ANALYTICAL EDITION
August 15, 1942
ments or phases as in alloys, unless, for example, a tissue is injected with thorotrast or some other heavily absorbing snbstance. The photographs here were made with the cbaracteristic K a radiation of copper exactly as with metals. Very soft general radiation generated at low voltages is useful for this type of specimen depending upon very slight differences in thickness of the same material in adjacent areas. Figure 10.4, shows in amazing detail the eye of a n ordinary house fly; Figure 10B,is the microradiograph of the smallest bones in the foot of a very small frog, taken through the web of the foot; Figure 11 illustrates the striking structures of white oak wood taken through radial, transverse, and tangential sections. The three photogaphs are highly characteristic of each type of wood at different ages, conditions of growth, drying, impregnation, and other treatments to which wood may be subjected. It is evident that very accurate measurements can be made even of cell-wall thicknesses.
Acknowledgments The authors gratefully acknowledge the assistance of former students in the X-Ray Laboratory of the University
683
of Illinois, in the preparation of specimens and photographs illustrated in this paper: specimen of Figure 4, W. J. Craig and E. C. Lauck; specimen and photograph of Figure 7 A , J. Hino; Figures 7B, SA, 10, and 11, E. J. Bicek; Figures 7C, 8B, 8C, and 9, W. M.Shafer.
Li
ited
(1) Clark,G.L.,Photo TechnZque, 1, NO. 12 (Deo. 1939). (2) Clark, G. L..and Shafer, W. M,, Tram. Am. SOC.Metals, 13. 732 (1941). (3) Daulillier, A,. C m p t . rend., 191, 1287 (1930). (4) Fournier, M. F.,Reo. mdt. 35, 349 (1938). (5) Goby, P.,Compt. ?end., 156,686 (1913). (6) Zbid., 180, 735 (1925). (7) Lamarque, P.,et al.. Ibid., 202, 684 (1936): Radiology, 27, 563 (1936): Compt. vend. soc. biol.. 123. 1051 (1936): Bull. histol. appl. physiol. path. tech. micioscop.. 14, 5 (1937); Aroh. soe. sCi. m4d. biol. Montpellier et Languedoc, 18,27 (1937); J . Rad.. 20,6 (1936). (8) Schupp, 0. E.,and Boiler, E. R., IND. END. C a ~ x . .30, 603 (1938). (9) Yoshida. U., m d Tanaka, H., M a . Coil. Sci. Kyoto Imp. Univ., AI^, 401 (1934).
Identification of Sugars By Microscopic Appearance of Crystalline Osazones W. Z . HASSID AND R. M. McCREADY Division of Plant Nutrition, University of California, Berkeley, C
caraonyutaws as a group form a number of chemical and give a variety of reactions greater perhaps than any other class of chemical compounds. However, because of the close similarity in properties of the various sugars, the identification of the different members is often VIE,
... .
.
identihuuSvLL yu.u uUbY.s L ... "..".. -"-.-..-erlvatlves is the microscope. The identification of pure sugars and certain sugar mixtures has been accomplished by crystallizing the sugar from saturated aqueous solutions upon the addition of precipitating agents . . (4, f%). . . . .The,- sugar -, is then . . identified . uAJ
I
,-
..
686
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
(6, IW, 14, 15). However, this method is objectionable in that impure samples often fail to crystallize, and a study of optical properties requires the use of the petrographic microscope, which may not be readily available in many laboratories. The phenylhydrazine reactions first introduced by Fischer (3) are still one of the most important tests for the identification of sugars. The osazones produced by various sugars are crystalline and give definite melting points by which the identity of the sugar may be established. I n dealing with unknown sugars, impurities may be present and if the available quantity is insufficient for further purification, advantage can be taken of the phenylhydrazine reaction. The osazones possess characteristic crystalline forms when observed under the microscope. Not more than 5 mg. of sugar is needed for this purpose. In the identification of a sugar, the unknown osazone may be compared with one prepared simultaneously from known sugars or with photomicrographs of osasones of different sugars. By observing the crystal form of the osazone under the low power of the ordinary microscope, a particular sugar may be tentatively identified. Further confirmation is desirable from melting point determinations or from a study of the optical properties, if a petrographic microscope is available (9, 16, 17). Although photomicrographs of a number of sugar osazones are shown by Morrow and Sandstrom (IO), the present authors found i t desirable to present a more extended list of phenylhydrazine derivatives of reducing sugars and of several important sugar derivatives. For this reason photomicrographs of phenylhydrazine derivatives of the commonly occurring hexoses, pentoses, niethylpentoses, disaccharides, uronic acids, and hexosephosphates n-ere prepared and are shown here ( X 4 5 ) .
Procedure To prepare the osazone 2 cc. of a neutral clear solution, containing 5 to 20 mg. of the sugar, are placed in a 15 X 150 mm. test tube with 0.3 gram of powdered sodium acetate and 0.2 gram of phenylhydrazine hydrochloride, and the tube is shaken until the contents are dissolved. The loosely stoppered test tube is then immersed in a boiling water bath for 30 minutes and after reacting is allowed to cool slowly a t room temperature. Sudden cooling by immersion in cold water should be avoided, since this causes distortion of the crystals. The osazones of the monosaccharides usually crystallize while hot. If the amount of the sugar is too small, however, crystallization occurs only after cooling. Since the osazones of disaccharides are soluble while hot, they can be separated from those of the monosaccharides by taking advantage of this difference in solubility. The monosaccharide is first identified when the solution is still warm; the solution is then filtered, and slowly cooled to allow crystallization of the disaccharide osazone. The osazone is identified by placing a drop of the solution with a pipet (a wide tip pipet should be used in order not to break up the crystals) on a slide and observing the shape of the crystals with a microscope at low power. There are certain limitations to the identification of sugars by the procedure. The osazone reaction with phenylhydrazine is not always a n absolute test of the identity of a sugar, as a number of sugars, having a common enolic form, yield the same osazone. Thus, the hexose sugars, d-glucose, d-mannose, and d-fructose, yield the same phenylosazone. Similarly, the same osazone is obtained from &galactose, d-talose, and d-tagatose. Other examples of sugars giving the same osazone are d-allose, d-altrose, and d-pseudofructose; d-idose, d-gulose, and d-sorbose. The pentose sugars, d-arabinose and d-ribose, yield one osazone; d-xylose and d-lyxose give another. Furthermore, the corresponding groups of sugars belonging t o the I-series, having the same configuration beyond the second carbon atom, also yield one and the same osazone, differing from the d-osazone only in the direction of
Vol. 14, No. 8
optical rotation. Additional tests should therefore be made in order to identify a sugar belonging in a particular enolic series. Glucose, fructose, and mannose, for example, may be distinguished from one another as follows: Glucose on oxidation with nitric acid (10) and neutralization with potassium carbonate or hydroxide produces crystals of potassium acid saccharate (Xo. 20): mannose forms an insoluble phenylhydrazone when treated with henylhydrazine hydrochloride and sodium acetate, as previous& described, and allowed t o remain in the cold (No. 5 ) ; fructose may be identified by the Seliwanoff reaction ( I ) . Galactose can be distinguished from its other enolic forms by preparing the I-tolylhydrazone derivative (6). &Gulose may be distinguished from d-sorbose and d-idose by the fact that, when oxidized with nitric acid, potassium acid saccharate is obtained. Sorbose, being a ketose sugar, gives the Seliwanoff reaction. In general, when a sugar is identified by the characteristic shape of the crystal of its osazone, the identification should be confirmed by some other test. Since in the last decade the hexosephosphates gained much prominence in the intermediary metabolism of animals and plants, photomicrographs of the phenylhydrazine derivatives of fructose diphosphate (Harden Young ester, 7 ) , and the glucose-6-phosphate (Robison ester, 13) are included. The fructose-6-monophosphate (Neuberg ester) gives the same osazone as the Robison ester. The latter is oxidizable with hypoiodite and thus can be distinguished from the former (8).
Remarks Sorbose produced a n amorphous osazone (No. 3). However, when this osazone was allowed to remain for 24 hours, the amorphous mass changed into crystalline needles (No. 4). When fructose diphosphate was heated with phenylhydrazine hydrochloride and sodium acetate for 15 minutes, diphenylhydrazine fructose diphosphate phenylhydrazone (KO.17) mas formed (7). Prolonged heating (one hour or more) caused hydrolysis of the phosphoric acid group on the first carbon atom, producing the same phenylhydrazine hexose monophosphate osazone as the Robison ester (No. 18). Galacturonic acid, when heated with the same reagents for 30 minutes, produced a derivative (No. 15) which was identical with the phenylhydrazine phenylhydrazone of galacturonate described by Siemann, Schoeffel, and Link (11).
Literature Cited (1) Browne, C. A., and Zerban, F. W., “Physical and Chemical Methods of Sugar Analysis”, 3rd ed., pp. 711-14, New York, John Wiley & Sons, 1941. (2) Denighs, G., Mikrochemie, 3 , 3 3 (1925). (3) Fischer, E., Ber., 17, 579 (1884). (4) Hudson, C. S., and Yanovsky, E., J . Am. Chem. SOC.,39, 1020 (1917). (5) Huntress, E. H., and Mulliken, S. P., “Identification of Pure Organic Compounds”, p. 78, Xew York, John Wiley & Sons, 1941. (6) Keenan, G . L., J . Wash. Acad. Sci., 16, 433 (1926). (7) Klein, G., “Handbuch der Pflaneenanalysa”, Vol. 11, p. 554, Vienna, Julius Springer, 1932. (8) Lothrop, R. E., and Holmes, R. L., IND.ENG.CHEM.,ANAL ED., 3, 334 (1931). (9) Morris, V. H., J . Am. Chem. Soc., 54, 2843 (1932). (10) Morrow, C. A . , and Sandstrom, W.hl., ”Biochemical Laboratory Methods”, 2nd ed., pp. 157-66, New York, John Wiley & Sons, 1935. (11) Niemann, C., Schoeffel, E., and Link, K. P., J . Biol. Chem., 101, 337 (1933). (12) Quense, J. A., and Dehn, W. M., IND.ENG.CHEX.,ANAL.ED., 11, 555 (1939). (13) Robison, R., and King, E. J., Biochem. J . , 25, 323 (1931). (14) Wherry, E. T., J . Am. Chem. Soc., 40, 1852 (1918). (15) Wherry, E. T., J . Wash. Acad. Sci., 18, 302 (1928). (16) Wright, F. E., J . Am. Chem. Soc., 38, 1647 (1916). (17) Wright, F. E., J . B i d . Chem., 28, 523 (1917).
