ANALYTICAL EDITION
October. 1945
ing several of the solutions containing the color and applying the phenylhydrazine test (3) to them. I n every instance a precipitate wm obtained. Four samples of genuine vanilla extract were analyzed by the method and in every instance the final solution was colorless, proving the absence of caramel. The results are shown in Table
VII.
637
and (3) per cent of caramelized concentrate in a volume of wine (Table IV). The method can be applied to all varieties of grape, fruit, and berry wine, distilled spirits, vinegar, vanilla extract, grape concentrate, and commercial caramel. It is not affected by substances usually present in those liquids, such as fruit and vegetable colors, wood extractive matter, or by certain coal-tar dyes.
SUMMARY
LITERATURE CITED
Caramel in wine, distilled spirits, vinegar, and vanilla extract is precipitated by organic acids and solvents in a form easily freed from other coloring matter. The purified precipitate is caramel and its identity can be corroborated by the usual tests. The quantity is determined in a Lovibond tintometer by color readings which are very accurate up to 20 units and when the manipulation loss in the method is corrected, the results of analysis are also very accurate. The use of analytical results in making calculations is shown by the examples given for (1) per cent of caramel color in the total color, (2)per cent by weight or volume of caramel in a liquid,
(1) Allen’s Commercial Organic Analysis, 5th ed., Vol. V, p. 384, Philadelphia, P. Blakiston’s Sons & Co., 1927.
(2) Assoc. Official Agr. Chem., Official and Tentative Methods of Analysis, 5th ed., p. 180 (1940). (3) Ibid., p. 252.
(4) Beyer, G. F.,J. Assoc. Oficial Ags. Chem., 26, 164 (1943). (5) Clapp, H.L,. Alcohol Tax Unit Laboratory, Philadelphia, Pa., unpublished. (6) Hackh’s Chemical Dictionary, 3rd ed., p. 165, Philadelphia, Blakiston Co., 1944. (7) Mulliken, S. S., “Identification of Pure Organic Compounds”, Vol. I, Color Standard Sheet A-Y-Tint 2, New York, John Wiley & Sons, 1904. (8)Von Elbe, Guenther, J. Am. Chem. SOC.,58,600 (1936).
Determination of Itaconic A c i d in Fermentation Liquors MORRIS FRIEDKIN Fermentation Division, Northern Regional RFrearch Laboratory, Peoria,
A method presented for the direct determination of itaconic acid in fermentation liquors containing other acids and glucose involves the use of aqueous bromine buffered at p H 1 .P to ensure the selective absorption of bromine b y the itaconic acid.
D
URIXG a recent study of itaconic acid production by strains of the mold Aspergillus terreus, a rapid and accurate method was developed for the determination of itaconic acid present in the fermentation liquors. This procedure involves a measurement of bromine absorption by itaconic acid in the presence of acid-buffered bromine water, and is based on the observation that, a t pH 1.2,aqueous bromine reacts with itaconic acid equimolecularly but does not react with glucose, the principal interfering substance in fermentation media. Heretofore, only slight consideration has been given to the determination of itaconic acid. Linstead and Mann (4), investigating the isomerization of unsaturated acids, used a bromometric method to determine itaconic acid in the presence of mesaconic acid. Calam, Oxford, and Raistrick (1) estimated the quantity of itaconic acid in fermentation liquors by simple alkaline titration and also by bromine absorption, using a method developed by Koppeschaar (3) for the determination of phenol. I n the examination of fermentation liquors, the assumption that total acidity values represent itaconic acid may not be correct, because the presence of acids other than itaconic acid has been shown by Calam et aE. ( I ) , who noted that several strains of A . lerreus produce succinic, fumaric, and oxalic acids. As used by the English group, Koppeschaar’s method gave fairly good results because their fermentation substrates contained low concentrations of glucose (5% or less); however, this method will not give valid results in the presence of moderate quantities of glucose, owing to the reaction of this subl;tance with bromine. The method developed by the author is a refinement of Koppeschaar’s procedure in that the bromine water is carefully acidbuffered, by means of a modification of Clark and Lubs’ standard buffer mixtures ( 2 ) , to ensure the selective akporption of bromine by itaconic acid in fermentation liquors containing glucose in concentrations m high as 15%.
111.
REAGENT Bromine Potassium bromide Potassium chloride 1.0 N HC1 Add water t o
1.0 ml. 3 . 0 grama 1.87 grama 48.5 ml. 500 ml.
Dissolve the bromine and potassium bromide in a small amount of water before adding the other constituents. The resultant pH is 1.2 * 0.1; no adjustment is required. Storage of the solution in a brown bottle in the refrigerator or in the dark slows the deterioration of bromine considerably, METHOD
One to 2 ml. of fermentation liquor are pipetted into a 125-ml. iodine flask and to the sample are added 50 ml. of acid-bufTered (pH 1.2) bromine water. The iodine flask stopper is watersealed to prevent loss of bromine vapor. After 10 minutes’ standing a t room temperature, the flask is placed in an ice bath. After 5 minutes, 5 ml. of strong potassium iodide solution (50 grams of potassium iodide in 100 ml. of water) are poured into the well surrounding the stopper. The stopper is then lifted carefully, so that the vacuum created by the previous cooling sucks the potassium iodide solution into the flask. After 10 minutes the released iodine is titrated with 0.1 N sodium thiosulfate, using starch indicator. The titer, T , of 50 ml. of bromine water (treated the same way as the sample) minus the titer, B, of unreacted bromine is equivalent to milliliters of 0.1 N itaconic acid; ( T - B ) 0.1 = milliequivalents of itaconic B ) 0.0065 = grams of itaconic acid in acid present or (T sample taken for analysis.
-
EXPERIMENTAL RESULTS
The reactivity of aqueous bromine with glucose and with itaconic acid is decreased upon lowering the pH. As shown in Table I, at p H 1.25 the bromine-glucose reaction cannot be detected during the first 15 minutes and is still negligible a t 20 minutes. At pH 3.0 the reaction is faster and a t pH 4.9 it is considerably accelerated. ‘At pH 1.25 the bromine-itaconic acid reaction is complete within 15 minutes. Results in Table I1 indicate the quantitative nature of this reaction. A standard solution of 1 N itaconic acid was prepared containing 5% glucose. As shown in Table 111, four determinations
I N D U S T R I A L A N D E N G I N E E R I N G C H EM I S TRY
638 Table
PH 1.25 3.00 4.90
1.
Effect of pH and Time on Bromine-Glucose Reaction
(Sample 2 ml. of 5% glucose solution) Bromine Absorption, G r a m of Itaconic Acid per 100 MI. 5 min. at room tem- 10 min. at room tam- 15 min. at room temperature and 6 min. perature and 5 min. at perature and 5 min, at at 0' C. 00 c. 00 c. 0.00 0.00 0.13 0.00 2.80
0.07 3.31
0.26 3.96
I
Vol. 17, No. 10
value. The remaining 10% of the itaconic acid in the mother liquor WM recovered as the monohydrated calcium salt. The discrepancy between total acidity and itaconic acid content (Table IV) aa determined by the bromination method sugf3-b the Presence Of appreciable quantities of.acids other than itaconic in some of the culture liquors. Studies directed toward the isolation and identification of these acidic substances have been successfully prosecuted and w ill be reported in forthcoming communications from this laboratory. DISCUSSION
according to the described method resulted in an average deviation of 1.5%. The following substances in the concentrations indicated were found not to interfere, within the experimental Bccuracy of the method. They include most of the acids commonly found in mold fermentation liquors. Two-milliliter samples were analyzed in each case except that of glucose, in which case only l ml. was analyzed:
The main advantage of the bromine absorption method is that itaconic acid can be determined directly in fermentation liquor containing glucose and many acids known to be mold metabolites. Aconitic acid, an unsaturated acid which has been suggested 89 a possible precursor of itaconic acid, does not interfere.
Table 1N 1N 1N 1N
15% glucose
Saturated fumaric acid 1 N succinic acid 1 N lactic acid 1 N citric acid 1 N d-gluconolactone
1N
malic acid tartaric acid oxalic acid acetic acid aconitic acid
111.
Determination of Itaconic A c i d in Presence of Glucose
(Sample: 1 ml. of 1 N itaconic acid containing 5% glucose, equivalent to 6.50 grama of acid per 100 ml. Reaction time: 15 minutes, 10 min. at room temperature; 5 minutei at 0' C., pH 1.2) Detn. A B C D
Bromine Absorption, Grams
of Itaconic Acid per 100 MI. 6.63 6.37 6.50 6.37
In Table I V are tabulated the results of analyses of several typical samples of fermentation liquor in which the total acid was determined by alkali titration and the itaconic acid by the described bromination procedure. The applicability of the bromiAnother advantage is that bromine absorption values, when nation method to the analysis of liquors of nrideb varying comcompared to total acidity values, indicate relative purities of position is evidenced by sample analyses (Expt. 799-33) after experimental fermentations with respect to itaconic acid produc2 and 14 days' mold growth. Samples taken after 2 days' growth tion. As has been shown, a low purity index indicates the showed 11.1%residual glucose and very little total acid. Alkali presence of acids which do not react with bromine under the titration, expressed as itaconic acid, was 0.091 gram per 100 ml., conditions of the method. but no itaconic acid was indicated by bromine absorption. After 14 days' mold growth, the glucose concentration had fallen to 1.87y0 and Table IV. Analyses of Fermentation Liquors the total acid had increased to 3.20 grams per 100 Blank Itaconic Total ml. (expressed as itaconic acid). The bromine Minus Acid by Acid by Alkali Purity Reaidual value after 14 days indicated 2.97 grams of itaExpt. No. Blank Titer Titer Bromination Titration Index Glucose Ml.of0.1 N Ml,o/O.l N conic acid per 100 ml., representing purity index of sodrzlm throaul- acid/%ml. G. of acid/ G. of itaconic acid by bromine absorption fate sample 100 ml. acid/100 ml. G./100 ml. 93.5. (Purity index = x 799-33 acid by alkali titration 2 days" 3 5 . 2 3 5 . 2 o 0 0.091 ... 11.1 4days 35.1 33.0 2.1 0.68 0.78 87.2 7.34 100. This index is used as a measure of purity 34.6 29.4 5.2 1.69 1.82 92.8 5.05 6 days on the assumption that the unknown acids have 8 ddays a y s 3 54 ,. 51 227,3 6.6 7 ., 52 8 2.44 2.79 87.4 3.00 2.67 2.90 92.1 2.84 approximately the same equivalent weight &s 12days 35.0 26.2 8.8 2.86 3.22 88.8 2.27 14 days 34.8 25.6 9.2 2.99 3.20 93.4 1.87 itaconic acid.) 3.06 3.61 84.8 0.35 Sample 8OO-prZC gave a bromine value of 3.06 34.8 25.4 9.4 70.1 389-19A 15.8 4.45 11.35 3.69 5.26 2.25 grams of itaconic acid per 100 ml. SB compared to a a Separate flasks. total acid value of 3.61 grams of acid per 100 ml. (purity index, 84.8). As a check on the titration method, 2.757 grams of crystalline itaconic acid were isolated from 100 ml. of sample W M C by concentration. Earlier survey analyses with aqueous bromine buffered a t pH This corresponds to a 90.1% recovery based on the bromine 3.0 resulted in apparent purities exceeding 100%. These high values were usually associated with fermentations that showed much pigmentation and low acid production. Though no pub rity index above 100 was encountered when the analysis was conTable II. Effect of Time on Bromine-Itaconic A c i d Reaction at ducted at pH 1.2, unsaturated pigments should be considered as p H 1.95 possible interfering substances in evaluating the results of the (Sample: 1 ml. of 1 N itaconic acid, equivalent to 6.50 grams of acid per 100 bromine absorption method. ml.) Reaction time at room temperature minutes 5 Reaction time'at ' 0 C., minutes 5 Bromine absorption, grams of itaconic acid per 100 ml. 6.21
10
15
5
5
6.50
6.57
25 5 6.54
2 hours
19 hours
5
5
6.60
6.65
LITERATURE CITED (1) Calam, C. T., Oxford, A. E., and Raistrick, H., Biochem. J.,33,
1488 (1939). (2) Clark, W. M . , and Lubs,H. A., J . B i d . Chem., 25,479 (1916). (3) Koppeschaar, W . F., 2. anal. Chem., 15, 233 (1876). (4) Linstead, R.P., and Mann, J . T. W., J . Chem. Soc., 1931, 723.
