V O L U M E 22, NO. ?, J U L Y 1 9 5 0 filtrate is allowed to take place slowly with occasional stirring while the solution cools to room temperature. Crystallization is completed a t 4' C. for 16 hours. The crystals are collected on a fritted disk, washed three times with 50-ml. portions of ice-cold 1N hydrochloric acid, and sucked free of excess liquid. Residual water and hydrochloric acid are removed in 24 hours in an evacuated desiccator over solid potassium hydroxide. The yield is approximately 94% of the starting material. When dry, the product is stored in an amber bottle.
60 mm. gas pressure, measured a t the 0.5-ml. marked in the Van Slyke-Neil1 manometric apparatus ( 7 ) , in excess of the normal blank analysis in the ninhydrin carbon dioxide method (6). After purification, two treatments with Korit, each followed by crystallization, a product was obtained with properties as described above and which no longer contributed to the gas pressure of the blank ninhydrin carbon dioxide analysis.
Pure ninhydrin is very pale yellow with a greenish tint, is completely odorless, and dissolves in water to give a clear light yellow solution. Ninhydrin purified as above turns pink a t 125' C., shrinks with loss of water of hydration, becomes deep purple red at 139-140" C., and melts sharply with decomposition a t 241 ' C. (uncorrected). Corrected for stem temperature the melting point is 247" C. Ruhemann (8)reported similar observations and found a melting point of 239-240' C. (uncorrected). Teeters and Shriner ( 5 )recorded a value of 241-243 ' C. but did not state in their publication whether this was a corrected melting point. Samples of ninhydrin have been found which contribute 50 to
( 1 ) Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J . , 3 8 , 2 2 4 (1944). ( 2 ) Moore, S., and Stein, W. H., J . B i d . Chem., 176,367 (1948). (3) Ruhemann, S.,J. Chem. Soc., 97,1446 (1910). (4) Stein, W. H., and Moore, S., J . Biol. Chem., 176,337 (1948). (5) Teeters, W. O., and Shriner, R. L., J. Am. Chem. Soc., 55, 3026 (1933). (6) Van'Slyke, D. D., Dillon, R. T., MacFadyen, D. A., and Hamilton, P. B., J . Biol. Chem., 141, 627 (1941). (7) Van Slyke, D. D., and Neill, J. M., Ibid., 61,523 (1924).
LITERATURE CITED
RECEIVED September 0, 1949.
Kjeldahl Microdetermination ROBERT M. SILVERSTEIN A N D ROBERT PERTHEL, JR. Stanford Reseerch Institute, Stanford, Calif. HREE improvements with respect to accuracy and conTvenienoe in the micro-Kjeldahl procedure have been made. The first modification is designed to eliminate the occasional, but extremely annoying, suck-back of material into the steam generator, or of distillate and receiving aoid back into the distillation flask. This is accomplished by means of a simple, allglass check valve on the steam generator.
type in which the steam generator surrounds the distillation flask; there are numerous occasions when interruption without suok-back of a steam distillation in a Pozzi-Escot a paratus (7) may be desirable. At the completion of a KjeldahPdistil+ tion, the flame is removed, and the distillation flask emptied u1 the usual way by holding a fingertip over the tip of the valve.
In Figure 1, the valve is shown on a Kirk-type Kjeldahl apparatus ( 8 ) . It consists of a solid,.ground-glass ball, a ground ring seat 1 mm. in width, and four mdentations placed to allo: approximately a Zmm. lift. The seat is ground in with a 45 taper brass grinding tool. This valve is applicable to steam distillation setups in general, and is particularly effective on the n
M
,-moo 0
r7mm00
The practice in this laboratory had been to immerse the delivery tip (8 mm. in outaide diameter) of a Kirk apparatus in 5 ml. of 2% boric acid in a tilted 50-ml. Erlenmeyer flask. Distillation had been carried out with the tip immersed for 3 minutes, and then for 1 minute with the tip above the liquid ( 5 ) . The slightly low results obtained with pure ammonium sulfate samples indicated incomplete absorption of ammonia. Therefore, in order to facilitate absor tion of ammonia and still use the smallest possible amount of goric acid, a small bulb was blown on the delivery tip and five holes were unched therein with a hot, 1-mm. tungsten wire (Figure 2). !his bulb was immersed in 5 ml. of boric acid in a 20 X 150 mm. test tube. The use of the test tube provided a greater depth of boric acid, and permitted distillation of a definite volume (appropriate marks on the test tube) rather than the indefinite amount which varied with the rate of distillation during the fixed time interval. Eight milliliters were collected with the ti immersed and 2 ml. with the ti raised. A quantitative transfer to a suitable titration flasE with two 1-ml. washings brought the total volume t o about 17 ml. Figure 2
U
Figure 1 Diagram of Check Valve
An investigation carried out in this laboratory into the cause of frequent, slightly low results obtained on standard samples led to the second modification of the Kirk apparatus. Willits, John, and Ross (8) obtained "slightly low nitrogen values" on pure nitrogeneous compounds, using the Kjeldahl macroprocedure of the kssociation of Official Agricultural Chemists. These results were accounted for by assuming that all the ammonia was not caught by the trapping liquid. Subsequent use of a Goessman trap ( 1 ) on the receiver gave higher and more nearly theoretical results.
950
ANALYTICAL CHEMISTRY
Theoretical results were obtained, thus confirming on a micro scale the observations of Willits and co-workers. Although these authors and others have reported blanks due to alkali entrainment (and considerably reduced them with a new connecting bulb), this is not a problem in micro-Kjeldahls, using either a Kirk or a Parnas and Wagner apparatus (6). The third modification consists of a simplified titration procedure. The use of boric acid as a receiver in micro-, semimicro-, and macro-Kjeldahl procedutes is widespread. Ordinarily, standard hydrochloric or sulfuric acid is used to titrate the trapped ammonia. Niederl (6) uses 0.01 N potassium biiodate as a receiving solution, and back-titrates with standard alkali. In this laboratory, the advantages of boric acid and potassium biiodate are combined. Because potassium biiodate is a primary standard as well as a strong stable acid, the use of a base either for titration or standardization is eliminated. The receiving liquid is 2% boric acid, to which has been added sufficient bromocresol green-methyl red mixed indicator (3)to give a faint pink color when 5 ml. of this solution are diluted to 17 ml. with distilled water. Five milliliters of this solution are pipetted into a test tube, and the distillation is carried out as
described above. Titration is carried out with 0.01 N potassium biiodate solution ( 4 ) to an end point determined by comparison with the boric acid-indicator solution (5 ml. diluted to 17 ml.). ACKNOWLEDGMENT
The authors are indebted to Catharine M. Brown for the analyses involved in developing these modifications. LITERATURE CITED
Goessman, C. I., Mass. Agr. Expt. Sta., Bull. 54 (1898). (2) Kirk, P. L., IND.ENG.CHEM.,ANAL.ED.,8, 223 (1936). (3) Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” p. 451, New York, Macmillan Co., 1946. (4) Niederl, J. B., and Niederl, V., “Micromethods of Quantitative Analysis,” 2nd ed., p. 54, New York, John Wiley & Sons, 1942. ( 5 ) Ibid., p . 73. (6) Parnas, J. K., and Wagner, R., Biochem. Z.,125, 253 (1931). (7) Poszi-Escot, M. E., Bull. soc. chim., 31, 932 (1904). (8) Willits, C. O., John, H. J., and Ross, L. R., J . Assoc. Ofic. Apr. Chemists, 31, 432 (1948). (1)
RECEIVED June 27. 1949.
Optical-Crystallographic Identification of Sulfanilamide BRIGGS J. WHITE, NORMAN F. WI’IT, JOHN A. BILES, AND CHARLES F. POE University of Colorado, Boulder, C o b .
ANY of the chemical tests for sulfanilamide have not been specific. As an extension of similar studies in this laboratory (9, 6, 8, Q), an optical study of several derivatives of sulfanilamide was undertaken, in order to facilitate the identification of this compound. PREPARATION AND ANALYSIS
The Schiff bases were prepared by the reaction of equimolecular amounts of sulfanilamide and aldehyde in ethyl alcohol, the mixture being warmed for about 5 minutes to ensure complete reaction ( 2 ) . The product was then removed by filtration and crystallized usually from alcohol. The crystals &ere washed with either acetone or alcohol, depending on their solubility. Several compounds were prepared which did not form crystals of sufficient size to be of use in the determination of their optical-crystallographic properties. Except for a description of the crystal appearance of the Schiff bases from benzaldehyde and cinnamic aldehyde (15),no optical data are found in the literature. Several of the sulfanilamide-cinchona addition compounds &ere prepared, following the methods described in the literature (10). Anils with sugars also were prepared by the method described in the literature (8). The melting points of the purified compounds were determined on a “bloc-Maquenne” apparatus. No stem correction was made. Nitro en was determined by the Gunning method ( 1 ) . Concentrated aydrogen peroxide solution was used to hasten the decomposition of some of the compounds according to the method proposed in this laboratory (7). The optical-crystallographic properties of the com ounds were obtained by the use of a petrographic microscope. Tge refractive indexes were determined by the immersion method; the interference figures were used as a means of determining the optical orientation of the crystals. The most usual orientations are noted, in order to facilitate the use of the optical pro erties in the detection of sulfanilamide. The temperature a t whicx the indexes were determined was 25’ 1 C. O
EXPERIMENTAL RESULTS
The data obtained are peported in Table I. Guided by them a method of identification based on the optical-crystallographic properties of p-chlorobenzylidene sulfanilamide has been devised. To a small amount of the unknown on a microsco e slide, a drop of water and a drop of a saturated solution of phorobenzalde-
hyde are added. The slide is warmed gently over a small flame. After crystals have formed, the excess water is removed by a microfilter, and the crystals are dried carefully over a small microflame. The crystals are mounted in liquids having indexes of refraction of 1.655 and 1.703. The former index corresponds to the index beta. The crystals will have a higher index of refraction than 1.703 in one position of extinction. An alternative procedure is suggested, in order to obtain c stals that are large enough for optical-crystallographic study, wyen the concentration of the sulfanilamide is low. One of the cinchona alkaloid addition compounds is used in this test. Five milliliters of an alcohol or water solution of the substance, which is thought to be sulfanilamide, are placed in a small beaker, and t o this are added a few milliliters of a dilute solution of uinine bisulfate. The solution is heated slowly until there is o a y a small volume, and then i t is cooled for 15 minutes below room temperature. A small portion of the crystals is transferred to a glass slide. The crystals are washed with alcohol and then with acetone; the excess liquid is removed in each instance. The crystals are air-dried and warmed slightly to make certain that they are completely dry. Two slides of the crystals are prepared, one set of crystals mounted in an immersion liquid of refractive index 1.657 and the other set mounted in one of refractive index 1.668. The crystals become invisible in each immersion liquid at the correct position of extinction. Large crystals are obtained which give characteristic acute bisectrix interference figures. The other optical properties, which are listed for this compound in Table I, also may be determined. If additional confirmation is desired, other derivatives given in the table may be prepared and tested in a similar manner. POLYMORPHISM OF SULFANILAMIDE
-4ttempts were made to isolate different crystalline phases of sulfanilamide. Three phases were found and are described. A. Sulfanilamide Monohydrate. Sulfanilamide monohydrate was obtained by the method described by Kienle and Sayward (6) by letting a cold (20’ C.) saturated solution of sulfanilamide crystallize in a refrigerator a t 4 ” C. Transformation to anhydrous sulfanilamide is rapid a t 25” C., especially if the humidity is not high. System, orthorhombio Sign, positive Common orientation, Bxa Elongation, plus or minus
