Assav of Benzaldehvde J
J
Use of Hydroxylammonium Sulfate and Aqueous Sodium Hydroxide MARCELLE SCHUBERT AND JANET G. DINKELSPIEL, Research Laboratory, Benzol Products Co., Newark, N. J.
T
HE strength of benzaldehyde, either pure or in solution,
can be estimated with varying degrees of accuracy by several methods. The official methods of the ilssociation of Official Agricultural Chemists (I) recommend determining the benzaldehyde as the phenylhydrazone. Though this method gives the most reliable and constant results, i t is time-consuming, and the phenylhydrazine must be freshly distilled (though if stored in the refrigerator i t will keep for about 3 weeks) ; the precipitate must stand overnight, the filtration requires considerable time, and in vacuo at 70" C. the precipitate requires 3 hours to dry. Furthermore phenylhydrazine can cause a very disagreeable allergy, resulting in swelling and intense itching of the fingers or other parts, and eventual peeling of the skin. The bisulfite method originally proposed b y Ripper (8) has been discussed by Kolthoff (5, 6) and Donnally ( 3 ) . Benzaldehyde is added to a known excess of sodium bisulfite, and the sodium bisulfite not used in forming the benzaldehyde addition compound is titrated with standard iodine solution. This method is not, in the authors' experience or according to the literature, satisfactory even for pure material. It is awkward and inconvenient, and the authors have found i t to he decidedly unreliable. The third method, recommended b y the United States Pharmacopoeia (9), involves the titration of the hydrochloric acid liberated b y benzaldehyde from a solution of hydroxylammonium chloride. The reaction of aldehydes with hydroxylammonium chloride was first studied b y Cambier and Brochet (a),who established the quantitative relationship between the liberation of the acid and the moles of aldehyde present. Since then, wherever the method has been applied, the chloride has always been used. I n dilute solution there is an error of 7 to 9 per cent ('7). Besides being expensive, the chloride is unstable, dissociating into free hydrochloric acid and hydroxylamine. The acid liberated from the hydrospl-
TABLE I. Assay 7
BENZALDEHYDE
Pharmacopoeia method0
%
%
243
84.3 85.8 92.6 92.2 95.0 95.5 98.3 97.0 97.3 99.2
86.0 85.8 92.6 92.6 95.3
243 244 233
PL12 PL13 151 152 153 154
6
OF
Benzaldehyde Found A. 0. A. C. method
Bntch KO.
... . .
I
94: 1
..
..
91.5 91.8 91.7
97.8
..
98.0 97.7 94.2 94.4
.. ..
95:9 96.0
..
.. .. .. .. ..
.. ..
NHaOH.HC1, 96% C&OH.
9 6 :5
..
9i:4 96.5
..
Authors' method
ammonium chloride by benzaldehyde is titrated using alcoholic potassium hydroxide with bromophenol blue as indicator (4, 9). It was claimed (4) that unless the alkali was added drop by drop, results would be low. The authors first compared the results obtained b y the phenylhydrazone and the hydroxylammonium salt methods and found that they checked. Next preliminary experiments using aqueous sodium hydroxide instead of alcoholic potassium hydroxide showed that the aqueous alkali was entirely satisfactory. They then found that i t was not necessary t o add the alkali drop b y drop. Finally, as a result of the search for a better indicator, they found that tetrabromophenol blue gave an end point much more quickly perceived, although i t does not affect the extreme deviation in the results. Two years ago the Commercial Solvents Corporation put hydroxylammonium sulfate on the market, and as this salt is stable and cheap, the authors immediately decided to test its usefulness in the assay of benzaldehyde. The hydroxylammonium sulfate as furnished b y the Commercial Solvents Corporation is entirely satisfactory, and can be used without further purification, though i t may be necessary to filter the solution to make i t brilliantly clear. If it is desired to recrystallize the reagent, boiling in 50 per cent methyl alcohol with a little Darco G6O gives excellent results. The authors' experiments may be summarized as follows (see Table I) : Hydroxylanimonium sulfate can be used in place of the unstable and expensive chloride. Aqueous sodium hydroxide can be used in place of alcoholic potassium hydroxide. The sodium hydroxide can be run in rapidly to within 2 to 3 cc. of the end point in less than one minute. A single titration, after sufficient practice in catching the end point, requires only 1.75 minutes. Tetrabromophenol blue gives an end point more quickly perceived than in the case of bromophenol blue (9),though the error is about the same. The extreme deviation in the results obtained depends largely on how frequently such titrations are run. When run daily, the extreme variation may be, on an average, only 0.2 to 0.3 per cent, while xeekly titrations immediately raise the error to as much as 0.6 to 0.7 per cent. The extreme deviation has thus been reduced from 1 to 1.5 per cent (alcoholic potassium hydroxide and bromophenol blue) to a maximum of 0.6 to 0.7 per cent.
%
Procedure
... .
Solutions required: 1.25 per cent hydroxylammonium sulfate in 50 per cent methyl alcohol, 0.1 N standard aqueous sodium hydroxide, and 0.1 per cent tetrabromophenol blue in 50 per cent methyl alcohol. The 50 per cent methanolic solution of hydroxylammonium sulfate is acid and must be neutralized. For six batches of benzaldehyde which are assayed in triplicate, 600 ml. of solution are made. Three milliliters of tetrabromophenol blue indicator are added, turning the solution a clear green. About 12 to 15 ml. of 0.1 N sodium hydroxide are required to change the color to blue. Since the indicator is dichroic, the color in bulk is different from that of a small quantity; therefore alkali is added until the color of a 30-ml. sample poured into a 75-ml. Erlenmeyershaped flask is a clear pale blue. The solution should be neutralized immediately before use, because if it stands for more than 2 hours, sufficient acid is liberated by hydrolysis of the hydroxylammonium sulfate to change the color of the indicator and so make the results of the benzaldehyde assay high. A 5-ml. Lunge weighing pipet is used to measure out the sample. In order further to reduce the danger of oxidation, the pipet is filled nearly full. The taper is fine enough so that about 10 drops give 0.2 gram. The weight of the pipet after weighing out the first sample can safely be used as the weight of the pipet be-
.. .. .. .. ..
9i:s 92.1 92.2 97.6 98.1 97.5 94.2 93.8 94.0 95.5 95.8 96.0 96.5 96.3 96.5 96.6 96.4 96.3
0.1 iV alcoholic KOH, bromophenol blue.
154
ANALYTICAL EDITION
February 15, 1942
fore the next sample, and so on, without introducing an appreciable error. The benzaldehyde is dropped into a 75-ml. Erlenmeyer-shaped glass-stoppered flask containing 30 ml. of neutralized hydroxylammonium sulfate solution, and the flask is gently swirled. Samples lower than 90 per cent benzaldehyde usually go into solution with difficulty.
Acknowledgment T h e authors gratefully acknowledge the cooperation of the Commercial Solvents Corporation in furnishing samples of hydroxy~ammon~um sulfate,
Literature Cited
Titration Standard 0.1 N sodium hydroxide is run rapidly into the solution until within about 2 to 3 ml of the end point, then a few drops a t a time until the color is gre&-blue, and finally dropn7iseuntil the color is again the pale blue. The absence of direct sunlight is desirable. This part of the assay is almost exclusively responsible for any error. The results obtained will be more truly representative if the samples are kept in glass-stoppered Pyrex bottles whose lips can be thoroughly cleaned. Xl. of 0.1000 N SaOH X 0.0106 weight of >ample
155
loo = % benzaldehyde
(1) AssoC. OfficialAm. Chem., Official and Tentative Methods Of Analysis, 4th ed., p. 314, 1935. (2) Cambier, and Brochet, A., Compt. rend.? l20, 449 (1895). (3) Donnab’, L. H., IBD.ENG. CHEM*?ANAL. ED., 5 ? 91-2 (l933). (4) Givaudanianl p‘
(5) Kolthoff, I. M., and Furman, N. H., “Volumetric rlnalysis”, 1st ed., Vol. 11, pp. 450-2, New York, John Wiley & Sons, 1929. (6) Kolthoff, I. M., and Rosenblum, C., “Acid-Base Indicators”, 1st English ed. (tr. from 4th German ed.). D. 123. New York. Macmalan Co., 1937. (7) Neubere. C.. and Gottschalk. A.. Biochem Z . . 146. 167 11924). (8) Ripper,YTfonatsh., 22. 1079 (1900): (9) U. S. Pharmacopoeia XI, p. 247 (1936).
Colorimetric Determination of Phosphorus in Biological Materials RUTH ADELE KOESIG AKD C. R. JOHNSON, University of Texas, Austin, Texas
ISSOS’S ( 3 ) method for the estimation of phosphorus has recently been adapt’ed to the determination of this element in iron, steel, and iron ores (4, 6 ) . During use of the method in food analyses in this laboratory, the fact became evident t’hat its range, sensitivity, and precision could be increased considerably in this application, where limiting interferences are rare. For this purpose the effect of time on the development of the yellow phosphovanadiomolybdate at various concentrations has been studied, the optimum acid concentration has been found, the range over which Beer’s law holds precisely has been determined at three wave lengths of light, and spectrophotometric calibration data have been obtained with nine series of standard solutions. The results of these experiments have been condensed in a convenient procedure for the rapid and precise determination of phosphorus in foods and other biological materials, and this procedure has been tested b y comparison with official gravimetric methods.
Apparatus and Reagents
,
A Coleman Model 10-S spectrophotometer was used to measure transmittance values. The spectral band width was 30 mp. Matched square cuvettes were used to hold the reference liquids and test solutions. The dark current adjustment was frequently checked during the measurements, which were made a t temperatures between 25” and 30’ C. Two independently prepared sets of reagents were used. 15 N nitric acid, 6 N hydrochloric acid, 9 N perchloric acid, and 4 N aqua ammonia were purified by distillation methods. Standard potassium dihydrogen phosphate solution containing 0.1000 mg. of phosphorus per ml. was made by dissolving 0.4393 gram of twice recrystallized and vacuum-dried material in distilled water and making 1.0 1 liter. Another standard solution containing 0.1000 mg. of phosphorus per ml. was made by dissolving 1.351 grams of silver phosphate (8)in 5 ml. of 15 N nitric acid and diluting to 1 liter. Ammonium vanadate solutions were made by dissolving 2.346 grams of vacuum-desiccated ammonium metavanadate in 500 ml. of hot water, adding 10 ml. of 15 N nitric acid, and diluting to 1 liter. One sample of this salt was prepared by double recrystallization of reagent grade material with centrifugal draining; the other was a sample of atomic weight purity. One ammonium molybdate solution was prepared as described by Willard and Center ( 5 ) , using vacuum-dried molybdic acid purified by precipitation with 6 N hydrochloric acid, followed by two crystallizations from water, with centrifugal draining. A second solution of this reagent was a 10 per cent solution of ammonium molybdate shown to be free of phosphate
and chloride by blank tests; this was used with the systems containing silver. Fresh molybdate solutions were prepared frequently, as required.
Summary of Calibration Experiments I n obtaining calibration data, opportunity was taken to study the effect of various factors on the precision and accuracy of the method. Thus, while test solutions for the calibr$ions mere made by the same method described below for the analyses, standard phosphate solutions were used instead of digested samples, various concentrations and mixtures of nitric and perchloric acid were tried, and each system was kept under observation for a much longer time. Different combinations of solutions from the two sets of reagents were used, to eliminate constant errors. I n Table I are given the median transmittances from about 1200 calibration observations made approximately 30 minutes after color development. Mathematical treatment of the data as a whole showed t h a t the best agreement with the medians in Table I for all phosphate concentrations studied was obtained with systems containing 5 ml. of 9 N perchloric or 3 ml. of 15 LV nitric acid per 100 ml. Delayed precipitation took place in all standards and blanks which contained only 2 ml. of 9 -Irperchloric acid per 100 ml., in the standards containing 10 ml. of 9 A‘ perchloric acid and 5 mg. of phosphorus per 100 ml., and in some corresponding solutions containing equivalent amounts of nitric acid, but not in systems with intermediate acid concentrations. I n such systems transmittance readings were sufficiently constant after 10 to 30 minutes for precise measurements: transmittances were almost invariably lower after 12 t o 24 hours, but rarely more than 1.0 unit lower. The minimum drop in transmittance occurred in systems containing 5 ml. of 9 N perchloric acid per 100 ml. On two counts, therefore, this is the optimum acid concentration. However, neither time nor acid concentration is extremely critical: transmittance readings taken from 10 minutes to many hours after making u p the test solutions to 100 ml. with 2 ml. to 10 ml. of 9 N perchloric acid or equivalent amounts of nitric acid or nitric-perchloric acid mixtures give approximately correct results, even when precipitation of molybdic acid or the yellow complex is eventually to occur.
