ANGE IN THE L FIELD JAMES C. WXNTERS AND ROBERT IUJNIX Kesircous Products Diuision, Rohrn and Hnas Company, Philadelphia, P a .
‘rlt
o general exchanger t j pes-cation exchangers deriFing activity from sulfonic acid groups a n d a n i o n exchangers possessing polyamino s u b s t i t u e n t s as active exchange centers-are responsible for m a n j of t h e purification a n d isolation processes a n d a n a l ) tical procedures now i n general use i n t h e pharmaceutical field. EFen greater expansion of t h e application of exchange p h e n o m e n a t o t h e purification of products possessing phj siological or biological acti5 i t 3 is p r d i c t e d , moreoier, b j t h e performance of two new s)nthetic adsorbents. One resin, Amberlite IR C-50, is a high c a p a c i t j , bead-form c ~ r b o x j l i cacid t j p e cation exchanger; t h e o t h e r , known as krnberlite IR 4-400, i q a 9tronglj basic
anion exchanger. The advantages of these t w o resins in pharmaceutical processing haFe been d e m o n s t r a t e d b j applying t h e m t o a m i n o acid separations, nllraloid recm ery, isolation of t h i a m i n e , a n d t h e concentration o f miscellaneous c o m p o u n d s s u c h a s adenine, pyridine, hydrazine, and a m m o n i a . New ion exchange m e t h o d s a r e described for concentrating a n d separating a m i n o acids i n t o t h e i r varioiis charge groups. Nigh capacity, elution elficienq , a n d buffering ability were found t o be t h e o u t s t a n d i n g properties of Amberlite I R C-50, whereas strong basicitj a n d salt-splitting capacity were determined t o be t h e two m o s t interesting features of Amberlite IR A-400.
-
ISCE the early work of Folin (8) and \Vhit,ehorn
ion exchange principles have been utilized to investigate many problems in the drug industry. Prior t o the development of synt,hct,ic adsorbents, siliceous gel zeolites were eniployed extensively in pharmaceuticnl research, particularly in the development of simplified analytical control procedures. Typical methods based on exchange adsorption techniques include the determination of ammonia ( 8 ) , adrenaline and epinephrine ( I b ) , and vitamin B, (fI). With the advent of synthetic exchaiigerj in 1940, the scope and versatility of the ion cxcharige process increased considerably. Anlino acids, vitamins, allraloids, and antibiotics a r ~ ariiong the many physiologically active products which have baeri isolated or purified with synthetic resins. These applications and others of interest in drug processing are described in some detail by Applean-cig ( 1 ) in a recent extensive revics~. AMINO ACID §El’ .\RATIONS
One of the more perplexing problems n-hich for years has o m fronted the pharmaceutical industry involves the isolation and purification of the natural amino acids. Since the first cominercial appearance of synthetic exchangers, various investigators have explored their usefulness in solving lhis difficult riddle. In 1942, Block (3) and Freuderiberg ( 0 ) reported successful laboratory usv of sulfonic acid type cation exchangers t o separate the basic amino acids (arginine, histidine, and lysine) from protein hydrolyzates. Later, Wieland (f6),Cannan ( 6 ) , Eiiglis and Fiess ( 7 ) , Cleaver et al. ( 6 ) , and Block (2) reported further work viith both cation and anion adsorbents in separating into different charge groups the basic, neutral, and acidic amino acids. Use of resin exchangers in amino acid analyses, as ~vellas large scale isolation procedures, has progressed dowly, however, because of certain inherent disadvantages in the cation and aiiion eschanger types now commercially available. Although the development of the sulfonate adsorbents overcame the objectionable feature of siliceous exchangers in that they are stable in acid media and can be uscd in the hydrogen form, the acid strength of the synthetic cation exchangers is so great, that very high concentrations and extremely large volumes of strong, mineral acids are required to effect desorption of organic bases from the resin (12). In contrast, the commercial anion wchangers are weak bases and exhibit greatest affinity for the hydroxyl ion. Therefore, they show appreciable capacity for anion adsorption only in acid media (pH range 1 to 5 ) and their effec,-
460
tiveness in amino acid isolations from protein hydrolyzates is limited t o the dicarboxylics, glutamic, and aspartic acids. Cannan ( 4 ) has provided a lucid dem-iption of an ideal method for utilizing ion exchangers to separate amino acids i n t o groups of the same charge type:
X basic resin (IZOII) n-ould be added to the hydrolyzate of a protein l o bring the p H close to the isoelectric point of the inorc basic amino acids (pH 9 to 10). This would remove t,he dicarboxylic acids (and perhaps, srnall amounts of neutral amino acids), together with the acid which had been used t o effect hydrolysis. The filtrate would then be treated with an aci$ic. resin (RH) t o reduce the pH to about 4 t o 6, thereby removing the basic amino acids and ammonia. It might be possible, indeed, to conduct this step in two stages. Addition of RH t o p1-I 8 should remove arginine arid lysine and a further addition to pII 5 should adsorb the less strongly basic histidine. The solut,iori which remained would contain the neutral amino acids uncontaminated by foreign reagents and could bc direciJy submitted to chromatographic analysis in charcoal or other columns. Thr basic and acidic amino acids could be recovered from the resins as hydrochlorides by elution with hydrochloric acid. This attractive scheme requires an acidic resin with weaker acrid properties than the sulfonic acid resins and a basic resin with stronger basic properties than those presently in use.
CAPACIIV IN MILLIEQUIVALENT$ P i R GRAM
Figure 1. pH T i t r a t i o n Curves for Weak a n d Strong Acid Type Cation Exchangers Titration in 1 IV potaaeium chloride soltition
INDUSTRIAL AND ENGINEERING CHEMISTRY
March 1949
The recent availability of a strong base anion exchanger and a weak acid cation exchanger complete a series of exchangers which meet the requirements of Cannan's ideal amino acid separation scheme. One of these products, designated commercially as Amberlite I R (2-50, is a high capacity, unifunctional, carboxylic acid type cation exchanger produced as white spherical particles. Behaving as a typical weak acid, Amberlite I R C-50 possesses buffering capacity in the pH range 6.0 to 8.0. The exchange properties of this new resin are demonstrated clearly in Figure 1 which compares the titration curves of the free acid form of Amberlite I R C-50 with the hydrogen derivative of Amberlite IR-100, a commercial sulfonic acid type cation exchanger. The second new exchanger, Amberlite I R A-400, is a strong basic anion exchanger produced as light-tan granular particles. Containing readily exchangeable hydroxyl groups, Amberlite I R A400 is essentially unifunctional, easily splits neutral salts, and effectively neutralizes the weakest acids-namely, silicic, boric, phenol, and hydrosulfuric. A comparison of the titration curves of Amberlite I R A-400 and Amberlite IR-4B, a typical weak-base anion exchanger, is shown in Figure 2. ADSOHPTION O F BASIC AMINO ACIDS BY AMBERLITE IR C-50
To establish the optimum operating conditions for Amberlite I R C-50 in this application, the individual amino acids first were employed. Beoause of the high cost of pure lysine, histidine, and arginine, microexchange columns were used to minimize the quantities of the various amino acids required for capacity determinations. Two-ml. pipets served quite satisfactorily as exchange columns. A diagram of the apparatus is shown in Figure 3. Adsorption studies were performed using an exhausting flow rate of 0.268 ml. per minute per ml. of exchangematerial. Effluent characteristics were ascertained with a ninhydrin spot test sensitive to a microgram of amino acid. The analysis was performed in the following manner: Two drops of the column effluent were adsorbed on a sheet of Whatman No. 40 filter paper and a drop of ninhydrin solution (20 grams per liter) added to the center of the moist spot, The paper was then dried on a hot plate while being held 1 to 2 inches from the heated surface. The intensity of the developed blue color was compared with standards prepared by a similar technique. Table I gives the results of the investigation, The amino acid
461
I
concentrations employed were approximately 1 mg. per ml. of solution. To convert a column of AmberIO-MILLILITER lite I R C-50 to a mixed BURET salt-acid form, a normal solution of sodium acetate buffered with acetic acid was employed. The treatRUBlER TUBE ment involved first converting the resin t o the sodium form with 4% sodium hydroxide, followed by treatment with the buffer 1-MILLILITER GRADUATED solution (3 volumes per PIPET volume of resin) a t a low I]ESIN BED rate of flow. The last portion of the buffer was allowed to remain in contact with the exchanger for 15 minutes. Then -!,he residGLASS CHIP ual solution was rinsed Figure 3. Micro-Ion Exfrom the resin bed with change Assembly deionized water. The data reveal pretreating the resin with a buffer a t p H 4.70 is necessary to achieve adsorption of all the basic amino acids. Lysine and arginine only are adsorbed if the exchanger is buffered to pH 7.0 prior to use. Figure 4 depicts the C/Co (effluent concentration + influent concentration) curves to be expected for the adsorption of lysine, arginine, and histidine by Amberlite I R C-50. Buffering the exchanger is necessary to achieve appreciable cation exchange and yet maintain appropriate balance between the salt and acid forms of the resin. If the salt-acid ratio is not controlled carefully, hydrolysis of the salt form of the exchanger will cause the p H of the influent solution to rise above the iqoelectric points of the basic amino acids where they no longer exist in cation form. Further examination of Table I reveals that the neutral and acidic amino acids are unaffected by Amberlite I R C-50 under the conditions recommended for complete adsorption of the basic amino acids. One of the most important features of the carboxylic acid type exchanger, its affinity for the hydrogen ion, is extremely useful in effecting desorption of the bound amino acids. Two ml. of 0.1 N hydrochloric acid per ml. of resin facilitates complett: removal of the adsorbed lysine, arginine, and hi'stidine. Of commercial significance is the high capacity Amberlite I R C-50 exhibits for the basic amino acids. It is apparent from the data of Table I that 1 cubic foot (40 to 48 pounds) of the exchanger will hold approximately 2 pounds of histidine, 7.5 pounds of lysine, and 9 pounds of arginine a t saturation. While this investigation was in progress, a report by Hems et al. (10) describing performance of several ion exchange resins
L-
TABLE I. BASICAMINOACIDADSORPTION O N AMBERLITE I R C-50 CapacityAmino Acid Histidine Lysine Arginine Histidine 1
I
I
I
I
1
Lysine
Figure 2. pH Titration Curves for Weak and
Strong Base Anion Exchangers
Titration in 1 N potassium chloride solution
Arginine Leucine Glutamic aoid
Column Pretreatment H-Rosin H-Resin H-Resin Column buffered pH 5.75 pH 5.30 p H 5.0 pH 4.7
Column buffered, p H 7 . 0 Na-resin Column buffered, PH 7.0 Na-resin pH 4.70 buffer PH 4.70 buffer
Leakage,
%
25
25
25 10 5-10 2-10 0-3 ca-100
0-2 30 100 100
MK./
G./
Cu. Ft. Very low Very low Very low
h11.
Very low T'ery low 510 18 35 980 120 3400 Very low 150 4248 Nil Nil
Nil Nil
462
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 41, No. 3
acids achieved in desorption. However, for a cornmercial process it is recommended that a dilute mineral acid such as sulfuric be employed. ROLE OF AMBERLITE IR-4B IN AMINO ACID SEPARATIOKS
Since Amberlite I R A400 is so strorigly basic that all amino acids but arginine are adsorbed b,v, the resin, aweak-base type anion exchanger t,o facilitate separation of the dicarboxylic (glutamic and aspartic acids) from the neutral (monoaminomonocarboxylic) amino acids should be useful. A resin of this type has been employed previously for the quantitative isolation of glutamic and aspartic acids from a protein hydrolyzste (b), MiLllLITERS OF SOLUTION I H P O U O H P U I PER MllllLITER O I RESIN , and, merely as a check, Amberlite IR-4B, a typiFigure 4. Effluent Concentration Histories cal weak-base anion exchanger, was evaluated C o l u m n adsorption of histidine, lysine, a n d arginine by Amberlite IR C-50 for the adsorption of leucine and glutamic acid. By employing the identical technique used with -4mberlite I R -4-400, it was found that the capacity of Amberlite TABLE II. -4DRORPTION O F AXIN0 ACIDS BY .$MBERLITE I R A-400 IR-4B for leucine was nil and for glutamic acid, 100 mg. per mi. (OH) (2830 grains per cubic foot). Thus, a weak-base anion exchanger Capa c 1t y may be utilized to separate the neutral from the acidic amino Amino pH (160Leakage, Mole/ Mg./ G./ llcid electric) 73 RI1. ml. cu.ft. acid fractions in a protein hydrolyzate. Elution of glutamic acid Arginine 100 10.8 0 0 0 from Amberlite IR-413was found to be as simple as its removal 9.7 Lysine 5 0.165 26.1 73 9 from Amberlite I R A-400. 4 fifleenfold concentration of the Histidine 7.6 Trace 0,242 54.8 1550 6.0 Leucine None 0.513 67 2 1990 amino acid %vasachieved by treatment with a 0.1 ?odium aro3.0 Glutamic acid None 0.645 94.8 2690 tate-acetic acid buffer a t pII 4.0. in amino acid adsorption was published. This work included evaluation of a carboxylic exchanger, Zco-Karb 216. The resin showed some promise, but low capacity precluded any consideration of its commercial application. Sulfonic acid exchangers also were examined, and although they exhibited good adsorption capacity, the amino acids were removed from the exchanger with great difficulty.
COMPLETE AMIRO ACID SEPARATIOV SCHEME
Careful assessment of the various resin exchangers heretofore described leads to formulation of several schemes for separating
TABLE 111. RECOVERY OF ~IISCELLANEOUB SUBSTANCES WITH AMBERLITEIK, C-50 Capacity
AMBERLITE IR A-400 IN AMINO ACID SEPARATION
With Cannan's ideal amino acid separation scheme in mind
( 4 ) , the behavior of Amberlite I R A 4 0 0 toward various amino acids was investigated. Evaluations were conducted using techniques identical to those previously described for the study of the carboxylic acid cation exchanger, Amberlite I R C-50, in ainino acid separations. Prior to treating the resin with the appropriate amino acid, Amberlite IR A-400 was converted to the hydroxyl form (free base) by leaching with 4% sodium hydroxide. Residual caustic was removed from the adsorbent by rinsing with deionized water. Table I1 summarizes the results of the investigation. These data indicate that Amberlite I R A-400 in the hydroxyl form is capable of adsorbing all of the amino acids having an isoelectric point below pH 10. That is, the resin has a definite adsorption affinity for all amino acids except arginine. This is shown more clearly by the C/CO curves illustrated in Figure 5 . From the tabulated information it is apparent that with Amberlite I R A-400 a clear-cut separation of lysine from arginine can be achieved, an operation which is more difficult to perform with Amberlite I R C-50. After exploring the adsorption characteristics of the anion exchanger, elution of the amino acids was investigated using various desorbing reagents. Water saturated with carbon dioxide removed the adsorbed amino acids, but large volume^ of solution were necessary. Sodium acetate-acetic acid 0.1 N buffer (pH 4.0) proved to be a most efficient eluting reagent as indirated by a ten- to twentyfold concentration of amino
Substance lorrn Recovered of IR C-60 Quinine sulfate Na Nicotine Nicotine Thiamine hydrochloride Na Thiamine hydrochloride Na Na Adenine sulfate Adenine H Pyridine H Hydrazine H Ammonia H Sodium hydroxide H
iia
10
20
30
40
Solvent
H 2 0
HJO Hz0 H20 CzIlaOH
W
nil.
cu. f t .
1320
37,400
100 0-2 5-8 25 100
....
, . . .
385
53,s
..
100 0 0-2 0
i4
80
10,950 1,510
,...
....
,
, . . . '
51.2 78 13
0
70
-
G.1
Mg./
'A 2
€130
HzO HzO HzO CzHrOH x20
60
Leakage,
90
MllliLlllRS OF SOLUTION THXOUGHPUT PER M l l l l l l T E R OF RESIN
Figure 5. Effluent Concentration Histories C o l u m n adsorption of a m i n o acids by Amberlite IR A-400
3'35 1,450 2,210 369
IW
March 1949
INDUSTRIAL AND ENGINEERING CHEMISTRY
and isolating amino acids from protein hydrolyzates. Not only is it now possible to follow Caunan’s suggested method, but it is also feasible to employ an alternate approach. Figure 6 depicts separation of a n amino acid mixture into the three charge groups (acidic, basic, and neutral) and also illustrates a technique for separating the basic amino acids from each other. The method as outlined will not effect quantitative separation of the various amino acids since it is known that the weak-base anion exchanger can not remove all of the glutamic and aspartic acids present in a mixture containing arginine, histidine, and lysine. Nevertheless, the scheme indicated a different treatment of a knotty problem which to date has defied practical commercial solution.
PROTEIN HYDROLYZATE
463
’
A AMBERLITE
1
IRC-SO
buffered pn 7.0
.
hirlidine and neulrds porr
mnd I m i n e adsorbed
arginine
lhroush
4
AMBERLITE IRC-50 buffered pH 4.7
1
i
desorb w i l h
nu I
.c
AMBERLITE IRA- 403 histidine
wtlh HCI clule w i l h a(e(mle buffer
ADSORPTION OF MISCELLANEOUS SUBSTANCES B Y AMBERLITE IR C-50
The high exchange capacity and elution efficiency exhibited by Amberlite I R C-50 for the basic amino acids led immediately to a n investigation of the resin’s behavior toward a number of miscellaneous organic compounds. The isolation of aIkaloids and vitamin B1 is of considerable interest, for the commercial sulfonic acid cation exchangers have been used in such applications with only limited success because of the previously described difficulties experienced in removing the products from the exchanger after adsorption (12). The use of Amberlite I R C-50 for isolating and concentrating these complex organic bases is summarized in Table 111. Quinine, nicotine, thiamine, and pyridine were analyzed nephelonietrically using phosphotungstic acid. Hydrazine and ammonia were determined by Nessler’s tests. The C/Co curves obtained from the above information are shown in Figure 7. These data show that it is necessary to select the proper derivative of the exchanger to obtain satisfactory adsorption. Whether the hydrogen or sodium form of the resin is employed for an application depends entirely on the nature of the compound to be adsorbed. Substances present in solution in the form of salts are picked up more readily if Amberlite I R C-50 is in the salt form, whereas those compounds existing as free bases are adsorbed more easily if the resin is employed in the hydrogen form. These observations are consistent with the acid-base charactcristics of the carboxylic acid groups in the exchanger. When the resin is utilized as the free acid, provision must be made to remove acid formed during exchange before any appreciable adsorption capacity will be realized. If the compound or compounds being exchanged are relatively strong bases, this prerequisite can be met. If they are weak bases, some difficulty will be experienced, for hydrolysis of the resin salt during exchange effects formation of the strong base, sodium hydroxide. I n such
Figure 6.
Separation of Amino Acids w i t h Amberlites IR (2-50 and IR A-400
cases, the hydrogen derivative of the resin will be preferred. For weak bases ( ~ K B lo), adsorption will be negligible under any conditions. Another significant point shown by Table I11 is the exchange activity of Amberlite IR C-50 in ethanol. The resin’s ability to swell in solutions such as water, ethyl alcohol, acetone, isopropyl alcohol, dioxane, and ethylene glycol indicates the adsorbent may possess appreciable capacity in all these solvents. CONCLUSIONS
The present investigations point to the properties possessed by two new synthetic exchangers, Amberlite I R C-50 (weak acid) and IR A-400 (strong base), in the adsorption and recovery of a variety of compounds mainly of interest in the pharmaceutical field. Coupled with the sulfonic acid cation exchangers and weak-base anion adsorbents, Amberlite I R C-50 and IR A-400 extend appreciably the scope and flexibility of ion exchange as a unit process. ACKXOW LEDGMENT
The authors are pleased to acknowledge the assistance of Ruth Barry in laboratory studies and the aid of F. X. McGarvey, R. P. Goodale, and R. E. Tenhoor in constructing the graphical illustrations. BIBLIOGRAPHY
(1) Applezweig, N., Ann. N . Y . Acad. Sci., 49 ( 2 ) , 295 (1948) (2) Block. R. J.. Chem. Revs.. 38. 501 (1946). (3j Block; R. J.; Proc. Soc. kzptZ. Bioi: M e k , 51, 252 (1942). (4) Cannan, R. K., Ann. N . Y . Acad. Sci., 47 ( 2 ) , 1 -.-5-4(1946). (5) Cannan, R. K., J . Biol. Chem., 152,401 (1944). \----,
(6) Cleaver, C. S., Hardy, R. A., Jr., and Cassidy, H. G., J. Am. Chem. SOC.,67, 1343 (1945). ENG.CHEM., (7) Englis, D. T., and Fiess, H. A., IND.
MltlltllERS OF SOLUTION lHROUGHPU1 PER MltlltllfR OF RESIN
Figure 7.
Effluent Concentration Histories
Column adsorption of thiamine, nicotine, and quinine by Amberlite IR C-50
36, 604 (1944). (8) Folin, O., and Bell, R. D., J . Biol. Chem., 29,329 (1917), (9) Freudenberg, K., Walch, H., and Molter, H., Naturwissenschaften, 30, 87 (1942). (10) Hems, B. A., Page, J. E., and Walker, J. B., J . SOC.Chem. Ind., 67, 77 (1948). (11) Hennessy, D. J., and Cerecedo, L. R., J . Am. Chem. SOC.,61, 179 (1939). (12) Herr, D. S., IND.ENG.CHEM.,37, 631 (1945). (13) Myers, F. J., U. S. Dept. Commerce, OTS Rept., PB 42802 (1946). (14) Topp, N . E., Brit. Intelligence Overseas Service Doc. 1901, 569, 35, H. M. Stationery Office, London (1946). (15) Whitehorn, J. C., J . Biol. Chem., 56, 751 (1923). (16) Wieland, Die Chemie, 56, 213 (1943). (17) Wieland, T., BeT., 77, 539-41 (1944). RECEIVED August 9, 1948.
