method in conjunction with the usual thin layer chromatographic sample size. Resolution was comparable at the 20-60 pg level; those transitions labeled poor were readily distinguishable from the instrument noise level. As seen in Figure 1, the poorest transitions tended t o resemble second order transitions. However, at 100 pg, all the samples produced well defined transitions. The water of hydration in the Silica Gel G binder was found to mask transitions from approximately 120” to 150” C. Preliminary heating above 150” C, cooling, and reheating removed this difficulty for some samples, but several apparently volatilized at the first pass. The use of Silica Gel G taken from an area very close t o the unknown spot was instituted as a reference. While the dehydration endotherm did not completely disappear in most cases, the sample and reference were sufficiently well balanced so that it was not difficult to observe superimposed transitions. It is not known what the exact effects of a n extreme dilution might be on the melting characteristics of the material under study. Most of the materials analyzed produced lower, although usually consistent, melting points than the literature values. Some interactions, such as dilution or reaction with the water, seemed to have affected the values obtained for adipic acid. As the sample size increased from 20 t o 60 pg, the melting point rose from 141 O t o 149” C.
The three phthalate isomers gave no apparent transitions due t o sublimation before the melting point could be reached. A sealed sample holder would eliminate this difficulty, but the instrument at hand is not capable of this. Future work will be performed with an instrument using a sample pan that can be sealed against volatile loss. Quantitative data will also be available, adding specific heat t o melting point as an obtainable parameter. One of the more interesting observations was the conversion on heating of dl-malic acid t o I-malic and fumaric acid (1). This conversion was observed in samples heated in capillaries and not in those heated on a watch glass. This phenomenon proved useful in detecting an estimated 2 of malic acid present as an impurity in maleic acid. A very useful technique for the identification of many dicarboxylic acids by differential thermal analysis of their thin layer chromatographic spots has been demonstrated. Further investigation is under way to apply this procedure to other polymeric and resinous components. RECEIVED for review July 20, 1967. Accepted September 28, 1967. (1) E. H. Huntress, “Identification of Pure Organic Compounds” Wiley, New York, 1941, p. 100. (2) Lange’s “Handbook of Chemistry,” 9th ed., McGraw-Hill,
New York, 1956.
A Colorimetric Method for Determination of Glycols Kenneth C. Leibman and Elsa Ortiz Department of Pharmacology and Therapeutics, Uniwrsity of Florida Medical School, Gainesville, Florida
IN THE COURSE OF INVESTIGATIONS on the metabolsm of unsaturated compounds, a general method for the determination of glycols was required, A procedure for assay of glyceric acids and related compounds has been reported (1, 2), in which the acidic glycol is oxidized with periodic acid, and the p-nitrophenylhydrazone of the resulting carbonyl acid is prepared, extracted, and measured colorimetrically. Starting from this method, we have devised a general procedure for the estimation of neutral glycols which can be applied, with appropriate modifications, to the assay of a number of different dihydroxy compounds. EXPERIMENTAL
Reagents. SULFURIC ACID,10N. SODIUMMETAPERIODATE, 0.1M. Store in a brown bottle in the dark. THIOACETAMIDE, 0.867M. Dissolve 650 mg of thioacetamide in distilled water and dilute to 10 ml. Prepare fresh daily. 2,4-DINITROPHENYLHYDRAZlNE HYDROCHLORIDE. Dissolve 100 mg of 2,4-dinitrophenylhydrazine in 100 ml of 2N hydrochloric acid. CHLOROFORM. Reagent grade. General Procedure. To a 15-1111 glass-stoppered centrifuge tube are added in the following order 2 ml of the aqueous sample solution, 1 ml of 10N HzS04, and 1 ml of 0.1M N a I 0 4 , mixing thoroughly after each addition. The tube is kept unstoppered at an appropriate temperature for a (1) E. Juni and G. A. Heim, Anal. Bioclzem., 4,143 (1962). (2) Ibid., p. 159.
32601
definite period of time (Reaction A). If this temperature is above ambient, the tube is placed in an icebath for 5 minutes at the end of the reaction. Thioacetamide solution (0.5 ml) is added, and the tube is shaken gently. After standing at room temperature for 5-10 minutes, the contents of the tube are mixed thoroughly for 30 seconds with a vibrating mixer. One-half milliliter of the 2,4-dinitrophenylhydrazine hydrochloride solution is added, the solution is mixed, and the tube is kept unstoppered for a measured time at a certain temperature (Reaction B). Chloroform ( 5 ml) is then added, and the tube is stoppered and shaken vigorously several times; the liquid is allowed to drain down and the pressure is vented between each shaking. The tube is centrifuged at about 1000 X G for 5 minutes. Sulfur usually collects at the interface as well as at the bottom of the chloroform layer. With a capillary pipet connected via suction flask to a vacuum source, the aqueous layer and as much as possible of the material at the interface is aspirated. A 3-ml volumetric pipet is introduced into the chloroform layer; use of a Propipette controller with appropriate manipulation of the “E” port and bulb to maintain a positive pressure in the pipet prevents entry of any residual water or interface material into the pipet. After wiping the exterior of the pipet carefully with a tissue, a sample of the chloroform extract is transferred to a 1-cm cuvet and the absorbance is measured at an appropriate wavelength in a spectrophotometer. The spectrophotometric blank consists of an extract obtained by applying the identical procedure to 2 ml of a solution similar to the experimental sample, but containing no glycol. The absorbance is compared to those measured on extracts obtained in like manner from solutions of known concentration of the reference glycol. VOL. 40, NO.
1, JANUARY 1968
251
Table I.
Conditions for Specific Applications of the Method. Method of Reaction A Glycol Melting point preparation* temperature Absorbancec & S.E. cis-1,ZDihydroxyindane 98.0-98.5" A 80" C 0.417 =I= 0.011 154-1 55" B trans- I ,2-Dihydroxyindane 80' C 0.416 i~0.014 cis- 1,2-Dihydroxycyclohexane 97.0-97.8a A Room 0.705i 0.011 trans- 1,2-Dihydroxycyclohexane 103.&104.8" C Room 0.700=k 0.011 65.5-66.5" D Phenylethylene glycol Room 0.6323~ 0.021 Reaction A was run for 60 minutes, and Reaction B at room temperature for 45 minutes, in each case. * Glycols were prepared: A, by permanganate oxidation of the respective cyclenes (3, 4); B, by the action of sodium carbonate on indene bromhydrin ( 5 ) ; C, by alkaline hydration of cyclohexene epoxide (6); D, by recrystallization of a commercial product (K & K Laboratories). c Absorbance at 375 mp obtained when method was applied to 100 mpmoles of glycol. Standard curves were linear at least to absorbances of 1.0 in ail cases. Standard errors were calculated from data of standard curve. (I
In the event that the absorbance is too high for accurate measurement, the experimental and blank extracts may often be diluted with chloroform. A linear relationship has been demonstrated for dihydroxyindane over at least a 10-fold range of dilution. For other glycols, this relationship should be tested before use. RESULTS AND DISCUSSION
Conditions for Specific Glycols. Table I shows the sensitivity of the method when it is applied to various glycols under conditions recommended for their assay in extracts derived from biological tissues. The wavelength of maximal absorbance in all these cases was 375 mp. Identical results were obtained for cis and trans isomers of the two cycloalkanediols tested. The method was calibrated in the case of phenylethylene glycol by applying it to benzaldehyde (reagent grade), which is produced from this diol by periodate oxidation. In addition, blank reaction mixtures were extracted with portions of chloroform containing measured amounts of the 2,Cdinitrophenylhydrazone (mp 240.5-41.0") of benzaldehyde, and the absorbances were measured. In this way, the overall conversion of phenylethylene glycol to benzaldehyde 2,4-dinitrophenylhydrazonewas 101%. Variables in the Reaction. The two parts of the method displayed differing dependences upon temperature when applied to various glycols. When Reaction A was carried out at 34" C, no aldehyde was obtained from truns-1,2dihydroxyindane in 1 hour, and the yield from the cis isomer was approximately 15% that achieved at 80" C. At 60" and at 100" C, yields were roughly half those at 80" C for cisindanediol. In the cases of the cyclohexanediols, however, the opposite temperature effect was seen in Reaction A, and very low yields were obtained a t 80". No changes in the final results occurred when the dihydroxycyclohexane isomers were oxidized at room temperature for periods of up to 3 hours, but after 90-minute oxidation at 80" C, the final absorbances were lower for both cis- and trans-indanediols than after 60 minutes. The second reaction, that of hydrazone formation, also exhibited some temperature dependence. The final result was increased as the temperature of Reaction B was increased, with a maximum at about 70" C for dihydroxyindane and a t about 50" C for dihydroxycyclohexane. The ratio of
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ANALYTICAL CHEMISTRY
absorbances after hydrazone formation a t the optimum temperature and those after Reaction B was run at room temperature, were 1.42 for indanediol and 1.26 for cyclohexanediol. This was useful, however, only when the method was applied to simple aqueous solutions. When the sample contained material derived from extracts of biological tissues, the results were quite erratic when the hydrazone formation step was conducted at elevated temperatures, and it is therefore recommended that Reaction B be run a t room temperature in such circumstances. The derivatizing reagent used by Juni and Heim in their colorimetric method for acidic glycols ( I ) was p-nitrophenylhydrazine. We have found that with the neutral glycols used in the present work, the results were some 40% higher and more reproducible when 2,4-dinitrophenylhydrazinewas used in Reaction B. With either hydrazine reagent, satisfactory extraction of the neutral hydrazones could not be effected with ethyl acetate (the solvent of choice for the acidic hydrazones); chloroform afforded rapid and quite reproducible extraction. Application to Biological Samples. The method has been used to assay glycols as products of in vitro metabolic reactions. The reactions were stopped by making the incubation mixtures 0.4M with respect to perchloric acid. Excess perchloric acid was precipitated by neutralizing with KOH. The supernatant fluids were extracted with a n appropriate solvent, aliquots of the extract were evaporated to dryness, and the residues were taken up in water and assayed. When the assay procedure was applied t o standards which had been mixed with control tissue extracts, the result was about 10% lower than when simple aqueous solutions of standards were employed. Therefore, standards were always prepared in a medium similar t o that of the sample.
RECEIVED for review September 5,1967. Accepted November 2, 1967. Supported in part by U. S. Public Health Service Grant No. UI 00453 (formerly O H 00251). (3) M.F.Clarke and L. N. Owen, J. Chem. SOC.,1949,p. 315. (4) J. S. Brimacombe, A. B. Foster, M. Stacey, and D. H. Whiffen, Tetrahedron, 4, 351 (1958). (5) H. D. Porter and C. M. Suter, J. Am. Chem. SOC.,57, 2022 (1935). (6) C. Van Loon, Diss. Tech. Hoogeschool De& (1919); C.A., 17, 1956 (1923).
