus was purified and a column containing p-aminophenyl-a-L-fucopyranoside (26). Several procedures have been described recently for activation of synthetic organic polymers and inorganic supports, An aldehyde-activated polyacrylamide support (26) was prepared by Fiddler and Gray (27) from a commercially available aminoethyl polyacrylamide gel (24). Direct reductive amination of glyceraldehyde gave the N-2,3-dihydroxypropyl derivative (25) which was activated immediately prior to use by periodate cleavage. The aldehyde content of the matrix could readily be controlled by the amount of periodate added. Protein immobilization to the matrix was accomplished by direct reductive amination with sodium cyanoborohydride to give stable, secondary amine linkages (27), and unreacted aldehydes were subsequently reduced with sodium borohydride. Excellent efficiencies of coupling and retention of activity were observed, and the immobilized proteins were exceedingly stable to chemical removal. Powdered polyacrylonitrile (28) was used as a support for protein immobilization by Feuerstein and Geyer (28). Activation of the support was accomplished by treatment with methanol and HC1 gas, and after thorough washing to remove HC1, the resulting imidate hydrochloride (29) was coupled to Lens culinaris hemagglutinin via an amidine linkage (30).








i'l c;.' '



Support-?--NH 0 Polyacrylamide-C-






A similar approach by Brown et al. (24) yielded functionalized polyacrylamide-agarose copolymers. In this case, polymerization was accomplished in the presence of acrylic acid derivatives containing carboxyl groups well separated from the double bond. The acrylic acid monomers used in this study, 6-acrylamidohexanoic acid (22) and N-methyacryloylglycylglycine (231, C H2 =CH-C-N




- Protein

Modified silica gels are being increasingly used as affinity matrices. A glycerolpropylsilane-bonded silica (31), formed by reacting silica with y-glycidoxypropyltrimethoxysilane (291, was used by Ohlson et al. (30) for the immobilization of ligands for use in high performance liquid affinity chromatography. Periodate oxidation of 31 gave the aldehyde-activated derivative (32), to which amine ligands were coupled by reductive amination (33). Silicas containing amine functionalities (34) have been prepared by reaction with 3-aminopropyltriethyoxysilane (31),and these matrices likewise offer many possibilities for ligand immobilization. An adsorbent containing the ligand Blue Dextran 2000 (35) Silica-0-Si










was prepared by Anderson and Jervis (32) by reacting the cyanogen bromide-activated dextran with aminoalkylated silica. Aminoalkylated silica was also used by Brown et al. (33) as an intermediate in the preparation of the succinyl thioester derivative 36.



were copolymerized in the presence of agarose, N,N'methylenebisacrylamide and, in some instances, acrylamide, t o give copolymers possessing hydrophobic and hydrophilic spacer groups, respectively. The suitability of these copolymers for use in affinity chromatography was demonstrated by the purification of lactate dehydrogenase and wheat germ agglutinin on columns containing immobilized p-amino-











30 N H -Ligand









II (CH;i!zC/I -S ( C H ~ ) Z C O ~ H


Amine-containing ligands, including proteins, were unexpectedly covalently bound to this derivative without activation, and were partially released in a thiol-containing form after ANALYTICAL CHEMISTRY, VOL. 52, NO. 5, APRIL 1980

11 R








p r o t e i n , p H 7-75




9 S ep harose-

0 -C

-N H (CH 2 ) z N H C

0 1 M difhinnite, pH 9








-N H - P r o t e i


0 P o l y a c r y latnide-

II-N H (CH2)zN H z


1) g l y c e r o l d s h y d e




-N H ( C H 2 ) 2 N H C H 2 CI H C H 2 0 H I





25 0

0 Polyacrylamide-






Pol y a c r y l a m i d e -








H ( C H 2 ) z N H (CH2)zN H-










1 ) Ligand-NHp 2)NoBHq






-( C H 2 ) 3 0 C H 2 C H z N H -L



treatment with neutral hydroxylamine. The utility of this procedure as a method for ligand immobilization remains in question, however, because of the lack of knowledge about the chemical events that are occurring. LIGAND MODIFICATION T h e chemical modification of a ligand is desirable when it does not contain suitably reactive groups for coupling, when coupling t o a matrix cannot be carried out in a chemically well-defined manner, when solubility problems preclude the use of otherwise workable coupling reactions, or when linkage to the support or spacer arm cannot otherwise be accomplished in a regio- and stereoselective manner. The strategies employed in ligand modification are to either incorporate a 12R


chemically reactive group (or spacer arm and chemically reactive group) that can be coupled to a matrix of known reactivity, or to incorporate a group that will allow copolymerization of the modified ligand into the affinity resin. There are many well-established methods for accomplishing these types of modification, and they will not be repeated here as recent comprehensive summaries are readily available ( 2 , 34). A few more recent examples will be presented, however, to illustrate these methods. The covalent attachment of carbohydrates to insoluble supports has been a very active area of investigation because of the utility these materials have for the isolation of proteins (lectins, antibodies, glycosidases, other enzymes) which bind carbohydrates. A very simple procedure for preparing this


. - IC H ~ O A C




^ I

1 ) NoOMe. M e O H



43 CH20H






type of affinity column was first described by Gray (35),and affinity columns prepared by this procedure were later demonstrated by Baues and Gray (36)to be very effective for the isolation of lectins. T h e basis of this procedure is the direct reductive amination of reducing disaccharides to amine gels in aqueous solution with sodium cyanoborohydride. T o illustrate this procedure, the disaccharide lactose (37), a commercially available aminoethyl polyacrylamide gel (24), and sodium cyanoborohydride are combined in aqueous phosphate buffer a t neutral or slightly alkaline pH.

HoHv .=; / H.OH


+ H2N(CH2)2NHC--Polyacrylamide 24




p H 7-9




H2N H (CH2)zNHC -Pol y a c r y l a r n i d e ' H OH Q r q


The allyl glycoside (40) was prepared by treatment of L-fucose (39) with 3% HCl gas in allyl alcohol. This procedure is effective for the preparation of allyl a-glycopyranosides derived from a variety of monosaccharides, including 2-acetamido sugars, but it is not effective for the synthesis of allyl p-glycopyranosides. The latter are prepared by condensation of allyl alcohol with the appropriate acylglycosyl bromide in the presence of silver carbonate, and subsequent deacylation (41). S-Glycosyl polyacrylamide gels analogous to 41 were prepared by Pipkovi et al. (42)and were shown to also be effective affinity adsorbents for lectins. These derivatives were ineffective, however, as glycosidase affinity adsorbents. 5'-Glycosyl polyacrylamide gels with longer spacer arms were prepared by Lee et al. (43)as is illustrated below for preparation of the P-galactopyranosyl derivative (44). Controlled addition of N,N'-methylenebisacrylamide to a solution of the 1-thio-sugar derivative (42) gave a mixture of mono- and diaddition products from which the monoaddition product (43) was readily separable. Deacetylation of 43 and copolymerization with acrylamide and N,N'-methylenebisacrylamide gave 44. The strategy of ligand immobilization via incorporation of a polymerizable functionality into the ligand has been used for the preparation of many types of affinity adsorbents. Polymerization of polyacrylamide in the presence of N acryloyl-4-amino malachite green 145) and .V-acryloyl-4'aminophenyl neutral red (46)


38 Coupling of the disaccharide to the gel occurs because cyanoborohydride anion selectively reduces the Schiff base formed between the reducing sugar and the amine groups of the gel, yielding N-1-(1-deoxylactito1)aminoethyl polyacrylamide (38). After the desired degree of substitution has been achieved, excess amine groups are blocked by selective N-acetylation. Affinity resin 38 was found to be very effective for the isolation of lectins from peanut and castor bean. T h e above procedure is very effective for incorporating carbohydrate ligands into gels provided a suitable disaccharide is readily available. In other cases it is necessary to synthesize the desired carbohydrate liggnd. In such cases, the allyl flycoside procedure of Hofej6i and Kocourek (37)is particuarly effective. This method has been exploited by these workers (38, 39) and others (40) for the isolation of a wide variety of lectins. As an illustration of this procedure, a polyacrylamide gel containing 0-a-L-fucopyranosyl residues (41) was prepared by copolymerization of allyl a-L-fucopyranoside (40) with acrylamide and N,N'-methylenebisacrylamide (37). HOCHpCH=CH2 HZC H =c H 2 HO @H'ou











I = CHp






e2C I-

45 0





gave adsorbents for nucleic acid fractionation (44). Polymers containing 45 were specific for the adeninethymine base pairs of double-stranded DNA and those containing 46 were specific for guanosine-cytosine base pairs. Proteins have also been immobilized by copolymerization, and the principles involved in the preparation of derivatives suitable for copolymerization were recently reviewed by Jaworek et al. (45). As an example of this technique, hog stomach peptone was immobilized by Hofejgi (46) by copolymerization of its maleic acid half-ester with acrylamide and N,N'-methylenebisacrylamide. The adsorbent was used to isolate a lectin from Ulex europeus; however, its capacity for the lectin was rather low. ANALYTICAL CHEMISTRY, VOL. 52, NO. 5, APRIL 1980



T h e chemical modification of a ligand is often undertaken to incorporate a spacer arm of appropriate length and a functional group that will allow its specific covalent attachment to a preformed matrix. Wright e t al. (47) prepared affinity adsorbents for hexokinases (48) by coupling various N-aminoacylglucosamine derivatives (47) to CNBr-activated Sepharose.




C -C H2C H 2s H






+ -NH-




NHCO(CH~), - N H ~


41 ( n = 2 , 3 , 5 , 7 )

54 CH20H I

which was coupled to the mixed disulfide of agarose (53), obtained by reacting the mercaptohydroxypropyl ether of agarose with 2,2'-dipyridyl disulfide. Removal of protein from the gel (54) was accomplished by reduction of the disulfide linkage with dithiothreitol, and the thiolated gel was recycled by treatment sequentially with 2,2'-dipyridyl disulfide and the thiolated enzyme.


T h e modified ligands were prepared by condensation of the competitive inhibitor glucosamine and the N-trifluoroacetyl derivative of the appropriate spacer amino acid in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, followed by removal of the N-trifluoroacetyl protecting group with aqueous piperidine (48). An affinity adsorbent for adenosine deaminase was prepared by Rosemeyer and Seela (49) by the carbodiimide-catalyzed coupling of 2',3'-0-[ 1-(2-carboxyethy1)ethylidenelnebularine (51) to 6-aminohexylagarose. The cyclic 2',3'-O-acetal (51) was prepared from 6-thioinosine (49) SH




0 0



50 1H2,Raney Ni



T? 0


x H02CCHzCHz CH3 51

by treatment with ethyl levulinate in the presence of HC1 and ethyl orthoformate, followed by successive saponification and catalytic hydrogenation with Raney nickel. Two final examples are given to illustrate the incorporation of specific reactive groups into larger ligands. In order t o apply the technique of affinity chromatography to DNA:DNA and DNA:RNA hybridization reactions, Dickerman e t al. (50) prepared adsorbents containing sheared-single-stranded DNA. T h e DNA was first modified by a limited coupling reaction with 4-diazobenzoic acid, which forms addition compounds with some of the nucleic acid bases, and the resultant modified DNA was coupled through its benzoic groups to the amino groups of an aminopentane Sepharose derivative using a water-soluble carbodiimide. Finally, Carlsson et al. (51) have introduced a method for the reversible, covalent immobilization of enzymes which requires the introduction of thiol groups into both the protein and support. Treatment of a protein with methyl 3-mercaptopropioimidate gave the thiolated derivative (52) 14R


53 CNBr-activoled Sepharose

OH 49









LITERATURE CITED (1) Sundaram, P. V. J . Solid-Phase Biochem. 1976, 1 , 101-103. (2) Turkovi, J. "Affinity Chromatography"; J . of Chromatography Library Elsevier: New York, 1978; Vol. 12. (3) Mosbach, K. I n "Advances in Enzymology"; Meister, A,, Ed.; John Wiley and Sons: New York, 1978; Vol. 46, pp 205-278. (4) Mosbach. K.. Ed. "Immobilized Enzymes", in "Methods in Enzymology"; Academic Press: New York, 1976; Vol. 44. (5) Hoffmann. 0. 0.; Breitenbach, M.; Koller, F.; Kraft, D.; Scheiner. 0.. Eds., "Affinity Chromatography. Biospecific Sorption: The First Extensive Compendium on Affinity Chromatography as Applied to Biochemistry and Immunochemistry"; Pergamon: Oxford, 1978. (6) Kennedy, J. F. Adv. Carbohydr. Chem. Biochem. 1974, 29, pp 306-405. (7) Ax&, R.; Porath, J.; Ernback, S. Nature(London) 1987, 214. 1302-1304. (8) Wilchek, T.; Miron, T. Mol. Cell. Biochem. 1974, 4, 181-187. (9) O'Brien, T. A.; Schrock, H. L.; Russel, P.; Blake, Robert, 11; Gennis, R. B. Biochim. Biophys. Acta 1978, 452, 13-29. (IO) Cuatrecasas, P. J. Bioi. Chem. 1970, 245, 3059-3065. (11) Joshi. V. K.: Shahani. K. M.: Kilara. A,: Waaner. F. W. J. Chromatoor. 1979, 176, 11-18. (12) Schnapp, J.: Shalitin, Y. Biochem. Biophys. Res. Commun. 1978, 70, 8-14. (13) Bethell, G. S.;Ayers, J. S.;Hancock, W S.; Hearn, M. T. W. J. Bioi. Chem. 1979, 254, 2572-2574. (14) Sundberg, L.; Porath, J. J. Chromatogr. 1974, 90, 87-96. (15) Uy, R.; Wold, F. Anal. Biochem. 1977, 81, 98-107. (16) Desai, N. N.; Allen, A. K. Anal. Biochem. 1979, 93, 88-90. (17) Kucera, J. Collect. Czech. Chem. Commun. 1979, 44, 804-807: Chem. Abstr. 1979, 91, 30159. (18) Lang, T.; Suckling, C. J.; Wood, H. C. S. J . Chem. Soc. Perkin Trans. 1 , 1917, 2189-2194. (19) Linnas, T.; Mikelsaar, R.; Nutt, N.; Kirret, 0. Eesti NSV Tead Akad. Toim. Keem., Geoi. 1978, 27, 46-48; Chem. Abstr. 1978, 88, 211502. (20) Danner. J.; Lenhoff, H. M.; Heagy, W. J . Solid-Phase Biochem. 1978, 1 . 177-188. (21) Porath, J.; Sundberg, L. Nature New Bioi. 1972, 238, 261-262. (22) Young. H. M.; Leon, M. A. Carbohydr. Res. 1978, 66, 299-302. (23) Schnaar. R. L.; Lee, Y. C. Biochemistry, 1975, 1 4 . 1535-1541. (24) , , Brown. E.: Racois. A.: Boschetti. E.: Coraier. M. J . Chromatoor. 1978. 150, 101-110. (25) Brown, E.; Joyeau. R.; Boschetti. E.: Moroux, Y. J. Chromatogr. 1978, 150. 111-117. (26) -Racois. A.; Boschetti, E. Biochimie 1978. 60, 193-196. (27) Fiddler, M. B.; Gray, G. R. Anal. Biochem. 1978, 86, 716-724 (28) Feuerstein, H.; Geyer, G. Z.Med. Laboratoriumsdiagn. 1978, 19, 114118; Chem. Abstr. 1978, 89, 175,858. (29) Regnier, F. E.; Noel, R. J. Chromatog. Sci. 1978, 14, 316-320. (30) Ohlson, S . ; Hansson, L.; Larsson, P.-0.; Mosbach, K. FEBS Lett. 1978. 93, 5-9. (31)659-66 Robinson, 1. P. J.; Dunnill, P.; Lilly, M. D. Biochim. Biophys. Acta 1971, 242,


(32) Anderson, P. A.; Jervis, L. Biochem. SOC. Trans. 1978, 6. 263-268. (33) Brown, R . J.; Swaisgood, H. E.; Horton, H. R . Biochemistry 1979, 18, 490 1-4906. (34) Jakoby, W. B.; Wilchek, M., Ed. "Affinity Techniques", in "Methods in Enzymology"; Academic Press: New York, 1974; Vol. 34. (35) Gray, G. R. Arch. Biochem. Biophys. 1974, 163, 426-428. (36) Baue3 R. J.; Gray, G. R . J. Biol. Chem. 1977, 252, 57-60. (37) Hoiejsi, V.; Kocourek, J. Biochim. Biophys. Acta 1973, 297, 346-351. (38) Hoiej9 V.; Kocourek, J. Biochim. Siophys. Acta 1978, 538, 299-315. (39) Hoiejsi, V. J. Chromatogr., 1979, 169, 457-458. (40) Sutoh K.; RosenfeM, L.; Lee, Y. C. Anal. Biochem. 1977, 79, 329-337. (41) Hoiejg, V.; Kocourek, J. Methods Enzymol. 1974, 34, 361-367. (42) Pipkovi, J.; Hoiejg, V.; Kocourek, J. Biochim. Biophys. Acta 1978, 541, 515-520.

Anal. Chem. 1980, 52, 15R-27R (43) Lee, R . T.; Cascio, S.;Lee, Y. C. Anal. Biochem. 1979, 95, 260-269. (44) Bueneman, H.; Mueller, W. Nucleic Acids Res. 1978, 5 , 1059-1074. (45) Jaworek. D.; Botsch, H.; Maier, J. Methods Enzymol. 1976, 44,195-201. (46) Hoiejgl, V. Blochim. Siophys. Acta 1979, 577, 389-393. (47) Wright, C. L.;Warsy, A . S.;Holroyde, M.J.; Trayer, I . P. Biochem. J . 1978, 775, 125-135.

(48) Hoiroyde, M. J.; Chesher, J. M. E.; Trayer, I . P.; Walker, D. G. Biochem. J . 1976, 153, 351-361. (49) Rosemeyer, H.; Seela, F. Carbohydr. Res. 1979, 74, 117-125. (50) Dickerman, H. W.; Ryan, T. J.; Bass, A . I.; Chatterjee. N. K . Arch. Biochem. Siophys. 1978, 186, 218-234. (51) Carlsson, J.; AxBn, R.; Unge. T. Eur. J . Biochem. 1975, 59, 567-572.

Ion Exchange and Liquid Column Chromatography Harold F. Walton Box 2 15, University of Colorado, Boulder, Colorado 80309

This review includes journals available to December 31, 1979. T o report the last two years of liquid chromatography is a formidable task. No area of chemical analysis has advanced so fast. I have heeded the request of ANALYTICAL CHEMISTRY’S editors and emphasized principles and methodology, citing enough representative examples to compile Table I and 11, tables that readers have found useful, but which make no pretense to be comprehensive. Many of the papers cited in the tables illustrate new techniques and interesting points of theory. Readers concerned with specific applications should consult the book list ( A l - A 8 ) or the reviews (Bl-B14). There have been several meetings and international symposia devoted wholly or in part to liquid chromatography. Volumes 158 and 165 of the Journal of Chromatography comprise the proceedings of two international conferences on chromatography; volume 149 of the same journal reports the proceedings of the Third International Conference on Liquid Column Chromatography, held in Austria in 1977. The Fourth International Conference was held in Boston in 1979, and its proceedings appeared in the Journal of Chromatography, Vol. 185. A U S - J a p a n Seminar on Advanced Techniques of Liquid Chromatography was held in Boulder in 1978; it was informal; several of the papers have appeared in the open literature and are cited in this review, and a summary of the seminar, by A. P. Graffeo and N. H. C. Cooke, was published in the Journal of Chromatographic Science [ 1979,17, 2021.

ION EXCHANGE General. Ion-exchan e chromatography is becoming more efficient. Advantage is ieing taken of conventional gel-type resins in small, uniform particles for the chromatography of inorganic ions (123,161,421)as well as for organic compounds; here, the advantages of low crosslinking (287, 51 7) and the effect of inorganic counterions on the retention of uncharged organic solutes (517 ) are noted. Macroporous and surfacesulfonated resins are being used (421);their preparation and properties are described (162,460). “Ion chromatography”, which uses resins whose ion-exchange function resides in a thin surface film, is very popular (A7,B10,118,161,186,271, 292,320). The method is especially useful for anions, and very low concentrations can be measured, down to parts per billion if concentrator columns are used (523). In its orthodox form, “ion chromatography” depends on conductometric detection, preceded by a “suppressor column” to remove ions of the eluent. The suppressor column can be eliminated if a suitable ion-selective detector is used (161). Detectors that respond to some ions and not others make ion-exchange chromatography more practical. Anion-exchange separation of transition-metal ions in hydrochloric acid, with a concentration gradient and atomic absorption detection, is a good example (209). Studies of the distribution of metal ions between resins and various com lexing solutions continue. Cation exchange in concentratefHC1-HC10, (359) provides means of separating easily hydrolyzed ions like titanium and zirconium; in tartrate 0003-2700/80/0352-15R$Ol .OO/O

and succinate (90, 91) many elements are separated; anion exchange has been studied in HBr-HN03 mixtures (4611, thiocyanate (447),and malonate media (61),and cation exchange in oxalic-hydrochloric acid mixtures has useful possibilities (365). A new aspect of ion exchange is the widespread use of paired-ion chromatography, in which the stationary phase is a hydrophobic packing and the mobile phase contains longchain, hydrophobic ions of opposite charge to the ions being separated. It is now clear that highly hydrophobic counterions are incorporated into the surface of the packing, making i t essentially an ion exchanger. Paired-ion chromatography, developed for or anic ions, has now been used for inorganic species like halijes, azide ions and oxy anions of nitrogen, sulfur, and the halogens (404). Paired-ion chromatography will be discussed a t greater length below. New ion-exchanging materials, organic and inorganic, continue to be reported, but less frequently than before. I n o r g a n i c Exchangers. Among the new materials are bismuth tungstate (402),antimonates of thorium (103),nickel and cobalt (399,401),arsenate and vanadoarsenate of tin(1V) (400, 481), and zirconium arsenophosphate (448). Older materials have been studied more intensively, particularly crystalline zirconium phosphate (B3, 73, 81, 1 2 7 ) and crystalline antimony pentoxide (2-4). On the latter, selectivity orders are reported for alkali and alkdne-earth ions and for transition-metal ions; they depend on the degree of loading. Tin dioxide exchanges both anions and cations; its mode of action is peculiar (233-235), and it is specially selective for scandium (86)and lithium (218). Activated carbon impregnated with tin dioxide combines the selectivity of an inorganic exchanger with the physical, hydraulic properties needed in chromatography (218). Most inorganic exchangers are either slow to react, or are soft and powdery, or disintegrate easily; hence, their use in columns is limited. Other exchangers whose special uses are noted are hafnium and thorium phosphates (25, 104), cerium(II1) oxalate (81, tungsten(V1) oxide and zirconium tungstate (102),zirconium molybdate (165),and copper(I1) ferrocyanide (2611. Aluminosilicate molecular sieves were used to separate glucose and fructose (531),and hydrous oxides of aluminum, titanium, and zirconium removed traces of heavy-metal ions from water (306). C h e l a t i n g a n d Special Resins. A polymer with 8hydroxyquinoline functionality, previously described, has been used for several metal separations and is specially selective for vanadium (505). Starting with macroporous crosslinked polystyrene, amide groups (374, 390), arsonic acids (1461, dithiocarbamate (26),thio lycolate (385),oxime (264,463)and nitroresorcinol units (4637 have been attached; t,he products have the selectivities expected from those of the parent substances in solution. Oxime and thioglycolate resins, for example, are selective for mercury and copper; nitroresorcinol is selective for iron(III), copper, and cobalt. A cellulose-based exchanger carrying salicylic acid absorbs iron(II1) and uranium(VI), and one with hydroxyphenylazo-2-naphthol units @ 1980 American Chemical Society

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