Fluorometric Estimate of CoumestroI on Paper Chromatograms A.
L. LIVINGSTON, E. M.
BICKOFF, JACK GUGGOLZ, and C. RAY THOMPSON’
Western Regional Research laboratory, Western Utilization Research and Development Division, Agricultural Research Service, Department of Agriculture, Albany, Calif.
U. S.
b This method of analysis has been applied to solutions of pure coumestrol and its limitations have been studied. Factors considered include: suitable solvents and suitable grades of filter paper; interrelationship of concentration of solution, volume applied to paper, and measured intensity of fluorescence; size of aperture of photocell of fluorometer; and stability of the fluorescence on the developed chromatograms. Quantities of coumestrol in the range from 0.2 to 1.0 pg. can be estimated with a standard deviation of about 7% of the amount present.
T
of coumarin compounds under ultraviolet light is well known, and the blue fluorescence of coumestrol (2) was utilized by Lyman and coworkers (4) for detection of the compound in extracts of forages which had been applied to paper chroniatograms. However, their method was only semiquantitative and it was still necessary to employ a biological test for estrogenic activity, in order to make a quantitative estimate. This study describes the development of a method for the quantitative nieasurement of coumestrol on paper chromatograms, including the effect of developing solvents, the grades of filter paper, size of aperture of the photocell of the fluorometer, size of spots applied to the paper, and the precision of measurement. HE FLUORESCENCE
EXPERIMENTAL
Materials and Apparatus. STANDSOLUTION. A stock solution containing 500 pg. of pure coumestrol per ml. in acetone was prepared and suitable dilutions were made. FILTER PAPER. Sheets of Whatman paper, grades 1, 2, 3MM, 4, 5, 7, 11, 20, 31, 40, 41H,42, 44, 50, 52, 54, and Schleicher and Schuell No. 589 red ribbon were evaluated. DEVELOPINGSOLVENTS. The solvents were all of reagent grade. CHROMATOGRAPFLIC APPARATUS.RecARD
Present address, University of California, Riverside, Calif. 1620
ANALYTICAL CHEMISTRY
tangular glass jars, 12 inches square by 24 inches high, with tight-fitting ground-glass covers, were used as chambers. The cylinders of filter paper were supported by hexagonal glass racks. FLUOROMETER. The design of the fluorometer was described by Bailey ( I ) . Two circular diaphragms, 8 and 30 mm. in diameter and prepared from heavy black paper, were used to reduce the aperture of the photocell below its normal value of 37.5 mm. The galvanometer was used a t its maximum sensitivity for the 8-mm. diaphragm and a t 0.22 maximum sensitivity for the larger photocell apertures. These combinations of galvanometer sensitivity and photocell aperture gave readings similar i n magnitude for a given amount of coumestrol. Procedure. For quantitative studies, paper chromatograms were prepared from sheets of Whatman S o . 1 papcr, 40 cm. high by 57 em. wide. Two- to 10-pl. aliquots of the pure coumestrol solution were spotted on marks 5 cm. apart along a line 11 cm. from the bottom edge of the paper using self-filling micropipets. Where multiple applications were made, the spots were dried between applications. After development of the papers by the ascending technique for 16 hours a t room temperature, they were airdried in a hood for 24 hours. The intensity of fluorescence was measured in the fluorometer by moving the fluorescent spot over the aperture until a maximum galvanometer deflection was obtained. The net fluorescent reading of an individual spot consisted of the maximum reading obtained for that spot minus the average value of the blanks on each side of the spot. Paper strips were prepared and spotted with solutions of pure coumestrol and developed by the ascending technique in acetic acid-water (1 to 1). After drying, the chromatograms were observed under the ultraviolet lamp (3660 A.), and the sharpness of the coumestrol spot was noted. The intensity of fluorescence of coumestrol on the most suitable chromatograms was then measured on the fluorometer. RESULTS AND DISCUSSION
Solvent Systems. More than 20 different solvent combinations were investigated for development of paper chromatograms. Most of these solvents had been utilized by other
workers in studies ( 3 ) involving paper chromatography of coumarins and flavones. Of the initial 20 reagent grade solvents tested, 15 were rejected because of the elongated coumestrol spots formed after development of the chromatograms. Five solvent systems gave round, sharp, fluorescent spots of coumestrol on the chromatograms. However, for later application to chromatograms containing extracts of forages, solvents giving coumestol R,’s of about 0.5 to 0.6 would be desirable. Conibinations of isopropyl alcohol and water, 22 to 78 or 60 to 40, gaye R,’s of 0.24 arid 0.80, respectively, while combinations of water-acetic ncid1.2-butanediolj 86:10:6 or 86:20:6, gave Rl’s of less than 0.3. Therefore, these four solvent systems Jvere also eliminated. The best chromatograms in thcse studies were prepared with acetic acid and water ( 1 to 1). This combination gave a sharp fluorescent spot lvith an RI of 0.9. and was used in the following studim. Chromatographic Papers. Chromatograms prepared from 17 different grades of filter paper, with 50% acetic acid as the developer, were visually examined under the ultraviolet lamp. Five of these grades, Whatman Nos. 1, 7 , 41H, 54, and Schleicher and Schuell KO. 589 red ribbon were selected for further studies on the basis of sharpness of the coumestrol spot. The R, values for coumestrol were very similar on the five papers and measurements on the fluorometer gave similar background readings. However, Whatman Kos. 1 and 54 and Schleicher and Schuell No. 589 red ribbon gave a greater response on the fluorometer for a given amount of coumestrol than did the other two grades of filter paper. These three papers were approximately equal in their chromatographic qualities, and the final choice of Whatman No. 1 was made on the basis of availability. To study the variation of background fluorescence, spots of pure coumestrol were made on the two marks, 10 cni. from each side and 11 cm. from the bottom edge of four sheets of Khatman No. 1 paper. After development and drying, marks yere made every 5 cm.
between the two developed counlestrol spots. The fluorometer was adjusted to a reading of 5 for the first blank and the values of the other blank spots were measured. The values for the 32 spots on the four chromatograms ranged from 3.5 to 5.5 deflection units with a standard deviation of 1 0 . 5 unit. Relationship of Volume and Concentration. I n routine analysis i t is time-consuming t o concentrate and then make dilutions of a given sample. A simpler procedure is t o make multiple applications per spot. TO examine the reliability of this procedure] a pure coumestrol solution was systematically diluted. Aliquots, 2-11., of these solutions were then spotted on several sheets of Whatman No. 1 paper with multiple 2-11. spots of the most dilute coumestrol solution. In addition] increasing volumes of the most dilute solution were spotted USing one application per spot with increasing sizes of pipets. After development and drying, the chromatograms were read on the fluorometer. The results are shown in Table I. The same fluorescence values were obtained, per given quantity of coumestrol, by both the multiple spot and concentration procedures. However, when larger volumes of the dilute coumestrol solution were applied, using the single spot method, the fluorescence values were decrcascd. This may be due to the larger size of the developed spot such that the 30-mm. diaphragm no longer included all of the spot area. Therefore, the simplest and most accurate procedure for assaying a sample nt more than one level would be multiple spot application with the same pipet used in spotting the standard coumestrol. Measurement of Fluorescence. Initially] no diaphragm was used over the photocell. Visual observations made under an ultraviolet lamp showed that 1.0 pg. of coumestrol on a developed chromatogram gave an elliptical spot 26 to 2s nmi. in length. Since the aperture of the photocell (37.5 mm.) was considerably larger than the diameter of the largest spot measured, the possibility existed that ixompletcly separated impurities in forage extracts might contribute to the readings. Accordingly, the two circular diaphragms were prepared for alternate use. The 8-mm. opening measures only the arc5 of maximum fluorescence in the spot, whereas tlic 30-mm. opening and the open photocell permit the measurenirnt of fluorescence from the entire spot. When the logarithms of the coumestrol concentrations were plotted against tlle logirithms of the correspcding gitlvariometer deflections, a linear relni ionship was obtained for the two larger apertures (Figurc 1). Slight curvltturo was found when the values
Table 1.
Relationship of Volume, Concentration, and Fluorescence on Developed Paper Chromatograms with 30-Mm. Diaphragm
(Each value represents the average of four determinations on separate sheets) Coumestrol Concentration Method Multiple Spots Single Spot Amlied. Volume. Volume.' Deflection Volume. pl. ' Deflection pg. rl. ' Deflection Pl. .I
0.2 0.4 0.6 0.8 1.0
I
2 2 2 2 2
13.4 26.5 37.6 45.8 57.0
2 3 4 5
2 x x x x
13.4 26.9 37.1 45.5 57.4
2 2 2 2
obtained by use of the 8-mm. aperture were plotted. Stability of Chromatograms. T h e fluorescence of amino acid chromatograms is relatively stable under atmospheric conditions (5). Coumestrol chromatograms gradually decrease in intensity of fluorescence when stored in the dark at room temperature (Table 11). To confirm that the difference between the 1- and 10C-day values was not due to changes in the sensitivity of the fluorometer, several new chromatograms were prepared and the fluorescence was determined a t the same time as the measurements were made on the
2 4 6
13.4 25.0 35.0
ii
53.4
II. Stability
Table
of Coumestrol Chromatograms
(Average value of four separate spots) Galvanometer Deflections with cou30-Mm. Diaphragm mestrol, 1 2 100 1 pg. day days days day" 16 30 41 52 62
0.2 0.4 0.6 0.8 1.0
15.5 28.5 39.5 50.5 59.5
11 21.5 29.5 38 45.5
15 29.5 43 51 62
e Prepared 100 days after initial chromatograms.
I
I
02
...
04
06
08
IO
Cournestrol, micrograms Figure 1 . Fluorescence intensity of coumestrol on paper chromatograms as a function of quantity in developed spot A.
With 3 0 - and 37.5-mm. apertures
E. With 8-mm. aperture
Table 111.
Precision of Fluorometric Measurement of Coumestrol Galvanometer Readings with 30-Mm. DiaDhragm
Meana Std. dev., =k CwR. of variation, yo
1.0 Pg. Opr. A Opr. B 62.2 62.0 3.0 3.1 4,8 5.0
0.6 pg. Opr. B 40.7 40.4 3.0 2.6 7.4 6.4
Opr. A
0.2 pg. Opr. B 15.7 16.2 1.3 1.0 6.4 8.0
Opr. A
-4verage value of 10 separate spots.
VOL. 32,
NO. 12, NOVEMBER 1960
1621
100th day of the initial chromatograms (Table 11, column 4). Coumestrol chromatograms lose 25 to 30% of their fluorescence when stored at room temperature for 100 days. Precision of Measurements. T h e reproducibility of the fluorescence measurement of a given coumestrol spot with the fluorometer was good. Slight variations in measurement occurred from one spot to another, and i t is recommended t h a t the measurements from at least four spots be averaged to obtain a more reliable
value. Measurements presented in Table I11 are typical of fluorometric readings of coumestrol on paper chromatograms. In this study two operators measured the fluorescence of the coumestrol spots using the 30-mm. aperture. The method is reproducible from one operator to another. LITERATURE CITED
(1) Bailey, G. F., ANAL.CHEM.32, 1726 (1960). (2) Bickoff, E. hl., Booth, A. N., Lyman, R. L., Livingston, A. L., Thompson,
C. R., DeEds, F., Science 126, 969 (1957). R. J., Durrum, E. L., Zweig, G., Paper Chromatography and Paper Electrophoresis," 2nd ed., p. 379, Academic Press, New York, 1958. (4) Lyman, R. L., Bickoff, E. M., Booth, A. N., Livingston, A. L., Arch. Biochem. Biophys. 80, 61 (1959). (5) Ven Horst, H., Tang, H., Jurkovich, V., ANAL.CHEM.31, 135 (1959). RECEIVEDfor review April 11, 1960. Accepted August 8, 1960. Mention of specific products does not imply endorsement by the Department of Agriculture over others of a similar nature not mentioned.
(3) Bl:ck,
Precipitation Chromatography Diffusion and Precipitation of Metal Sulfides on Agar Gel Columns JAMES D. SPAIN Department of Chemistry and Chemical Engineering, Michigan College of Mining and Technology, Houghton, Mich.
b When certain metal ions are allowed to diffuse into a buffered agar gel containing sulfide ions, sharply defined colored bands of precipitate develop whose relative positions are determined largely by their solubilities. The name precipitation chromatography is used to describe this procedure because the apparent separation of bands results from a selective reversible distribution between the fixed nonmobile precipitated phase and the mobile ions in solution. The procedure described provides a method whereby metals which produce insoluble sulfides may b e identified either separately or in mixtures by a single simple procedure employing the simplest of apparatus.
A
LTHOUGH diffusion techniques are
recognized as fitting into the larger category of differential migration analysis ( 8 ) , they have so far had limited application because of the poor resolving power of the method in general. Antelman (1, 2) showed that certain mixtures of metal ions could be partially separated by simple diffusion in gelatin on Petri dishes. He employed a variety of developing reagents following diffusion to render the disusion zones visible. A slightly different technique was used by Veil ( 9 ) , who incorporated the precipitant right in the gel and identified metal ions by t>heir characteristic chromate and iodide precipitates. More rccently, 2 precipitant Containing agar gel-impregnated filter gaper was t ~ ~ p l o y pin d the chromatographc separation ci anioiis by ICrishnamurti and Dhareshwar (61. They
1622
e
ANALYT!CAL CHEMISTRY
stated that the sequence of bands was related to the solubilities of the precipitates that formed. Because of its potential as a qualitative procedure, a modification of these techniques has been investigated for possible use in the analysis of the cations which form insoluble sultides. I t is presented here because of its
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Figure I. Appearance of sulfide bands under ideal conditions
extreme simplicity and its possible application where the use of complicated laboratory apparatus is impracticale.g., in the field identification of geological specimens. PROCEDURE
Preparation of Gel Columns. Agar gel was prepared in batches of 200 ml. by putting 4 grams of agar agar (U.S.P. grade shreds) in 100 ml. of distilled water to swell and leach away soluble pigmented material (ea. 30 minutes). The water was discarded and the agar was added t o 200 ml. of boiling water containing 54 grams of sodium acetate trihydrate. This mixture was boiled and stirred for approximately 5 minutes and filtered hot through a thick pad of glass wool into a stoppered storage flask. The columns were prepared from 6mm. soft glass tubing that was cut into 12-cm. sections, constricted a t one end and fire polished a t the other. Portions, 2&ml., of agar gel were softened in boiling water and mixed with 3 drops of ammonium sulfide (Baker and Adamson, light reagent solution). Tubes were filled in the same n-ay as for a pipet by dipping the constricted end into agar and removing when about z/3 full. The constricted IoJTer tip \yas cooled with ice until sufficientll- hard and the column n.as placed upright for approximately 1 hour for complete setting of tlie gel. Application of Sample. Soli:t.ons of n e t a l ians w e r e diluted w i t h 6 M hydrochloric acid until t h e 5n:ii concentration of each mr,tal inn ~,Y proximatrip 5 rug. ?er ml. ':hi tion K::S transfeyred tc t h e top of the c,oli;nin t o a d t p t h of ahG!it 0 :. c n i . I he eojcn-ir:s i ~ t a!lon = ~ ?ti t o dta:id ifi m
