877
NOVEMBER 1947 to about 94 ml., and add 1 ml. of 7.5% ammonium molybdate solution. After 5 minutes, add 4 ml. of tartaric acid and mix. Add 1 ml. of the l-amino-2-naphthol-4sulfonicacid reagent and dilute to 100 ml., if necessary. Make the transmittancy measurement after 20 minutes. Water may be used in the reference cell. DETERMINATION OF ARSENIC
I n acidic solution arsenate ions condense with molybdate ions to form molybdiarsenic acid, which may be reduced t o a stable heteropoly blue (7, 8, 17, 18, 13, 87, 18,81). Effect of Variables on Color Development. Only the effect of arsenic concentration and the nature of the absorption spectrum were investigated.
A definite volume of the standard arsenate solution (0 to 20 ml.) was transferred by means of a buret to a 50-ml. volumetric flask and water added to bring the volume to about 30 ml. Twenty milliliters of the molybdate-hydrazine sulfate reagent were added, the volume was adjusted to 50 ml. with water, and the contents of the flask were thoroughly mixed. The flask was then immersed in boiling water for 10 minutes, removed, and cooled rapidly. If necessary, the meniscus was adjusted to the mark with a few drops of water and the transmittancy measured a t 840 mp. Arsenic Concentration. The blue color produced is proportional to the concentration of arsenic for 0 to 3 p.p.m. of arsenic when the transmittancy is measured a t 840 mp. Discussion. The procedure used for determining arsenic is the same as that developed for determining phosphorus. Figure 3 shows the absorption spectrum obtained for one concentration of arsenic. These data substantiate Sultzaberger’s statement that maximum absorption occurs a t about 840 mp. Although a maximum absorption band is found for low concentrations of arsenic, the nature of the curve a t the blue end of the spectrum differs from that for higher concentrations. This is caused by the presence of quinquivalent molybdenum ions which are formed by the reduction of the excess molybdate reagent. As the concentration of arsenic is increased, the amount of excess molybdate ion reduced is less, as evidenced by sharper bands of minimum absorption at about 440 mg. Recommended General Procedure. SAMPLE. Weigh or measure by volume B sample containing an amount of arsenic such that the final soluton contains not more than 0.15 mg. of
arsenic per 25 ml. of solution. This solution should be made neutral to litmus. Dilute t o 100 ml. DESIRED CONSTITUENT.Transfer a 25-ml. aliquot t o a 50ml. flask and add 20 ml. of the molybdate-hydracine sulfate reagent and sufficient water to bring the meniscus to the mark. Mix thoroughly. Immerse the flask in a beaker of boiling water for 10 minutes, remove, and cool rapidly. Again shake the flask and adjust the meniscus to the mark with a few drops of water if necessary. Measure the transmittancy a t 840 mp in 1-cm. cells. LITERATURE CITED
Alimarin and Iwanoff-Emin, Mikrochmie, 21, 1 (1936). Berenblum and Chain, Biochem. J . , 32, 286 (1938). Boyle and Hughey, IND.ENG. CHEM.,ANAL. ED., 15, 618 (1943).
Brabson, Harvey, Maxwell, and Sohaeffer, Ibid., 16, 705 (1944). Bunting, Ibid., 16, 612 (1944). Cotton, I b a . , 17, 736 (1945). Deemer and Schricker, J . Assoc. Oficial AOT. Chem., 16, 226 (1933).
DenigBs, Compt. rend., 171, 802 (1920). Fontaine, IND. ENG.CHEM.,AN,+. ED., 14, 77 (1942). Hague and Bright, J . Research Natl. BUT. Standards, 26, 505 (1941).
Hybbinette and Sandell, IND.ENG.CHEM.,ANAL. ED., 14, 715 (1942).
Isaacs, Bull. SOC. chim. bioi,, 6, 157 (1924). Kahler, IND.ENG.CHEM.,ANAL.ED., 13, 536 (1941). Kitson and Mellon, Ibid., 16, 128 (1944). Ibid., 16, 466 (1944).
Lindsay and Bieienberg, Ibid., 12, 460 (1940). Maechling and Flinn, J. Lab. Clin. Med., 15, 779 (1930). Morris and Calvary, IND.ENG. CHEM.,ANAL. ED., 9, 447 (1937).
Olsen, Gee, McLendon, and Blue, Ibid., 16, 462 (1944). Pavelka and North, Mdkrochemic, 16, 239 (1934). Poluektof, Z . anal. Chem., 105, 23,(1936). Pons and Guthrie, IND. ENG.CHEY.,AAAL.ED., 18, 184 (1946). Rodden, J . Research Natl. BUT.Standards, 24, 7 (1940). Schwartz, IND.ENG.CHEM.,ANAL.ED.,14, 893 (1942). Stoloff, Ibid., 14, 636 (1942). Straub and Grabowski, Ibdd., 16, 574 (1944). Sultzaberger, Ibid., 15, 408 (1943). Truog and Meyer, Ibid., 1, 136 (1929). Woods and Mellon, Ibid., 13, 760 (1941). Wu, J . Bwl. Chene., 43, 218 (1920). Zinzadae, Ann. A Q T O ~(N.S.) ., 1, 321 (1931). RECEIVED November 18, 1946. Abstracted from a portipn of the dissertation presented by D. F. Bolt5 to the Graduate School of Purdue Unirersity in partial fulfillment of the requirements for the degree of doctor of philosophy.
Determination of Aconitic Acid in Sugarhouse Products J. A. AMBLER AND EARL J. ROBERTS Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U . S . Department of Agriculture, New Orkans, La. The decarboxylation method for determining aconitic acid has been successfully applied to sugarhouse and sugar refinery products. The only interfering substance found was sulfur dioxide which was readily remaved from the decarboxylation gases by w-ashingwith potassium dichromate solation.
H
.
ERETOFORE the only method for determining aconitic acid in sugarhouse products has been by long extraction with ether, titrating the dried extractives with standard alkali (3, 4,IO), and correcting for the oxalic acid (8) which may be present. Ether-soluble waxy and fatty substances (4)and pigments make the titration uncertain and often practically impossible. ’The method is too time-consuming for practical use in the sugarhouse. The decarboxylation method of Roberts and Ambler ( 7 ) is not subject to these difficulties, has been shown to be highly accurate when applied to solutions containing sucrose (‘7) and, especially when used with the carbon dioxide absorption and titration tube of ‘Roberts ( 6 ) , requires but a short time for completion. When
adapted to sugarhouse and refinery products as described below, the method has been valuable in the further development of processes for the recovery of aconitic acid from sugar-cane molasses (1, 9). Since the two methods are based on totally different properties of aconitic acid, comparative determinations on identical products were made as a means of disclosing a t least some of the possible interferences in the decarboxylation method. As a further check on both methods, the titrated solutions of the ether extractives were dried and decarboxylated directly.
.
EXPERIMENTAL
To make certain that the samples for the two methods were of like composition liquid materials containing suspended matter
.
V O L U M E 19, NO. 1 1
878 were thoroughly mixed and appropriate samples for both methods were taken immediately and as rapidly as possible. The size of the samples taken was governed largely by the Brix of the various types of material tested. The quantities used for the decarboxylation tests are those which have been found most satisfactory for actual assay. Decarboxylation Method (7). No change in the decarboxylation procedure was necessary, except when sulfur dioxide was present, the interference of which is discussed below. JUICES. I t is advantageous to clarify juice (2) with lime and heat before taking the sample. Sufficient acetic acid mas added to a 500-ml. portion of the clarified juice to lower its pH to approximately 6.0 and the solution was heated on the steam bath. Fifty milliliters of a saturated solution of neutral lead acetate solution were added with stirring and the precipitate was collected and decarboxylated as described by Roberts and Ambler (7). SIRUPSAKD MOLASSES. d quantity of the material sufficient to contain 20 to 25 grams of Brix solids was weighed and diluted with distilled water t o about. 200 ml. If necessary, sodium hydroxide or acetic acid solution \vas added to adjust the pH to between 5.5 and 6.2. The unfiltered solution was heated on the steam bath and treated with 50 ml. of saturated neutral lead acetate solution. The precipitate of lead salts was collected and decarboxylated by the usual procedure. R A W S U G ~ R STwenty . grams of sugar were dissolved in about 200 ml. of distilled water and heated on the steam bath. Fifty milliliters of saturated neutral lead acetate solution were added and the precipitate was collected and decarboxylated as usual. SOLID.%CO~~ITATE. One or 2 grams of the air-dried solid were decarboxylated directly with acetic acid and potassium acetate. Extraction.Method (2,.4, IO). JUICES. A 100-ml. portion of the clarified juice was acidified with 10 ml. of concentrated hydrochloric acid and extracted with ether for 16 hours. Aconitic acid was determined in the dried (vacuum, 70’ C.) ether extract (?, 4).by adding 5 ml. of benzene and about 80 ml. of water and titrating x-ith standard alkali (phenolphthalein). The titrated solution was evaporated to dryness on the steam bath in a 280ml. Erlenmeyer decarboxylation flask, and the aconitic acid in the dried salts was determined by direct decarboxylation ( 7 ) .
Table I. Aconitic Acid in Sugarhouse Products Determined by Decarboxylation and by Extraction Samde No.
Material
Aconitic Acid Found By deBy extraction carboxylTitraDecarboxylation ation tion
%
Brix
Remarks
7 0 . 7 0
Brzx
Brix
1 2 3 4
Crusher juice Mixedraw juice Cane sirup A Cane sirup B
1.20 0.71 0.48 1.96
1 . 1 6 (av.) 0.73 0.46 1.90
1 . 1 7 (av.) 0.50 1.95
Contained
5
Cane sirup B
....
1.96
1.93
Contained
6
7 8 9
A molasses B molasses Final molasses Table molasses
3.54 3.94 5.42 2.48
3.20‘L 3.93 5.40 2.27
3.55 3.89 5.30 2.20
10
Table molasses
2.40
2.01b
2.39
11
Cooking molasses 3 , 3 0 A Cooking molasses 5 . 0 2 B Cooking molasses 4 . 4 0 B Cooking molasses 3 . 9 4 C 13.86
......
sn.
Y V .
so2
3.29
3.28
Contained sn. K2CnO7 wash used Contained
4,23
4.15
Contained
--1
12 13 14
15 -_
16 17 18
19 20 21 22 5
b c
d
.
RawsuzarC
DISCUSSION
The data obtained from representative samples of various sugarhouse products,. of commercial sirups and molasses, of refinery products, and of aconitate separated from molasses are given in Table I. With most of the products, the aconitic acid values obtained by all three procedures agree closely. Examples of the interference of ether-soluble pigments, \\axes, and fats in the extraction method are shown in samples 6, 10, and 22. No case of interference attributable to the “uronic acids” found in sugar cane by Browne and Phillips ( 3 ) was encountered. There is no indication of interference by the small amount of citric acid which has been reported in sugar-cane juices (8) and molasses ( 6 ) . Oxalic acid and oxalates are not decarboxylated in the aconitic acid procedure ( 7 ) and therefore, when present, would cause the titration values of the extractives (2) to be higher than both the decarboxylation values. INTERFERENCE OF SULFUR DIOXIDE
The only interfering substance detected was sulfur dioxide in products such as edible and cooking molasses, samples 9, 12, and 14. Values for aconitic acid by decarboxylation of the lead precipitate from these samples xere higher than those obtained by either the titration or the decarboxylation of the extractives. In these samples, lead sulfite precipitated n-ith the lead aconitate and reacted with the acetic acid-potassium acetate reagent to form free sulfur dioxide which passed into the alkaline absorbent and resulted in falsely high values for carbon dioxide. Blank tests with lead sulfite demonstrated that this interference can be prevented by using a saturated, acidified (sulfuric acid) solution of potassium dichromate for washing the gases evolved by the decarboxylation procedure. These products gave checking results when the dichromate wash was used (samples 10, 13, and 14). Although sulfur dioxide was declared on the labels of samples 4, 5, and 11, the amount present was insufficient to cause interference in the decarboxylation. Since sulfitation processes are frequently used in sugarhouses, the dichromate wash solution should be used when assaying products from mills known to use sulfur dioxide. LITERATURE CITED
$01
4,31
4.38
3.87
3.81
,.....
0.087
0.087
0.043 0.51
0.035 0.53
0.49
0.75
0.81
1.16 1.18 0.59 51.5
0.57 58.3
Af?&ion siyC Affination sirup, 0.85 2nd remelt Affination black1.25 strapC 1.15 Affination sirupd 0 .61 Sorgo sirup Crude aconitate 5 8 . 5
so2
SIRUPSAND MOLASSES.Fifteen grams of sirup or molasses were diluted with distilled water to about 100 ml. and acidified with 15 ml. of concentrated hydrochloric acid. The solution was extracted with ether and the ether extract treated as described under juices. RAWSCGARS. Ten grams of sugar were dissolved in distilled water to make a volume of about 100 ml. The solution was acidified with 10 ml. of concentrated hydrochloric acid and extracted with ether. The aconitic acid in the extract was determined by titration as described under juices, but not by decarboxylation because of the small quantities found. SOLIDACONITATE.A suspension of 0.5 gram of the air-dried material in 100 ml. of distilled water was acidified with 15 ml. of concentrated hydrochloric acid. The resulting solution was extracted with ether and the aconitic acid determined as described under juices.
......
1.20
,.....
Re-extraction of substrate gave 0.19% as aconitic acjd. Re-extraction of substrate gave 0.18% as aconitic acid. From Puerto Rico. From Cuban raw sugar.
KzCrzO, wash used Contained
so9
KtCrzOi wash used
(1) Anon., Sugar Bull., 23, No. 8 , 16; 23, No. 19, 173 (1945); Intern. Sugar J., 47, 112 (1945). (2) Balch, R. T., Broeg, C. B., a n d Ambler, J. A., Sugar, 40, No. 10, 32 (1945): 41, No. 1, 46 (1946). (3) Browne, C. A , , and Phillips, M., Intern. Sugar J., 41, 430 (1939). (4) McCalip, M.A., a n d Seibert, -4.H., Ind. Eng. Chem., 33, 637 (1941). (5) Nelson, E. K., J . A m . Chem. SOC.,51, 2808 (1929). (6) R o b e r t s , E. J., 4N.4L. CHEM.,19, 616 (1947). (7) Roberts, E. J.. a n d Ambler, J. A , Ibid., 19, 118 (1947). (8) T a n a b e , T., Rept. God. SUQW Ezpt. Sta., Tainan, Formosa, No. 4, 33 (1937). (9) Ventre, E. K., Ambler, J. A , , Byall, S., a n d H e n r y , H. C., U. S. P a t e n t 2,359,537 (Oct. 3 , 1944). (10) Yoder, P. A, J . Ind. Eng. Chem., 3, 640 (1911). RECEIYEDFebruary 24, 1947. Agricultural Chemical Researrh Dlvisipn, contribution 202.
8
