spray, the use of certain fluorescent dyes permitted a degree of qualitative differentiation, and subsequent spraying with sulfuric acid was not interfered with. Inspection of Table I reveals several interesting qualitative differences in the response of various sterols to the fluorescent dyes employed. When safranin was used, cholesterol, ergosterol, and 7-dehydrocholesterol (with intact sterol B rings) were not visible under 254-mp U.V. light, whereas vitamins DP, D3-dihydrotachysterol, and D3D N B (with open sterol B rings) appeared as dark absorbing spots against a pink fluorescent background. Cholesterol could readily be differentiated from sterols more closely related to vitamin D by several different dyes. For example, with acridine yellow, the yellow fluorescence observed with vitamins DI and D3, dihydrotachysterol, ergosterol, and 7-dehydrocholesterol under the 365-mp lamp was transformed to dark absorbing spots under the 254-mp lamp, while cholesterol retained fluorescence. With each dye shown in the table escept Auramine 0, the ester, vitamin D3-3,5-dinitrohenzoate, appeared as a dark absorbing spot under 365-mp
illumination while the free sterols were generally fluorescent. 254-mp illumination of the same T L C plates, however, revealed most of the sterols as absorbing spots, with the exception of cholesterol which was either not visible or gave a reaction differing from the other sterols. Although attempts to demonstrate the presence of vitamin D by TLC in organic solvent extracts of saponified mammalian blood or tissue or in cod liver oil have for us been unsuccessful, the high concentrations of vitamin D in tuna liver oil (1) have permitted the direct visualization of vitamin D in certain samples run directly on thin layer plates. When the developed TLC plates after sulfuric acid spray were heated on a hot-plate, the cholesterol and cholesterol esters in each of the oil samples characteristically showed up initially as pink spots which then turned brown, in contrast to the vitamin D (and DHT standard) which only became brown. The greatest concentration of vitamin D was present in sample No. 4, with visible quantities in the other tuna oil samples. The halibut oil contained a barely perceptible vitamin D spot and nothing could be seen in the cod liver oil sample. From comparisons of relative spot intensities it was estimated that one
tuna oil contained in excess of 1 ug per pl. (40,000 I.C./gm) which is equivalent to the most potent oils described ( I ) , while in contrast, another tuna oil contained less than 0.1 pg per pl. Because vitamin DP and D, run with identical Rf’s in all the TLC systems tested, it was impossible to assess the identity of the vitamin D present in these tuna liver oil samples. LITERATURE CITED
(1) Bills, C. E., in “The Vitamins, Tol. 11,” W. H. Sebrell, Jr., and R. S. Harris, eds. p. 165, Academic Press, New York, 1954. ( 2 ) Davidek, J., Blattna, J., J . Chromatog. 7, 204 (1962). ( 3 ) Norman, A. W., IleLuca, H. F., ANAL. CHEM.35, 1247 (1963). ( 4 ) Schachter, D., Finkelstein, J. D., Kowarski, S., J . Clin. Invest. 43, 787 (1964). PHILIPS. CHEN,JR.
Department of Radiation Biology School of Medicine and Dentistry University of Rochester Rochester, 5 . Y. This paper based on work performed under contract with the U. S. Atomic Energy Commission at the University of Rochester Atomic Energy Project, Rochester, N . Y.
Inhibition of Polymer Formation in Dimethoxypropane-Induced Esterification by Use of Dimethyl Sulfoxide SIR: The use of 2,2-dimethoxypropane (DMP) for the rapid promotion of the classical Fischer esterification was first reported by Lorette and Brown (1). Since this first report, little data have appeared in the literature describing the use of this reagent in esterification reactions (3, 4). Recently Mason and Waller described a method for the D M P induced transesterification of fats and oils (e). These authors indicated the effectiveness of D M P in promoting rapid and quantitative esterification without recourse to elevated temperatures. The only objection to this otherwise excellent technique is the formation of polymers originating from the D M P by acid catalyzed condensations. We wish to report the use of dimethyl sulfoxide as a suitable inhibitor of DAMPpolymerization.
Reagents. 2,2-Dimethoxypropane obtained from Eastman Organic Chemicals and redistilled from 78’ to 80” c. Methanolic HCl was prepared every two days by bubbling dry HCl into methanol until the concentration was lOyo by weight. Methanol was spec100
90
82
-
80
$ 70 t
$a 60 --
0.05%
NO DIMETHYL SULFOXIDE
EXPERIMENTAL
Apparatus. Colorimetric studies were performed with a Bausch & Lomb Spectronic 20 colorimeter (Bausch & Lomb Optical Co., Rochester, N. Y.). Gas chromatographic data were obtained with an .\erograph HyFi chromatograph. 302
ANALYTICAL CHEMISTRY
\
50
0
60 120 180 240 XH) 360420400 TIME (minutes)
Figure 1. Effect of dimethyl sulfoxide concentration on inhibition of DMP polymerization
troquality from Matheson Coleman & Bell Chemical Co. Other chemicals were reagent grade. Procedure. Appropriate amounts of dimethyl sulfoxide were added to each of five tubes containing 0.5 ml. of D M P , and the volume in each tube was adjusted to 5 ml. with 10% methanolic HCI to give the following dimethyl sulfoxide concentrations: I%! 0.5%, 0.25%, 0.1%) and 0.05%. A sixth tube contained 0.5 ml. of D M P and 4.5 ml. of 10% methanolic HCl, but no dimethyl sulfoxide. Colorimetric readings were taken every 30 minutes over an eight-hour period a t a wavelength of 525 mp. Two special mixtures were also prepared for gas chromatography as follows: Mixtures containing 50 mg. each of octanoic, decanoic, and succinic acids were placed into two separate 10ml. reaction flasks. The acids were dissolved in 4.5 ml. of 10% methanolic HCl, and 0.5 ml. of DXlP was added to each tube; finally 0.0125 ml. of dimethyl sulfoxide was added to one of the tubes. After 12 hours 1-p1. aliquots from each tube were injected directly into the gas chromatograph. All experiments described above were carried out at ambient temperature (74’ F.).
I
I
I
20
15
Figure 2. formation
1
5 TIME (minutes) IO
0
No inhibition of polymer
Column, 200 feet, 0.03-inch i.d. coated with 10% Ucon HB 2000. Temperature, 135' C. Sample h e , 1 pl.
RESULTS AND DISCUSSION
Inhibition of D M P polymerization by different concentrations of dimethyl sulfoxide is illustrated in Figure 1. Complete inhibition of polymer formation occurs with a concentration of 1% DMSO for a t least eight hours; further
observation indicated however that a faint yellow color appeared after 20 hours. Where dimethyl sulfoxide was absent, a high percentage of polymer had formed after just two hours, and after eight hours the solution had become dark brown. It has been our experience with the majority of mono- and dicarboxylic acids tested that, esterification reactions which included D M P were loo'% complete within four hours. A concentration of dimethyl sulfoxide as low as 0.25% effectively inhibits significant polymer formation. If the time of reaction for a particular esterification is known, a concentration of dimethyl sulfoxide could be chosen which would completely prevent polymer formation for the duration of the reaction. Figure 2 shows the interference which may be caused during a gas chromatographic analysis by the presence of D M P polymers. Figure 3 represents identical conditions to Figure 2 with the exception that dimethyl sulfoxide had been added to the esterification procedure prior to gas chromatography. Other compounds which were tested as possible inhibitors of the reaction described above and found to be ineffective were: pyridineN-oxide, di-n-hexyl sulfoxide, di-n-hexyl sulfone, and trimethylamine oxide. Tetramethylene sulfoxide, however, inhibited D M P polymerization, but was only 500/, as effective as dimethyl sulfoxide. LITERATURE CITED
(1) Lorette, N. B., Brown, J. H., J . Org. Chem. 24, 261 (1959).
I
20
15
IO
5
TIME (minutes)
Figure 3. Inhibition of polymer forrnation b y dimethyl sulfoxide Chromatographic conditions as in Figure 2
(2) Mason, M. E., Waller, C. R , ANAL. CHEM.36, 583 (1964). ( 3 ) Tove, S B., J . Nutr. 75,361 (1961). ( 4 ) Waller, G. R., Symposium on Gas
Chromatography, 16th Southwest Regional Meeting of A.C.S., December 1-3, 1960, Oklahama City, Okls. P. G. SIMMONDS ALBERTZLATKIS Chemistry Department University of Houston Houston, Texas
Use of Sorbitol as Internal Standard in Determination of D-Glucose by Gas Liquid Chromatography SIR: I n adapting the method of Sweeley, Bentley, Makita, and Wells ( 4 ) to the quantitative analysis of D-glucose in commercial corn sugar and corn sirups, several difficulties arose. Sample size could not be controlled well enough to allow direct peak-area measurement. In addition a film, thought to be silicon dioxide, was deposited on the flame detector, greatly reducing its sensitivity. Peak areas were 30% higher in some cases when measurement was made directly after cleaning the detector. An internal standard was then sought. For a material to classify as a n internal standard it should not be present in the sample being analyzed ( 1 ) . I t should be eluted near the sample's components, and the ratio of its peak area to the components' should be near unity. Sorbitol (D-Sorbitol or
D-glucitol) fulfilled these requirements, and its use overcame the difficulties discussed above. A typical chromatogram is seen in Figure 1, which shows sorbitol being eluted between CY-D- and 0-D-glucose. With a total analysis time of '/* hour, up to 16 samples can be analyzed in a normal working day. The new procedure is considerably more rapid than present paper chromatographic techniques, and it offers the obvious advantage over the numerous D . E . (Dextrose Equivalent) methods in directly analyzing a material for its glucose content. EXPERIMENTAL
Reagents. Hexamethyldisilazane was obtained from Peninsular Chemresearch, Gainesville, Fla. ; trimethylchlorosilane from General Electric
Co., Silicone Products Division, Waterford, N. Y . ; a-D-glUCOSe, 0-Dglucose a n d D-sorbitol from Pfanstiehl Laboratories, Inc., Waukegan, Ill.; Enzodex, anhydrous dextrose and corn sirup solids, from Grain Processing. Corp., Muscatine, Iowa. Materials were used as supplied unless indicated otherwise. Apparatus. An derograph A-600-B chromatograph equipped with hydrogen flame ionization detector (Wilkens Instrument and Research, Inc., Waln u t Creek, Calif.) attached to a Leeds and Northrup Speedomax H recorder was used. Columns. During most of this study a 6-ft. X 0.25411. 0.d. coiled, stainless steel column packed with 370 SE-52 on 100- to 120-mesh Chromasorb P was employed. It was obtained from Applied Science Labs, State College, Pa. Operating Conditions. T h e column oven was maintained a t 180' C VOL. 37, NO. 2, FEBRUARY 1 9 6 5
303
