V O L U M E 26 NO
5, M A Y 1 9 5 4
tate were treated in fluxes containing varying percentages of potassium nitrate. After the melts cooled, they were treated in the usual manner, and finally ignited to constant n-eight. Table VI11 describes these experiments. The increase in weight is marked, reaching approximately 46 nig. per gram of barium sulfate at a higher ratio of nitrate to flux. The weight decrease beyond this point is probably due to the partial decomposition of nitrate a t the higher conceiitrations, sinrc nitrogen oxides were liberated when these samples were leached. I t is tentatively suggested that nitrate be removed before precipitation by repeated tieatmcnt n i t h hot, concentrated hydrochloric acid.
849 Joseph Greeuspan for many helpful discussions. bcknowledgnient is a1.o made to Isidore hdler for spectrographic analyses. LITERATURE CITED
.Irerell, P. R., and Walden. G. H., J . .4m. Chem. Soc., 59, 906 (1937).
Booth, H. S., Pollard, E. F., and Rentschler, M. J., Ind. Eng. Chem., 40, 1981 (1948).
and Thompson, W.S., ISD. ENG.CHEW.,ANAL. ED.,1 1 , 206 (1939). Hints. E., and Weber, H., 2. anal. Chem., 45, 31 (1906). Johwton. .J.. and Adams, L., J . A m . Chem. Soc., 33, 829 (1911). Kolthoff, I. AI., and Rlaciievin, W.II.,J . Phys. Chem., 44, 921 (1040).
COhCLCSION
Sodium and chloride ions can br quantitatively ienioved f i om barium sulfate in one purification by a flux containing equal amounts of potassium chloride arid sodium chloride. Leaching with slightly acidified barium chloi ide solution yields the theoretical quantity of pure barium sulfaic. Sitrate cannot be removed in one purification. ACKNOW LEDGXIEhT
One of the authors (J. S.) wishes to thank Roland Ward and
Kolthoff, I. AI., a,nd Sandell, E. H., “Textbook of Quantitative Inorganic .Iiialysis,” 3rd ed., p. 112, New York, Macmillan Co.. 1952. Popoff, S.,and Seuman, E. W., ISD. ENG.CHmi., ANAL.ED., 2, 45 (1930).
Schmitt, G. ,J., B.S. thesis, Polytechnic Institute of Brooklyn, June 19.50. Schneider, F., and Rieman, W.,J . -4711. Chem. Soc., 59, 354 (1937).
Walden, G. 11.. and Cohen, 11. U., Ibid., 57, 2691 (1935). Walton. G.. and Walden, G. R . , I b i d . , 68, 1742 (1946). RECEIVED for review August 24, 1953. A c c e i ~ t r dFebruary 10, 1954.
Determination of Residual Crag Herbicide 1 and Its Hydrolysis Products on Food Crops J. N. HOGSETT and G. L. FUNK Carbide and Carbon Chemicals Co., Division o f Union Carbide and Carbon Corp., South Charleston, W. V a .
The increasing use of sodium 2-(2,4-dichlorophenox~)ethyl sulfate, Crag herbicide 1, for the weed control of various agricultural crops has necessitated the development of analytical procedures for the determination of spray residues. A procedure is described for the quantitative estimation of as little as 0.018 mg. of Crag herbicide 1 on food crops by measuring the intensity of the colored complex formed with methylene blue chloride. Application of the procedure to the determination of 2-(2,4-dichlorophenoxy)ethanol and 2,4-dichlorophenol, hydrolysis products of the herbicide, after a preliminary sulfation is also described. Data are presented to illustrate the applicability of the procedure to many food cropg. In addition to the determination of Crag herbicide l, the method of sulfation and colorimetric measurement of the resulting sulfate, suggests a sensitive method for the determination of many long-chain alcohols.
S
ODIUM 2-(2,4-dichlorophenoxy)ethylsulfate.
c1 c i a - 0 - c
H?CH~-o--so~N~
Crag herbicide 1 (6, 7’). an effective new chemical for weed control, has the unique property of being noninjurious to plants when sprayed or dusted directly on the foliage a t the concentrations that will kill weed seedlings in the soil. I t is activated upon contact with the soil and is very effective for the control of shallow-rooted, broad-leaved plants and grass roots. Microorganisms in nonsterile soil convert the herbicide to 2-(2.4-dichlorophenosy)ethanol and 2-(2,4-dichlorophenoxy)acetic acid, which are active plantgrowth regulators ( I O ) . Its application and
effectiveness are facilitated by virtue of its water solubility and its surfactant properties. Crag herbicide 1 is commercially available as a powder with a minimum purity of 90.0% by weight. The compound is used in dilute concentrations, 0.4 to 0.5’% in water, which is sprayed upon the soil. Because of the widespread use of synthetic herbicides, fungicides, and insecticides, i t has become necessary to develop sensitive methods of analxsis for their detection on food crops. To ensure adequate protection for the public from possible health hazards arising from the use of these chemicals, adequate procedures for the determination of spray residues in trace amounts must be developed so that reliable data can be obtained for toxicological studies. During an extensive herbicide-spray program, accurate methods of analysis for low concentrations of Crag herbicide 1 and ita hydrolysis products, 2,4-dichlorophenol and 2-(2,4-dichlorophenoxy)ethanol, were needed. Their determination by measuring total aromatic chlorides was rejected because of a lack of selectivity of the method. -4few methods of analysis for chlorinated agricultural chemicals have been reported. None of the three procedures for DDT as outlined by Fahey and Rusk (S) proved applicable to the analysis of Crag herbicide 1 or its hvdrolysis products The residue control of 2.4-D has been successfully developed 15 ith the use of Swanson’s bioassay method ( 9 ) , Bandurski’e spectrophotometric method ( 1 ). and two colorimetric methods-the method of Freed (Q), involving the substitution of chlorine in 2,4-D by an amino group \’i ith the subsequent detection of the amino group with sodium 1.2-naphthoquinone-4-sulfonate. and the method of Marquardt and Luce ( 8 ) ,involving the reaction of 2,4-D with chromotropic acid (l$-dihydroxynaphthalene-3.6-disulfonic acid) in concentrated sulfuric acid. The methods of Swanson, Bandurski. and Freed could be applied to the residue analysis of Crag herbicide 1 and its hydrolysis products, hut all three lack sensitivity and ease of application,
850
ANALYTICAL CHEMISTRY
The method of Marquardt and Luce applies only to the analysis of aromatic acids. The proposed analytical method for Crag herbicide 1 is based upon the observation of Jones ( 6 ) that sodium alkyl sulfates form complexes Tyith methylene blue chloride that are chloroform soluble. I n brief, the procedure consists of the following steps: The treated and control plant materials are extracted with water. An aliquot of the aqueous extract is taken for the determination of parent compound (Crag herbicide 1) and the remainder is reserved for the determination of its hydrolysis products. The first aliquot is added to an aqueous solution of methylene blue chloride and any complex formed is extracted into chloroform. The absorbance of the chloroform solution of the treated material a t a wave length of 650 mp is compared with that of untreated material, and the concentration of Crag herbicide 1 is obtained from a calibration curve. The aliquot reserved for determination of the hydrolysis products is first extracted with chloroform to remove those products. The concentrated extract is treated with a dilute chlorosulfonic acid solution in chloroform to convert the hydrolysis products to their corresponding sulfates. This reaction solution is shaken with water, which immediately hydrolyzes the excess chlorosulfonic acid and extracts the water-soluble organic sulfates. The final water solution is treated in exactly the same manner as the original aliquot for the determination of the parent compound, except that the absorbance is measured a t 640 mp (see notes on procedure). Reference is made to a second calibration curve to obtain the concentration of 2-(2,4-dichlorophenoxy)ethanol plus 2,4-dichlorophenol. APPARATUS AND REAGENTS
Apparatus. A Beckman Model B spectrophotometer with 1-cm. absorption cells and glass covers and 250-ml. Soxhlet extractors, equipped with 33 X 94 mm. thimbles, were used. Reagents. METHYLEKEBLUE CHLORIDESOLUTIOK.Dissolve 0.050 == 0.005 gram of Eastman Kodak reagent-grade methylene blue chloride indicator in 1 liter of distilled water. Carefully add 10 nil. of concentrated C.P. sulfuric acid and 50 grams of anhydrous C.P. sodium sulfate. Mix the solution until the salt is dissolved. CHLOROFORM. C.P. or equivalent. METHAKOL.C.P. or equivalent. SODIUM 2-(2,4-DICHLOROPHENOXY)ETHYL SL-LFATE (98.0% by weight, minimum). Purify by recrystallization from a 50y0 mixture of water and methanol. To determine the purity of the reagent as measured by surface active titration (2), add an aqueous aliquot of the material to a two-phase system of equal volumes of chloroform and methylene blue chloride solution. Titrate the mixture with a standard 0.005N cationic agent, such as cetyl pyridinium bromide, As the titrant is added and the mixture shaken, the blue color migrates to the water layer. When the color intensities of both layers match exactly when viewed in transmitted light, the end point has been reached. An anionic agent of known purity or a highly refined sample of Crag herbicide 1 should be used as a primary standard for determining the normality of the cationic agent. 2 - ( 2 , 4 D I C H L O R O P H E X O X Y ) E T H A X O L (98.0% by weight, minimum). To determine the purity of the reagent as measured by hydroxyl content, heat a 3.0-gram sample for 1 hour on a steam bath in 25 ml. of 2 . 0 s acetic anhydride in pyridine. Treat a blank of 25 ml. of the reagent in like manner, and hydrolyze the excess reagent of both blank and sample with distilled water. Titrate both blank and sample with 0.5N alcoholic potassium hydroxide to a phenolphthalein end point. The titration value of blank minus sample is equivalent to the amount of hydroxyl present in the sample. CHLOROSULFOXIC ACID REAGENT. Pipet 3 ml. of Monsanto reagent grade chlorosulfonic acid into 100 ml. of chloroform. Prepare a fresh solution daily. Caution. Chlorosulfonic acid is a hazardous chemical which reacts violently on contact with water. I t may cause severe burns. Do not pipet by mouth. PROCEDURE
Preparation of Crag Herbicide 1 Standard Calibration Curve. Dissolve exactly 0.200 gram of the Crag herbicide 1 in a 1000-ml. volumetric flask and dilute to the mark with distilled water. Transfer 1-, 2-, 5-, lo-, and 20-ml. aliquots of this dilution into 100-ml. volumetric flasks and again dilute each to the mark with
distilled water. Transfer a 5-ml. aliquot of each final dilution into a 250-ml. separatory funnel containing 25 ml. of the methylene blue chloride reagent and 50 ml. of chloroform. Use 5 ml. of distilled water as a blank. Shake the contents of the funnel and allow the layers to separate and stand a t room temperature for 15 minutes. Determine the absorbance of the standard against the blank a t 650 mM, Plot a calibration curve of absorbance versus milligrams of Crag herbicide 1, using the values obtained from each dilution. I n accordance with Beer's law, a straight line is obtained if the molar concentration of methylene blue chloride exceeds that of the Crag herbicide 1 by a t least 2 to 1. The absorbance developed per milligram was 5.2. Preparation of Standard Calibration Curve for Hydrolysis Products. Dissolve exactly 0.200 gram of the 2 4 2,4-dichlorophenoxy)ethanol in 1 liter of chloroform. Introduce 1-, 2-,5-, lo-, and 20-ml. aliquots of this dilution into 100-ml. volumetric flasks and dilute each to the mark with chloroform. Transfer a 10-ml. aliquot of each final dilution to a 250-ml. separatory funnel. Use 10 ml. of the chloroform as a blank. Pipet 10 ml. of the chlorosulfonic acid reagent into the funnel. Swirl the funnel vigorously and let stand for 2 minutes. Add 25 ml. of water and again shake the funnel several times, venting after each agitation. Allow the layers to separate and discard the lower or chloroform layer. Filter the upper or water layer through KO.1 Whatman filter paper into a clean 250-ml. separatory funnel containing 25 ml. of the methylene blue chloride solution and 50 ml. of chloroform. Wash the filter paper with an additional 5 to 10 ml. of distilled water and add the washings to the funnel. Shake the contents of each funnel and allow the layers to separate and stand a t room temperature for 15 minutes. Determine the absorbance of the standard against the blank a t 640 mp. Plot a calibration curve of absorbance versus milligrams of 2-(2,4-dichlorophenoxy)ethanol,using the values obtained from each dilution. This curve also follovis Beer's law and a straight line is obtained if a 2 to 1M excess of methylene blue chloride is used. The absorbance developed per milligram was 1.85. Sample Preparation. PRELIMINARY TREATMENT.Most of the fresh food crops can be prepared by grinding in a suitable mill grinder before addition to the Soxhlet thimbles. Corn, cucumbers, egg plant, cabbage, and potatoes should be cut into slices before addition to the grinder. It is unnecessary to grind fresh strawberries and grapes. Foods that cannot be analyzed before spoiling can be preserved by drying 100 to 200 grams of the treated and untreated sample in a vacuum drying oven. Heat the samples a t 50" C. until all moisture is removed. The samples may be stored indefinitely in this condition. Before analysis add the dry sample to a Waring Blendor and dry blend for 5 minutes. Transfer the blended sample to a Soxhlet thimble using the distilled water reserved for the Soxhlet extractor. STRIPPING RESIDUESFROM THE FOODCROPS. Transfer 150 ml. of distilled water to each of two 250-ml. Soxhlet extractors equipped with 33 X 94 mm. thimbles. Reserve one of the extractors for a control. If a number of samples are to be analyzed, a series of extractors placed on a strip heater will facilitate the analysis. Introduce a sufficient amount of treated and untreated sample into separate thimbles so that each is conveniently filled. For dried plant materials use 15 to 20 grams Feighed to the nearest 0.1 gram. For fresh materials use a proportionately heavier sample. Apply heat to the extractors and allow the extractions to continue until a total of three siphonations have occurred in each flask. When the extractant has cooled, filter through Whatman No 1 filter paper using a suitable suction flask and funnel. Applications of Procedures. CRAGHERBICIDE1. Transfer a 23-ml. aliquot of each filtered extract into 250-ml. separatory funnels and continue the procedure as outlined for the preparation of the Crag herbicide 1 standard calibration curve. HYDROLYSIS PRODUCT^. Transfer a 75-ml. aliquot of each filtered extract into 250-ml. separatory funnels and add 100 ml. of chloroform. Shake the contents of each separatory funnel for several seconds and allow the layers to separate into two distinct phases. Draw off the lower or chloroform layer and filter through Whatman S o . 1 filter paper into separate widemouthed bottles. Carefully evaporate the chloroform extract to 10.0 =t1.0 ml. on a suitable steam bath. A previous calibration of each bottle facilitates the estimation of the correct volume. Allow the samples to cool and continue the procedure as outlined for the preparation of the standard calibration curve for the hydrolysis products beginning with the addition of the chlorosulfonic acid reagent.
851
V O L U M E 2 6 , NO. 5, M A Y 1 9 5 4 Notes from Applications of Procedures. It is essential to allow both treated and untreated samples to continue for the same number of siphonations when the residues are extracted from dried plant materials. Unequal amounts of extraneous material extracted from dried plant materials may affect the results because of charring when the chlorosulfonic acid is added to the chloroform layer for the determination of the hydrolysis products. A large amount of extraneous material in the extractant, indicated by a brown-colored solution, may also cause an emulsion to form in the chloroform layer when the hydrolysis products are extracted especially if vigorous agitation has been employed. If emulsions continue to develop with a particular type of sample, a reduction in the sample size will often eliminate the difficulty.
Table I.
Sample Corn
Onions
Analysis of Synthetic Samples
Crag Herbicide 1 Mg. Mg. Recovery, added found % 83 0.024 0.020 93 30 28 56 42 75 38 39 103 95 76 72 59 61 36 95 38 36 76 87 114 115 61 68 90 84 93 AV. 92 Std. dev. 117 0.056 0.044 56 48 38 30 38 35
25 26 26 26 60 Av. Std. dev.
79 86 79 92 75 104 108 108 108 98 94 113
0.048 0.052 48 52 81 84 81 84 37 28 37 32 24 20 41 44 19 16 160 150
108 108 104 104 76 87 83 107 84 94 96 i12
0.024 0.026 24 26 24 '26 38 33 38 30 38 30 18 15 18 18 61 48 72 72 Av. Std. dev.
108 108 108 87 79 79 83 100 79 100 93 113
0.048 0.050 81 70 18 20 18 16 24 20 164 134 81 134 81 122 167 169
104 86 111 89 83
0.076 76 76 24 24 24
100 103 99 108 108 83 100 100 64 109 97 114
0.048 0.046 48 51 48 52 24 26 24 16 37 36 108 106 108 124 160 160
0.024 0.021 30 30 56 49 38 35 76 77 61 69 38 35 76 74 61 66 90 84 Av. Std. dev.
88 100
0.048 0.046 60 60 96 72 120 120 64 76 48 52 60 70 32 32 96 92
0.24 0.015 56 33 30 20 38 26 76 45 61 25 56 56 61 56 30 25 90 60 Av. Std. dev.
63 59 67 68 59 41 100 92 83 67 70 i17
24 ~.
18 ._
24 24 24 24 61
A8par a gus
Lima beans
90
90 66 06
Soybeans
Strawberriea
-
2- (2,4-Dichlorophenoxy) ethanol Mg. hlg. Recovery, added found % 0.048 0.064 133 60 66 110 32 36 113 96 78 81 120 120 100 64 68 106 100 96 96 120 98 82 64 88 138 96 104 108 107 5 18
0.076 78 75 26 26 20
90 ..
36 36 61 Av. Std. dev.
38 92 101 113 92 97
108 93 97
83 165 151
101
104
1 30
96 106 108
108 67 97 98 115 100
99
114
94 100 75 100 119 108 117 100 96 101 113
58 0.048 0.100 60 63 96 140 120 120 96 140 120 118 180 156
208 107
146
100 146 98 87
127 i43
When the chloroform layer in the hydrolysis products determination is evaporated, care should be exercised to exclude any moisture which would cause hydrolysis of the reagent. The evaporation should not be carried to dryness, as excess charring of the residues on glass will cause erroneous results. The chlorosulfonic acid reacts immediately with the hydrolysis products and the reagent should not be allowed to remain in contact with the chloroform extractant too long because of the possibility of charring any extraneous plant materials present. The reaction time of the control should be approximately the same as that of the sample. Calculations. Aliquots taken for analysis are considered fractions of the recovered volume. Residues found are calculated as parts per million based on the weight of either the fresh or dried material. Results calculated on a dry-weight basis may be calculated on a fresh-weight basis if the quantity of water removed from the dried material is known. Mg. in total sample X 1000 = p.p.m. as Crag herbicide 1 Grams of sample or 2-(2,4-dichlorophenoxy)ethanol EXPERIMENTAL DATA
To evaluate the method of analysis, the data shown in Table I were obtained. Dried plant materials were treated with 100 ml. of an aqueous dilution of each component and redried by heating for 24 hours in a vacuum oven a t 50" C. and 2-to 4-mm. pressure. The sample size used was approximately 20 grams of the dried material, which is equivalent to 100 to 200 grams of the fresh food crop. The concentration range of the Crag herbicide 1 was 1 to 5 p.p.m. on the dry basis, equivalent to 0.1 to 1.0 p.p,m. on the fresh basis. I n the case of the hydrolysis products the concentration ranged from 1 to 10 p.p.m. on the dry basis or 0.1 to 2.0 p.p.m. on the fresh.
Table 11. Maximum Residues Obtained on Food Crops Crag Herbicide 1 P.P.M., Calcd. on' Fresh Wt. Basis 0.4 hylene blue chloride-rganic sulfate complex also are verj- stable. However, it has been observed that these solutions are subject to a slow color increase when included with the hydrolysis products of chlorosulfonic acid. In the determination of the hydrolysis products it is advisable to measure the color intensity of the complex within I hour after its extraction into chloroform. The presence of other sodium alkyl sulfates as residue? would interfere in the determination. However, most of the common insecticides, fungicides, and herbicides such as D D T , 2,4-D, lime, lead arsenate, and sulfur do not interfere. Some nonionic detergents used in conjunction with common sprays are known to interfere in the determination of hydrolysis products, but these materials are used in relatively small concentrations in spray applications. There is no apparent loss of the Crag herbicide 1 or hydrolysis products during drying of the food crops in the vacuum oven. Several samples of the condensate recovered in the drying process were analyzed and, within the precision of the method, no indication of the herbicide or its hydrolysis products were found. The principle source of error in the method is due to the extraneous materials extracted from the food crops. This is especially t,rue of the analysis of dried materials. When analyzing dried samples for 2-( 2,4-dichlorophenosy)ethanol it' is essential to run a control determination on unPprayed material. Duplicate blank determinations agreed to *0.010 absorbance unit when the sample size was limited to 20 grams. The presence of interfering materials in the extract is indicated by an excessive amount of charring upon the addition of the chlorosulfonic acid reagent. This condition usually occurs when 40 or more grams of dried sample have been allowed to extract for more than three siphonations. The interference is more prevalent in the case of dried strawberries, beans, onions, and asparagus. I t can be substantially reduced by limiting the sample size and number of siphonations. However, even under these circumstances the strong coloration of the extract encount,ered in the analysis of dried strawberries substantially reduced the accuracy and precision of the determinations. Interference is less pronounced in the determination of the Crag herbicide 1 in dried materials, although it is still significant in the analysis of dried strawberries. In some cases an emulsion is produced in the chloroform layer, which usually can be broken by adding more water and shaking the liquids vigorously. This procedure does not adversely affect the results. Very little interference was noted in analyzing fresh crops,
V O L U M E 26, NO. 5, M A Y 1 9 5 4
853
inclu ling straw.berries. If the samples can be analyzed before decomposition occure, any convenierit &le weight may be used and a blank determination on unsprayed crops is not esseiit.ial. The average antl standard deviations of the per cent recovery on the synthetic samples in Table I are a measure of the accuracy antl precision of the method. Except in the case of the dried stran-berries. the method appears to be inherently accurate and free from constant. error. The 3 a limits of sensitivity of the method calculated on the basis of 100 grams of the fresh material is about +0.2 p.p.m. for the r r a g herbicide 1 determination and fO.4 p,p.m. for the tltltrrmination of the h?-drolysis products. This means that if the sample contains 0.2 p.p,ni, or more Crag herbicide 1, there is less than one chance in 100 that analysis will shon- no herbicide t o he present. Conversely, there is an equally sm:rll chance that samples contriining no herhiride will show niore than 0.2 p.1i.m. by anal! '
Cheniicals Co., especially J. -4.Lanibrech and his associates of the R e s e a i ~ hand Development Department, and R. H. Wellman, J . B. Harry. A. J. Vlitos, and associates of the Biological Research Group. The authors are also indebted to E. F. Hillenbrand. Jr., K. D. Dunn, and J. B. Johnson of the Methods Development Group arid members of the Physical Properties Group for their valuable suggestions. LITERiTURE CITE11
, Botan. Gat., 108,446-9 (1947). Eptoii. S.R., Trans. F a ~ a t l a ySoc., 44,226-30 (1948). E'ahey. J . E.. and Rusk, H. W., .Ix.AT,. CHEM.,23, 1826 (1951). Freed, T. M.,Scir?ice, 107, 98-9 (1948). Jones. .J. 11.. J . Sssoc. Ofic.i l g r . Chemists. 28, 398-409 (1945). King. 1,. J., Lanibrech, ,J. -1.. and Finn. T. P., Coritrihr. Royce Thornpso?i Inst., 16, SO. 4, 191-208 (1950). Lanihrech, J. .I., I-,S.Patent 2,573,769 (Nov. 6, 1951). .\lai,quardt, R. P., arid Luce. I:. S-., .Is.AL. ( ' H E M . , 23, 1194 (1951).
Swanson, C . P., Botau. G'az., 107, 507-9 (1916). Vlitos, 1.*J., ('orifi,iba. B o l p Tlionipsorz Inat.. 17, S o . 2. 1"-49
ACKNOWLEDGNIENT
(1953).
The authors gratefully :icl.;nowledge the assistance of the personiicl of the various laboratories of Carbide and Carbon
RECEIVED
f o r review .July '2'3 1933.
d e c e p t e d February 3 , 1034
The Fluorescence Reactions of Steroids JOSEPH W. GOLDZIEHER, JOHN M. BODENCHUK
The Southwest Foundation for Research
and
PHILIP NOLAN, Farrand
I'roniising results w i t h fluorescence methods in the microdetermination of estrogens indicated the talue of a basic in\ estigation of the fluorescence reactions of steroids generally, both for quantitatipe technique4 and for the relation to steroid molecular structurr. Results indicate that the product resulting from heat treatment of the steroid in concentrated sulfuric acid, irradiated with 436-1np light, gives maximum fluorescence in most instauces. In general, the greater the number of hy drox>lgroups, the greater the fluoresrrnce energ?. Steric molecular configuration affects both the rharacteristics and intensit? of the spectrum. 4n aromatic A-ring greatly increases fluorescenre. The data obtained permit prediction of the t?pe of steroid from which fluorimetric methods can he de+eloped and supply the basic data, comparable to absorption curves in colorimetr>, for the deielopment of such methods.
.IX\-strong acids such as sulfuric, phosphoric, trichloioacetic. or formic wid, as well as methyl sulfate are known to react mith steroids (4, 6, Q), yielding colors or fluorescence \\ hich can be used in qtudies of molecular structure or for quantitative techniquee. The exact conditions of such reactionsthe presence of water, alcohol, or metals such as zinc or antiniony (If, I9)-are of critical importance and small changes of these conditions may make a great difference in the natuie and u-efulness of the result. Miescher (16) has studied the reaction ot steroids with sulfurlc acid and a large number of aromatic nldehydes and has shown that under these circumstances the CIS 17-hydroxyl group is strongly chromogenic while the trans configuration is not. The same has been found in the calciferol serw q using trichloroacetic acid and aldehydes (18). Zaffaroni (23) iiicubated steroids with concentrated sulfuric acid a t room temppernture for 2 hours and showed that the absorption spectra ti11 h developed were of great value in establishing the identit)
& Education, San Antonio, r e x .
O p t i c a l Co., N e w York,
N. Y.
of coniyouriilr isolatcd fi~onit)iological sources. Csing sulfuric. acid and phenylhydrazine, Porter and Silber (20) have devt,loprcl a quantitative micromethotl for 17, 21-dihydrosy 20-kctosteroids. Linford snd Paulson (16) h a w studied a number of steroids incubated \\-ith conc.entrated sulfuric acid and brought to ~-:iriousdilutions with absolute alcohol: undcr these conditioris. sperific alisorption hands were found for aroniatic and IIonaI'oniatic C-3 h\-drosXl groups. Formic acid has also been shos.11 t o he mildlj- chromogenic (4)hut detailed studies have not as >xlt appeared. The reaction of phcnolic steroids (estrogens) with phosphoric, and sulfuric acids hiis received a great deal of a t t m tion (a, R d ) , though perh:ips niore with regard to fluorescenc.r than to color formation. Reacliona yielding fluorescence rather than color have been used u-idely for quantitative measurement of estrogen (1, 7 , 10. 13) and recently for corticosteroids (8, Sf). Progress in tlik field has h e n hmipered considerably by the technical difficulties encountered in the determination of fluorescence spectra, am1 Goldzieher, Bodenchuk, and Solan have discussed elsewhere (12 ) the problems inherent in measurements made by serial intrrference filters or vpectrographic techniques. Severtheless, careful studies of estrogen fluorescence have been published by Bates and Cohen ( f ) , Rompiani (S), Engel et al. ( 7 ) , Linford ( I d ) , and others. Certain of these difficulties have been circumventetl by use of the instrumentation described by Goldzieher et al. ( 1 2 ) . There is evidence in the literature that qualitative (structural) information can he ohtained from the study of fluorescence; for example. Bellet ( 2 ) has shown that digit,oxin andgitosin (glycosides with a C-16 hydroxyl or keto group) form a fluorescent dianhydro compound on heating with phosphorir acid, whereas the analogous nonos:-genated compounds failed to fluoresce. Such information emphasized the need for the investigation of both qualitative and quantitative aspects of fluorescence reactions. Sulfuric. phosphoric, and possibly formic acids seemed to he the most promising reagents. Experimental conditions, however. xere lesp easy to select. .I\ series of preliminary experiments
