ANALYTICAL CHEMISTRY
878 procedure as outlined above would appear very satisfactory for application to both routine and research vitamin -4bioassays. ACKNOWLEDGM EXT
hPP”eciation is expressed to lv. J. SRanson and H . -4. R i s k for their expert technical assistance in this investigation. LITERATURE CITED
Ames, S. R., Risley, H. d..Harris, P. L., ASAL.CHEM.26, 1378 (1954). .\mes, S. R., Swanson, K. J., Harria, P. L., J . Am. Chem. Soc. 77, 4134 (1955). Ibid., p. 4136. Bliss, C. I., “Suggested Revision of the USP Biological Assays for Vitamins -4and D , ” hnimal Kutrition Research Council, Nov. 15, 1948. Bliss, C. I., in “Vitamin Methods,” P. Gyorgy, ed.. vol. 11, pp. 560-75, Academic Press, Kew York. 1951. Ibid., pp. 93-7. Booth, V. H., J . Xutrition 48, 13 (1952). Brenner, S., Brookes. 31. C. H.. Roberts, L. J., ICid. 23, 459 ( 1 942).
(9) Finney, D. J., “Statistical 3Iethod in Biological Assay,” pp. 187-211, Hafner Publishing Co., New York, 1952. (10) Fov. J. R.. Moraareidee. K.. ANAL.CHEM.20.. 304 (1948).
(11) Guerrant, N.B.; J . NGtrition 37, 37 (1949). (12) Guggenheim, K., Koch, W., Biochem. J . (London), 38, 256 (1944). (13) johnson, R. bf.,B ~c. A , , ~~ , . ~ ~h , ~ i ~14, ~ ~361 h(19.17). ~ ~ ~, (14) Lemley, J. M., Brown, R. A , , Bird, 0. D., Emmett, -1.D., J .Vutritbn 33, 53 (1947). (15) Pharmacopeia of t h e United States, “USP Vitamin .1 Heferenue Standard, Instructions for Use,” May 18, 1948. (16) Pharmacopeia of the United States, vol. XIV, Mack, Easton. Pa., 1950. (17) Sobel, A . E.,Sherman, AI., Lichtblau, J., Snow, Y.. Kramer. B.. J . Yutrition 35, 225 (1948). (18) Wood, E. C., Analyst 71, 1 (1946). (19) Wood, E. C., Finney, D. J., Qitart. J . Pharm. and Pharrnacol. 19, 112 (1946). (20) World Health Organization. Tech. R e p t . Ser. 3, 4 (1950). RECEIVED for reriew October 2 5 , 1954. Accepted Februarj- 1, 1956. Paper XI11 of a series entitled “Biochemical Studies on Vitamin h.” The previow paper of this series ( I ) appeared in 1934. Communication S o . 214, Researoh Laboratories of Distillation Products Industries, Divislon of Eastman Kodak Co.. Rochester, S . T.
Use of Periodic Acid for Detecting and locating Ethylenic Unsaturation ASIMA CHATTERJEE and SUBHENDU GHOSH MAJUMDAR University college o f Science dnd Technology, Calcutta,
Periodic acid has proved to be a promising reagent in the detection and location of both terminal and exocyclic double bonds in organic molecules. Like ozone, this per acid oxidizes unsaturated compounds by splitting grouping. The resulting products are the -C=Ccarbonyl derivatives which can be readily characterized and estimated as their 2,4-dinitrophenylhydrazonesor dimethones. The yield of the aldehydic or ketonic fragments thus obtained corresponds to that calculated from the number of double bonds present.
T
H E first important appllcation of the use of periodic acid as a selective oxidizing agent for a-glycols was made by RIslaprade (8, 9) in 1928. It is now being extensively employed in the field of organic reactions to detect various functional groups-viz , -C“OH-CH(XHZ)-
-CO-COZH
-CHOH-CHO
CO?H-CH:-CO?H
C‘HO-CO-
COIH-CHI-CHO
-CO-CO-
CHJOH-CH (SHz)-CO?I-I
CHO-CH:OH
H,S-CH,-CH,-CH,OH
a3 well as the active methylene linkage in citric and malic acids and the like. The use of periodic acid in the analysis of organic compounds and in determination of their structure has become of increasing importance. Several review articles ( 3 , 5-7, 10) on the reagent have been published. Hoirever, a careful survey of the relevant literature reveals t h a t the osidizing action of periodic acid on unsaturation in organic molecules has not yet been studied. The present investigation was undertaken with a view to developing, if possible, a suitable method for identification and location of double bonds in unsaturated molecules. Oxidation eupeliments with periodic acid have
lndia been studied in various unsatuiated systems rThen the carbon to carbon double bond is split, resulting in carbonyl compounds (Table I). Volatile carbonyl fragments are then isolated by steam distillation and subsequently identified as their 2,4dinitrophenylhydrazones or dimethones. Both iodine and iodic acid are produced in this reaction. The presence of a large amount of iodic acid in the reaction products indicates that the Rlalaprade reaction has been operative after the formation of a-glycol. The mechanism by which iodine is produced is not fully understood. It appears to come from the direct reduction of periodic acid, because no iodine is produced when iodic acid is refluxed with unsaturated conipounds. Further work on this is in progress. MECHATISM OF REACTION
When periodic acid is used in a large excess to oxidize double bonds, i t is converted mainly to iodic acid (about 84%) and in a secondary measure to iodine (Table 11). The yield of iodic arid has been found to increase with an increasing amount of periodic acid, but the yield of iodine increases with decreasing amounts of the per acid. Thus, i t is not possible to account for the double bond reaction by the production of either iodic acid or iodine. The ovidation of the double bond by periodic acid appears t o proceed with the formation of an a-oxirane compound ( 1 2 ) (11) as observed in the oxidation of double bonds by perbenzoic and perphthalic acids. The epoxy compound thus formed (11) is transformed into a-glycol (12). Excess periodic acid present in the medium then initiates the Xfalaprade reaction producing carbonyl derivatives (IV). (See structural formula.) The epoxide (11) may also be produced by the ozonization effect of periodic acid according to Smith and Duke (If ): 2 H:O:H
-
HJOB
[O:H]+
+ [:O:Hl-
+2H+
+ 2e-
_I
+ 2 H + + 2 e - +HIOJ + 3HzO 6(OH+) --+ 0 3
+ 3HzO
,
879
V O L U M E 2 8 , NO. 5, M A Y 1 9 5 6 Table I.
Periodic Acid Oxidation of Unsaturated Compounds Oxidation Product CsHsCHO
Compound CsHsCH=CH.CO%H Cinnaiiiic acid
Yield, % Identification 58 2,4-Dinitrophenylhydrazone* 1ii.p. 234’ C.
CsHsCHO
/=CH.CeHa N.HC1
98
2.4-Dinitrophenylhydrazone, n1.p. 234’ C.
95 55 55 95
Dimethone, m.p. 188-189’ C. Dimethone, m.p. 188-189’ C. Dimethone,m.p. 188-18QeC. Dimethone, m.p. 188-185° C .
f5H8 Glycosine hydrochloride ( 1 , s )
Quinine hydrochloride Cinchonine hydrochloride Cinchonidine hydrochloride Quinidine hydrochloride Cupreine hydrochloride (demetliylquinine h>-drochloride)
Table 11. Quinine Hydrochloride, Gram 0.6112 0.4662 0.4662
0.4858 0.2Fti5
H-CHO H-CHO H-CHO H-CHO
Oxidation Products of Periodic Acid Periodic Acid Added. Grains
Iodic Acid Produced. Grams
Iodine Liberated, Gram
I-nreacted Periodic ticid, Grain’’
3.9790 1,2730
3 0756
0.3177
0 1197
0,4552
0.4598
1.2760 3 9790 2.3880
0.4541
0.5185
2,9625 1 . ti220
0.20 0.1210
... ...
0.45 0.64
Excess periodic acid was estimated in presence of iodic acid according
t o tlie niethod of Fleury and Lange
(4).
EXPERIRI E h T A L
Oxidation of Cinnamic Acid. One gram of cinnamic acid n as dissolved in 250 ml. of m ater, to n hich 6.0 grams of periodic. acid ae added. The reaction miyture n-as left a t room temperature for 4 hours, then steam distilled. Benzaldehyde was produced in 9800 vield, along with a copious amount of iodine. The benzaldehyde was extracted from the steam distillate with 100 ml. of ether, which was then washed Kith an aqueous solution of sodium sulfite and water to remove iodine. Benzaldehyde m-aa obtained from the ether extract by concentrating the extract in a n atmosphere of nitrogen. T h e benzaldehyde gave 1.809 grams (98y0 of theory, melting point 234” C.) of orange-red crystals of 2,4.-dinitrophenylhydrazone x-hen reacted M ith 2,4dinitropheny lhydrazine.
DISCUSSION
The results indicate that periodic acid, like ozone. can be used for the detection and location of a carbon to carbon double bond (terminal as well as exocyclic). The reaction involves cleavage of the double bond, resulting in carbonyl derivatives. During this oxidation of the double bond, periodic acid is reduced, the eud products always being iodine and iodic acid. Iodine does not originate from iodic acid, but from the direct reduction of periodic acid, the reaction mechanism of which is not yet clearly understood. The production of iodine and iodic acid is not quantitative t o account for the double bond reaction. The osidation grouping with periodic acid appears to reaction a t the -C=Cproceed with the formation of an a-oxirane derivative. I t is then converted into a-glycol, lvhich undergoes the Malaprade reaction with an excess of periodic acid present in the medium. However, this per acid does not react v i t h a n aromatic unsaturation. Cleavage of the double bond with periodic acid proceeds smoothly with water-soluble substances, but may present some difficulties with n-ater-insoluble compounds. L I T E R A T U R E CITED
(1) Chatterjee,
(2) (3) (4) (5) (6)
(1)
I - I€+
V
Oxidation of Quinine. One gram of quinine hydrorhloride was added t o 6.09 grams of periodic acid in 250 ml. of water. The mixture vas kept for 4 hours a t room temperature, then steam distilled. The distillate containing formaldehyde was treated as above. The ether extract of the distillate was concentratrd and treated n-ith an alcoholic solution of dimedone until dimethone separated (yield, 0.76 gram). The dimethone crystallizes from alcohol in colorlem needles (melting point 188189” C.). Formaldehydeliberated from quinine was identified as its 2,4-dinitrophenylhydrazone (melting point 155” C.). Oxidation of Other Unsaturated Compounds. \Then osidized in a similar manner, the hydrochlorides of quinidine, cinchonine, cinchonidine, and cupreine produced formaldehyde in 94 to 95y0 yield. Glycosine hydrochloride, under the same experimental conditions, liberated benzaldehyde ( 1 , 2 ) .
.L,llajumdar, S. G., J . Am. C h e m .
SOC.7 5 , 4365 (1953). Ibid., 76, 2459 (1954). Fleury, P. F., China. anal. 35,197 (1953). Fleury, P. F., Lange. J., J . pharm. chim., [SI 1 7 , 107 (1953). Hudson, C. S., “Organic Reactions,” Roger hdams, ed., vol. 11, chap. 8, Wiley, S e w York, 1947. Jackson, L. E.,Ibid.. p. 341. (7) Lange, J., “Action de l’.\cide Periodique sur les Polyalcools,” Les Editions Vega. 43 Rue Madame. Paris. 1933. (5) AIalaprade, L., Bull. SOC. chim. 141 43,683 (1928). (9) AIalaprade, L., Conzpt. rerid. 186,382 (1928). (10) Smith, G. F., “Analytical Applications of Periodic * k i d sild Iodic iitid and Their Salts.” 5th eti., G. Frederick Smith Chemiral Co., Columbus, Ohio. (11) Smith, G. F., Duke, F. R., ISD. ENG.CHEM.,ANAL.ED. 15, 120 (1943). (12) Swern, D., Chern. Rer3.45,26 (1949). I
!
I
unu
-
RECEIVEDf o r review M a y 5 , 1955. cepted February 18, 1956.
4c-
