Table
II.
Effect of Incubation Time on Color Development"
Time, hlinutes
Sample 0 004 0 240 0 540 0 538
0 5
10 20
Absorbance Blank 0 004 0 004 0 006 0 006
Net 0 000 0 236 0 534 0 532
Xeasurements made at 635 mw
Table V. Effect of Relative Amounts of Amyl Alcohol and Ethyl Alcohol on Color Development"
Amyl Ethyl Alcohol, Alcohol, Absorbance hI1. MI. Sample Blank Net 4.0 .. 0.520 0.006 0.514 3.0 1 . 0 0.540 0.006 0.534 2.0 2.0 0.480 0.018 0.462 1.0 3 . 0 0.300 0.088 0.212 Measurements made a t 635 mp. Table VI.
Table 111. Effect of Temperature on Color Development"
TpPf C. 26 30 35 40
a
Absorbance Sample Blank Net 0 260 0 002 0 258 0 493 0 005 0 488 0 540 0 006 0 534 0 638 0 004 0 634 60 0 650 0 004 0 646 90 0 919 0 004 0 915 Measurements made at 635 mp.
Table IV. Effect of Concentration of Sodium Carbonate on Color Development"
Absorbance Sample Blank Net 0.423 0.004 0.419 0.540 0.006 0.534 0.520 0.008 0,512 0.441 0.008 0.433 Measurements made at 635 mp.
hol, and 1 ml. of distilled water were added, and the solution was shaken thoroughly, and then allowed t o stand for 5 minutes to permit the salts to settle. The amyl alcohol layer was removed, and the color was read at 636 mp in a Beckman spectrophotometer
F. WITTER
hIenadiSbsorbone Reance Recovered, covery, (3635)" -, 76 5 0.027 5.0 100.0 10 0.053 9.9 99.0 50 0.268 49.6 99.2 100 0.532 99.6 99.6 a Corrected for blank. Blank run on mold extract alone (3635 mp for blank was 0.002 n-hich remained constant in all cases).
Color Development"
2.CDinitrophenylhydrazine, Absorbance hI1. Sample Blank Set 0 08 0 250 0 004 0 246 0 10 0 540 0 006 0 534 0 15 0 540 0 005 0 535 0 20 0 548 0 004 0 544 a Measurements made at 635 mp.
against a blank containing all reagents except Menadione.
ratio of the concentrations of amy1 alcohol and ethyl alcohol, and the concentration of 2,4-dinitrophenylhydrazine. The addition of more than 1 nil. of water made the solution very turbid. Table VI1 presents the percentage recovery of different amounts of Mensdione added to the mold extract. I t shows that the method is useful in estimating the amounts of hIenadione in mold extracts. CONCLUSION
Tables I and I1 show that the rate of color formation depends on the concentration of Xenadione and the period of incubation. Table I11 shows the effect of temperature on the formation of the color complex. Even though the intensity of the color increased with temperature of incubation, it was observed that the color formed a t 35" C. was stable for 24 hours; the color that developed at higher temperatures was more intense at the junction and erratic results mere obtained. Tables IV, V, and V I show that the rate of color formation depends on the concentration of sodium carbonate, the
Spot Test for 3-Hydroxy ROBERT
Nenadione Added,
2,4-Dinitrophenylhydrazine on
RESULTS
Sodium Carbonate, hII. 0.2 0.3 0.4 0.5 a
Effect of Amount of
Table VII. Recovery of Menadione Added to Mold Extract
The results obtained in these experiments show that this method can be used to estimate Menadione in the range of 2 to 300 y. It remains to be seen whether this method will accurately measure natural vitamin K (K, and K2) in plant or animal tissues. LITERATURE CITED
(1) Sovelli, A., Science 93,358 (1941). (2) Reddy, D. V. S., Srinivasan, V., Current Sci. (India) 17. 22-3 11948). (3) Sathe, Vanamala; Dave, J.' B., 'Ramakrishnan, C. V., Nature 177, 276 (1956).
RECEIVED for review December 13, 1955. Accepted June 25, 1956.
Steroids
and SHELLEY STONE
Department of Biochemistry, School o f Medicine und Dentisfry, University of Rochester, Rochesfer 20, N. Y.
b Steroids
containing the 3-hydroxy-
A-5 groups give a characteristic pink test when the paper is heated a t 80" C. for 8 minutes after spraying with phenol and perchloric and molybdic acids. The presence of a keto group or a double bond in position 7 interferes with the test. Fructose and its derivatives give a purple color.
156
ANALYTICAL CHEMISTRY
S
specific for the various functional groups of the steroids are needed. It has been found that steroids containing the 3-hydroxyl A-5 groups form a characteristic pink color when heated a t 80' C. on paper that has been sprayed with phenol and then with perchloric and molybdic acids. The steroids can be detected a t concentraPOT TESTS
tions as low as 5 y per 10 pl. (The spot area is 1.8 sq. cm.) EXPERIMENTAL
Reagents. C.P. phenol is saturated with water at room temperature. This reagent is used for about 2 weeks and is kept in the dark. The Hanes-Isher-
Table 1.
Color Reactions with Molybdic Acid in Presence of Phenol and Perchloric Acid
of substance in 10 pl. of isoamyl alcohol benzene (1: 1) or water applied to paper! Spot Test Compounds Tested Pink after 8 minutes at 80'; fades Cholesterol, cholesterol palmitate, A-8-androsteneto blue after 15 minutes to 1 A -6-pregnene-3P,2 l-diol-20-one, 30.,17a-dio1, hour at room temperature ~-~-pregnene-313,21-diol2 0- on e-2 1- a c e t a t e . A -S-pregnene-3a,21-diol-20-one-3-acet a t e, 1-5pregnene-3@-01-20-one, A -j-andr ostene-36, 173diol-diacetate, diosgenin S o color after 8 minutes at 80", 11-Dehydrocorticosterone, corticosterone, 1Tahydroxycorticosterone,ll-dehydro-lia-hydroxyand no color or light bluish green after 1 hour at room temcorticosterone, testosterone, l7a-hydroxyprogesterone, pregnane-3,20-dione, allopregnane-36,perature 17a-diol-20-oneL androstane-3a,lia-diol, KOandrostene-3@,1I p-diol-i-one, AS-androstene33,17P-7-one-17-acetate, A-5-androstene-3p,li3diol-7-one-diacetate, cholic acid [20
A-16-Progesterone, A-4-pregnene-lia,20,21-trioi- Light brown color after 8 minutes at 80"; brown to green color %one, deoxycorticosterone, A-6-pregnene176,20,21-triol-3-one, A-4-cholesten-3-one, alloafter 2 hours at room temperature pi egnan-21-01-3,20-dione, pregnane-3,12,20t i ione, pregnane-3a,21-diol-20-one, a-estradiol Light yellow before heating; bright green after heating 8 minutes at
Ergosterol
80
Monopalmitin, tristearin, stearic acid, oleic acid, distearyl lecithin, sphingomyelin, liver phospholipide
KO color or light brown after 8 minutes at 80"; no color or faint blue after 1 hour at room temperature
A4scorbicacid
Intense blue after 3 minutes at 80"
Orthophosphate
Immediate blue color
a-Ket oglutaric acid, sodium pyruvate, osaloacetic acid
S o color after 8 minutes at 80'; blue color after 2 hours at room temperature
Fructose, sucrose, inulin hlalic acid, succinic acid, glycogen, starch, glucose, arabinose, mannose, galactose, lactose, xylose, maltose, sorbitol, inositol, dulcitol, rhamnose
Purple color after 8 minutes at 80"
wood reagent ( 1 ) for phosphate esters mas modified by using one half the original amount of perchloric acid, and contained 1 gram of C.P. ammonium molybdate and 2.5 ml. of 607, perchloric acid in 100 ml. of 0.1N hydrochloric acid. T h e sugars studied nere obtained from Kutritional Biochemicals. T h e steroids used ere purified samples obtained through the courtesy of Anthony J. Izzo, Frank Ganis, and Paul Kurath of this lledical School. The spot tests !!ere carried out on K h a t m a n S o . 1 paper. Procedure. After the spots containing 5 to 40 y per 10 pl. (spot area 1.8 sq. cm.) of the compound under investigation have dried, the paper is sprayed n i t h phenol saturated with water. T h e paper is allowed to dry a t room temperature for 1 hour and then heated a t 75' for 10 minutes. (If this step is omitted, excessire charring of the paper takes place after the next step. However. the evaporation of phenol must not be too complete; otherwise no test is obtained.) The dried paper is then sprayed with the modified reagent of Hanes and Isherwood (1) and immediately heated in a n oven at 75" to 80". The development of the color is noted xt 3-, 5-, and 8-minute intervals.
S o color after 8 minutes at 80"
Then the papers are taken out of the oven and the color of the spot is noted. RESULTS AND DISCUSSION
In Table I are presented the spot tests given by a variety of compounds n-hen heated with phenol and molybdic and perchloric acids. Steroids that have the 3-hydroxy-1-5 group but not a n additional keto group or a double bond a t position 7 give a transient pink color after heating at 80"for 8 minutes. Because the 3-hydroxy esters of the reactive steroids also react, it is possible that the ester linkage is broken under the conditions of this test. The addition of a double bond a t position 7 as in ergosterol interferes with the test, as a bright green is produced instead of a pink color. The presence of a keto group a t position 7 suppresses color development. It is possible to use this reaction to detect steroids on chromatograms. For example, cholesterol 17-asdetected when run a t concentrations ranging from 10 to 100 y per 10 pl. in the octanol-lutidine-acetic acid (90: 5 : 5 Y./v.) or the methanol-lutidine-acetic
acid (4: 16: 1 v., v.) hohents described by Narinetti and Stotz (3). The specificity of previous color reactions employing perchloric acid phosphomolybdic acid (6),or silicotungstic acid ( 2 ) is not the same as that of the present test. Pontius (4)found that estrone reacted with perchloric acid and phenol, whereas in the present test an aromatic ring A does not substitute for the 1 - 5 group, as is shown by the negative reaction given by CYestradiol. Kritchevsky and Kirk ( 2 ) found that hydrosy steroids with or without a double bond in the 5- position and other steroids reacted with phosphomolybdic or silicotungstic acids. L-nder the conditions in which the steroids containing the 3-hydroxy1 A-6 groups give the characteristic transient pink color, some of the other steroids give a light brown color, but this reaction, unlike the test for the 3-hydroxyl groups, is not dependent on the presence of phenol. Other lipides such as tristearin, phospholipide, fatty acids, and monopalmitin did not interfere in this color test. Ascorbic acid and sugars containing fructose form blue colored compounds after being heated in the oven at SO", hut these carbohydrates do not interfere in the spot test for 3-hydroxyl A - j steroids, as they should not be found in extracts containing the steroids. However, if they are present, they can easily be removed b y soaking the paper for 10 minutes in an excess of distilled water. Fructose or its compounds were the only sugars of the large number tested which gave a color with molybdic acid. Hence under appropriate condition. thiq color reaction could be used a. a test for fructose or other keto sugar>. Likevise, these spray reagents could be used for the detection of a-keto acids 011 appropriate chrornatograms.
(e),
ACKNOWLEDGMENT
This work n a s supported by grant B-679 of the Sational Institutes of Health, Vnited States Public Health Service, Department of Health, Education and Kelfare.
LITERATURE CITED
(1) Hanes, C. S., Isherwood, F. A., Suture 164, 1107 (1949).
(3) Kritchevsky, D., Kirk, XI. R., Arch. Biochem. Biophys. 35, 346 (1952). (3) Marinetti, G. V., Stotz, E., J . Am. Chem. SOC. 77,6668 (1955). (4) Pontius, D., 2. physiol. Chem. 298,268 (1954).
RECEIVEDfor review April 5, 1956. Accepted November 6, 1956. VOL. 2 9 , NO. 1 , JANUARY 1957
157
