Figure 3. The Teflon band gives the most plates at low throughputs. The pressure drop and holdup with the Teflon band are lower than those of the Hyper-Cal but are slightly higher than those with the metal spinning band. Comparisons a t partial reflux of these columns are shown in Figure 4. The boiling point curve represents both distillations. The refractive index curves show that the Teflon band is equivalent to both reference columns.
1.X
Figure 4. Comparison of miniature columns
1.41
1.4E
8.
c
d 1.44 z
CONCLUSIONS
The higher efficiency of the Teflon band results from specific design features: more blades, larger band core, and more points of contact with the reflux on the column wall. These features cause better mixing, shorter vapor diffusion path, and smoother band rotation. I n addition, higher optimum band speeds are attained because the Teflon band causes little frictional heat. Use of a Teflon band incorporates all of the advantages of low pressure drop, small holdup, and a large number of plates in a single column. Hence, for many distillations the choice between packed and band columns is no longer necessary. The design principles evolved in the present study should also apply to columns of larger diameter. I n such columns more than four blades might further improve efficiency. LITERATURE CITED
(1) Beatty, H. A , Calingaert, George, I n d . Eng. Chem. 26, 504-8 (1934). (2) Chemical and Powder Products, Inc., Kew York, N. Y . , Bull. CP554, Sect. B (1955). (3) Donnell, C. K., Kennedy, R. M., I n d . Eng. Cliem. 42, 2327-32 (1950). (4) Fenske, XI. R., Ibid., 24, 482-5 (1932). (5) Griswold, John, Zbid., 35, 247-51 (1943).
W
? U
2
1.42
W
1.40
1.38
1.36 DISTILLATE. ML
Table II.
Effect of Blade Edge Width
Width a t Blade Edge, Mm.
ThroughPut, Ml./Hour
0.92
15 30 60
133 113 85
0.64
Plates
0.92
0.64
Pressure drop, rnm. Hg 140 114 85
0.37 0.49 0.75
Murray, K. E., J. Am. Oil Chemists’ SOC.28, 235 (1951).
Nerheim, A. G., Dinerstein, R. A., ANAL.‘CHEM.28, 1029 (1956). Piros, J. J., Glover, J. A., U.S. Patent 2,608,528 (Aug. 26, 1952); Chicago Section, ACS, One-Day Technical Meeting, Jan. 24, 1947. Podbielniak, Inc., Chicago, Ill., Bull. A-3 (1953).
Podbielniak, W. J., IND. ENG. CHEM., ANAL. ED. 13, 639-45 (1941).
0.31 0.39 0.66
0.92
0.64
Holdup, ml. 2.4 2.7 2.9
2.2 2.3 2.6
(11) W‘eissberger, A., “Technique of Organic Chemistry,” Vol. IV, p . 215-19, Interscience, New Yorf, 1951. (12) Ibid., pp. 229-31. (13) Winters, J. C., Dinerstein, R. A., ANAL. CHEM.27, 546 (1955).
RECEIVEDfor review July 23) 1956. Accepted May 11, 1957. Divlsion of Petroleum chemistry, 130th Meeting, ACS, Atlantic City, N. J., September 1956.
p-ToIuenesuIfonic Acid Spot Plate Test for Steroids EMANUEL EPSTEIN, WILLIAM 0. MADDOCK, and A. J. BOYLE Departments o f Medicine and Chemistry, Wayne State Universify, Defroif 2,
A rapid, convenient color test for steroids avoids the charring often encountered with the use of sulfuric acid. p-Toluenesulfonic acid as a spot test reagent does not char steroids in the course of the test and yields colors which are frequently different and generally more intense than those produced by sulfuric acid. The spot test described appears to add another method of identifying steroids which 1548
ANALYTICAL CHEMISTRY
Mich.
is easy to control. The resulting melt presents a more intense fluorescence than is exhibited by sulfuric acid.
G
ENERALIZED color tests for ster-
oids as analytical aids have been confined largely to sulfuric acid and antimony trichloride. The references t o these and similar reagents are too voluminous to be mentioned here. I n recent years a reaction involving
perchloric acid was submitted by Tauber ( 2 ) . This reagent yielded various colors for many types of steroids. The work reported involves p-toluenesulfonic acid as a convenient and definitive steroid reagent in a melt-spot test reaction. MATERIALS
Spot plate, porcelain, white.
Color and Fluorescence Reactions of Steroids
Table 1.
Compound Lst d i o l - l i p
Estrone Estriol Epiandrosteronc. De hydroepiandrostc.roiIc Dehydroepiandrostrrone acetate .indrosterone Testosterone 17-Hydroxycorticosterone Cortisone Cortisone acetate l'regnan-3a,20a-diol Pregnan-3a,20a-diol glucuronoside
Color, 2 to 5 min. Yellow Yellow Pink-yellow Light pink Orange Red None Pink Yellow-brown Red Red Yellow Light brown
Using p-Toluenesulfonic Acid Color, 10 min. Fluorescence Deep orange Brilliant pink Bright orange Bright yellow Orange Bright yellow Red-brown Green Lavender-brown Yellow-green Yellow-green Lavender-brown Pink Red Blue and pinka White-green Deep yellow-brovm White-green Yellow-red Blue-p-hite Yellow-red Blue-white White-green Pink-yellow Brown Light green
'
Using Coned. Sulfuric A4cid Color, 5 minutes a t 110' to 120" C. Fluorescence Pink-yellow Brilliant blue Brilliant green-bluv Green-pink Light yellow Brilliant green Yellow-orange Yellow Blue Light green Green-blue Light green Light orangc Light green Light yellow Light green Brown Green Green-brown Light green Yellow-brown Light green Orange Light green Yellow-orange Light green
~5~16-Pregnadien-3p-ol-20-onc A53'6-Pregnadien-3p-ol-20-one
Red Red-brown
Deep red-brown Red-brown
White-pink Pink
Red-brown Red-brown
Light green Light green
A 4-Pregnene-20p-ol-3-one
Yellow-green Yellow-green Red Green
Yellow-red Green-yellow Deep red-brown Blue-green .
Bright green Light green-yellow Red Green
Pink Yellow Brown Red-lavender
Light Light Light Light
Light green-brown
Green
Bright blue
Yellow-brown Green
Deep blue Yellow-green Brown Brown Red-brown Green-brown Red Yellow-brown Green-brown
Red Bright green Pink Pink Bright white-pink Bright white-pink Bright white-pink White Bright yellow-green
Brown Red-brown Yellow-orange Red-brown Yellow-brown Orange Brown Light pink Yellow
acetate
Pregnan-3p-ol-20-one A5-Pregnen-3p-ol-20-one acetate A5-Androsten-178-acetoxy-3-
rthylene cyclic hemithioketal lIethyl-A6-3p-hydroxy etiocholenate
A4~7-22-Isospirostadiene-3-one Deep lavender 7-Dehydrodiosgenin acetate Deep yellow-brown Kryptogenin acetate Yellow-red Ergosterol acetate Deep red-brown Cholesterol Red Stigmasterol Red Cholesteryl-methyl ether Red Sitosterol Red Progesterone Yellow-brovm a Heterogeneous mixture of colors.
Hot plate, capable of a 110 to 120" C. setting, such as Tenico, Model 1900A. p-Toluenesulfonic acid, pure, Eastman Kodak Co. Fluorescence testing lamp, ultraviolet, long wave. Steroids. METHOD
d small quantity of steroid, 10 to 100 is placed in the depression of a spot plate and is overlaid with about 20 mg. of crystalline p-toluenesulfonic acid. If the steroid is in solution, i t may be pipetted into the spot plate depression and the solvent evaporated before the acid is added. The spot plate is brought to 110 to 120" C. and maintained a t that temperature so that the reagent (melting point, 104 t o 106" C.) begins to sinter in about 2 minutes and melting i- c,omplete within 3 t o 4 minutes. Color is developed usually when melting first occurs and develops further n i t h additional heating. y,
The steroids tested developed characteristic colors and fluorescence. Other nonsteroidal materials either developed no color or color without fluorescence. I n Table I are listed the steroids tested and the color and fluorescence characteristics which were noted visually. As little as 3 y of estrone may he detected hy color or 0.5 y by fluores('CI1W.
DISCUSSION
All tlie hteroids tested
I\
itli p-tolueiie-
sulfonic acid yielded distinctive colors, most of them of great intensity and purity. Almost all the steroids were highly fluorescent when tested with a n ultraviolet lamp. In most instances the colors developed with p-toluenesulfonic acid were similar to those obtained using concentrated sulfuric acid, as shown in Table I. Examples of difference, however, may be cited. Estradiol-17 & estrone, and estriol developed orange colors directly with p-toluenesulfonic acid, whereas when sulfuric acid was used the primary yellow color had to be diluted with water to produce a n orange or pink color (Kober reaction). Heating with sulfuric acid was stopped at the end of 5 minutes because purity of color seems to decrease after that time. Some steroids showed obvious differences, while others exhibited more subtle variations in color. The most striking characteristic of p-toluenesulfonic acid as a spot test reagent may be its fluorescence-yielding properties. The steroids were fluorescent after reaction. While most fluorescences were of exceptional brilliance, only the most striking fluorescences qualified a s brilliant and bright. The fliioreswncw induced by sulfuric acid did not. in tlic niaiii, rvhibit this propert!~ under the conditions used. Thiq \I :is due prohabl\. to quenching or selfnbsorptioii plienoniena. For none of the coiiipouiids tcstcd
green green green green
Green Light pink Bright blue Light pink White-green Yellow Green White green Light green
is the sulfuric acid test more sensitive in the production of color. I n general, fluorescences are more intense with p-toluenesulfonic acid. One of the disadvantages of the concentrated sulfuric acid test is that discoloration may occur, which results in a loss of color quality and sensitivity. Feigl (1) describes a reaction similar to t h a t reported here. Methylenedisalicylic acid is used as a reagent for the detection of small amounts of low melting sulfonic acids, among them p-toluenesulfonic acid. Feigl regards this as a solid-solid, solid-melt, or solid-gas reaction and not the result of sulfur trioxide derived from pyrolysis of the molten material. ACKNOWLEDGMENT
FMmdiol-l7p, estrone, and estriol were supplied by the Schering Corp. Most of the other steroids were furnished by Carl Djerassi, Wayne State University. These steroids were used without further treatment. LITERATURE CITED
(1) Feigl, F., "Spot Tests," Vol. 11, pp.
184-6, Elsevier, Amsterdam, 1954. (2) Tauher, H., ASAL. CHEW 24, 1494 (1952). R E C E I ~ E Dfor rrviea January 5 , 1957.
icrepted IIav 27, 1957. Supported by Research Grab A-121-C from the Division of Research Grants and Fellowships of the Xational Institutes of Health. VOL. 29,
NO.
10, OCTOBER 1957
1549
