ANALYTICAL CHEMISTRY
118 and digestibility of blood meal also decrease with increase in time and temperature of drying, the extinction coefficient may be used as a measure of the relative value of a specimen of this product as a feed. The extinction coefficient was used to determine the amount of hemoglobin in meat scraps, which, by official definition, should contain no more blood than “might occur unavoidably in good factory practice”. I n only 7 of 28 samples was the hemoglobin content over 1.5%. More study will be needed before a standard for hemoglobin in meat scraps can be proposed.
LITERATURE CITED
(1) Assoc. American Feed Control Officials, Inc., Official Publication, 1946. (2) Morris, S., Wright, N. C., and Fowler, A. B., J . Dairy Research, 7, 97 (1936). (3) Reiser, Raymond, and Fraps, G. S., IND.ENG. CHEM.,AXAL. ED., 14, 851 (1942). (4) Saywell, L. G., and Cunningham, B. B., Ibid., 9. 67 (1937). (5) Van SIyke, D. D., J . Bid. Chem., 33, 127, (1918). (6) Winter, A. R., Ohio Agr. Expt. Station, Bull. 436 (1929). (7) Wong. S . Y., J . Bid. Chem., 77, 409 (1928).
Quantitative Method for Aconitic Acid and Aconitates E. J. ROBERTS AND J. A. AMBLER Bureau of Agriculturaland Industrial Chemistry, Agricultural Research Administration, U.S . Department of Agriculture, New Orleans 19, La.
A method is described for determining aconitic acid and aconitate by decarboxylation in boiling potassium acetate-acetic acid solution. The determination may be completed in 1.5 hours for dry samples or in 2.5 to 3 hours for solutions. Interfering substances have been studied and are discussed. The decarboxylation is quantitative and the accuracy of the method is dependent on the accuracy of determining carbon dioxide.
I (e,
N T H E course of recent studies on the recovery of insoluble aconitate from sorgo (23, 24, 25) and sugar-cane products S, 24), a new and rapid quantitative method for aconitic acid and aconitates was developed. Heretofore, the most accurate method of general applicability for determining this acid has been by quantitative extraction with ether, as used by Yoder (as), McCalip and Seibert ( I S ) , and Balch, Broeg, and Ambler ( 3 ) . However, the extraction is t o o time-consuming for practical work, and is accurate only in the absence of all other nonvolatile acids which may be removed from acidified aqueous solutions by prolonged ether extraction, Ventre (2%) adapted the color reaction of Fdrth and Herrmann (10) to obtain approximations of the amount of aconitic acid in dilute solutions such as sugar plant juices and liquid sugarhouse products. The method has the usual disadvantages and limitations of colorimetric methods. Polarographic and conductivity methods (15,19,21,22) are applicable only in a very few special cases. Umbdenstock and Bruins (22) devised an ingenious indirect method for aconitic acid in the presence of citric, itaconic, and citraconic acids. Aconitic acid has been decarboxylated by pyrolytic methods (6, 16) which are not suitable for general analytical procedures. Balch, Rroeg, and Ambler (S) found that dilute aqueous solutions of acoriitic acid evolved carbon dioxide during evaporation by heat unless the concentration was conducted a t reduced pressures and temperatures, This decarboxylation of aconitic acid in aqueous solution is too slow to be of use as an analytical procedure. However, it was observed that, although aconitic acid is stable in hot glacial acetic acid and may be recrystallized from this solvent ( 5 ) , some aconitates evolved carbon dioxide rapidly when boiled in this medium. Preliminary experiments showed that the decarboxylation was very rapid with the very soluble alkali aconitates, slow with the slightly soluble cadmium, calcium, and calcium-magnesium salts, and extremely slow with the very insoluble lead salt. When, however, an excess of potassium acetate was added to suspensions of these more insoluble aconitates in acetic acid and the mixtures were heated to boiling; the aconitates rapidly dissolved and the decarboxylation proceeded a t a speed approximating closely that of the decarboxylation of the alkali aconitates, [For r6sum6s of solubilities and reactions of salts in acetic acid, see reviews by Evans (9), Davidson ( 7 ) , and Hall (21).]
Quantitative studies in the apparatus described below showed that the decarboxylation in the presence of potassium acetate is complete in from 30 to 40 minutes and that from each mole of aconitic acid (or each aconitate radical in an aconitate) one mole of carbon dioxide is formed, according t o the equation C&O6 +. C02 C5Hs0,. The nonvolatile product remaining in the acetic acid solution after removal of the cations with sulfuric acid (8) and evaporation of the acetic acid was a yellow, very hygroscopic mixture from which it was possible to isolate a small amount of itaconic acid, melting point 160-1 ’ C.
+
Apparatus. An electric hot pIate was the source of heat. A 250ml. Erlenmeyer flask was connected with (1)a tube through which a stream of carbon dioxide-free air was introduced above the surface of the liquid in the flask, and (2) a short reflux condenser. The top of the condenser was connected with a wash bottle containing distilled water for removing acetic acid vapors from the air-gas mixture. The wash bottle was connected in turn with a suitable carbon dioxide-absorption train. DECARBOXYLATION
For Dry Solids. Weigh out and transfer to the flask 1 to 2 grams of aconitic acid, or 2 t o 5 grams of aconitate. Add 75 to 100 ml. of “empyreuma-free” glacial acetic acid, some boiling chips, and 5 or 10 grams of potassium acetate, using the larger amount for insoluble aconitates. Connect the flask with the air inlet tube and the condenser and attach the wash bottle of water. Pass a slow stream of carbon dioxide-free air through the apparatus for 15 to 20 minutes. Start heating the acetic acid mixture and attach the carbon dioxide-absorption train. Continue the passage of the slow stream of carbon dioxide-free air while the reaction mixture is refluxed for 1 hour. Then disconnect the absorption train and determine the carbon dioxide absorbed. The total time required for the analysis is 1.5 hours. For Aqueous Solutions. Take an amount of solution which contains between 0.5 and 2.0 grams of organic acid calculated as aconitic, and dilute with carbon dioxide-free water to a volume of about 200 ml. Adjust the pH of the solution to,a value of 6.0 to 6.2, using sodium hydroxide or acetlc acid solution as necessary. Add with stirring 50 ml. of saturated neutral lead acetate solution. Collect the precipitate on a strong qualitative filter paper fluted to fit a Hirsch or small Biichner funnel and precoated Tyith a thin layer of finely divided acid-washed asbestos. Wash paper and precipitate once with water, and drain completely by suctibn. Cut off the suction and fill the paper with acetone, carefully puddling the precipitate, so that the acetone penetrates it thoroughly. Drain off the acetone and repeat with a second portion. Finally, drain the acetone through by suction and continue the suction
119
V O L U M E 19, NO. 2, F E B R U A R Y 1 9 4 7 until the acetone has completely evaporated and the paper is dry. Complete the desiccation by heating at 100' to 105" C. for 0.5 hour. Place the paper and precipitate in the decarboxylation flask, add 100 ml. of acetic acid, 10 grams of potassium acetate, and some boiling chips, and proceed with the decarboxylation as described in the preceding paragraph. Determination of Carbon Dioxide and Calculations. The carbon dioxide may be absorbed and determined by any standard method. For the work reported here, it was absorbed in sodium hydroxide solution and determined volumetrically by precipitation of the carbonates with barium chloride solution and titration of the residual alkalinity according to Reid and Weihe's modification (18) of Winkler's method (20). For this and similar volumetric methods, 1 ml. of N N a O H ~ 0 . 0 8 7gram of CeHaOs. For gravimetric methods of determining mg. of C&IW,O,. carbon dioxide, 1mg. of C02~3.954
Table I.
D e t e r m i n a t i o n of Aconitic Acid by Decarboxylation
(COXdetermined volumetrically, using N HC1 except i n the 3rd a n d 4 t h analyses, in which 0.1 N HC1 was used) Sample Aconitic acid Method A for solids
Method B for solutionsa KHnAcon ( 1 1 ) Caz.(Ca,Mg).Aconn.6H~O( 1 ) CdsAconz.GHz0 (f1 )
Weight Grams
2 000 2 000 0 2000 0 1000 1 000 1 000 0 200 2.000 5 000 3 000
rlconitic Acid Found ExtracDecartion boxylation Grams Grams
... ... ...
... ...
.
I
.
1 . k lb 3.100 1.323C
2.001 2.001 0,2005 0.1009 1.001 1.001 0.203 1.640b 3,100 1.3256
Difference Grams %
+0.001 +0.001 +0.0005 +0.0009
+0.001 +0.001 +0.003 -0.001 0.000 +0.002
4-0.05 +0.05 4-0.25 +0.90 +0.10
+O.l0 +1.50
-0.06 0.00 +0.15
Aconitic acid dissolved in 16 Brix sucrose solution. b Theoretical, 1.642 grams: deviation by extraction, -0.001 gram, or -0.06%; by decarboxylation, -0.002 gram, or -0.13%. e Theoretical, 1.326 grams: deviation b y extraction, -0.003 gram, or -0.237,; by decarboxylation, -0.001 gram, or -0.07%.
INTERFERENCES
Water. Water retards the reaction, as was shown by a series of three comparable experiments in which 50ml. portions of glacial acetic acid, 95y0 acetic acid, and 90yo acetic acid were used to decarboxylate in each case 1 gram of aconitic acid in the presence of 1 gram of potassium acetate. Carbondioxide equivalent to 1.001 gram of aconitic acid was obtained in 50 minutes with the glacial acetic acid, and in 90 minutes with the 957, acid, whereas with the 90% acid the carbon dioxide evolved in 2 hours was equivalent only to 0.80 gram of aconitic acid and the decarboxylation was still progressing a t a slow rate. Alcohol. In the presence of alcohol the evolution of carbon dioxide stops before the amount of carbon dioxide equivalent t o the aconitic acid is obtained. This is probably due to esterification of a portion of the aconitic acid in the anhydrous medium. Organic Acids. Malonic and acetonedicarboxylic acids were rapidly decarboxylated, the latter yielding two molecular proportions of carbon dioxide, when tested by this procedure. The presence of acids of these types has not been reported in sorgo or sugar cane. Galacturonic acid (2.00 grams) in boiling potassium acetateacetic acid solution gives 537, of the theoreticalamount of carbon dioxide in 1hour. Since uronic acids are normal constituents of plants and have been reported in sugar-cane products by Browne and Phillips ( 4 ) , the extent of this interference and the possibilities of overcoming it will receive further study. S o decarboxylation took place when thi- procedure was applied to oxalic, succinic, maleic, fumaric, itaconic, citraconic, mesaconic, malic, lactic, tartaric, citric, tricarballylic, glutamic, aspartic, aminoacetic, or phthalic acids. (Since acceptance of this paper it has been found that citric acid and some citrates yield a small amount of carbon dioxide under certain conditions when boiled with the acetate-acetic acid reagent. The extent of this possible interference and the conditions under which it occurs are being studied and mill be discussed at a later time.) Cystine gave no carbon dioxide, but the solution darkened and gave off hydrogen sulfide. Carbohydrates do not yield carbon dioxide in this procedure, Inorganic acids and salts. When heated in acetate-acetic acid solution, salts of easily volatile inorganir acids, such as chlorides, nitrates, and carbonates, yield acidic vapors which pass through the wash bottle into the absorption train. Hydrogen chloride from chlorides can be trapped effectively by washing the gas with solutions of silver sulfate or nitrate. Sitrates, even in small quantities, not only evolve acidic oxides of nitrogen, but also in the anhydrous medium cause the formation of carbon dioxide by oxidation of the aconitic acid and the decarboxylated residue. Hence, the decarboxylation method is not applicable in the presence of nitrates.
Determination of Aconitic Acid in Presence of Carbonates and Bicarbonates. When aconitates and carbonates are present in solution, acidify the solution strongly with acetic acid and remove carbon dioxide by aeration a t room temperature. Neutralize the solution and proceed with the precipitation and decarboxylation of the lead salt as previously described. When solid aconitates are contaminated with carbonates, two determinations of carbon dioxide are nececeary. 1. For total carbon dioxide, set up the apparatus with a separatory funnel whose stem leads into the decarboxylation flask. Proceed with the decarboxylation as described, but a t the end of the decarboxylation period allow the mixture to cool until refluxing stops, keeping the absorption train attached and maintaining the current of carbon dioxide-free air a t such a rate as is necessary to prevent backward suction through the apparatus. Add 100 ml. of carbon dioxidefree water through the separatory funnel, and reflux the diluted mixture for another 30 minutes in order to assure complete decomposition of the carbonates. Determine the total carbon dioxide evolved and calculate as per cent aconitic acid. 2. Determine the carbon dioxide from the carbonates by dissolving a second sample in hydrochloric acid and aerating a t room temperature (14) for 1 hour. Calculate the carbon dioxide obtained as per cent aconitic acid. For the pei cent actual aconitic acid, deduct this value from the value obtained by the modified decarboxylation procedure. By this combmition of procedures, a mixture of 30 parts of crude calcium-magnesium aconitate (54.23Yc aconitic acid) and 1 part of powdered calcium carbonate (reagent grade) gave total carbon dioxide equivalent to 58.09% aconitic acid and carbonate carbon dioxide equivalent to 5.617, aconitic acid. Actual aconitic acid was, therefore, (58.09 - 5.61 = ) 52.48yo which 30 agrees with the aconitic acid content of the mixture, - X 54.23%
31
or 52.487,. Oxidizing Substances. Copper aconitate (17) was tested by this method and gave excessive and variable quantities of carbon dioxide. When the reaction mixtures cooled, red cuprous oxide settled to the bottom of the flasks, indicating that oxidative reactions had taken place and were responsible for the high carbon dioxide values observed. For this reason, the decarboxylation method is not applicable to material containing cupric, silver, mercuric, or other oxidizing compounds which are soluble in hot acetate-acetic acid solution. DISCUSSION
A few of the results obtained with pure aconitic acid, melting point 184" C., and aconitates of known composition are given in Table I. The accuracy of determination is dependent upon that of the carbon dioxide determination. Because of the weight rela-
120
ANALYTICAL CHEMISTRY
tionship involved in the reaction CpH606 +COS, 174:44, the accuracy decreases with decrease of the weight of sample taken as shown in the cases of the smaller amounts of aconitic acid taken. This weight ratio is a critical factor in choosing the weight of sample and the method t o be used for determining carbon dioxide. Except when the amount of sample available is so’ small that it contains less than 0.25 gram of aconitic acid and an accuracy of less than 1 mg. is desired, the gravimetric method, with its elaborate absorption train, offers no advantage over the simpler volumetric method applied t o samples containing from 1 to 3 grams of aconitic acid. The use of the larger samples, especially of impure or crude products, is often desirable in order to reduce the magnitude of errors due t o sampling. The method has been found to be valuable in assaying laboratory preparations of aconitic acid and aconitates. I t s application and adaptation to sugar plant juices and sugarhouse products will be studied as soon as these seasonal materials are available. LITERATURE CITED (1) Ambler, J. rl., Turer, J., and Keenan, G. L., J . Am. Chem. Soc., 67, 1 (1945). (2) Anon., Sugar Bull., 23, No. 19, 173-4 (July 1, 1945); Intern. Sugar J., 47, 112 (1945). (3) Balch, R. T., Broeg, C. B., and Ambler, J. A , , Sugar, 40, No. 10, 32 (1945); 41, No. 1 , 46 (1946). (4) Browne, C. A,, and Phillips, M., Intern. Sugar J., 41,430 (1939).
(5) (6) (7) (8) (9) 1 1
Bruce, W. F., Org. Syntheses, 17, 1 (1937). Crasso, G. L., Ann., 34, 63 (1840). Dsvidson, A. W., Chem. Revs., 8, 175 (1931). Davidson, A. W., J . Am. Chem. Soc., 50, 1890 (1928). Evans, W. V., Chem. Revs., 8, 167 (1931). Fiirth, O., and Herrmann, H., Biochem. Z . , 280, 448 (1935). Guinochet, E., Compt. rend., 94, 455 (1882). Hall, N. F., Chem. Revs.. 8, 191 (1931). McCalip, M. A., and Seibert, 8 . H., IND.ESG. CHEM., 33, 637 (1941); Sugar Bu7Z., 19, No. 17, 84 (1941).
MacIntire, W. H., and Willis, L. G., J. IYD.ENG.CHEY.,7, 227 (1915).
Malachowski, R., Bull. Intern. Acad. Polonaise, 1931A, 369. Pebal, L., Ann., 98, 67 (1856). Pickering, S. U., J . Chem. Soc., 101, 178 (1912). Reid, J. D., and Weihe, H. D., IND.EXG.CHEM.,ANAL.ED.,10, 271 (1938).
Schwaer, L., Collection Czechoslov. Chem. Commun., 7 , 326 (1935). Scott, W. W., “Standard Methods of Chemical Analysis”, 5th ed., pp. 2265-6, New York, D. Van Nostrand Co., 1939. Semerano, G., and Sartori, L., Mikrochemie, 24, 130 (1938). Umbdenstock, R. R., and Bruins, P. F., ISD. ENG.CHEY..37, 963 (1945).
Ventre, E. K., Sugar J., 3, No. 7, 23 (1940); Ventre, E. K., and Paine, H. S., U.S. Patent 2,280,085 (April 21, 1942). Ventre, E. K., Ambler, J. A., Byall, S., and Henry, H. C.. U. S. Patent 2,359,537 (Oct. 3, 1944). Ventre, E. K., Ambler, J. A., Byall, S., and Paine, H. S., IND. ENG.CHEM.,38, 201 (1946). Yoder, P. A., Ibid. 3, 640 (1911). AGRICULTURAL Chemical Research Division Contribution S o . 190
Constant Ratio Still Head L. E. LLOYD AND H. G. HORNBACHER, The Dow Chemical Company, Midland, Mich. Several constant reflux ratio laboratory still heads have been devised which have given improved column efficiency and more reproducible distillations with less operator attention than former types. These heads are based on the principle of two condensing surfaces in parallel-one to give reflux, the other product. These still heads have been called Corad heads, from Constant RAtio heaD. A bibliography on laboratory still heads is included.
I
N T H E past 25 years, laboratory distillation technique and equipment have been considerably improved. Kot less than three dozen articles have appeared in the literature describing different laboratory still heads; a t the end of this article is a bibliography of important references. Several of the earlier articles (2, 10, l a , $1, 31, 37, 38, 40, 43) described dephlegmators, but in the past 10 or 15 years most of the suggested still heads have been of the total condenser type. Of this latter type, most of the authors describe units in which the product delivery rate is controlled by some sort of adjustable orifice, such as a stopcock or needle-type valve. Typical is the Booty head shown in Figure 1. For routine distillations such heads have several operating difficulties and undesirable characteristics. The following shortcomings are inherent in laboratory still heads which depend on throttling of product for reflux control:
1. To obtain the desired reflux ratio, the stopcock (adjustable orifice) must be laboriously and painstakingly adjusted t o deliver only a small part of the condensate as product. 2. The actual reflux ratio a t any given moment can be ascertained only by counting drops of reflux and product-a timeconsuming process which often is little better than a guess. 3. Unless extreme precautions are taken in drying both the apparatus and the still charge, a small amount of water collects in the still head a t the beginning of each distillation. When these water droplets reach the partially closed stopcock (throttling valve) , they usually refuse to pass because of interfacial tension forces and thus seal the product line. When this happens, the stopcock must be opened t o permit the water to pass and then readjusted to give the desired reflux ratio. This opera-
tion may have to be repeated two or three times, thereby consuming considerable operator time. 4. The reflux ratio changes directly with any change in distillation rate, since the product take-off rate remains fixed. This is an important factor, for even a small reduction in reflux ratio will give a noticeably decreased column efficiency; and unless the operator watches his heat input closeIy, the distillation rate may drop as much as 50% in the latter part of the distillation with a corresponding reduction in reflux ratio. 5 . When distilling multiple liquid-phase mixtures, as for example, steam distillations, satisfactory reflux control is virtually impossible with a head using a throttling orifice. 6. Any approach to good reproducibility in analytical distillations can be obtained only a t the expense of almost constant operator attention. 7. The need for frequent checking and adjustment of.the reflux ratio on adjustable orifice heads increases the probability of errors and the need for reruns. l l o s t of these difficulties are avoided in still heads of the intermittent take-off type (f, 1.2, 15, 51). These heads, however, require timers which are an added expense and which sometimes fail in the middle of a distillation. Some of these heads have excessive holdup (1, 15). To avoid the above-mentioned difficulties, the authors devised a laboratory still head based on a principle not previously applied: The use of two condensing surfaces in parallel, one t o furnish product,, the other reflux. This is in direct contrast t o the dephlegmator-final condenser arrangement which represents two condensing surfaces in series. If, in the case of the parallel condensing surfaces, the heat transfer characteristics are the same for each surface and the vapor paths are comparable,
