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, South 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 O F 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.
