V O L U M E 2 4 , N 0.
OCTOBER 1952
calcium oxalate as described by Willard and Furman ( 5 ) . Thoium oxalate appears to be effectively precipitated along with the rcalciuni under these conditions. Use a filtering crucible (Selas No. 3010). After heating in the electric muffle furnace for conversion of the oxalate to carbonate, weigh the crucibles and contents. Place the crucibles in Gooch filtering funnels with the outlets leading into 15 X 125 mm. test tubes on which graduation marks a t 6 and 10 ml. have been scratched. Add from a pipet 1 ml. of 3700 hydrochloric acid solution to each, allowing the acid to run down the sides of the crucibles. Turn on gentle suction and wash each crucible with a very fine stream of redistilled water until the volume of solution in the test tubes is nearly 6 ml. Dry and weigh the empty crucibles. Remove the test tubes from the filtering funnels, add 2 ml. of a lO7’ solution of hydroxylamine hydrochloride to each, and boil gently for about 1 minute. Add 5 drops of 3i7’ hydrochloric acid and 1 ml. of a 0.1% solution of the sodium salt of l-(o-arsonophenylazo)-2-naphthol-3,6-disulfonic acid. Dilute all solutions to 10 ml. Prepare a reference solution for use in the spectrophotometer by suspending in 10 ml. of redistilled water an amount of calcium carbonate equivalent to twice the average weight of calcium carbonate found in the above four samples. Add 1to 1hydrochloric acid dropwise until 10 drops in excess of the amount needed to dissolve the carbonate are present. Add 4 ml. of 10% hydroxylamine hydrochloride solution and 2 ml. of the dye solution, and dilute the resulting solution to 20 ml. Compare the absorbancy of each of the four samples, two of u hich have 10 micrograms of added thorium, against the reference solution by use of a Beckman ?\lode1DU spectrophotometer.
1625 Set the slit opening a t 0.0355 mm. and the wave length a t 545 mp. Determine cell corrections by comparing the absorbancy of the reference solution in different cells. Determine the amount of thorium present by the relative absorbancy of the “spiked” with the plain samples when compared to the reference solution. Analysis of Synthetic Samples. A series of synthetic samples was prepared to test the determination of thorium in the presence of other materials. The results of these determinations are presented in Table 111. LITERATURE CITED
(1) Hall, R. H., U. S. Atomic Energy Commission, AECD-2437 (Piov. 12, 1948). (2) Kuznetsov, V. I., J . Gen. Cheni. (C.S.S.R.), 14, 914-19 ( 1 9 4 4 ) . (3) Reed, S. A.. Byrd, C. Pi.,and Banks, C. V., U. S. .Itoniic Energy Commission, AECD-2565 (.-lpril 18, 1949). ( 4 ) Thomason, P. F., Perry, SI. h.,and Ryerly, TT, SI,,ANAL. CHEM.,2 1 , 1 2 3 9 (1949). ( 5 ) TTillard, H. H., and Furman, Pi. H., “Elementary Quantitative Analysis,” 3rd ed., pp. X39-4:3, Sew York. D. Van Kostrand
Co.. 1940. RECEIVED for review March 30, 1962. Accepted June 16, 1962. Based on work done for the Atomic Energy Commission under contract AT(10-1)-310 xith Idaho State College.
Qualitative Detection of Urea in Commercial livestock Feeds GR4EME L. B4KER AND LEON 13. JOHNSON D e p a r t m e n t of C h e m i s t r y Research, Montana S t a t e College Agricultural Experiment Station, Boaeman, M o n t .
.XSY state l a ~ require s that commercial livestock feed con-
IM
taining nonprotein nitrogen be so labeled and its equivalent crude protein value be given. Urea is the most common source of nonprotein nitrogen employed and is usually determined b~ means of a urease digestion followed by a Kjeldahl-type distillation of the ammonia released as recommended by the Association ot Official Agricultural Chemists ( I ) . This method is too timeconsuming to be employed for the sole purpose of qualitativel\ checking for unreported nonprotein nltrogen, especially when a large number of samples is involved. Many methods have been proposed for the detection of urea. but none can be conveniently applied to the problem encountered in commercial feed analj The method proposed by Sanchez (11-13) is too tedious for routine application. The method of Fearon ( 3 ) is not advantageous since guanadine, creatine, and gelatin all give results similar to urea. A second method of Fearon ( 4 ) results in a golden-yello~v color as a positive test This is difficult to read because of coloration of many feed e\tracts. Schemes using urease and detecting the released ammonia by means of acid-base indicators have been proposed ( 2 , I O ) , but are rather difficult to control in laboratory operation. involving many samples. A method which detects the released ammonia with a silver-salt test paper (6) has the same objections. The problem of detecting urea in animal and vegetable nitrogen o ~ 3materials was investigated by Moore and White (8). Their method involved the uqe of bromine water and caustic soda to release nitrogen from the urea. A positive test resulted in thc effervescence of nitrogen. The method required close observation and would be difficult to conduct when many samples are to be tested. -1rapid, qualitative method has, therefore, been devised which may be used as a preliminary check for urea in a feed sample The method utilizes the normal urease digestion followed by a teqt for ammonia b) means of a modified Sessler reagent. The use of a modified Sessler reagent as a qualitative test was suggested by the use of Nessler reagent for the quantitative determination of urea in hlood and urine. The reagent as used in
the pi esent piocedure is considerably more concentrated than that applied by Folin and Wu ( 5 )and others ( 7 , 9 ) . REAGENTS
Urease (Jack Bean), Arlington Brand. Sodium hydroxide, 6 S. Modified Nessler reagent. Dissolve 28 grams (0.5 mole) of potassium hydroxide, 166 grams ( 1 mole) of potassium iodide, and 227.5 grams (0.5 mole) of mercuric iodide in 500 ml. of distilled Tvater. PROCEDURE
A 0.3- to 0.5-gram sample of the feed to be checked is added to 10 ml. of distilled water in a 15-ml. centrifuge tube, and to this is added approximately 0.01 gram of urease. The materials are shaken vigorously in order to dissolve any urea that may be present in the feed and then centrifuged a t 2500 r.p.m. for about 20 minutes. Approximately 1 ml. of the supernatant liquid is decanted into a test tube and diluted to 10 ml. with distilled water. Three to five drops of the modified Nessler reagent are added to the diluted sample, followed by the addition of 1 nil. of 6 N sodium hydroxide. The presence of urea will be indicated by the production of a red-brown precipitate of dimercuric ammonium iodide (XHgzI), or by an orange to red-brown coloration of the solution, in cases of low urea concentrations. DISCUSSION
This method has been used to check different commercial feeds for urea. I n the preliminary study with 120 different commercial feeds, the feeds were first examined by the approved official method ( 1 ) for urea determination, to ensure the validity of the tests made by the method. Fourteen of 120 samples contained urea, and all were readily detected by the suggested method. The urea content in the feeds examined ranged from 1.5 to 13.37’ equivalent crude protein. To establish the sensitivity of the test, trials were made on feed samples adulterated with varying amounts of urea. Urea vias detected positively in all samples n hich contained a t least 0.5% urea (1.570 equivalent crude protein) in the original feed sample. The procedure is designed specifically for feed analy If it is t o be utilized for other purinpoqeG n heregreater sensitivity i q deqired, the sarrple neight and
ANALYTICAL CHEMISTRY
1626
aliquot may be increased. The modified Nessler reagent, when used as described, will detect 0.025 mg. per ml. of urea in solution. The sodium hydroxide solution is added after, rather than with, the modified Sessler reagent because sharper, more immediate results are obtained in this manner. It is important that the results be noted immediately upon addition of the sodium hydroxide, since low urea concentrations are best noted by the color change which occurs a t this time. The presence of ammonium salts in feeds will give a positive test by the method described. These salts can be distinguished from urea by a preliminary test employing the modified Nessler reagent without the prior addition of urease. The modified Sessler reagent will precipitate protein in many feed extracts, but the protein precipitate offers no interference since its color differs greatly from the red-brown dimercuric ammonium iodide. The characteristic color someitmes forms without the formation of a visible precipitate of dimercuric ammonium iodide. This is also a positive test for urea and occurs with some feeds containing low concentrations of urea.
LITERATURE CITED
(1) Assoc. Official -4gr. Chemists, “Official Methods of Analysis,” 7th ed., Washington, D. C., 1950. (2) Ruchanan, G. H., 2nd. Eng. Chem., 15, 637 (1923). (3) Fearon, TV. R., Analyst, 71, 562 (1946). (4) Fearon, TV. R., Sci. Proc. Roy. Dubltn Soc., 22, 415 (1941). (5) Folin, Otto, and \Vu, Hsien, J . Bzol. Chem., 38, 81 (1919). (6) Ishler, N. H., Sloman, Katherine, and Walker, M.E., J . dssoc. Offie. Agr. Chemists, 30, 670 (1947). (7) Jlarenzi, A. D., Anales farm. y biogulim. (Buenos Aires), 16, 3 (1945). ( 8 ) Moore, H. C., and White, Robert, Ind. Eng. Chem., 19, 264 (1927). (9) RIosto, 0. V., and Romano, -4.C., Anales farm. y bioquim. (Bitenos Awes), 16, 52 (1945). (10) Pinsuccen, L., Biochem. Z.,132, 242 (1922). (11) Sanchez, J. ri., Ann. chim. a n d chim. appl., 18, 64 (1936). (131 Sanchez, J. A., Chimie & industrie, 37, 869 (1936). (13) Sanchez, J. A,, Seman med. (Buenos Aires), 1930, I, 1484. RECEIVEDfor review April 21, 1952. Accepted July 9, 1952. Montana State College, Agricultural Experiment Station, Project 162, Paper 268, Journal Series.
Improved Method of Preparing Sulfonated 1-Naphthol for Carbohydrate Tests ARTHUR W. DEVOR’ Department of Chemistry, S o u t h Dakota State College, Brookings, S . D .
H E usual Molisch reaction can be modified by presulfonation of the 1-naphthol t o give better results when testing for a very small amount of carbohydrate (1). Even when highly purified 1-naphthol (melting point 95-96’ C.) is used, the sulfonated product is somewhat colored (8). No solid sulfonated product was obtained by this method and the water solution of the reagent contained considerable sulfuric acid. The objectives of the present investigat’ion were to obtain a white sulfonated 1-naphthol product which could be made up in water t o any desired concentration and t o investigate the interference by colored impurities. EXPERIMENTAL
Sulfonation of 1-Naphthol. Twenty-eight to 30 ml. of concentrated sulfuric acid was added t o a small beaker containing 15 grams of purified 1-naphthol (product of the J. T. Baker Chemical Co.). This mixture was warmed slightly (the reaction is exothermic) and continually stirred until the sulfonated product formed a pasty mass. After standing several hours (usually overnight) all large lumps were broken up, 25 ml. of warm to hot distilled water was added, and the hot mixture was stirred until a solution was obtained. The resulting hot solution was set aside in a cold place (refrigerator) for 16 hours or longer. After crystallization was complete, as much as possible of the dark colored liquid (fraction B) was removed by filtering with suction through coarse fritted glass. The nearly x7hit.e crystals (fraction -\) were mixed (while in the filter) with 1 or 2 ml. of cold distilled water and the washings were drawn off as much as possihle. Yield was about 20 grams. The procedure for highly purified 1-naphthol (melting point 9,?-96O C.) was the same as for purified 1-naphthol. Yield was about 26 grams of fraction A . The same procedure was used for technical 1-napthhol, except that 20 grams of technical 1-naphthol was used in place of the 15 grams of purified material and a longer time was allowed for sulfonation as well as for crystallization. Yield was about 10 grams of fraction A. SULFONATION TECHNIQUE
The sulfonated product dissolves readily in warm to hot (not boiling) uater. The resulting solution should not be heated. Sufficient time is needed for fraction A to crystallize. I t is better to allow the mixture to stand a t room temperature until crystallization stops (a day or two) and then to place the material in the refrigerator overnight. Sometimes seeding is necessary. Crystallization must be slow in order to obtain large crystals. The filtration process should be carried out a t as low a temperature as possible, so that the crystals do not redissolve. The crystalline product (fraction -4) should be packed in the filter so as to ensure a more complete separation from fraction B. Washing removes some of the colored fraction as well as most of the adhering sulfuric acid. Since the sulfonated product is very soluble in water, not more than 1 or 2 ml. of water should be used, or the yield of fraction A nil1 be greatly reduced. It is not always neceqyary to wash the product when highly purified 1-naphthol is used. Fraction A can be dried over sulfuric acid, but drying is not adviqable because an insoluble material forms during the drying period. U S E OF FR4CTIONS A AYD B
\\’hen used for tests as described in previous reports ( 1 , d ) , 8 to 10 grams of fraction ?I is dissolved in 100 ml. of water. The water solutions of the two fractions were studied in the Beckman spectrophotometer. All solutions of fraction il were clear and nearl\T coloiless, while the solutions of fraction B were very dark in color. It !vas proved that such dark colored impurities cause some inteiference even for pure carbohydrate solutions. It is impossible to use dark colored reagents for visual tests. Fraction A should be stored in a water solution and exposure to hright light should be avoided. Any insoluble material which forms during storage can he easily filtered off.
It is not necessary to allow the sulfonation mixture t o stand for more than 1 or 2 hours, except when technical 1-naphthol is used. However, the yield is lower when less time is allowed for this sulfonation. The mixture muRt he stirred continually until sulfonation is nearly complete ( a t’hick pasty mass is formed). The sulfonation occurs more rapidly when the mixture is warmed, but overheating will cut down the yield. 1 Present address, Department of Physiological C h e m i s t r y , Ohio State University, Columbus, Ohio.
LITERATURE CITED
(1) Devor, A. IT., J . Am. Chem. Soc., 72, 2008 (1950). (2) Devor, A. W., and Kamstra, Leslie, Proc. S o . Dah. Acad. Sci., 30 (1951). RECEIVED for review January 19, 1952. Accepted June 14, 1952. W o r k supported in part b y Statewide Service.
