on these diffusions rcwived S a C ’ I T I ’ .J:~cwljsoii, Boston University, Boston, certificates from Randbook I’ulilislim, 11:~~s. Inc., for their satisfactory solution of an undergraduate research program: Larry LITERATURE CITED ii. Teter, Fresno Stat,e Collogc~,Fresno, Calif.; Richard ill, ,james 1 ~ . (1) .liltelman, &I., ANAL. CHEM. 26, 1218-19 (1954). ”Id G. ’’-ilkerson, ( 2 ) Harris, F. E., S a s h , L. K., ZhicE., 22, University of Texas, Austin, Tex.; I552 (1950). Gloria nefina, Paula Fetsko, and Con( 3 ) Leupin, O., Iluggli, P., Helv. Chim. Acta 2 2 , 1170-7 (1939). stance St* Joseph ( 4 ) Kicholas, R. E. H., Rimington, C., Biochem. J . 48, 306 (1951). Emmitsburg, N d . ; and Saniuel Hirsch
(5) Strain, H. H., 3Iurphy, G. IV., .IY.~L. CHEM.24, 51 (1952). (6) Veil, S., Compt. rend. 198, 1854 (1934). (7) Zhid., 199, 611 (1934). (8) Zhid., p. 1044. RECEIVEDfor review March 7 . 1058. Accepted December 31, 1958. ?ivieion of Analytical Chemistry, Symposium on Separation Processes through Differential Migration Analysis, 134th Meeting, .4CS, Chicago, Ill., September 1958. Work done under the auspices of the National Cooperative Undergraduate Chemical Research Program (NaCCRP).
Carrier Displacement Chromatography on Ion Exchange Resins DONALD 1. BUCHANAN Radioisotope Service, Veterans Adminisfration Hospital, West Haven, Conn., and Department o f Biochemistry, Yale University, New Haven, Conn.
b Certain volatile bases assume intermediate positions in the order of displacement of amino acids on cation exchange resins and thereby convert partial into complete separations. Acids can do the same on anion exchange resins. A paramount criterion in the search for these selective agents or carriers is the dissociation constant, but aromaticity and size are also considered. Some of these carrier substances may be used in a second way. If a resin is first saturated with an appropriate acid or base, amino acids may separate by differential migration, if displaced into and through the zone of saturated resin. This method of column development has distinct advantages over those using buffers or strong acids. The need for clarification of the terminology and the mechanisms of ion exchange chromatography of arnpholytes are discussed. solutes that bind firmly to an inimobile phase are able to strip conipletely or displace more weakly bound substances, zones “push” one another down a column all a t the same rate, the filial displacing agent acting like a piston ( 3 ) . This is diagrammed in Figure 1, n here thc curves represent concentrations of solute along a column. El en L\ lirii individual affinities for the stationary phase differ greatly (-4 and B ) , pure zones are a h ays separated by a miwd zoiie, the lower limiting size of which is governed by the mechanical imperfections of column operation. It is theoretically impossible to recover any substance completely iii pure form. HEX
Mixed zones tend to be larger when relative affiities differ to a lesser degree ( B and C). Often solutes compete almost equally for the stationary phase (D and E ) and nearly all of one or both substances is mixed xith the other. Unlike most types of chromatography, the relative size of a mixed zone does not decrease as less material is chromatographed. Conversely, addition of more material does not increase the absolute sizes of mixed zones beyond certain maxima and the relative quantity of mixed material therefore becomes less. Because of these characteristics displacement chromatography of amino acids has been successful when very large mixtures-e.g., 280 grams of hydrolyzed egg albumin (25)-are separated. Even M hen two solutes differ but slightly in their affinity for the solid phase ( D and E , Figure 1) they may be separated by displacement chromatography if enough of a third substance n i t h a n intermediate affinity is interposed. Such interposed displacers, called “carriers,” have been used in absorption chromatography (9, 12, 15, 17,18,28, 34). Recently (5) carriers were used to expand the methodology of Partridge and coworkers (20-27) in the separation by displacement chromatography of mixtures of amino acids on ion exchange resins. The carriers mere nonamphoteric bases and acids, all easily separated from the amino acids by evaporation or ion exchange. The observation of navies ( 7 ) that with bases as me11 as acids the stronger displace the \T ealier on ion exchange columns made the search for carriers
much easier. Although the displacement sequence of a group of substances cannot always be predicted from their dissociation constants, (24, 25), experience is a reliable guide in evaluating the magnitudes of the effects of molecular size and aromaticity in altering the expected displacement order. Some groups of three or more amino acids have dissociation constants and other properties so nearly alike that when the group is chromatograplied by displacement there is little tendency for one to act as a carrier to separate the others. Because of this the hope of finding other carriers to separate thebe groups of very similar amino acids is not great. However, the individual members of such groups are often separable by diff erential migration, if a resin column is first regenerated with the carrier initially used to displace the group and the amino acid mixture is then displaced through the column. In this case the carrier becomes a competitive developer. Partridge and Brimley (22, 2;) had demonstrated that some amino acids that fail to separate on a sulfonic acid resin may sometimes do so on an anion exchanger. These workers (23) a n d others (10, 19) had also improved some separations by raising temperatures. Cooling may also inzprore a separation (5). These factors were brought t o bear in developing the empiricallJsuccessful schemes (5) for separation of synthetic amino acid mixtures. The present paper illustrates the resolution of a n acid hydrolyzate of protein. VOL. 31, NO. 5, MAY 1959
833
EXPERIMENTAL
Hydrolysis. R a t muscle protein was precipitated, washed with 1% picric acid, and dried. After correction for picric acid content and ash, the sample weighed 2.34 grams. It was hydrolyzed by refluxing 24 hours in 25 ml. of 6N hydrochloric acid. After one treatment to near dryness in a rotary evaporator, the residue was redissolved in 50 ml. of water and filtered. Chromatography. The mixture was then subjected t o chromatography as described in Figure 2, which is a flow diagram of the separation plan with projections of the paper chromatograms of the fractions of effluent, all taken a t 20-minute intervals. The type of resin of each of the six columns, the volumes and lengths of the resin beds, the displacing solutions, the temperatures of operations, the fraction numbers, and the volumes of the pooled effluent appear with the appropriate paper chromatograms. The latter here developed with l-butanolglacial acetic acid-water solvent (4: 1 : 1). Chromatograms of the long ninhydrin-negative “breaks” between and preceding some amino acids were deleted from the figure, but the volume of discarded effluent is designated. Many of the technical details have been described (6). If desired, very wide separations can be attained with carriers. This is emphasized in the primary fractionation, which was carried out on a column larger than previously used and twice the usual quantity of each carrier (30 mmoles). The first two carriers, l-isopropylpyrazole and pyrazole (each 0.2M), were first added, followed by the amino acid mixture. When the third carrier, pyridine (O.lM), was added, a precipitate, presumably of tyrosine and cystine, appeared in the resin bed. No attempt had been made to precipitate these slightly soluble amino acids before the hydrolyzate was put on the resin. After about 300 ml. of water, added to the pyridine, had run through the column, the precipitate redissolved and the remaining carriers were added in 0.1M solution. The n-ater accounts for the large volume of effluent between Groups I and 11. Seemingly because this separation was carried out during very warm weather, cystine, which had always emerged a t the end of Group I1 and had trailed across the pyrazole break, now appeared in Group 111. The advantage of this is evident, and probably warrants the use of a heated column. In three secondary separations columns of Dowex 50 were first saturated with one of the three weak bases, 1-isopropylpyrazole, pyrazole, or nicotinamide; each had been used as a carrier in the preliminary fractionation. For
834
ANALYTICAL CHEMISTRY
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the two remaining separations Dowex 2 was first saturated with the weak acid, succinimide. The amino acid mixtures were displaced into the zone of carrier-resin from a short segment of hydrogen form resin in the case of the Dowex 50 columns or from hydroxide form resin in the case of the Dowex 2 columns. This sharpens and concentrates a mixture before it enters the zone where separation will occur. The further separation of Group I was as given ( 5 ) , except that a trace of hydroxyproline appeared well separated from proline. Threonine and serine separation was improved. The separation of Group I1 was unchanged, except that no attempt was made to separate the small final mixture of alanine and glycine by recycling. It is clear from the combined results of this separation and the previous one (6) that Group 111, cystine, phenylalanine, tyrosine, and tryptophan all separate completely on Dowex 50 saturated with nicotinamide. Recovery. The yields of twice recrystallized amino acids appear on Figure 2. Recovery from the crude protein was about 55% when corrected for the water of hydrolysis. This was lower than previously found for synthetic mixtures and lower than would be expected from a purer protein. Impurities such as carbohydrate, known to be present in the residue, not only detract from the true sample weight but also catalyze the decomposition of some amino acids during hydrolysis ( 2 ) . Because this separation was merely to acquire enough of each amino acid for an isotope analysis, little effort was made to get high recoveries of the more abundant amino acids. Tissue Extracts. This method has been applied with success to the isolation of free amino acids from picric acid extracts of several 1-kg. batches of rat muscle. With little extra labor several substances not present in protein may be obtained in pure form from these extracts. Taurine, creatine, creatinine, anserine, and carnosine are the more abundant of such compounds. Taurine passes through the original desalting column (4) and can be recrystallized directly after the effluent is reduced in volume. Creatine follows
Figure 1. Diagram of l n !‘“E’ concentrations in zones v YE3 A to E in displacement c h r o m a t o g r a p h y and \ effect of carriers in \ promoting separation
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pyrazole in the primary separation and is mixed with Group 111. Much of it crystallized directly from the effluent and the rest may be crystallized by holding back the amino acids of Group I11 on a small column of Dowex 2-hydroxide, reducing this effluent to small volume, and then adding alcohol. Creatinine follows Group I11 and may be obtained in an analogous manner. Carnosine and anserine follow histidine and the three are mixed. They may all be separated by passing the mixture through a column of Dowex 50 saturated with picoline or with imidazole (details to be published). DISCUSSION
Selection of Carriers. The practical aspects of carrier displacement chromatography on ion exchange resins are straightforward. Although one can distinguish a t least three factors that determine the order of displacement of a group of substances from a cation resin exchange column, the most important is basic strength. Partridge and Brimley (S4, 25) found that if a margin of 0.2 pK unit is allowed, all but one of 24 amino acids emerge from a cation exchange resin in the same order as the relevant pK values (the dissociations that convert the positively charged ampholytes into neutral molecules). The exception was phenylalanine, which came off more than a full pK unit “late.” Tyrosine and tryptophan are retarded even more (6), the latter (pK,, 2.38) not being very cleanly displaced even by pyridine (pK, 5.2). Aromaticity is, therefore, associated with the most conspicuous “anomalies” (26) in the displacement order. Size is well correlated with smaller deviations-Le., those that seem equivalent to 0.1 to 0.3 pK unit. Ammonia (pK, 9.27) cleanly separates lysine (pKz, 8.95) and arginine (pKn, 9.04), while valine (pK1, 2.32), a larger molecule, follows alanine (pK1,2.34). To select a carrier that may be useful in separating two compounds, one searches for a substance that has a dissociation constant between the two? the allowance given for size and aromatic character is dictated by preceden; and experience. The carrier compound
t
PRIMARY S E P A R A T I O N GROUP
I
- DOWEX
GROUP
5 0 - X 4 , 2 0 0 MESH;
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C O L U M N - I I O C M , 150 M L ;
GROUP
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DOWEX 2 - X B , 100-200 M E S H SUCCINIMIDE FORM
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GLUTAMIC ACID
210
2 2 3 0 ML
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77 M L t
96 M L
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2-XB. 3 0 - 3 2 ’ C SUCCINIMIDE FORM
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-
/1
SERINE 164 MG
I
DISPL- 0.1M A C E T ~
I
OOWEX 50, 200 MESH P Y R A Z O L E FORM
t C O L - 9 5 CM, 90 M L 0 I M PYRIDINE, 45’C
135 MGI
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................................................. l
