920
ANALYTICAL CHEMISTRY
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Table 111. Elementary Analysis and llolecular Weights Siibstance Phenol Oxidized plirnol Creaols Oxidized o-cresol Oxidized m-cresol Oxidized p-cresol Dlhydroxybenzenes Oxidized resorcinol Oridized catechol
Time of Oxidation, Xin. 0 1
76.61 75.68
6.43 4.11
Oxygen, % (by Diff.) 16 96 (calcd.) 20 21
n
77 43
1
76 4 i
7.46 5 52
14 80 (calcd.) 18 01
3 1
zs.49 ,a.ni
5.59 5.28
3
7 i 33
5 94
17 92 17.71 16 73
74.61 65 47 52 70 56 23
5.18 5 50 3.51 3.43
20 21 29 08 (calcd.) 43 79 40 24
inoo 0
1
1
Carbon,
%
Hydrogen,
co
Mol. Wt. (Benzonhenone)
trodes was used. An empirical equivalent weight iq used in the calculations. When the gravimetric procedure is used, the precipitate is allowed to settle and is washed with water a number of times by decantation, after which the entire precipitate is transferred t o the sintered glass filter. The precipitates may be dried in a vacuum oven at 50" C. or overnight in a vacuuni desiccator containing Anhydrone. The gravimetric factor is empirical. Typical results are shown in Table I. NATURE OF PRECIPITATE
From a comparison of the number of equivalents per mole of phenol found volumetrically and the gravimetric factor it can be seen that the precipitate differs from phenol in empirical formula by having fewer hydrogen atoms (Table 11). The elementary analyses on the precipitates also hear out this observation, although the indication is that a small amount of oxygenation may also have occurred (Table 111). The molecular weight determinations show that polymerization has occurred in the precipitation process (Table 111). These were done by the freezing point depression of benzophenone. It is noteworthy that the number of equivalents per mole of phenol approaches the number of ortho- and parahydrogen atoms in the phenol molecule. The high color of the precipitates indicates quinone linkages; it appears then that hydrogens must be absent from hydrosyls as well as from the ortho or para popi-
94
ion
i40 108 ,580
tions. On the average, then, there must either be about one ortho- or parahydrogen left untouched, or quinone formation takes place only in part. The reason for the incomtdeteneas of the osidation is probably a solubility phenomenon A possible typical product of phenol which u ould contain the possible groups would be the following:
620
540
...
510 540 5-20 110
A further experiment was done to support this sort of formula. .4 large excess (10 grams) of phenol was added to 250 nil. of 0.liV cerium(1V) sulfate. The resulting dark brown precipitate was extracted with hot water. From the filtrate of the estraction, colorless crystals ( 3 mg.) of 0,O'-dihydrosybiphenyl were obtained. This dimer of phenol was identified by its double melting point [the hydrate melted a t 71" C., and upon further heating, the anhydrous substance formed and melted a t 108" C. Literature values are 71" to 73" and 109' C., respectively (;)I, This shows that polymerization could take place in the manner described above. LITERATURE CITED
Baker, R.,and Brown. S . ,J . Chem. Soc., 1948, 2303. (2) Roeseken, J., L l e t r , C. F., and Pluim. J . . Rec. frnr. chim.. 54, (1)
343 (1933).
(3) Duke, F. R., and Smith, G. F., ISD.E s c . C'HESI.. ;\SAL. ED., 12, 201 (1940). (4) Furman, S . H., and Wallace, J. H., J . Aru. C'hern. Soc., 52, 1443 (1930).
Kraemer, G., and Weissberger, R.. Ber.. 34, 1663 (1901). ( 6 ) Pummerer, R., and Frankfurter, F.. Ibid.. 47, 1472 (1913). (5)
RECEIVED for review November 2, 1953. Accepted January 29.1954. Contribution No. 300 from the Institute for Atomic Research and Department of Chemistry, Iowa State College, .iiiies, Iowa. \Vork was supported in part by the Ames Laboratory of the Atolnic Energy Coiiiii~i~sion.
Spray Reagent for the Detection of Carbohydrates R. U. LEMIEUX and H. F. BAUER N a t i o n a l Research Council, Prairie Regional Laboratory, Saskatoon, Sask., Canada
T
HE detection of sugars and their derivatives on filter paper
after separation by chromatography ( 2 , 4)01' elcctrophoresis ( 8 , has been accomplished in a variety of W:L,I.S (f-.;, 8). The pul)lished methods have one or more of thc following diwdvantagcs: limited applicability, low sensitivity, production of fading spots, cumbersome and time-consuming proredures, and rspid conromitant discoloration of the background, The present met'hod circumvents most of these disadvantages in being simple, adequately sensitive for most purposes, arid applicable to nearly nll sugar derivatives. The authors have discovered that a slightly alkaline (pH 7 . 2 ) aqueous solution of periodate and permanganate forms an almost ideal spray reagent for general use in carbohydrate chemistry. The presence of a periodate- or perniangariate-reducing substance 0 1 1 the paper results in the formation of a greenish yellow spot, usually in a short period of time. The hackground retains the
permanganate coloration for more than 1 hour at rooin temperature because of the slow attack of the paper by the reagent and the ability of periodate under the conditions used t o regenerate the permanganate. I'acsu, Mora, and Kent (8) have detected carbohydrates on chromatograms by spraying with an aqueous solution of permanganate and Buchanan, Dekker, and Long (31 have used pcriodate to convert carbohydrates to aldehydes whic7h were subsequently detected with Schiff's reagent. The present method differs fundamentally from these techniques in that it depend? not only on the fact that both periodate and permanganate usually osidize carbohj-drates readily but also on the fact that in alkaline medium periodate is able to regenerate the perni:inganate from the reduced state formed on its osidation of an organic compound. The permanganate-regeneration reaction has been studied in this laboratory and it K I P established ( 7 1 that periodate
V O L U M E 26, NO. 5, M A Y I 9 5 4
921
can oxidizc ni:ingaiintc [tho first product formed on the reduction of pc’rmanganate by an organic compound ( 5 ) ] t o permanganate. This observation has led to the development of a useful olefinsplitting reagent ( 7 ) . Since manganate decomposes t o mangancse dioxide and permanganate in acid medium, the regeneration of the permanganate by the periodate come8 to a stop once the p l l of the system has dropped t o about 6 ( 7 ) . Thus, the fact that the periodate oxidation of carbohydrates other than cellulose usually liberates considerable amounts of formic acid also favors the action of the new spray reagent. The usefulness of the reagent for detecting substances which reduce periodate Flightly or slowly probably depends mainly on the unreactivity of thc insoluhlc cellulo~e compared to dissolved compounds.
to 3 y of glucose, sylose, ascorbic acid, sorbose, maltose, cellobiose, and olefins-such as crotonic acid-are readily detected. The reagent requires 5 t o 8 y of substances such as glucuronolactone, 3-methylglucose, mannitol, erythritol, ethylene glycol, xylonolactone, and tartaric acid for positive detection. Ten t o 15 y of substances which reduce periodate only slowly or to a small extent are required. Such materials are, for example, p-methyl glucopyranoside, sucrose, trehalose, pentaerythritol, 2,3,4,6 - tetramethylglucose, 2,3 butanediol, and lactic acid. The time required for the appearance of a spot varies with both the amount and chemical nature of the substance. Forty to 60 minutes may be required for the appearance of spots for small amounts of substances such as trehalose.
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EXPERIMENTAL
LITERATURE CITED
The spray reagent is prepared freshly before use by mixing 4 parts of 2% aqueous sodium metaperiodate and 1 part of 1% potassium permanganate in 2% aqueous sodium carbonate solution. h b o u t 1 ml. of the reagent is applied to each 50 sq. cm. of Whatman Yo. 1 paper and the sprayed paper is left at room temperature. It, is advantageous to hang the sprayed paper in a covered jar for development if the atmosphere is either contaminated or arid. If the paper is washed under the water tap after the spots have finished appearing and before the cellulose has discolored the permanganate background, a permanent record of the chromatogram is obtained in the form of brown spots on an almost n-hite backgrountl. RESL-LTS AND DISCUSSION
The new reagcnt compares favorably in sensitivit,y with many of the reagents commonly used t o detect reducing sugars. Two
.lbdel-.\khet~. 31.. and Smith, F.. J . .4m. Cheni. Soc., 73, 5859 (1951).
Block, 11. J., Le Strange. R.. and Z a e i g , G., “Paper Chromatograuhv.” DD.i8-91. Sew Tork. Academic Press. Inc.. 1952. Rurothe photometer cell with the mercury. Because most organic substances have an appreciable absorption in the ultraviolet region of the spectrum, the presence of organic impurities in the prepared pad may result in a spurious reading for mercury. When heating of the pad is accompanied by the generation of smoke. the presence of a n organic impurity is apparent and the analysis is discarded. I n those cases where there ie no visible evidence of pad contamination, it has been
necessary to apply the procedure to a mercury-free sample if the photometer reading is to he attributed unequivocally to the presence of mercury. PROCEDURE
-4relatively simple procedure has been found useful in these laboratories for detecting the presence of interfering substances in the photometer cell. rlfter the reading using the mcrcury photometer has been obtained, the cell is immediately transferred t o a Beckman DU spectrophotometer, and a second reading of absorbance is made a t 2537 A. using a slit width of 0.8 mm. to give a spectral slit width of 32 ,4. A special cell compartment has been built for the spectrophotometer to accommodate the mercwy cell. RESULTS 4 Y D DlSCUSSIOh
JIercur) vapor has no appreciable absorbance under these conditions. since it abEorbs only a narrow line a t 2537 A., which represents a small fiaction of the total energy in the wave lengths represented in the effective band. Interfering organic suhstances. on the other hand. generally have rather broad a h s o r p tion bands and produce a substantial absorbance both in the photometer and as measured by the Beckman instrument. ilccordingly, the amount of extraneous absorbance at 2537 A. can he approximately measured in the Beckman instrument and applied as a correction to the mercury reading. The accuracy of such a correction obviously depends upon the nature of the absorption curve of the contaminant within the hand limits. and the correction becomes exact when the effective absorbance betn een the hand limits is equal to the absorbance at 2537 A.
