ANALYTICAL CHEMISTRY
1064 rubber bands are put in place to hold the cover securely and mercury is added through the fillin tube until the well is filled. The filling tube is sealed with a rub%er policeman. After the apparatus is filled (an operation requiring about 5 minutes), it is completely immersed in a water bath or other container where the bulbs will be a t the same temperature. The volumes of the solutions are determined at appropriate intervals by temporarily rotating the apparatus so that the solutions drain into the measuring arms (1). When the volumes become constant, the molecular weight is calculated from the formula
where M , V , and W are, respectively, the molecular weight, volume of solution, and weight of the reference compound, and Mi, VI, and W1 are the corresponding values of the unknown. RESULTS
The data in Table I illustrate the accuracy obtainable with the apparatus. Errors of the order of magnitude shown in the table could be caused by an uncertainty of 0.01 ml. in reading one of the solution volumes. The apparatus has also been used successfully for the determination of the molecular weights of research compounds, with chloroform, tetrahydrofuran, and methylene chloride used as
Table I. Apparent Molecular Weight of Ambenzene Reference Compound Benzoic acid, NBS.!40 Acetanilide, NBS 141 Acetanilide, NBS 141
T ~ ~ Molecular ~ . , Weight C. Found Theory
Weight, Mg.
Solvent
15.02
Ethylether
25
181.8
18 84
Acetone
39
183.0
182.2
4.702
Acetone
39
181.4
182.2
182.2
solvents. The rate of distillation of the solvent appears to be the same in the new and in the original apparatus. The apparatus described is easily and quickly filled by technicians having no glassblowing experience, and its use causes no sacrifice in accuracy of results or time required to reach equilibrium. The type of closure designed for this apparatus should find use in other applications where a nonlubricated, vacuum-tight closure is required. LITERATURE CITED
(1) Clark, E. P.. 1x0. ESG. CHEM.,ANAL.ED.,13, 820 (1941). (2) Signer, R., Ann., 478, 246 (1930). RECEIVED for revierv December 26, 1951. Accepted February 18, 1952
Photometric Determination of Aconitic Acid with Potassium Permanganate SHERMAN R. DICKMAN, University of Utah, Salt Lake City, U t a h
N T H E course of a study of the enzyme aconitase it was deIcitric sirable to measure the formation of cis-aconitic acid from either acid or isocitric acid as substrate. A satisfactory chemical method for this purpose would be sensitive to microgram quantities of cis-aconitic acid and insensitive to relatively large quantities of either citric or isocitric acid. The procedure developed fulfills these criteria, is rapid, and makes possible the estimation of aconitic acid with but little interference from many other types of compounds. The method is based on the reaction of the ethylenic group with potassium permanganate in dilute metaphosphoric acid solution. The decrease in the absorbancy of the permanganate is determined with a spectrophotometer, and the concentration of aconitic acid is calculated by means of a standard curve. Although the reaction of ethylenic substances with potassium permanganate has been known for many years, difficulties in controlling the extent of the reaction have made quantitative applications almost impossible. Lauer and Makar ( 4 ) ,for example, were unable to utilize the partial oxidation of aconitic acid by acidified permanganate solutions because of the separation of manganese dioxide a t low temperatures and the fading of the end point a t temperatures up to 80". Stamm ( 6 ) devised an alkaline permanganate reagent containing barium ions for the complete oxidation of organic compounds. The very insoluble barium manganate is formed as a reduction product and thus the formation of manganese dioxide is prevented. Excess potassium permanganate is determined volumetrically. The photometric measurement of the absorbancy of permanganate solutions serves as a very sensitive index of permanganate concentration. Goldblith and Proctor (9)added potassium permanganate to solutions of hydrogen peroxide acidified with sulfuric acid, in the determination of catalase activity. The concentration of excess permanganate was determined photometrically. The absorbancy of the solution did not remain constant for over 1 minute, however, because of the rapid formation of manganese dioxide. In the present work it was found that 6.6 X lop5M solutions of cis-aconitic acid which contained sulfuric acid or trichloroacetic acid (TCA) were readily oxidized by 2.67 X 10-3 N potassium
permanganate. The reaction mixture retained the permanganate color, but a brownish discoloration, probably due to the formation of manganese dioxide, became noticeable almost immediately. M ) were not oxidized by this Citric acid solutions (6.6 X concentration of permanganate under these conditions, but on the addition of catalytic amounts of manganese ions to the reaction mixture, an induced oxidation of the citric acid was observed. If a mixture of aconitic acid and citric acid in the above N potassium listed concentrations was treated with 2.67 X permanganate, the solution was rapidly and completely decolorized even a t 0". These observations indicate that the lower valency states of manganese ions act as electron carriers in the induced oxidation of citric acid, whether they are added directly or formed in the course of the aconitic acid reduction of potassium permanganate. Similarly, Mew, Stafford, and Waters (6) have found an induced oxidation of alcohols by potassium permanganate in acid solutions containing ferrous sulfate. In order to prevent these induced oxidations of hydroxy compounds in the analysis of aconitic acid, the inorganic acid in the reaction mixture was changed to metaphosphoric acid, an excellent metal-complexing agent in acid solutions. Two effects of this substitution were noted: (1) An aconitic acid-potassium permanganate-metaphosphoric acid mixture remained a clear pink-Le., manganese dioxide formation was prevented (Table I, lines 3 and 5),and (2) the induced oxidation of citric acid m-as completely inhibited (Table I, lines 4 and 6). REAGENTS AND MATERIALS
Stock Solution of Metaphosphoric Acid. Metaphosphoric acid pellets, anal tical reagent grade (30 grams), are dissolved in io ml. of distilgd tvater with mechanical shaking. The solution is stored a t 4' and is prepared fresh every few days. Stock Potassium Permanganate. Potassium permanganate solution (0.1 N ) is prepared and stored according to Kolthoff . and Sandell ( 3 ) . Working Potassium Permanganate Solution. One milliliter of the potassium permanganate stock solution is diluted to 26.0 ml. with water. This solution is unstable and is prepared just Drior to use. The addition of 1 ml. of 10% metaphosphoric acid to the dilute permanganate solution greatly refards- manganese dioxide formation.
V O L U M E 24, NO. 6, J U N E 1 9 5 2
1065
Table I. Effect of Metaphosphoric Acid on Oxidation of Aconitic Acid and on Induced Oxidation of Citric Acid by Potassium Permanganate Aconitic Acid
Citric Acid
pM.
pM.
0 0 0.10 0.10 0.10 0.10
0 1.0 0 1.0
0
1.0
50%
10%
0.004 N KMnOl M1.
Bbsorbancy a t 5300 A" 12 min. 20 min.
TCA M1.
HPOn M1.
0 0.10
0.10 0
1.00 1.00
0.73 0.73
0.73 0.72
0.10 0.10
0 0
1.00 1.00
0.62 0
0.546 0
0 0
0.10 0.10
1.00 1.00
0.59 0.59
0.57 0.56
6 min.
0.73 0.73 0.54b 0
0.55 0.55
Time after addition of KSfnOa. b Solution discolored by formation of MnOz.
a
The family of curves obtained with cis-aconitic acid solutions when readings were carried out 10, 20, 30, 40, and 50 minutes after the addition of the potassium permanganate is shown in Figure 1. The lines were fitted to the experimental points by the method of least squares. The standard deviation of each line is small enough to permit a waiting period of convenient duration before the absorbancies are determined. If the reaction mixture is maintained a t O " , the rate of decolorization is reduced considerably-for example, the 0.1micromole cis-aconitic acid solutions had an absorbancy of 0.60 =k 0.01 in the period 10 to 60 minutes after the addition of thp permanganate and 0.2-micromole aconitic acid showed an absorbancy of 0.48 f 0.01 during the 20- to 40-minute period Known quantities of allyl alcohol, itaconic acid, crotonic acid, maleic acid, or fumaric acid were substituted for aconitic acid in the standard procedure and the absorbancy of the ewes3 permanganate was determined after 20 minutes. The concentration of each unsaturated substance plotted against absorbancyielded a straight line. The slopes of these lines indicate that there are wide differences in the rate of oxidation; allyl alcohol was most s l o d y oxidized and aconitic acid most rapidly.
Table 11. Effects of Various Ions on Absorbancy of Aconitic Acid-Citric Acid-Potassium Permanganate iMixtures
0.
0.10
0.20
TCi
p.11.
MI.
0 0.10 0.10
0 0 1.0
0.10
..
0.10
0.10
.. ..
0.10
0 1.0
0.10 0.10
....
0.10 0.10
0.10
507
0.10
0.10
PzO, MI.
0.73 0.52
..
0
0.51 0
0.73
0 73 0 50 0
0.29 0.29
0.22 0.22
012 012
Table 111. Recovery of Aconitic Acid
PER 1.50 M L
Time in minutes between sddition of potassium permanganate t o cuvette and measurement of absorbancy is indicated b y number at end of each line. Standard deviation of points about line = = d 4 Y - Yc)2 N - 2 ?I = observed absorbancy wc = calculated absorbancy .V = number of determinations m = a t 10 minutes = 0.011: a t 20 minutes = 0.009; a t 30 minutes = 0.011. a t 40 minutes = 0.010: at 50 minutes = 0 01; absorbancy unit
0 1M
Citric Acid
pJJ.
MICROMOLES OF ACONITIC ACID
Figure 1. Determination of cis-..lconitic Acid
0 4 M NaF M1.
Aconitic Acid
Min. after addition of 1.0 M1. of 0.004 X KRlnOL 6 12 20Absorbancy at 5300 A
Protein Soln.
JI1. 0 60 0 50
Aconitic Acid Sdded pM.
0 10 0.0
Total KhInOc Reducing Substances (L
'VI.
0 23 0 13
Aconitic Acid Recovered
cb 100
~~
Working Solution of Aconitic Acid. One milliliter of 0.1 ilf cis-aconitic acid solution is diluted to 100 ml. with distilled water. This solution contains 1 micromole per ml. cis-Aconitic acid anhydride (melting point 77-78 O ; neutral equivalent found 52.1, calculated 52.0) was prepared from transaconitic acid by the method of Anschutz and Bertram ( 1 ) and was recrystallized from benzene. It dissolved readily in water to form a light yellow solution which became colorless on neutralization. PROCEDURE
An aliquot which contains 0.05 to 0.2 micromole of aconitic acid is placed in an optically calibrated 10 X 75 mm. cuvette and 0.10 ml. of 10% metaphosphoric acid Bolution is added. If the solution to be analyzed is alkaline or is buffered above pH 3, a larger quantity of metaphosphoric acid may be required. The solution is diluted with water by means of a pipet to a total volume of 0.50 ml. One milliliter of 0.004 N potassium manganate is added to the cuvette, and the solution is m i x e f r i inversion, and allowed to stand away from direct sunlight for 20 t o 50 minutes. The absorbancy of the solution a t 5300 A. is determined us. a distilled water blank in a Coleman, Jr., spectrophotometer. Since the reaction does not attain a definite end point a t a given time, it is advisable to include two different concentrations of a standard solution of aconitic acid in each set. These values can be used in constructing a standard curve.
EFFECTS O F IONS ON RATE OF OXIDATION OF ACONITIC ASD CITRIC ACIDS
The inclusion of sodium fluoride in the aconitic acid-potassium permanganate-trichloroacetic acid reaction mixture prevented manganese dioxide formation. When citric acid was also present, however, its induced oxidation was not prevented by fluoride ions (Table 11, lines 2 and 3 ) . The presence of pyrophosphoric acid likewise prevented manganese dioxide formation, but the oxidation of aconitic acid was found to proceed much more rapidly than in the presence of metaphosphoric acid. Pyrophosphoric acid resembled metaphosphoric acid in inhibiting the induced oxidation of citric acid (Table 11,lines 4 and 5). EFFECTS O F ORGANIC COMPOUNDS ON DETERMINATION O F ACONITIC ACID
As potassium permanganate is known to oxidize a large number of compounds other than unsaturated substances, representative materials were added to the 0. I-micromole aconitic acid-metaphosphoric acid-potassium permanganate reaction mixture. One-micromole quantities of the following compounds were without effect on the absorbancy of the solutions 20 minutes after the addition of the potassium permanganate; a-ketoglutaric acid, malic acid, tartaric acid, pyruvic acid, citric acid, isocitric acid, malonic acid, oxalic acid, fructose, and glucose. The presence of ascorbic acid or oxalacetic acid in 1.0-micromole quantities resulted in rapid decolorization of the permanganate. T h e n the
ANALYTICAL CHEMISTRY
1066 amount of glucose was increased t o 100 micromoles, it still was not oxidized under these conditions. However, 10 micromoles of oxalic acid decolorized the permanganate.
statistical treatment of the data, and John Ilemp and H. P. Iiato for technical assistance. LITERATURE CITED
RECOVERY OF ACONITIC ACID FROM A PROTElh SOLUTION
cis-ilconitic acid (0.1 micromole) was added to a protein solution and the mixtures were deproteinized by the addition of metaphosphoric acid, final concentration 3%. The suspension was centrifuged and the permanganate-reducing materials of the supernatant solution were determined. The date of Table 111 indicate that complete recovery of the added aconitic acid was obtained. ACKNOWLEDGMENT
The author wishes to thank Lowell Woodbury for aid in the
(1) Anschiitz, R., and Bertram, TT.. B c r ~ 37, , 3967 (1904). (2) Goldblith, S. A,, and Proctor, E. E., J . Bid. Chem., 187, io5
(1950). (3) Kolthoff, I. JL, and Sandell, E. B., "Textbook of Quantitative Inorganic .4nalysis," p . 592, Sew York, Maomillan Co., 1946. (4) Lauer, K., and RIakar, S. 31.,.Is.~L. CHEY.,23, 587 (1951). ( 5 ) JIerz, J. H., Stafford, G., and K a t e r s , K. A., J . Chern. SOC., 1951, 638. (6) Stamm, H., Alzgew. Chem., 47, 791 (1934). R E c E I r E D for review October 1, 1931. Accepted January 16, 1952. supported in part by a grant f r o m t h e U. S.Public Health Servxe.
'Kork
Densities and Refractive Indices for Diethylene Glycol-Water Solutions GORDON M A C B E T H I
AND
A. RALPH THOMPSON
University of Pennsylvania, Philudelphia, Pa.
S A means of determining the compositions of aqueous
A diethylene glycol ( p , 8' - dihydrosyethyl ether) solutions,
measurements were made on a number of solutions of known composition for density a t 35" C. and for refractive index a t 25" C. Although values of these properties for the purified glycol have been reported in the literature ($), for aqueous solutions only graphical representations of the specific gravity using commercial grade materials could be found ( 1 i. I
I. I O
,
I
The water content of this purified diethylene glycol, as dereimined by Karl Fischer reagent, ivas found to be 0.025% by weight, The value of the refractive index, nVl 1.4461, agrees exactly with the value reported by Rinkenbach ( 4 ) , and the specific gravity At,
,I:;
1.1183, is in close agreeinelit ii-ith previously reportell values ( 1 ) . This purified diethylene glycol, together with freshly boiled distilled water, n-as used t o prepare nine solutions of various glycol concentrations from 10 to 90 xyeight %. The materia!s were pipetted into 50-ml. ground-glass-stoppered Erlenmeypr flasks, the actual amounts being determined by weighing to 0.1 mg. on an analytical balance. All solution compositions, based on amounts of material weighed and accuracy of the weighing., are known t o within approximately 1 part in 30,000. _.
DENSITY MEA SUR EM ENTS
The density measurements were made using capped, 10-1n1. \Veld-type specific gravity bottles. These were equilibrated in a constant temperature bath maintained a t 35.00' i= 0.02" C. as determined by a calibrated thermometer. Duplicate deterniinations were made on each solution. All the weighings were reduced to values in vacuo and the absolute densities a t 35' C. were calculated as grams per millilit,er. 0
20
40 60 80 WEIGHT % DIETHYLENE GLYCOL
100
Figure 1. Absolute Densities of Aqueous Diethylene Glycol Solutions at 35'C.
I n an earlier paper (Z),the authors presented data on densities and refractive indices for the system propylene glycol-water. Purification of materials and methods of measurement were described, and limitations of the data discussed. For the system under consideration, sufficient differences were encountered to make description of these advisable.
The experimental results are listed in Table I . Snioothrd values obtained from a large scale plot similar to Figure 1 are presented in Table 11. An esamination of these data shows that up t o approximately SOY0 diethylene glycol, the composition should be measurable to xithin approximately 0.03 weight %, Beyond this, the slope of the curve decreases rapidly, as does the usefulness of density as a measure of composition. 1.45
PREPARATION O F SOLUTIONS
For use in preparing the solutions of known compositions, commercial grade diethylene glycol was purified by two successive distillations in a Todd column, 1 inch in diameter by 36 inches high, packed with 3 / , 6 inch glass helices. The absolute pressure was maintained a t 10 to 15 mm. of mercury, and a reflux ratio of approximately 20 to 1 was used in both distillations. The distillate came in contact only with glass, and drying tubes containing anhydrous calcium sulfate were used to prevent contamination with moisture during venting of the receiver. I n the first distillation, the middle 50% of the charge was collected, all within a 1" C. boiling point range. This material was then refractionated, the middle 50% being collected for use in making up the solutions. 1
Present address, The Dow Chemical Co, hlidland, hIich.
n x- 1.41 W
n
z W
> 1.37
a
a '
u. W a
1.33
b/
0
'
I
20 40 60 80 WEIGHT 5. DIETHYLENE GLYCOL
Figure 2. Refracti\e Indices of Aqueous Diethylene Glycol Solutions at 25' C.
100
