Aconitic Acid from Sugar Cane Products M. A. MCCALIP' AND ARTHUR H. SEIBERT2 Bureau of Agricultural Chemistry and Engineering, U. S. Department of Agriculture, Washington, D. C .
0.1 per cent of the total solids. Nelson (9) isolated from Puerto Rican final molasses 0.8 per cent of aconitic acid, calculated on sample (equivalent to approximately 0.9 per cent on solids). Von Lippmann (6) found aconitic acid in sugar beet products, and Beath (1) and others noted its occurrence in several species of native delphinium and aconitum. Identification of the acid by these investigators was mainly through its lead, zinc, and silver salts. Ried and co-workers (6,12) described its p-nitrobenzyl and phenacyl esters. Our investigation (7) of the sediment which has been encountered during recent years in first and second molasses (and sometimes in sirup) produced in the western portion of the Louisiana sugar belt, and of the scale which forms on the heating surfaces when the sugar liquors originating in this territory are concentrated, revealed the fact that the sediment and some of the unusual scales were composed largely of calcium aconitate. This led to an examination of Louisiana sirups for their content of aconitic acid, and to the finding that this organic acid is present in larger proportions than was formerly supposed. Hence it is possible that sugar cane juice may prove a convenient natural source of aconitic acid, the industrial development and utilization of which has been limited because the most convenient source heretofore has been the dehydration of citric acid. The opening up of such a natural source will stimulate study of this acid, which is a derivative of both succinic and fumaric acids, and of its derivatives, many of which may readily be converted intomaleic acid derivatives. Succinic and maleic acids and their derivatives are valuable intermediates in the manufacture of plastics and plasticizers. During the investigations of the sediments it was necessary to study more carefully the properties of aconitic acid, especially since various "melting points", ranging from 182' to 204' C., have been reported by previous workers and, therefore, identification by this means is uncertain. As Bruce (a) pointed out, the decomposition point obtained is dependent not only on the purity and dryness of the preparation, but also on the rate of heating and on the temperature a t which the capillary is introduced into the bath. Introduction of the charged melting point tube at approximately 10' to 12' C. below the melting point and 4 to 5 minutes before the melting temperature is reached causes the acid to melt a t a higher point. Melting takes place with decomposition and rapid evolution of gas; i t is thus difficult to obtain a sharp determination of the temperature at which melting occurs. Much of the variation in melting point reported by earlier workers is probably due to differences in technique in making the determination as well as to differencesin purity and dryness of the samples. We have found no evidence that the variations in melting point of the acid as reported by others may be due to the possibility that the natural acid is a mixture of the common, or trans, form of aconitic acid with varying proportions of the isomeric cis form. The latter melts a t 125' C. and is less stable than the trans form. Comparison
The cream-colored sediment occurring in sirup and first and second molasses tanks during recent years in certain areas of Louisiana was studied and found to consist principally of calcium aconitate. The sediment was analyzed, and a method of separating aconitic acid from it and from related materials and of purifying it are described. The isolated acid decomposed at 191.5' C. (194' C. corrected), and other characteristic properties are described which identify it as the trans isomeric form of aconitic acid. Refinery pan and evaporator scales were analyzed and found to contain aconitic acid. A simple test for the detection of aconitic acid in sediments and scales is described. The aconitic acid content of sirups made without chemical clarification from juices from two different types of cane grown in different localities was determined and found to range from 0.75 to 1.33 per cent on solids. Two samples of Louisiana final molasses were analyzed and found to contain 1.80 and 2.52 per cent aconitic acid.
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H E presence of aconitic acid in the juices of sugar-producing plants and in the products of sugar manufacture has been demonstrated qualitatively by many workers, but no quantitative determinations of the acid in such products had been reported previous to those of McCalip and Seibert (7). Behr (2) found i t in molasses, in muscovado sugar, and in cane juice. Parsons (10) detected it in sorghum juice from which, upon the addition of lime, the calcium salt separated on heating surfaces as a buff-colored, tenaciously adherent scale. Yoder (16) and Zerban (16) isolated aconitic acid from Louisiana sugar cane juice, and the former stated that i t is the predominating organic acid in the juice; from the data he presented it may be calculated that he obtained approximately 0.3 per cent on the basis of dry solids. Taylor (14) found i t in both healthy and diseased cane, and described the delicate color reaction it gives with acetic anhydride, a reaction which is characteristic and was later modified by Furth and Herrmann (4). Both Taylor and Yoder noted that the calcium salt is less soluble in hot water than in cold. Prinsen-Geerligs (11) found that a deposit centrifuged out of Cuban molasses contained a high percentage of calcium aconitate. Tanabe (IS) recently found that aconitic acid accounted for 90 per cent of the total acid extracted with ether from a large quantity of juice from cane variety P. 0. J. 2725. His recovery of aconitic acid may be calculated as roughly 1 Present address, Glenwood Qsrdens, 3-B Rochambeau Apartment, Yonkers, N. Y. 1 Present address, 815 School St., Houms, La.
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of the acid isolated in this study with synthetic trans-aconitic acid made by the Bruce method (3) showed that their properties were identical in all respects. The purest sample of the natural acid decomposed at 191.5" C. uncorrected (194" C. corrected). The synthetic acid gave the same decomposition point, which was not changed by admixture with the acid obtained from the sediments, scales, and sirups.
Extraction and Purification FROM SEDIMENT.Samples of the sediment collected during the 1938 and 1939 seasons were freed from adhering molasses by mixing with an equal weight of 50 per cent alcohol. The sediment was removed from this slurry by filtering and drying on a Biichner funnel. After this had been done three times, the cake was washed with 50 per cent alcohol until the washings were only slightly colored, and then was dried for 15 hours a t 70" C. in a vacuum of 28 inches (71.1 cm.). The dry material was ground, passed through a 50-mesh screen, and again dried for 5 hours. Average recovery was 44.3 per cent of insoluble dry sediment based on the original sample. The amount of 50 per cent alcohol necessary for satisfactory washing was equal to two and one half times the weight of the original sample. The sediment was only slightly soluble in water. The dry sediment collected during the two seasons had the following percentage composition: 1938 Sample 2.35 0.78 15.66 4.08 0.13
9.78 64.64
1939 Sample 3.58 0.66
13.58 1.25 Trace 11.98
65.21
Since practically no carbonates were found before ignition, these data indicate that the material consisted principally of an organic compound of calcium. An elemental analysis proved the absence of nitrogen, organically combined sulfur, and halogens. Forty grams of the dry sediment were treated with 65 ml. of concentrated hydrochloric acid and 80 ml. of water; the mixture was heated until most of the material had gone into solution, and was then filtered hot. When the hot filtrate was cooled, a small amount of material insoluble in the cold separated out and was removed by filtration. The cold filtrate was extracted with ether and the ether was evaporated, leaving a highly acidic organic compound which was recrystallized from hot glacial acetic acid until further recrystallization did not materially alter the melting point of the crystalline material. An alternate method of obtaining the acid was by boiling the dried sediment (20 grams) with concentrated hydrochloric acid (25 ml.) and filtering hot. Upon cooling the filtrate, the organic acid crystallized out and was purified as before by recrystallization from glacial acetic acid. FROMEVAPORATOR AND PAN SCALE. Samples of scale were collected from pan coils of a refinery during 1938 and 1939, and one sample of scale was obtained in 1939 from the evaporators of a raw sugar factory. Analyses of the refinery scale for the two seasons showed the following percentage composition based on dry material: 1938 Sample
1939 Sample
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Twenty grams each of the dry finely pulverized scales were acidified, atered, washed, and extracted with ether in the same manner as the sediment. A high percentage of the crystalline organic acid was obtained which showed the same melting point and reaction as the acid obtained from the sediment. The evaporator scale was also examined for the presence of this acid. By the same procedure of extraction with hydrochloric acid and with ether as was used for the pan scales, the same organic acid was obtained but in smaller proportions than from the pan scales. Scale deposits collected from evaporators which had just been boiled out with acid contained none of this organic material.
Identification I n view of the uncertainty of the melting point of aconitic acid discussed in an earlier paragraph, other means of establishing the identity of this acid were sought. In every test, applied synthetic aconitic acid made by Bruce's method ( 3 ) and the natural acid gave identical results. Both acids had neutralization equivalents of 58.55 and both contained an unsaturated linkage reactive to potassium permanganate. COLORREACTIONS.The addition of acetic anhydride to the acids brought about the color reactions discussed by Taylor (14). Since this test is simple and specific, it is repeated here in detail. To a few milligrams of the acid in a clean dry test tube is added 1.0 ml. of acetic anhydride. Upon heating carefully over a flame, a faint pink color first appears. On further heating the color changes progressively to reddish violet, to bluish green, and finally to brown. If the heating is stopped and the liquid is cooled as soon as the green color has developed, a separation of pigments may be effected by shaking the contents of the tube with 3 ml. of ether followed by the same amount of water, the ether layer becoming blue and the water layer yellow. Similar treatment as soon as the reddish-violet or magenta color has formed gives a blue ether layer and a red aqueous layer. The colors are unstable and fade on standing. Fiirth and Herrmann's modification (4) of this color test is the addition of a drop of pyridine to the acetic anhydride and aconitic acid mixture. Immediately, and without heating, the solution turns burnt orange, then changes through red to carmine to reddish purple or claret. These deep colors appear to be much more stable than those produced without the pyridine. To detect aconitic acid in sediment and scales, add 4 drops of glacial acetic acid to approximately 3 mg. of the washed. dry material and heat gently over a light flame until one half or more of the acetic acid has volatilized in order to free the aconitic acid. Then add 1 to 2 ml. of acetic anhydride and again boil carefully with constant shaking for about 2 minutes. If aconitic acid is present, the mixture first turns pink, then magenta, and finally claret. A drop of pyridine added after the acetic anhydride makes heating a t this stage unnecessary and speeds up the color changes, but causes the production of slightly redder colors. The effect of impurities on the color reaction is worthy of note. I n the presence of large amounts of foreign material, as in testing sediments and scale as just described, the color does not develop to a green, but stops a t the reddish-purple or claret color. With purer specimens of aconitic acid, such as that isolated from the sediments and scales and recrystallized from acetic acid, the whole range of color changes takes place. But these are apparently due to the catalytic effect of small amounts of impurities, such as calcium, sodium, potassium, and ammonia, for the purer the acid, the less perceptible are the color changes and the more rapidly is the green color produced. I n fact, it was found posaible to purify
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the synthetic and the natural acids to such an extent that, with minimum exposure to the air, they would not show the color changes. Exposure to the air or the addition of traces of the above mentioned substances would cause the acid to give the color reactions with acetic anhydride immediately.
Optical-Crystallographic Data on Aconitic Acid G. L. Keenan, who made the optical-crystallographic measurements, reported as follows: “The refractive indices have heretofore been reported by Nelson (8) on a sample of aconitic acid consisting of very minute crystals. The material here described is identical with that examined a t that time with the exception that it consists of much larger plates, enabling the determination of the refractive indices as more nearly representative of the true values. This material consists of large, irregular, colorless fragments showing brilliant polarization colors when examined in parallel light (crossed nicols). I n view of the fact that the plates all extinguish sharply with crossed nicols, it is apparent that ng is more or less perpendicular to their broad face so that interference figures could not be expected in convergent polarized light (crossed nicols). The refractive indices as determined by the immersion method are: na = 1.475; ng = indeterminable; ny =. 1.642; both *0.002.”
Quantitative Determination in Sediments and Scales To determine the aconitic acid content, 20 grams each of the calcium aconitate sediments and refinery pan scales were extracted with hydrochloric acid, and the acid solution was carefully extracted for 24 hours with ether in an automatic extractor. The residue left after evaporating the ether was extracted and washed with benzene to remove traces of grease, wax, and soluble foreign matter, dried at 70” C. under vacuum for 12 hours, and weighed. A weighed portion of this dry acid was titrated with standard alkali and the total weight of pure acid calculated. These benzene-washed extracts were found to be from 92 to 99 per cent pure acid. Twenty grams of the evaporator scale from the raw sugar factory were treated in the same manner, but since i t was probably mixed with material adhering to the surface of the evaporator after previous cleanings with acid, analysis of this scale could not be expected to give the true content of aconitic acid in the substance as it deposits on the heating surface. In this case the ether extract was so highly contaminated with soluble inorganic salts that it was necessary to purify it further by reprecipitating the calcium salt, which was then again extracted with acid and the acid solution extracted with ether. Even with this purification the determination of the aconitic acid content of this type of scale is considered to be only approximate. The percentages of pure aconitic acid found in these materials, based on the weight of dry sample, are as follows: Calcium aconitate sediment Refinery pan scale Evaporator scale
I
1938 Samples 56.21 25.50
...
1939 Samples 57.44 24.48 8.15
Aconitic Acid Content of Louisiana Sirups and Final Molasses Sirups made in Louisiana from cane juice which had been clarified by boiling and skimming without the use of lime or sulfur dioxide were tested for aconitic acid. Experiments had confirmed the findings of Yoder (16) that practically all of the acid present in the ether extract of the acid solution resulting from treating a slurry of the lead precipitate from the juice with hydrogen sulfide is aconitic acid; in the absence of a more reliable method for quantitative separation of aconitic acid from the small quantities of other material in
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the ether extract, the following procedure was adopted as giving approximate percentages satisfactory for comparative purposes: Four hundred grams of sirups of known solids content were diluted and treated with a suitable quantity of dry lead subacetate, agitated, and allowed to settle 2 hours before filtering and washing. The lead precipitate was made into a slurry with water and treated with hydrogen sulfide to free the acids from the lead. The lead sulfide was removed by filtration, and the filtrate and washings were concentrated under high vacuum and finally made up to a volume of 200 ml. Twenty milliliters of this solution were used for titration with standard alkali and for pH measurements, and the remaining 180 ml. were extracted with ether for 18 hours in an improvised automatic extractor similar to the one described by Yoder (16). The ether extract was dehydrated with calcium chloride which rendered some dark extracted material insoluble. The dehydrated extract was evaporated to dryness a t low temperature, and a buff-colored crystalline residue was obtained which was extracted and washed with benzene to remove traces of wax and other soluble foreign matter. The residue was then dried a t 70” C. under 28 inches of vacuum for 6 hours, cooled, and weighed. A weighed portion of this residue was titrated with standard alkali, and the acid present was calculated as aconitic, using the neutralization equivalent of 58.55. A weighed quantity of the dry residue was further purified by reprecipitation of the calcium salt, from which the aconitic acid was again extracted with hydrochloric acid and finally with ether. The aconitic acid recovered in several such purifications was found to vary only slightly in amount from that obtained by titrating the dry benzene-washed residue, and the color and purity were only slightly improved. Therefore, the long and tedious purification by reprecipitating the calcium salt and recovering the aconitic acid was dispensed with in subsequent work. The percentages of aconitic acid reported here were obtained by titrating with standard alkali a solution of the dried benzene-washed residue obtained by evaporating the ether extract to dryness. The titer so obtained was calculated to aconitic acid. While any other acid present in this residue would affect the percentage of aconitic acid, it is felt that the errors due to such acids are so small that for comparative purposes they may safely be ignored. TABLEI. ACONITICACID C O N m N T AND SODIUM HYDROXIDEI EQUIYALIUNT OF ACID PBR 100 SOLIDS OF SIRUPS FROM Co. 290 CANHI IN DIFFERI~INT LOCALITIES
District Red River Bayou Lafouche Miss. River Western St. Francisville
Cc. of N NaOH Required t o Neutralize Acid freed Grams by HzS beAconitic Aconitio fore ether Acid left i n acld Undetd. Acid(100 extn. extd. soln. extd. difference Sollds 34.12 16.81 14.58 2.73 0.853 37.57 10.96 14.99 5.62 0.880 42.98 18.46 15.48 9.04 0.904 47.65 15.70 21.31 10.64 1.250 49.33 13.11 22.78 13.44 1.330
Examination of several of the solutions after the aconitic acid had been extracted with ether showed that, on the average, only 38.3 per cent of the total acids originally present remained in the extracted solution, and that the acids left were mainly phosphoric and sulfuric. Table I shows the aconitic acid, the sodium hydroxide equivalent of acids originally present, extracted, and left in solution per 100 grams of solids when sirups made without chemical clarification from juice of Co. 290 cane grown in different localities were extracted according to the above procedure. Where several samples from the same locality were tested, the results recorded are averages.
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Table I1 shows comparative results obtained from sirups made from Co. 290 and C. P. 29/320 cane grown in the same locality.
sample clarified at the lower p H contained 2.52 per cent of aconitic acid, and the one clarified a t the higher pH contained 1.80 per cent, based on solids.
TABLE11. COMPARISON OF ACONITIC ACID CONTENTAND SODIUM HYDROXIDE EQUIVALENT OF ACID PER 100 SOLIDS OF SIRUPSFROM Co. 290 A N D C. P. 29/320 SUGAR CANE
The authors wish to acknowledge with thanks and appreciation the assistance rendered in securing samples by A. G. Kellar, Louisiana State University, and E. C. Simon, Louisiana Agricultural Experiment Station. Thanks are extended to E. K. Nelson of the Bureau of Agricultural Chemistry and Engineering, who made the saturation test on the aconitic acid, and to G. L. Keenan, of the Food and Drug Administration, Federal Security Agency, who made the optical-crystallographic measurements.
Acknowledgment
District Miss. River Western Red River
Cc. of N NaOH Required t o Neutralize Acid freed Grams by HIS bo- Acid left Aconitic Aconitic fore ether in extd. acid Undetd. Acid/lOO extd. difference Solids soln. Variety extn. 14.07 2.64 0.759 39.10 22.39 C. P.29/320 18.46 15.48 9.04 0.904 Co. 290 42.08 15.84 2.13 0.030 34.12 16.08 C . P.29/320 21.30 10.64 1.250 15.70 c o . 200 47.67 17.15 2.11 1.010 17.61 C. P.20/320 38.88 16.81 14.58 2.73 0.853 Co. 290 34.12
It is evident that there is a rather wide variation of aconitic acid content in the sirups examined, the smallest value being 0.75 per cent and the largest 1.33. The variation seems to be attributable to both the variety of cane and the locality in which i t was grown. The samples examined in this work are too few, however, to determine and evaluate the factors involved in bringing about these differences. Two samples of final molasses from the district which had the most trouble with the formation of sediment mere extracted by this method for aconitic acid. One sample was from a raw sugar factory which clarified its juices chemically at about 6.2 pH, and the other sample was from a factory which carried out the clarification a t around 6.8 pH. In each sample aconitic acid 'was present both in solution and as a suspension of calcium aconitate. The latter, which would not redissolve with moderate dilution, was recovered, acidified, and added to the solution to be extracted with ether. The
Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16)
Beath, 0. A,, J . Am. Chem. SOC.,48, 2155-8 (1926). Behr, A., Ber., 10,351 (1877). Bruoe, W. F., Org. Sunlheses. 17, 1 (1937). Furth, O., and Herrmann, H., Biochem. Z.,280, 448-57 (1935). Lippmann, E. 0. von, Ber., 12, 1649 (1879). Lyons, E., and Ried, E. E., J. Am. Chem. SOC.,39, 1727 (1917). McCalip, M. A., and Seibert, A. H., Div. Sugar Chem. and Tech., A. C. S., Detroit, Mich., 1940. Nelson, E. I
