*
The reactions of representative amino acids with various forms of synthetic ion-exchange materials are studied. All amino acids tested react with the hydrogen form of the cation exchangers. Using the column method, the capacities of two commercial exchangers, Amberlite IR-1 and Zeo-Karb H, for glycine, leucine, norleucine, phenylalanine, tryptophan, hydroxyproline, glutamic acid, asparagine, and lysine hydrochloride have been determined. A fairly uniform capacity of each individual exchanger, in terms of equivalents of the acids examined, suggests that their removal proceeds essentially by salt formation betw-een the basic amino group and the acid form of the exchanger. This conclusion is substantiated by the fact that static studies of the adsorption of glycine from solutions containing varying amounts of HC1 show decreasing removal as the pl3 is lowered, and by the fact that the calcium and sodium forms of cation exchangers either did not react or reacted slightly with amino acids. Addition of formaldehyde to the amino acid solutions had little effect upon their reaction with the hydrogen form of the exchangers. Adsorption of the nine amino acids by the hydrogen exchangers is studied by static methods also. Previous equations for treating the data are not entirely applicable, in part because of the diverse acidic groupings and consequent inhomogeneity of the exchangers. Anion exchangers rlmberlite IR-4 and De--4cidite react with dicarboxylic but not with monocarboxylic monoamino acids. Thus a separation of these two groups of acids may be expected. The basic amino acid hydrochlorides are not removed by the anion exchangers.--The photograph (reproduced through courtesy of The Resinous Products & Chemical Company) shows a commercial installation of three exchanger cells designed for special applications.
. D. T. ENGLIS AND H. A. FIESS University of Illinois. Urbana, Ill.
A
the distribution of the nearly neutral manoamino monocarboxylic acids was a matter of more uncertainty. A few preliminary experiments involving this subject were carried out in this laboratory by D u Puis (4). Bhatnagar and associates ( I ) , in studies of the mechanism of acid removal by resin exchangers, compared amino acetic acid with other acids. Block ( 2 ) reported an application of the exchangers to the removal of basic amino acidb derived from proteins. He subjected the acid hydrolyzate to the action of the anion exchangers and then passed the resulting solution through the cation exchanger in the hydrogen cycle. He finally eluted the basic amino acids from the cation exchanger with hydrochloric acid and separated them by a modified Kossel procedure. Myers ( 7 ) called attention to many interesting applications of ion exchangers, among which are mentioned their use in the isolation, purification, and recovery of physiologicslly active materials. I n this connection it has been found (S), in comparing the rate and extent of fermentation of the refined sirup from Jerusalem artichokes with that of the raw extract, that some materials essential to yeast development are removed by the exchangers and decolorizing charcoal. Wachtel and Cassidy (9) employed chromatography upon charcoal as a
PROCESS for the preparation of sirup from the extract of Jerusalem artichokes was described in a pievious paper ( 5 ) . The acidity necessary for the hydrolysis of the polysaccharides was developed by treating the extract with a cation exchanger operating in the hydrogen cycle, in which the metal cations of the naturally occurring salts were exchanged for hydrogen ions. After hydrolysis of the polysaccharides by heating the resulting solution under pressure, the acids, mainly organic, were removed by an anion exchanger. I n this manner the purity of the sirup was greatly increased by removing the salts; at the same time the nitrogen content was materially reduced. I n studying the distribution of the nitrogen fraction, it was found that the greater portion of the nitrogen-containing material was removed by the cation exchanger, and that a relatively smaller amount of the nitrogen compounds in the solution was taken out by the anion exchanger. This immediately raised questions as to how the different amino acids would respond to the exchangers and how the latter might be utilized in the separation of the amino acids. It was anticipated that the basic amino acids would be retained by the hydrogen exchanger and that the dibasic amino acids would be removed by the anion exchanger, but
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INDUSTRIAL A N D EN G INEEBIN G CHEMISTRY
means of separating amino acids. Their paper cited a number of references of this and other adsorbents for amino acids’. Since nitrogeneous compounds are present in many raw materials which will be given exchange treatment, some knowledge of the conduct of individual amino acids with ion-exchange materials as well as other adsorbents is of interest. Elution of these substances from the absorbing medium with the proper reagents may make possible the recovery of valuable by-products. Two methods are commonly used for studying the reactions of exchange materials with solutions. One method, termed “column exchange’’ or “dynamic exchange”, is based upon usual practice and simulates the process which takes place when an untreated solution percolates through a bed of exchange material by gravity flow. I n the laboratory the exchange material is placed over a suitable support in a glass tube, the tube is filled t o a desired height, and flow is regulated by a stopcock at the baee of the column. Static exchange is analogous to batch procedure which is also employed industrially. A quantity of exchanger is kept in contact with the solution being treated until practical equilibrium is attained between the ions or molecules undergoing exchange or adsorption. I n the present study the conduct of several amino acids with the exchange materials was studied by both of these general procedures. PREPARATION OF EXCHANGE MATERIALS
The synthetic cation-exchange materials used were a carbonaceous exchanger, Zeo-Karb H, and the resin exchangers, Amberlite IR-1 and Amberlite IR-100. The synthetic anion exchangers f e r e Amberlite IR-4 and De-Acidite. All cation-exchange materials were screened t o -20 4- 40 mesh. Those used for adsorption in the kydrogen cycle were treated with a number of portions of 5 % hydrochloric acid, with occasional stirring, over a period of 2 days. The materials were washed thoroughly with distilled water until free of hydrochloric acid and then air-dr:ed under comparable conditions for several days. The prepared exchangers were then stored in airtight bottles and constituted the stock samples. Calcium and sodium exchangers were prepared by treating the materials with 6% solutions of calcium chloride and of sodium chloride, respectively, following the procedure mentioned above. The anion-exchange materials were prepared by stirring the samples with portions of a 5% solution of sodium carbonate. These materials were air-dried for a shorter period than were the cation-exchange materials and were then similarly stored in airtight bottles. To calculate capacities on a comparative basis, the moisture content of the exchange materials used was determined by drying in a platinum dish at 105” C. to constant weight. The moisture content of the various samples is given in Table I. COLUMN ADSORPTION OF AMINO ACIDS
A sample of 2.50 grams of the cation exchan er or 2.00 grams of the anion exchanger was placed in a glass tu%eof such diameter that the depth of the bed was ap roximately 6 inches (15 cm.). The exchanger was supported &y a small plug of glass cotton, and the base of the column fitted with a glass stopcock. T h e top of the tube was widened to a diameter of approximately 1 inch (2.5 cm.) so that a small reservoir of liquid could be maintained above t h e evchange material. The exchanger column was connected with a large reservoir to avoid repeated filling of the column. 1 A publication by Freundenberg, Walch, and Molter [Nulurwidoenschujten, 30, 87 (194211 waa brought to our attention after this article had been submitted for publication. These investigators suggeated that acidic amino acids may be separated from other amino acids by anion exchangers: they also reported that cation exchangers adaorb all amino acids. In another
article which appeared while the present paper was being reviewed, R. IC. Cannan [f.Bid. Chem., 152,401 (1944)] demonstrated that the dioarboxylio amino acids mag.be meparated and estimated in protein hydrolysates with Aznberlite IR-4.
605
A 0.01000 M solution of the amino acid being studied was allowed to percolate through the exchanger at a slow rate of flow, approximately 6.5 to 7.0 ml. per minute. Experiments conducted with hpproximately a 30% faster rate of flow showed but a small decrease in reaction. The effluent was collected in 25-ml. portions and analyzed for the amino acid being examined. An attempt was made to include amino acids representative of various types. From the group of monocarboxylic monoamino acids were chosen the simplest member, glycine, and the closely analogous leucine and norleucine, in which the effect of an increase in chain length as well as chain branching might be evident. Other compounds of this classification were phenylalanine, hydroxyproline, and tryptophan. The latter two have a cyclic >NH group although the indole nitrogen of tryptophan is not basic. Of the basic amino acids, lysine was used in the form of the hydrochloride. Glutamic acid was chosen as representative of the dicarboxylic amino acids. I n addition, the amide of aspartic acid, asparagine, was studied. Some of the amino acids were C.P. or high-purity products obtained from reliable commercial sources. Others had been prepared or purified in the organic manufacturing laboratory a t the University of Illinois and their purities tested. Confirmation of purity is based upon the checking of the neutral equivalent of most of the acids by the titration method and obaervation of the molecular extinction value of the color complex as established with the spectrophotometer. The latter test would have indicated any gross error in the value for the NH2 group. It is reasonably certain that any errors from impurities in t h e compounds studied is within the error of the colorimetric method. The amino acids investigated were found t o be taken up from their solutions by the hydrogen form of the cation-exchange materials. One might anticipate that this reaction would proceed by physical adsorption or by salt formation with the basic amino group according to the equation
RH
+ NH&HR’COOH
a RNHaCHR’COOH
where RH is the acid form of the cation-exchange material, or by a combination of physical adsorption and chemical combination. Amino acid concentrations were determined by a modified Folin colorimetric procedure (6) employing sodium P-naphthoquinone-4-sulfonate as reagent: One to three milliliters, containing 0.0005 to 0.006 millimole of amino acid, were placed in a 50-ml. flask and diluted to approximately 20 ml.; the following reagents were added: 3 ml. of a solution of 0.05 N hydrochloric acid and 0.118 N sodium carbonate, and 2 mi. of a fresh 0.5% solution of sodium P-naphthoquinone-4-sulfonate. The flask and contents were allowed to stand in the dark for several hours; then 1 ml. of a solution of 25 grams of sodium acetate and 250 ml. of glacial acetic acid per liter, and 3 ml. of a 4% sodium thiosulfate solution were added. Colorimetric measurements were then made in a Cenco-Sheard spectrophotometer with a I-cm. cell and a slit width of 5 mp. The spectral transmittancy curves of the complex produced by a number of amino acids were identical and showed a characteristic absorption peak at 470 mp. Measurement was made within one hour after development of the color, for fading occurred after that time due to decomposition and to precipitation of sulfur from the thiosulfate. Within the range mentioned above, Beer’s law was found to hold a t 470 mp. Residual concentrations in millimoles per 25 ml. were determined by the colorimetric procedure. In Figure 1 residual concentrations in millimoles (or in milliequivalents upon the basis of the alpha amino group) as obtained in column adsorption are plotted against total volume exchanged. The presence of the amino acids in the effluent-e.g., the break-through pointsoccurred early in the adsorption curves obtained for the carbonaceous exchangers in contrast with those obtained for the resinous exchangers. By increasing the amount of exchanger used, the adsorption curves would be displaced to the right on the horizontal axis and break-through points would occur at a later
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EXCHANGERSOLUTIONS
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related to a limited degree with the acidity of the molecule being adsorbed. It might be expected that free lysine would be effectively removed. As the hydrochloride, it takes a place among the acids for which the exchangers have a lower adsorption.
AMBERLITE IR-l 2.50 GRAMS
0.01000 M
3.CLUTAMIC ACID 5.LEUCINE
EFFECT OF ACIDITY ON ADSORPTION BY HYDROGEN EXCHANGERS
7. NORLEUCINE
That the hydrogen exchanger absorbs amino acids, mainly by virtue of its properties as an acid, is apparently substantiated by a series of experiments involving the static adsorption of glycine in the presence of varying amounts of hydrochloric acid. These results are shown in Table I11 and Figure 2 . A marked reduction in the degree of adsorption is evident as the acidity is increased and the mass action effect of the hydrogen ions in solution becomes more pronounced. These results may be interpreted as evidence for a base exchange mechanism for the reaction:
9.PHENYLALANINE
U . W
E XC HANGER- ZEO
- KARB
H
,‘
I. TRYPTOPHAN 2 . PHENYLALANINE 3. HYDROXY PROLINE
4. 5. 6. 7.
ASPARAGINE LYSINE HCI GLUTAMIC ACID LEUCINE 8 . GLYCINE 9. NORLEUCINE
B.TOTAL
Figure 1.
VOLUME: O F EFFLUENT IN M L .
Column Adsorption of Amino Acids
RHi
‘ I
+ NHs+CHR’COOH + C1RXHBCHR’COOH + H + + ‘31-
as opposed to the simple salt formation previously indicated. Zeo-Karb H and Amberlite IR-1 show the same type of conduct. These observations are in accordance with the well recognized fact that, after adsorption, the amino acids may be eluted with hydrochloric acid. SODIUM AND CALCIUM EXCHANGERS
stage. When the exchanger was near saturation, residual concentrations approached the original concentration very slowly and resulted in a curve of little slope. Because complete saturation of the exchanger was approached slowly, for convenience it was arbitrarily assumed that saturation was reached when the effluent amino acid concentration was 93y0 of the original concentration. The capacities in millimoles and in grams of amino acid per gram of bone dry exchanger were calculated from the data of Table I on moisture content. The results are given in Table 11. The capacity of a unit weight of the same exchanger in terms of millimoles of amino acid adsorbed is nearly the same for a number of the compounds studied. The lowest capacity of both exchangers was shown for hydroxyproline. Amberlite IR-1 showed highest capacity for tryptophan and for phenylalanine; Zeo-Karb H showed greatest capacity for norleucine and leucine. The slopes of the curves for Amberlite IR-1 exchanger were steeper and somewhat more uniform than those for Zeo-Karb H, which may indicate a more homogeneous nature of the former adsorbent. If any generalization may be drawn, it is that Figure 2. the adsorptive ability of the exchanger is cor-
If the theory is correct that the retention of amino acids by a cation exchanger in the hydrogen form i6 primarily salt formation with the basic amino group, and if the sodium, calcium, or some other than the hydrogen form of the exchanger is employed, it
Static Adsorption of 0.005 M Glycine in the Presence oi Hydrochloric Acid
TABLEI. MOISTURECONTENT OF EXCHANGE MATERIALS Exchange Material Amberlite IR-1 (air-dried) Zeo-Karb H (air-dried) Amberlite IR-4 moist) Amberlite IR-4 {air-dried) De-Acidite (moist) De-Acidite (partly air-dried)
% Moisture
Cycle
H+ H.+
Anion Anion Anion Anion
23.3 17.3 46.0 16.3 70.4 65.4
T A B L E 11. COLUMN ADSORPTION O F AMINOACIDSBY EXCH.4NQE MaTERIALS IN HYDROGEN CYCLE (TOTAL CAPACITIES)
Bmino Acid, 0.01000 M Aqueous Solution Hydroxyproline Glutamic acid Lvsine HC1 Aiparagine Leucine Glycine Norleucine Tryptophan Phenylalanine
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Amberlite IR-1 Millimoles/ Grams/ 2.50 g., 1.00 bone %-y air-dried 2.44 0.167 2.71 0.208 2.88 0,274 2.95 0.204 3.35 0.229 3.43 0.134 3.47 0.238 3.74 0,398 3.80 0.327
Zeo-Karb H Millimoles/ Grams/ 2.50 g., 1.00 air-dried bone %y 1.48 0.094 1.81 0.129 1.66 0.142 1.62 0.103 2.25 0.143 2.05 0.075 2.33 0.148 1.66 0.164 1.77 0.141
EFFECT O F FORMALDEHYDE ON ADSORPTION BY HYDROGEN EXCHANGERS
T o test further the idea that the retention of amino acids was mainly by virtue of combination with the amino group, the properties of this group were modified by the addition of formaldehyde and the degree of adsorption was again tested. Formaldehyde is known t o react with the 2" group, and the reduction of its basicity in this manner makes possible a titration of the acid grouping of the a-amino acids by the well-known Sorensen method. The reaction of the formaldehyde is generally believed to proceed according to the following equation :
RCH--NH*
+ CHzO e RCH--N
COOH
CHzO
COOH
CH,OH
TABLE 111. STATIC ADSORPTIONOF GLYCINEIN PRESENCE OF
HYDROCHLORIC ACID
(0,400 gram of air-dried exchanger, 100 ml. of 0.005 M glycine solution)
Solvent HzO 0 001 N HC1 0,010 N HCl 0.10 N HC1 0.25 N HC1
Initial pH 5.48 3.35 2.19 1.08 0.68
Adsorption, Millimoles/100 M1. Zeo-Karb H Amberlite IR-1 0.238 0 324 0.224 0.317 0.131 0.226 0.027 0.053 0.020 0.021
will be anticipated that little neutral amino acid will be removed. Apparently this prediction is borne out by Table IV. The concentration of amino acids was the same as that in the column experiments (Table 11) for the hydrogen exchanger. In the Amberlite cation resin, the total capacity of the sodium or calcium forms for glycine and for norleucine is 1 to 2% of the total capacity of the acid exchanger for glycine and for norleucine; the total capacity of the sodium or calcium form for tryptophan is 5% of the total capacity of the acid exchanger for that amino acid. I n the Zeo-Karb H exchanger the total capacity of the sodium or calcium forms for glycine is 5 t o 8% of the total capacity of the hydrogen form; the total capacity of the two exchangers for norleucine is 15 to 16% of the total capacity of the acid form; the total capacity of the sodium or calcium forms for tryptophan is 25 to 26% of the total capacity of the acid form. These results give support to the idea that the binding of the amino acid by the exchanger is primarily chemical in nature, although physical adsorption may account for part of the total capacity, especially in the cases of tryptophan and norleucine. Sodium and calcium exchangers do not differ in their reactions with individual amino acids. Since the effluents from the calcium exchangers did not give a test for calcium ion, it may be assumed that no preliminary exchange occurred between the hydrogen ion of the amino acid and the calcium or sodium ion of the exchanger. on with higher concentrations of the amino acids may show some replacement. If this is evident. some meferential elution methods with sslt dolutions may be possible:
As representative amino acids for the test, glycine and glutamic acids were selected and adsorption experiments were carried out by the column method. The procedure was modified in that the solution of amino acid contained 2 to 4% formaldehyde. Results are shown in Figure 3. The effect of formaldehyde on adsorption was small. I n the case of Zeo-Karb H a small lowering of the total capacity was indicated. It had been expected that
GLUTAMIC ACID
250.
EXCHANGER
A. NUMBER
8.TOTAL
SOLVENT
OF 25-ML. PORTIONS
WITHDRAWN
VOLUME OF EFFLUENT I N M L .
Figure 3. Effect of Formaldehyde on Column Adsorption of 0.01 M Glutamic Acid and Glycine
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PROCEDURE FOR STATIC ADSORPTION
A 100-ml. solution of an aniino acid and 0.400 gram of exchange material were placed in a 250-ml. wide-mouth, glassstoppered bottle. The amino acid solutions varied in concentration from 0.0200 to 0.00125 X . The sealed bottles containing the materials were placed in a device which revolved a t a rate of 22 times per minute and kept the exchanger and solution in a slow, constant state of agitation. Preliminary experiments showed that practical equilibrium was attained before 1 hour had elapsed. However, the reaction was allowed to continue for 3 hours 7. HYDROXY PROLINE Q 1.20 8 GLUTAM IC A C I D or longer, and then the solution in contact. 0 E X C H A N G E R - A M B E R L I T E IR-1 9.LYSINE HCI with the exchanger was analyzed f o r residual amino acid. -l'&O 460 -lbO -1:20 -LOO -0.80 -0:60 - 0 4 0 -0.120 0.bo The data obtained on static adsorption / I of the nine amino acids are given in Table J-. Adsorption isotherms based on Equation 3, in which log [Ali, exchsnger or log adsorption was plotted against log [Ali, or log residual concentration, are given in Figure 4. It is evident that p , the slope of the isotherm, is variable and thus dependent upon the original concentration ; slope p of the isotherm obtained \5-ith Amberlite IR-1 and glutamic acid varied 4.LEUClNE from 0.25 to 1.06. I n similar cases, in the 5.LYSINE H C I U I 6.GLYCIN E reaction of Amberlite IR-1and hydrosy6 7.ASPARAGlNE proline, p varied from 0.39 to 0.82; in -1201 0 8.HYDROXY PROLINE the reaction of Amberlite IR-2 and trypt,o9,GLUTAMIC ACID phan, p varied from 0.06 to 0.33. -1.40 M'alton ( I O ) used Equation 2 in his -0.60 -0.40 -0.20 0.00 -IkO -160 -1.40 -1.20 -1.00 -0.80 study of sodium-hydrogen exchange with L O G R E S I D U A L C O N C E N T R A T I O N , M M. A C I D / I W M L . the carbonaceous exchanger, Zeo-Karb H. Figure 4. Static Adsorption of Amino Acids He found that the numerical value of p varied and explained it by postulating inhomogeneity in the Zeo-Karb H due to diverse acidic groupings, the nature of the compounds would be so altered by the addition including sulfonic, carboxylic, and phenolic. Thus some of the of formaldehyde that the adsorption would be markedly reexchangeable hydrogen is present as an acid with a very IOK duced. However, previous experiments had shown that even dissociation constant. at a p H of 3 only a slight lowering in adsorption occurred. ApSuch a postulate would explain the adsorption isotherms obparently the basicity of the amino acids was not decreased enough tained by the reaction of amino acids with Zeo-Karb H and Amto effect adsorption. berlite IR-1. With an increase in amino acid concentration, STATIC ADSORPTION BY HYDROGEN EXCHANGERS mass artion Fould cause a greater percentage of the acidic groups Myers and associates ( 8 ) a;plied the empirical Freundlich equation to static exchange data:
-
'
I
'
log u = log k
+ n log c
(1)
I
..
