Application of Differential Thermal Analysis to Thin Layer Chromatography Ronald V. Mangravite
Central Research Laboratories, Interchemical Corp., 1255 Broad St., Clifton, N . J .
CHARACTERIZATION OF DICARBOXYLIC ACIDS separated by thin layer chromatography is usually time consuming and difficult because of the relatively small amounts of material available. The author has applied the technique of differential thermal analysis to the identification of dicarboxylic acids, separated on silica gel plates, at the 20-60 pg level, without separation from the substrate. EXPERIMENTAL Apparatus. Differential thermal analysis was performed with a DuPont 900 Thermal Analyzer, using the 500" C cell, chromel-alumel thermocouples, and microcapillaries (1.5 mm). Thin layer chromatographic plates were prepared in the laboratory using Merck (Darmstadt) Silica Gel G at a thickness of 250 microns with a Brinkman-Desaga spreader. Procedure. Samples amounting to 20 to 60 pg of material as 0.5 solutions in acetone were spotted from a 4-pl pipet. After development, the plates were first air-dried briefly, then oven-dried at 110" C for 30-60 minutes, and then cooled to room temperature. A variety of visualization techniques were employed; the most common was exposure to iodine vapors in a tank and the outlining of the spot with a needle before the iodine sublimed. Ultraviolet light was employed in some instances. The use of parallel spots was also attempted; one was masked, the other sprayed with an indicator.
Table I.
Acid Adipic
Comparison of Literature and Experimental Melting Point Data
Literature ( 2 ) mp, C O
151-3
Observed mp, "C
142, 141 (20 pg), 144 (60fig) 130.5134.5(observed 134 Maleic 139, 142)b dl, 128-9 123-9 Malica 1.99-100 93-4 2867 286 Fumaric Itaconic 165-6 (observed 157)* 165, 174 189-90 never clear Succinic 135 Sebacic 134-5 sublimation 0-Phthalic 191 sealed tube 231 fast heating @ 210 none Isophthalic 347-8 ~....Terephthalic 425 sealed tube none
General resolution at small sample weights good poor
good good
poor poor poor poor ...
...
See discussion, re the formation of /-malic and fumaric acid on heating dl-malic acid. Melting points obtained on pure compounds by conventional differential thermal analysis.
250
ANALYTICAL CHEMISTRY
I T -C (COIIECTEO
FOR CHROMFL ALUMEL THERMOCOUPLES1
Figure 1. Representative traces of the onset of melting endotherms Heating rate; 10°/min; sensitivity; ( A t ) a-d: 0.1" C/inch, e--: 0.5' C/inch Silica gel zis. silica gel Adipic acid, 40 p g Malic acid, 20 pg Malic acid, rerun Adipic acid, 100-150 pg f. Itaconic acid, 100-150 pg a.
b. c. d. e.
After the sample was circled, a DuPont capillary tube already cut to 1 inch was used to scrape up all the substrate, tapping it down to the bottom of the tube as the mouth filled. A 5-7 mm diameter spot generally filled the capillary adequately. The same size circle was then marked on an unused portion of the same plate in line with the solvent flow (90' to the spreader direction to prevent thickness variations), and the reference tube was filled with the same amount of material. The thermograms were run at 10" per minute, 20" per inch, and from 0.5" to O.l"/inch dT (sensitivity). Usually the temperature was increased until the major endotherm occurred; then the sample was cooled and reheated to a higher temperature such that no further significant transition could be expected. Initially, it was helpful to run a silica gel us. silica gel trace on the same chart for comparison. If this procedure is followed the base line should not have any acute transitions from 120" to 140" C, although it was very difficult to avoid one around 185-90" C. RESULTS AND DISCUSSION
The data in Table I represent the onset of the melting endotherms. Most of the experimental work was done at the lowest levels of detection to determine the usefulness of the
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 an 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 32601
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 NaI04, 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.
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
