V O L U M E 2 7 , N O . 6, J U N E 1 9 5 5
965
appear to be improved, the use of a large excess of oxine did not seem advisable in the procedure. DISCUSS103
Completeness of Extraction. The extractions in the present work were made from aqueous solutions of pH 5 . 7 . Experiments were conducted n-hich showed that the extraction of aluminum was as complete at this pH as in the pH range of 8.0 f 1.5 recommended hy Goon and coivorkers ( 3 ) . The estraction of gallium v a s more than 907, complete after a single extraction and was essentially 1007, complete after the second extraction, from a solution of pH ,5.i, while in Sandell’s original method ( 7 ) , where selectivity rather than completeness was desired, the extraction from :i solution nf pH 2.6 to 3.0 was 70 t o 80% romplete. Intensity of Mercury Lines. D a t a given by the General Electric Co. on the U-1-2 pliotocheniical lamp ( 8 ) show that the intenxitie3 of the variow lines of wave length less than 5000 A. dec r e a x in tlie following oi,tier: 3650, 3131, 2530, and 4358 A. Of these, tlie 3650- and 4358-A. lines were found to lie most useful in esciting fluorcrcence so that, beside3 intrinsic line intensity, alxorption and scattering it1 the optical system and alisorption characteristics of the fluorrrcent compounds are responsible for the efficary of the 4358-.1. line over the more intense C 3 6 - and 313 L-.l. lines. Although the readings for a given standard solution were essentially constant over a 3- t o 5-hour period, the readings often differed by as much as l5Y0 from day to day (see Table 111). For that reason, the use of‘ quinine was adopted as a secondary standard. The observed variations may have been due to variations iu voltage and temperature of the mercury lamp or t o changes in the operating characteristics of the photomultiplier. Zinc Oxinate. Solid zinc osinate was prepared and found to be highly fluorescent a.hen tlisolved in chloroform solution. Its fluorescence spectrum is almost identical with those of gallium arid aluminum. Though mistures of zinc and aluminum osinates were determined after dissolution in chloroform, more complete results on zinc are not included in the present study because its
osinatc was not extractable from aqueous solution into chloroform. This behavior apparently is due to water of hydration in the zinc oxinate which renders it insoluble in chloroform, even though the oven-dried product is soluble.
Table 111. Day-to-Day Variations of ,ibsolute Values of Fluorescence Oxinate“ Quinine, P.P.lI. Quinine r\bsolate equir. 0.3 3.0 1 10.3 87.3 (i? .7 2.14 10.4 85.2 2 10.1 77.2 35 n 2 10 10.0 78 8 * Oxinate e x t r a c t s contained 1.7 p.p.m. of galliuni and 0.030 i1.p.m. of ali~~~iini~iir. Uar
ACKNOWLEDGMENT
The authors are indebted t o the Xtomic Energ), Commission for partiul support of this study. LITERATURE CITED
(1) ditken, E. H., and Preedy, J. R. K., J . Endoc~irtol., 9, 251
(19531. ~I
( 2 ) General Electric Co., Lamp Dirkion, Application Eng. Dept.,
Bull. LS-103 (June 1953). ( 3 ) Goon. E., Petley, J. E., ;\IcA\lullen, JV. H.. and Wlberley, S.E., h K . i L . CHEhI., 25, 608 (1953). (4) Huke, F.B., Heidel, K.H., and Fassel, V. A , J . Opt. SOC.Amer., 43, 400 (1953); A N h L . CHEM., 26, 1134 (1954). (5) Kolthoff, I. AI., and Sandell, E. B., “Textbook of Quantitative Chemical Analysis,” 3rded., p. 321, hIacmillan, S e w York, 1953. ( 6 ) Peattie, C. G., Ph.D. thesis, Massachusetts Institute of Technology,
Cambridge, Alass., 1952.
(7) Sandell, E. B., d K . i L . CHEhf., 19, G3 (1947). (8) U. S. Pharmacopoeia, XIV Rev., p. 772. Alack Publishing Co.,
Easton, Pa., 1950.
R E C E I V Efor D reriew August 11, 1954. Accepted February 11, 1955, Presented before tile Diyision of Analytical Chemistry a t tlie lSGtli 1Ieeting of the . ~ I I E R I C . A S CHEIII(.ILS O C I E T Y , S e w I-ork, Sel,tember 19%.
Adsorption-Dialysis, an Extraction Technique Use in Recovery of Amino Acids IRVING R. HUNTER, DAVID F. HOUSTON, and ERNEST 6. KESTER W e s t e r n Utilization Research Branch,
U. 5. D e p a r t m e n t o f Agriculture, A l b a n y 70, Calif.
.Sn extraction procedure useful for recovering amino compounds from biological materials involves the combined use of adsorption and dialysis. Slurries of an adsorbent and the material are separated by a semipermeable membrane and agitated. Amino acids and other substances of low molecular weight diffuse out of the material and through the membrane. When a cation exchange resin is used as an adsorbent, compounds containing basic groups such as amino acids are selectively removed from solution and nonbasic components are rejected. The concentration of adsorbable material in the dialyzate above the resin is accordingly reduced, permitting additional amounts to diffuse through the membrane. The adsorbed substances are easily recovered from the resin by elution with ammonia. The method yields considerably larger amounts of amino nitrogen compounds in higher concentration than extraction with water or aqueous alcohol.
F
REE amino acids occurring in small amounts in biological
substances must be separated before they can be analyzed by physical or biochemical methods. Estrartions with water or 80% alcohol have the disadvantage of dissolving relatively large amounts of other soluble ingredients and requiring large volumes of estracting liquid arid tedious evaporations. The procedure described herein was devised to overcome these difficulties. It has been used in an investigation of parboiled rice and may be generally applicable to biological materials. Modifications might be upel‘ul for compounds other than amino acids. Briefly, the method comprises concurrent dialysis and adsorption. Dialysis permits entry of compounds of low molecular weight into a compartment containing a resin that adsorbs one or more classes of compounds and rejects others, depending on type. Dialysis is continued until substantially all the compounds of a specific group have migrated from the starting material to the adsorbent. Gilbert and Swallow ( 1 ) used a similar system for purifying solutions of proteins and enzymes but vere
ANALYTICAL CHEMISTRY
965
not concerned with the identity and composition of adsorbed dialyzed material. I n the present study the desired results were obtained by enclosing an aqueous suspension of a cation exchange resin having a high adsorptivity for amino acids in a cellophane bag, placing the bag in a slurry of rice and water, and agitating the entire system. The adsorbed materials, mainly amino acids, were then oluted from the resin as a relatively pure concentrate suitable for further concentration or analysis.
steaming under 15 pounds of pressure, and drying before being dehulled and milled to the equivalent of white rice. Wheat Flour. A straighbgrade, unbleached flour milled from Pawnee wheat grown in Kansas. Rice and Potato Starches. Commercial products. Wheat Starch. An experimental product obtained from Northern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture, Peoria, Ill. PROCEDURE
I n a I-liter glaspstoppered cylinder are placed 100 grams of parboiled rice, mound to vasa a 20-mesh mreen, and a celloohme
APPARATUS AND REAGENTS
Dialysis Apparatus. Glass-stoppered cylinders of 1- or %liter capacity were mounted on the face of a revolving vertical disk with the aid of three-pronged clamps. During B test, the disk was rotated a t a speed of about 9 r.p.m., by a reducing gear and a '/e-hp. motor (Figure 1).
1 .
.. . . .
..
Figure 1.
Dialysis apparatus
Membrane. Nojsx cellophane smsage casing, z z / 8 x inch in diameter (Visking Corp.). Prior to use, a length of casing was easked in water, tied a t one end, and tested for leaks with nitrogen. Elution Column. Borosilicate glass tubes 77 em. long, 16 mm. in inside diameter. The height of the adsorbent w&s anproxi-
RESULTS AND DISCUSSION
Comparison of Adsorption-Dialysis with Water and Aqueous Alcohol Extractions. Since adsorption-dialysis was proposed far use in place of water or alcohol extraction, it was necessary first to know the advantages it offered over other methods, and particularly the added yields of amino nitrogen and the concentration in the recovered solids that adsorption-dialysis would give. The following procedures were used for the water and alcohol extractions in making comparisons.
reported in this study. This material F a s in the form of small beads most of which passed a 20-mesh U. S. standard sieve. To
i[y of the resin a t least 10 times, Excess &ali and fine mat&al
converted to the acid form i h t h i n ~ X C ~ RofS 5 % suifuric acid. About 50 meq. of acid per gra,m of resin was used in this trentment. The resin was then was!hed with distilled water until the mnshings no longer gave test for sulfate, drained, and air-dried prior to use. urior For other vortions of this stiidy, Dowex 2, a u anion exchanger of tho quah-nary ammonium 1,ype, was used in the basic form. Reagents. Ammonium hy droxide ( 5 N ) was prepared by dilutinz C.P. made 15N reaeenit with distilled water. .4lcahol(86%) wa6 prep&d bv dilutinn 4 volumes of absolute alcohol to 5 volumes with disti Sodium ethylmercurithiosali< This chemical was added as a in concentrations of about 0.01 MATERIALS
Parboiled Rice. A commercial sample of California-grown Caloro rice which had been par boiled by steeping in warm water,
mixing was acoGplished"bv rotating the container on the Aixine wheel-for 24 hours. Tho residue was allowed to settle and the supernatant liquid wan siphoned off. A 500-ml. aliquot of the extract was bailed far 3 minutes to arecioitate oroteins. then centrifuged and concentrated under a ;acuhm. Determinations were made of total nitrogen (Icjeldahl), amino nitrogen (Van Slyke), and total solids. A m m o m EXTRACTION. Conditions were identical with the aqueous extraction, except that 80% alcohol was used as an extractant, and the oxtract m m separated by filtration. The extracted rice was washed with three 100-ml. portions of 80% alcohol. The total filtmie was evaporated to drynefis under a vacuum and made up to volume for analysis. Table I summsriaes results obtained by the three extraction techniques. The ratio of amino nitrogen to total solids (column 5 ) indicates the relative concentyation of amino acids in the extracts. For a mixture of pure amino acids the percentage would be of the order of 9 to 10%. The advantage of adsorption-dialysis over either alcohol or water in separating amino acids from rice is apparent from an inspection of data in Table I. By tho use of this principle, more
961
V O L U M E 27, NO. 6, J U N E 1 9 5 5 than dou!ile the amount of nniino nitrogen is obtained, :v conipared n-ith alcohol extraction, and moreover it yields an extract containing far less extraneous matter in the total solids than either n-ater or alcohol extractions. .4similar series of experiments 11-asmade on a sample of Pawnee wheat flour. The results in Table I1 are parallel n-ith those obtninrd on rice.
was intimately niixed with Dowex 50 (acid form) by tumbling for 24 hours a t room temperature. The mixture mas filtered, and the resin was washed in the usual manner. The resin was transferred t o a column and elut'ed with 5-Yammonium hydroxide. The eluate was concentrated and analyzed, as xere also thP liquid in contact with resin before elution and the untreated liitlf of the dialyzate. Table IV summarizes the results of these test,s and analyses.
The use of Dowex 50 does not cause an increase in the amino nitrogen of the dialyzate contacted by the resin, since the suni of the quant,ities present in the liquid above the resin and adsortied Table I . Comparison of Three Extraction Techniques on it does not exceed appreciably the amount found in t,he original Total S , .Iiiiino S , Total Polidin, L%~!!C, dialyzate. Extraction llg.~100 XIg./100 3 1 ~ . / 1 0 0 Total Solids lletliod G . Rice G . Rice G. Rice 0 Effect of Ammonium Hydroxide on Amino Nitrogen Content 10 7 21n 4.9 19.7 Adsorption-dials sis of Eluate. Ammonium hydroxide was used to elute amino acids 9.8 3810 0 2ii Al,\Ieous 35.4 5.1 923 0 55 ROC.; alcollol 13.6 from resin because it could be easily removed by distillation. It u .-imounts given include 10 t o 50 iiip. of a iireicri-ative. was necessary to show, however, m-hether amino nitrogen analysis of the product n-as artificially raised by lingering traces of ammonium hydroxide in the concentrate, or by the presence of Tahle I I . Extraction of Pawnee R heat Flour artifacts containing amino nitrogen formed by reaction of ani.liiiino S . Total Solids, .Imino N , I: \ t i :IC t ion 3Ig. '100 3Ig. /I00 Total Solids inonia with other compounds such as carbohydrates. T h a t no Alettiod C:. Flour C;. Flour 3 error from either cause \vas introduced was indicated by eluting 2-15 .i .7 one portion of a resin from :in adsorption-dialysis test with SA\ 4595 n.1 1233 0.47 :tmnionium hydroxide and ailother ivith 5% sodium hydroxide. The caustic solution \vas neutralized to pH i , and both eluates \\-ere the,n concentrated under a vi~cuuni. Analyses showed 11.9 :tiid 11.6 nig. of amino nitrogen in the ammonia and soda eluates, Effect of pH on Validity of Adsorption-Dialysis Extraction. respectively. l h e minor difference in these values is within the 111 developing adsorption-dialysis as a method for separating limits of experimental error, which is &5y0. :imino acids from a biological substrate such as rice, it \\-as important t o know whether the product obtained represented uncquivocally the amino acids present in the niateri:ii extracted. Table 111. Comparison of idsorption-Dialysis Extractions If, during extraction, amino acids were formed hy hydrolyPis of Vsing a Cation Exchange Resin and a 3lixture of Cationproteins or peptides, the method would have no value. -1s pH is and Anion-Exchange Resins :in important factor in hydrolysis of proteins: and as the acid forin Total of the ion exchange resin used affects the pH of the solution in the .-iriclitY Of Total X, .4mino T, Solids, kinoS, Rice ~ l L l r r Y .\Ig./100 hlg./100 AIg./100 Total extraction system, tests weye made to determine: n-hether a pH Initial Final G. G. G. Solids Rire c7, Rice Resin DH DH Rice lowering of the liquid in contact 11-ith the rice caused amino acids ," Dowex 50 6 . 5 2 8 1 1 . 1 5 . 0 185 2.7 to form from the proteins and peptides of the rice and Tvhether Dowex 50 and Dowex2 8.5 ii.4 8.2 5.1 190 2 7 peptides of low molecular weight that might dialyze through the niembrane were broken don-n a t the surface of the resin. The pH of a parboiled rice slurry is usually in the range of 6.5 Table I\'. Effect of a Cation-Exchange Resin (Dowex 50) to 6.8. During adsorption-dialysis, the pH drops to about 2.5. on Adsorbable Amino Nitrogen in a Dialyzate of Parboiled Rice To sholv the effect', if any, of pH lowering on formation of amino Total S , Amino N, Total Solids, ttcids from rice protein, the follon-ing experiments were performed: lI&/lOO lIg./IOO Mg./IOO ~~
~~~~
Adsorpt,ion-dialysis by the outlined procedure. Adsorption-dial?-sis using a resin mixture consisting of equal weights of cation and anion exchange resin. to niaintain the pH of the water a t or near its original level. This cxperimcnt x - a ~ similar in all details t o thc regular procedure, except, that the resin consisted of 20 grams of Don-ex 50, :ication-exchanger in t h r acid form, and 20 gr:ims of Doxvex 2. an anion exchanger in tlie hasic form. T h e pH of the rice slurry hefore an(! after extraction \vas t:iken \\-it11 :I Beclman pH meter. The results of t h ( w tcst? :ire wniniarized in Talile 111. Co1uniii.q 5 nritl 7 indicnte t h a t thr pH of the slurr!- does not affect t h c qu:intit!- of nmiiio acidq ohtniried I?!- the extraction procedure from :i givcn (1u:tiitity of rirc nor its ratio to the extracted total solids.. A higher tOtd nitrogen was o1jt:tined \\-ith Dowex 50 alone t l i m n.itli tlie mixed resin?: the reason for tlii. difference is not clear, :ind thc tests n'c>renot rcplicnted. T o detcrminr in addition \\-hether lon-ering tlie pEI a t the surf:rce of thc ioti exchange refin increased the ainouiit of amino nitrogen availa1)lc and :iclsortwl from a dial>-zate,measurements w r e made of tlicb :mino nitrogen in a portion of fresh dialyzate :ind of the amino nitrogen adsorbed and unadsorhed by cation (.\-change resin from a second portion of the dialyzate. Three hundred grams of ground parboiled rice were mixed with 1500 ml. of water and dialyzed against 1500 ml. of water in cellophane bag? for 24 hours at, room temperatures. The liquid in the \lags \\-as then removed and conihined. On? half of the solution
lIateria1 Dialyzate Resin eluate Liquid above resin
MI.
19.5 12.5 5.8
311. 8.6
7.3 1.5
~
MI. 1434 185 I134
EFFECT O F PROCESS VARI.ABLES
The follon-ing studies were undertaken to evaluate the effect of several process variables on the adsorption-dialysis method. Particle Size of Rice. Four 100-gram samples of parboiled rice ground to pass 80-, 60-, 40-, and 20-mesh screens were extracted for 30 hours by the described adsorption-dialysis technique. The resulting extracts were concentrated arid analyzed for amino nitrogen. T h e results of these experiments are shown in Table V. The results shojr that' efficiency of extraction is independent of particle size n.ithin the range studied for a 30hour extraction. S o study as made of the effect of particle size of resin. Time of Dialysis. Samples of 100 grams of ground parboiled rice n-ere extracted by'aclsorption-dialysis for 4, 7.5> 16, and 23 hours. The extracts after suitable processing were analyzed for amino nitrogen. Results are shown in Table VI. It would appear from the figures in column 2 that under the conditions used, the amount of amino nitrogen recovered reached a near maximum a t 23 hours. As other unreported experinleiits showed that little addit,ional amino nitrogen was obtained Ijy prolonging extraction
ANALYTICAL CHEMISTRY
968 to i 2 hours, a 24-hour period was considered adequate and was
used thereafter. Fractional Elution of Resin Column. After an extraction of a parboiled rice sample for 24 hours by the adsorption-dialysis method, the resin was eluted with 500 ml. of 5 5 ammonium hydroxide incrementni.v. The first fraction consisted of the neutral liquid preceding the ammoniacal eluate. By following ~ indicated when the boundary with t'he rise of pH it T V clearlleluting liquid had been reached. Thereafter, successive 100ml. fractions were collected and analyzed. The results of this test are presented in Table VII.
Table
\-.
Effect of Particle Size of Rice on AdsorptionDialvsis Extraction
Particle Time, Size Hours 30 Through 20-mesh 30 Through 40-niesh 30 Through 60-mesh 30 Through 80-mesh
Table VI.
Amino X , I\Ig./lOO G . Rice 12.9 11.7 12 4 12 0
Total Solids, llg./100 G . Rice 147 140
Amino S , hfg./lOO G. Rice 6.6 7.9 10.0 11 4
Total solids
5%
149
.Imino T i 8 ~Total solids 8 8 8 8
3 9 4 4
Table \ l I . Distribution of Amino 3-itrogen and Total Solids in a Resin Eluate Total Solids, _ Amino _~_N , .imino S . Fraction
hf1. 100 100 100 100 100
6.
rice
SO% Alcohol
Total solids,
-~ N,
Total solids
ing./,lOO g.
7%
rice
Table IX. Comparison of Adsorption-Dialysis of Parboiled Rice Sample with and without Added imino Acids Total N , Amino N , nfg./100 3Ig./100 G . Rice G . Rice Sinele 24-hr.
Total Solids, hfg./100 G. Rice
-5.
Total solids
5%
ex-
a Solution of glycine, phenylalanine leucine, a n d \ d i n e containing 13.4 mg. of nitrogen (125 m g . of solids).
Table X .
7c
__
Mg./lOO G . Rice 0.2 9.3 0.3 0.08 0.05 0.05
mAmino S . mg./lOO
8.8 8 4 8 1 8 1
1R3
Total Solids. RIg./100 G . Rice 79 89 119 136
Resin-Dialysis .Imino K, Total Total Extrac- nig,/100 solids, tion g. mg./JOO solids, KO. rice C g. rice
&I=,
Effect of Time of Extraction on AdsorptionDialvsis Extraction
Time, Hours 4 7.5 16 23
Table VIII. Successive Extractions of Ground Parboiled Rice by Adsorption-Dialysis and by Aqueous Alcohol
Ng./lOO G.
Total solids
Rice 12.5 107.8 7.5 2.0 1.3 1.3
1.6 8.6 4.0 4.0 3.8 3.8
Ratio of Rice,to Resin 7.5 5.0 2 5 2 5 1 67 1 0
Effect of Varying Ratio of Resin to Rice on Extraction , Amino S , Total Solids, _Amino _ S- ~ AI g. / 100 G . Rice 30.3 21.8 11.6 11.0 11.1 11.2
lIg./lOO G . Rice 33 1 252 136 121 122 132
Total solids
%
9 1
8 0 8 5 9 1
:.;
7c
Apparently, the major portion of amino nitrogen is eluted by the first 100 ml. of ammonia (fraction 2 of table). This fraction and the third fraction represent 96% of the total amino nitrogen accounted for in the elution, including the forerunnings, and in most instances would be adequate for characterization tests. Multiple Extractions. T o obtain greatest yields of amino nitrogen compounds from rice either by the adsorption-dialysis method or by aqueous or alcohol extraction, repeated treatments are necessary. Fresh resin must be used in successive treatments by- adsorption-dialysis. The difficulty of recovering materials quantitatively from biological substances has been mentioned by other investigators-for example, Woodward and Rabideau ( 3 ) . Table VI11 s h o w the results of a series of extractions of parboiled rice bv the t n o methods under comparison. I t is observed that after the second adsorption-dialysis extraction, further removal of amino nitrogen compounds proceeds slowly. Little difference is shown between the amounts obtained in fractions 3 and 5 . Although one extraction does not completely remove amino acids from rice, the capacity of the resin t o adsorb acids is not fully utilized. Experiments have shown (Table IX) that if adsorption-dialysis extractions are made on two identical samples of parboiled rice, one of which contains an added amount of pure amino acids, the resin will adsorb additional acids almost equal to the amount added. Ratios of rice to resin of 2.5 t o 7.5 yielded amounts of amino nitrogen almost proportional to the ratio used (Table X). With ratios in the range of 1.0 to 2.5, the amino nitrogen obtained was practically constant. The percentage of amino nitrogen on the basis of total solids did not varv appreciably, regardless of the proportions of rice and resin.
Comparative Retention of Amino Acids by Three Types of Starch. The foregoing results may be explained on the basis that starch itself is an excellent adsorbent for amino acids. Moore and Stein ( 2 ) used starch to fractionate amino acids on a chromatographic column. Thus in an adsorption-dialysis eytraction of rice two adsorbing media, the resin and the starch of the rice, compete for the dialyzable amino compounds. Equilibria are established which may be likened to partition coefficients. They are undoubtedly different for various amino acids and, if some of the latter are adsorbed more selectively on the starch than on the resin, their recovery by this procedure will be more difficult than for others and may require multiple resin treatments. Tests mere made to determine the comparative retention of amino acids by starches of different origin in an adsorptiondialysis system. One-hundred-gram samples of rice, potato, and wheat starch were each slurried with 1 liter of water containing 23.2 mg. of amino nitrogen as amino acids and extracted by the adsorption-dialvsis procedure. Analyses showed that 82.5, 84, and 88.5% of the amino nitrogen of the acids added in the respective tests rvere transferred to the resin, indicating that the starches were not widely different in their retentive power for amino acids. ACKNOWLEDGMENT
The authors acknowledge n ith thanks the helpful advice of H. S. Olcott, and the assistance of Geraldine Secor, L. AI. White, Marion Long, and R . E. Ferrel in the analyses reported in this investigation. LITERATURE CITED
(1) Gilbert, G. A , and Swallow, A . J., Biochena. J . (London) 47, 502
(1950). (2) Moore, S., and Stein, W. H., J . BioZ. Chem., 178, 53 (1949). (3) Woodward, C. C., and Rabideau, G. S., Plant Physiol , 28. 535
(1953).
RECEIVED for review November 1 5 , 1954. Accepted January 29, 195,5. Mention of equipment and materials by trade name does not imply t h a t they are endorsed or recommended by t h e Department of Agriculture over others of similar nature not mentioned.
