Extraction of Ascorbic Acid from Plant Materials Relative Suitability of Various Acids J . D. POh-UTI>-G Western Regional Research Laboratory, Bureau of Agricultural and Industrial Chemistry, U. S. Department of Agriculture, Albany, Calif.
T
HE stabilization of ascorbic acid during it's extraction
introduce an appreciable error due t o bleaching of the dye. This optimum concentration mas determined for each acid as follo1vs :
and determination has long been a problem. X a n y different est'racting acids have been recommended, such as trichloroacetic acid b y Birch, Harris, and Ray (S), acetic b y Ressey and Kink (e),metaphosphoric by Fujita and Imatake (6), oxalic by K a t a n a b e ( I C ) , sulfosalicylic b y Okrent and Wachholder (13), nietaphosphoric plus trichloroacetic by Xusulin and Kiiig ( l a ) , metaphosphoric plus sulfuric b y Mack and Tressler ( I I ) , and various other mixtures. Of these extractants metaphosphoric acid appears t o be in most general use a t present, and has been found in this laboratory b y Loeffler and Ponting (9) to be satisfactory for extracting a wide range of plant materials. However, t'here is little information in the literature on the relative ability of the various acids to stabilize ascorbic acid solutions, and there is some difference of opinion as to rvhich acid is most satisfactory for the purpose.
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The rate of drift in galvanometer reading of the standard 1 9 mixture of acid and dye e as measured (with the Evelyn colorimeter) in the absence of ascorbic acid, using a series of different acid concentrations. That concentration of acid was selected which caused a drift in galvanometer reading of not over '/2 division per minute (1 division = 1 per cent transmission), since in the determination the readings are made in 15 seconds to the nearest division. Therefore, there was no error caused by fading of the dye. The acid concentrations selected are listed in Table I. These concentrations are only approximately correct, since a variation of about 5 per cent is not important. They are for use with pure ascorbic acid solutions and do not apply t o the extraction of plant tissue. In the latter case the acid concentrations can be at least doubled without causing bleaching of the dye. With t'he above criterion of suitable acid concentration, sulfuric acid can be ruled out a t once as unsat'isfactory, since itJhas t'he peculiar property of causing rapid bleaching of the dye even at concentrations too low to turn the dye red, and far too low to st'abilize ascorbic acid. I n determining the stabilizing effect of each acid on ascorbic acid t,he following procedure was employed :
Watanabe (14) first recommended oxalic acid a3 the best extractant for ascorbic acid, but in a later paper (15) he advised the use of a mixture of metaphosphoric and oxalic acids. Lyman, Schult,ze, and King (10) compared metaphosphoric, orthophosphoric, sulfuric, and hydrochloric acids as extractants in the presence of added copper and found metaphosphoric acid the best of this group. Willberg (16) compared the stability of ascorbic acid in oxalic, acetic, citric, and tartaric acids and found oxalic acid to be the best of the group. Krislinamurthy and Giri (8) compared the stability of ascorbic acid in many carboxylic and hydroxy acids in the presence of acetate buffer and copper, and found t,he stability greatest in oxalic acid. bIetnphosphoric acid was not included in this comparison. Krishnamurthy ( 7 ) studied further the retardation by oxalic acid of ascorbic acid oxidation (copper, iron, and enzyme-catalyzed), also employing acetate-buffered solutions of pH 5.6. He stated that the degree of retardation produced by oxalic acid is much higher than that produced by either metaphosphoric acid or sodium pyrophosphate at corresponding concentrations, but gave no supporting data. The experiments herein described were undertaken in order t o establish the relative merits of m'ost of the recommended acids and also other common acids as extractants of :tscorbic acid, with the possibility of finding a suitable subst'it,ute for metaphosphoric acid. $nother readily available acid would be desirable because of the present difficulty in obtaining metaphosphoric acid and because of its expense.
Approximately 8 nig. of crystalline ascorbic acid were xeighed and dissolved in acid of suitable concentration as determined above, to give 260 ml. of solution containing about 3.2 mg. of ascorbic acid per 100 ml. Tn.0 50-ml. portions were set aside in stoppered 250-ml. flasks at room temperature (about 23" C.). Since it, is a-ell knovn that copper catalyzes the oxidation of ascorbic acid, copper sulfate pentahydrate was added to another M. 100-nil. portion to give a cupric-ion concentration of This solution also was divided into 50-ml. portions and set aside. The remaining 50-ml. portion of the original solution was used to determine the pH and initial concentration of ascorbic acid. After 2-1 hours the residual ascorbic acid was measured in the
TIBLE I.
Experimental The method used for the ascorbic acid determinations in making these comparisons Tvas that of Loeffler and Ponting (9), in lyhich the ascorbic acid is determined in dilute unbuffered acid b y measuring the reduction of the dye 2,6dichlorophenolindophenol m-it'h a photoelect'ric colorimeter. Since the method employs a pure acid extractant without added buffer, the direct comparison of acids is simple and less interpretation of results is necessary than in the case of buffered extractants. Too high a concentration of any of the acids causes bleaching of the dye (in the absence of ascorbic acid) and therefore makes the determination difficult and subject to error because of the resulting drift in the galvanometer reading. Since the effectiveness of the various acids in stabilizing ascorbic acid in solution tends t o increase with their concentration, i t is desirable t o use the greatest concentration t h a t does not 389
IA05.sO F L k 5 C o R B I C r l C I D FROM P V R E
VIRIOUBACIDS
.Id
pH
0 , 5 % metaphosphoric 0 . 2 % oxalic
2.10
Iriitiai Ascorbic .Icid MY. % 3.26
1.86
3.24
0 . 0 7 % perchloric
1.84
3.24
1% citric
2.17
3.32
0.5$, tartaric
2.21
3.26
0 . 0 7 % nitric
1.88
3.23
5% acetic
2.30
3.28
0. 2vo maleic
1.97
3.32
0 . 5 % sulfosalicylic
1.64
3.26
0.2% trichloroacetic 1% lactic
2.07
3.22
2.01
3.26
0.07% hydrochloric 1 . 6 8 0. ;porioorthophos1.87
3.24 3.26
Water
3.29
4.27
Yo Copper Added .Iscorbic acid after hv. 24 hours loss Jfg.
%
3.16 3.18 3.14 3.16 2.76 2.78 2.58 2.46 2.42 2.46 2.50 2.50 2.16 2.34 2.05 2.22 1.79 1.77 1.71 1.72 1.39 1.33 1.34 1.17 0.45 0.45 0
SOLUTIOSS
IS
10-4 If Copper Added Ascorbic acid a f t e r Av. 24 hours loss
%
.vo.%
%
2.8
2.79 2.79 2.92 2.92 0.17 0.10 0.08 0.08 0.17 0.17
14,4
2.8 14.5 24.1 24.5 25.7 31.4 35.6 45.4 46.6 58.3 61.1
86.2 100
0.18
0.18 0.03 0.00 0.00
0.04 0.71 0.71 0.08 0.08 0.65 0.55 0.02 0.02 0.02 0.00
0
9.9 95.7 97.6 94.8 94.4 99.5 99.4 78.3 97.5 81.6 99.4 99.7 100
INDUSTRIAL AND ENGINEERING CHEMISTRY
390
solutions which had been set aside. The solutions without added copper were tested for copper with sodium diethyldithiocarbamate and found t o give negative results in all cases. The solutions were made up in water twice distilled from glass and giving no test for copper even when concentrated 1000 to 1.
Vol. 15, No. 6
orthophosphoric acid with a pH of 1.87 shows a loss of 86.2 per cent under similar conditions. Oxidation of pure ascorbic acid in water solution was complete at p H 4.27, although the water contained a copper concentration of less than 5 X 10-9 M . One hundred milligrams The results of these experiments are listed in Table I. of the ascorbic acid in 10 ml. of water gave a negative test for The only acids showing low losses with pure solutions of copper; this was about 300 times the concentration used in ascorbic acid are seen to be metaphosphoric and oxalic. the experiments. These acids therefore were used to extract the ascorbic acid From the results listed in Table I, it is evident that the from a series of vegetables and fruits in order t o check the only acids of the group listed which are satisfactory for the consistency with which the acids exerted a stabilizing effect extraction of ascorbic acid are oxalic and metaphosphoric. in different plant extracts and in the presence of ascorbic Oxalic acid seems to be the more effective of the two in inacid oxidase. hibiting oxidation in the presence of copper, whether ascorbic acid oxidase is present or not (Tables I and 11). K i t h some Twenty-five t o 50-gram sam les of plant tissue, depending on vegetable extracts in oxalic acid (Table 11) the loss of ascorbic its ascorbic acid content, were hended in a Waring Blendor for 5 acid in the presence of added copper was less than in its minutes in 450 ml. of extracting acid of concentration shown in Table I1 and suction-filtered. About 125 ml. of filtrate were absence. No explanation is offered for this, but i t appears obtained and two 50-ml. portions were set aside in 250-ml. glassto be a rather consistent phenomenon. I n the absence of stoppered flasks. The pH and original ascorbic acid were detercopper the losses of ascorbic acid in plant extracts were mined using the remaining 25 ml. of filtrate. In the case of generally a little less in metaphosphoric than in oxalic cabbage, broccoli, and strawberries more filtrate was obtained and two portions also were set aside with added cu ric ion at a acid. concentration of M. After 24 hours the ascor&icacid was I n a series of tests with plant extracts in 0.5 per cent oxalic measured in the flasks which had been set aside. The results of acid and 1 per cent metaphosphoric acid, the losses were these experiments are shown in Table 11. generally a little less in oxalic than in metaphosphoric acid. The concentration of either acid can be markedly increased in the presence of plant extracts without causing bleaching of TABLE 11. Loss OF ASCORBICACID FROM EXTR-LCTS OF PLANT the dye, b u t even in vegetables having a high p H it has not MATERI.4L.9 IN OXALlC AKD METAPHOSPHORIC ACIDS been found necessary to use a concentration greater than Ascorbic Acid 0.5 per cent for oxalic acid or 2 per cent for metaphosphoric p H of After 24 Av. Plant Material Extracting Scid Filtrate Initial hours Loss acid to pievent oxidation of ascorbic acid during blending. Me./IOO m1.0 7% Ordinarily a Concentration of 0.3 per cent for oxalic or 1 per Fresh cabbage 0.4% oxalic 1.79 3 56 3 14 3 16 11 5 cent for metaphosphoric acid is satisfactory, used in a ratio 0 . 4 % oxalic 4-Cu 1.79 3:56 3:18:3:20 1014 1% metaphosphoric 1.91 3.33 3.20,3.22 3.6 of 7 volumes of acid to 1 of plant material, or higher. This 1% metaphosphoric ratio has been found necessary to obtain extraction of the 2.95,2.95 1 1 . 4 cu 1.91 3.33 Frozen broccoli 0 . 4 % oxalic 1.87 3.52 3.12,3.14 11.1 ascorbic acid to equilibrium between the solid and liquid 0 . 4 % o x a l i c + Cu 1.87 3.52 3.18,3.16 9.9 1% metauhosphoric 1.97 3.37 3.08,3.06 8.9 phases (Loeffler and Ponting, 9). Ttaphosphoric Both oxalic and metaphosphoric acids prevent enzymic 3.37 1.97 2 . 8 8 , 2 . 9 6 13.4 1.67 Frozen lima 0 . 4 % oxalic 2.37 1 . 4 7 , 1.42 13.5 oxidation of ascorbic acid, as shown by an even lower loss in 2.41 beans 1% metaphosphoric 1 . 7 6 1.39.1.39 2 1 . 0 0 . 4 % oxalic Frozen peas 1.65 2.02 1.08,1.09 34.2 fresh cabbage extract, which contains a relatively large 2.17 1.11,1.17 25.0 1.52 1% metaphosphoric amount of ascorbic acid oxidase, than in extracts of peas or Frozen straw- 0 . 4 % oxalic 3.55 1.68 3.04.3.03 1 4 . 5 3.55 berries 1.68 2.97,2.97 16.3 0 . 4 % oxalic + Cu strawberries, which contain practically none. (The frozen 3.12 1 % metauhosuhoric 2.90.2.90 1.88 7.1 1% metaphosphoric vegetables listed in Table I1 were all blanched prior to freezcu 1.88 3.12 2 . 5 5 , 2 . 5 5 18.3 ing.) This is to be expected, since Ebihara (4) found no a Mg. of ascorbic acid per 100 grams of plant tissue = mg. per 100 ml. of activity of ascorbic acid oxidase below pH 4 and Engelhardt filtrate X 18.9 for cabbage, broccoli, and strawberries; and mg. per 100 ml. of filtrate X 9.8 for lima beans and peas. and Bukin (5) found none below a pH of about 3.5. As Lyman, Schultze, and King (IO) have shown, metaphosphoric acid buffers have no value in stabilizing ascorbic acid above a p H of about 3.3. This is in marked contrast t o Discussion the stability a t a high p H in buffers containing oxalic acid, As can be seen from the duplicate values listed in Table I, which has been pointed out b y Krishnamurthy (7). I n the reproducibility of results is satisfactory under the condiconfirmation of Krishnamurthy, the author has found almost tions employed and the values are comparable. With plant the same rate of oxidation in a 0.01 M ammonium oxalate extracts (Table 11) the losses in each acid are reproducible buffer at pH 5.6 as in the acid itself a t p H l.86-namely, and the initial and final values are comparable, but the about 3 per cent in 24 hours under the conditions described initial ascorbic acid values in one acid are not to be compared above-and for the measurement of ascorbic acid oxidase, a with those in the other acid because no effort was made to 0.01 M oxalate buffer of pH 6.0 is employed, in which no obtain completely uniform samples of plant tissue. With measurable nonenzymatic oxidation of ascorbic acid occurs uniform samples the ascorbic acid values are the same with in 20 minutes, even though considerable copper (up to 2 or either metaphosphoric or oxalic acid. I n either acid there is 3 p. p. m.) is present. no loss of ascorbic acid during blending for the usual 5 There is often a difference in turbidity of oxalic acid and minutes; in fact, a t the start of this Lvork a lo-minute blendmetaphosphoric acid extracts of fruit or vegetable tissue. ing period was used to promote Oxidation, but this method With some materials oxalic acid gives the clearer filtrate and was abandoned because the losses in metaphosphoric and with some the metaphosphoric acid extract is clearer. I n oxalic acids n-ere within the experimental error, even though either case the turbidities do not interfere in the colorimetric the temperature reached 45" C. method used. It is obvious that the stability of ascorbic acid solutions is Because of the instability of metaphosphoric acid, pointed not merely a function of pH but depends also on the nature out by Bessey (I), solutions of this acid must be made fresh of the acid (Table I). Thus metaphosphoric acid with a pH every few days. Oxalic acid solutions, on the other hand, of 2.1 shows a 2.8 per cent loss, in the absence of copper, and can be made in large quantities and stored indefinitely.
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14
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ANALYTICAL EDITION
June 15, 1943
Summary and Conclusion
Of t h e 13 acids compared as to their stabilizing effect on ascorbic acid solutions under conditions favorable to oxidation, only metaphosphoric and oxalic acids appeared suitable, these two acids being far superior t o any of the others and about equally satisfactory. It is concluded that oxalic acid may be safely substituted for metaphosphoric acid in the determination of ascorbic acid, thus providing a more stable, more easily obtainable, and less expensive extractant. Literature Cited (1) Bessey, O., J . B i d . Chem., 126, 771 (1938). (2) Bessey, O.,and King, C. G., Ibid., 103, 687 (1933), (3) Birch, T. W., Harris, L. J., and Ray, S. N., Biochem. J., 27, 303 (1933). (4) Ebihara, T., J . Biochem. ( J a p a r ~ )29, , 199 (1939).
391
Engelhardt, W. A., and Bukin, B. N., Bwkhimia, 2, 274 (1937). Fujita, A., and Iwatake, D., Biochem. Z., 300, 136 (1938-9). Krishnamurthy, P. V., J . I n d i a n Chem. Soc., 18, 201 (1941). (8) Krishnamurthy, P. V., and Giri, K. V., Ibid., 18, 191
(5) (6) (7)
(1941). (9)
Loeffler, H. J., and Ponting, J. D., IND.ENG.CHEM.,ANAL.
(10)
Lyman, C. M.,’Schktae, 13.D., and King, C. G., J . Bid. Chem.,
(11) (12) (13) (14)
Mack, G. L., and Tressler, D. K., Ibid., 118, 735 (1937). Musulin, R. R.,and King, C. G.. Ibid., 116,409 (1936). Okrent, A., and Wachhorder, K., Biochem. Z., 306, 6 (1940). Watanabe, K., J. SOC.Trop. Agr. Taihoku I m p . Univ., 8 ,
En.. 14. 846 (1942). 118, 757 (1937).
381
(1937). (15) Ibid., 9,’ 162 (1937). (16) Willberg, B., 2. Untersuch. Lebensm., 76, 128 (1938). BUREAUof Agricultural and Industrial Chemistry, U. S. Department of Agriculture, Outside Publication Series No. 3908.
Apparatus for Purification of Hydrocarbons bv Recrvstallization ., J
JOHN LAKE KEAY SI, University of British Columbia, Vancouver, Canada
D
URIKG the course of an investigation into the properties
of normal paraffin straight-chain hydrocarbons, it was necessary to synthesize various homologs and to obtain them in the highest possible state of purity. Purification was accomplished principally by repeated recrystallization from glacial acetic acid, since the higher paraffin homologs are slightly soluble in this acid at boiling temperatures and quite insoluble in the acid at room temperatures. A description of the apparatus used may be of particular interest to those wishing to prepare pure hydrocarbons. I n practice, the impure hydrocarbon was dissolved b y heating to the boiling point 1 to 3 grams of the hydrocarbon per liter of acid. Upon cooling, the hydrocarbon crystallized out in a mass of small, white needles. This procedure was repeated until the sample gave a constant melting point. I n some cases i t v a s found necessary t o repeat the crystallizations as many as tm-enty times, A the final melting point being approached asymptotically. I n order to facilitate the operation, which is rather cumbersome and tedious when carried out by the usual method, and to reduce the possibility of contamination from outside sources, the apparatus shown in Figure 1 was devised.
In this way it was possible to purify by recrystallization up to 15 grams of the hydrocarbon a t one time. When the acid was allowed to cool to room temperature, the hydrocarbon crystallized out and collected as a diffuse, white, cloudlike layer on top of the acid. Section F consisted of 10 em. of 2.5-em. bore Pyrex, joined to 2-mm. bore tubing. This tubing was connected through stopcock D to flask 1. The expanded section, F , was fdled with acidwashed glass wool and so packed as to provide a filter for the hydrocarbon crystals and whatever solid or insoluble impurity might be included with the sample being purified. With stopcocks C and A closed, suction was applied through B , transmitted through stopcock D, through E and F t o the bottom of flask 2. When all the mother liquor had been drawn into flask 1 from
Two 5-liter flasks were connected across a condenser, as shown. The hydrocarbon to be purified was dissolved in pure glacial acetic acid and poured into flask 2 through the condenser, vacuum being applied to the system through A and C. When this method was not practical, as in the case of hydrocarbons with high melting points, flask 2 was filled with the acid and the solid hydrocarbon was dropped down through the condenser. Flask 2 was then heated and the mixture allowed to reflux for several hours. 1
Present address, Powell River, B. C.
FIGURE 1. DIAGRAM OF APPARATUS
