Wrangell (4). At all of the levels checked, results between the treated and untreated portions were identical (Table I). As a cross-check, Kjeldahl nitrogen analyses were performed on treated and untreated samples a t two levels. Table I1 shows the close agreement obtained between the experimentally determined nitrogen content and the theoretical content calculated from aluminum recoveries. Because the bromine-methanol extraction is not specific for aluminum nitride but also leaves other nitrides in the residue (see Table IV), analyses of seven separate steel samples for aluminum nitride were conducted as aluminum determinations on the residues and calculated to aluminum nitride. Frequent x-ray analyses on representative samples were made to verify that aluminum was not present as any other compound. Results are
Table IV. Aluminum and Nitrogen Analyses of Bromine-Methanol Residues from Steel
Aluminum,
%
expt.
Aluminum Nitrogen, yc nitride, yo Ca1cd.a Expt. Calcd. ExpLC
0.077b 0.071b 0.049 0.031 0.049 0.037 0.043
0.040 0.037 0.025 0.016 0.025 0.019 0.022
0.044 0.044 0.027 0.016 0.026 0,020 0.021
0.117 0.108 0.074 0.047 0,074 0.056 0.065
0.121 0.115 0.076 0.047 0.075 0,057 0.064
shown on Tables I11 and IV. T o indicate completeness of recovery, nitrogen determinations are also shown on Table IV. The standard deviation on steel samples is not so good as on the aluminum nitride sample, probably because the purchased material is more homogeneous. Experience has shown that heat treatment and processing background have a direct bearing on the homogeneity of nonmetallic inclusions within the sample. LITERATURE CITED
The theoretical nitrogen values were calculated on the basis of the experimental aluminum results. b These samples were found by x-ray analysis to contain niobium nitride in their bromine-methanol insoluble residues, accounting for the higher than theoretical nitrogen recoveries. c Sum of experimental % A1 and experimental yo N,. a
(1)
Beeghly, H. F., ANAL.CHEM.21, 1513
11949). ( 2 j Zbid:, 24, 1095 (1952). (3) Ibid., p. 1713. (4) Hynek, R. J., Wrangell, L. J., ANAL. CHEM.28, 1520 (1956).
GERALDA. BAUER Research Laboratory The Carpenter Steel Co Reading, Pa.
Fluorescent Dyes and Thin Layer Chromatography Applied to Detection of Vitamin D and Related Sterols in Tuna Liver Oil SIR: Thin layer chromatography (TLC) methods with their well recognized advantages in resolution, speed and sample capacity have been successfully applied to the separation of vitamin D and related sterols (2-4). In this communication we wish to point out certain qualitative advantages in using some fluorescent dyes for the detection of sterol spots and present the successful demonstration of vitamin D in a naturally occurring biological source, tuna liver oil, run directly without extraction or pretreatment on TLC plates.
are shown the dyes found most useful among several dozen tested (National Analine Division, Allied Chemical Corp., Kew York, or Matheson Coleman & Bell Div., Matheson Co., Cincinnati, Ohio). Chromatography of tuna oil (supplied through the courtesy of Georg Lambertsen, Fishery Research Station, Bergen, Korway) was performed by spotting 5-111. samples directly onto silica gel G plates either alone or admixed with 10 pg. of crystalline vitamin D3 standard. After development in dichloromethane, the plates were sprayed with Auramine 0, viewed under ultraviolet light, then resprayed with sulfuric acid and heated.
RESULTS AND DISCUSSION
Most of the fluorescent dyes tested were of limited usefulness for detection of spots in paper chromatography because they stained the background so strongly that contrast between spots and paper was extremely poor. I n contrast, on thin layer plates, the background fluorescence was apparently suppressed by the silica gel and the sterols spots therefore stood out well against a generally weakly fluorescent background. illthough sensitivity was not so good as with the standard procedure of heating after sulfuric acid
EXPERIMENTAL
Sterols were run on 250-micron thick silica gel G (with binder) layers using dichloromethane as a developing solvent. Activation of plates by heating was not necessary. I n this system the R,’s vary somewhat from one run to another, as usually seen in TLC, but for a typical plate were: cholesterol, ergosterol, and 7-dehydrocholesterol, 0.18; vitamins Dz and DB, 0.32; dihydrotachysterol, 0.51 ; and vitamin D3-3,5-dinitrobenzoate, 0.97. Spots of 10-pg. were routinely run and the plates sprayed with the various fluorescent dyes (dissolved in methanol in concentration of 0.5 mg. per ml. After being sprayed, the plates were visualized successively a t 254 mp and 365 mp. The plates were then resprayed with 10% HZSO4in methanol, heated on a hotplate, and revisualized under the same lamps. I n Table 1
Table 1.
Dye Auramine 0 Auramine 0 HzSO~
+
Rhodamine 6G Brilliant yellow
Fluorescent Dyes on Thin Layer Plateso 365 mp Ergo 254 mp Dz, D3 7D,- BackDz,Da Ergo
BackD3ground Chol DHT DHC DNB ground Chol DHT 7-DHC DNB ... Y Y Y ... ,..
Y
...
Y 0
Y f
Y D
Y
...
D
f
D
6G Y ,.. Y Y D Y , D D D Acridine yellow Y Y Y D Y D D D Acridine orange V V V D V D D D Acridine red O O O O D P P D D D Brilliant acid yellow 8G Y Y D D D D Y f D D D D D D Fluorescein Primuline V B B B D B B B D f f D P . . D , . . D Safranin Pyronin Y O P P D Y P D D a Invisible; Y, yellow fluorescence; 0, orange fluorescence; D, dark absorbance; f, faintly observable; Y,violet fluorescence; P, pink fluorescence; B, blue fluorescence.
VOL. 37, N O . 2, FEBRUARY 1965
301
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.).
