Fluorometric Determination of Tin with Flavonol CHARLES F. COYLE and CHARLES E. WHITE University of Maryland, College Park,
Md.
b Flavonol produces a bright blue fluorescence with quadrivalent tin in 0.5 to 0.1N sulfuric acid solution. The reagent is sensitive to about 0.17 of tin(lV) per ml. Zirconium, fluoride, and phosphate ions interfere with the test. The qualitative test may be performed in water solution, but 33% dimethylformamide is preferred for the quantitative determination. The fluorescent complex has a band spectrum with its greatest intensity from 420 to 520 mp and a maximum at 470 mp, The ratio of tin to flavonol in the complex is 1 to 1 .
from the stock solution. Ten milliliters of water was mixed with about 75 ml. of 3N sulfuric acid in a 100-ml. volumetric flask; 2 ml. of the above solution was then added and the volume made to 100 ml. with 3N acid. FLAVONOL. The flavonol (3-hydroxyflavone) was prepared in the University of Maryland laboratories, but it is now available from Eastman Kodak Co. Solutions of 0.1 and 0.01% were made in redistilled 95% ethyl alcohol. A 0.1% solution of flavonol contains about 4.2 pmoles of flavonol per ml.
I
SULFURIC ACID. Solutions approximately 18X, 3N. and 1117were prepared by the dilution of C.P. concentrated sulfuric acid. The latter two were standardized by titration. N,N’-Dimethylformamide. The C.P. grade of N,N’-dimethylformamide (DJIF) from Eastman Kodak Co. was satisfactory without further purification. QCININESULFATESTOCKSOLUTIOH. Ten milligrams of quinine sulfate was dissolved in 1 liter of 0.liV sulfuric acid and 0.3 ml. of the solution was diluted to 25 ml. B-ith 0 . 1 s sulfuric acid for standardization of the fluorometer.
I
GENERAL PROCEDURE
T
and quantitative determinations of very small concentrations of tin are difficult. Phenylfluorone, the standard reagent for germanium, has been reported (6) as a colorimetric quantitative reagent for tin(1V). Quercetin, 3,3’,4’,5,7-pentahydroxyflavone, serves as a colorimetric reagent for zirconium (3) and tin (4, 6). Because flavonol (3-hydroxyflavone) is similar to quercetin in structure and is a fluorometric reagent for zirconium ( I ) , it was tested as a fluorescence reagent for tin. Both zirconium and tin(1V) produce a blue fluorescence with flavonol in sulfuric acid solution a t about the same acidity. This reagent provides a sensitive test for either element in the absence of the other one.
Preparation of Calibration Curve. From 0.1 to 0.6 ml. of the tin solution (1 pmole per ml.) was added to 7.5 ml. of iV,iY’-dimethylformamide with sufficient 3h‘ sulfuric acid to make 1 ml. of 3.0N sulfuric acid in each mixture. Two milliliters of 0.05% flavonol solution was added and the mixture was diluted to 25 ml. with water. This resulted in a 33% dimethylformamide solution about 0.121Y with sulfuric acid. The solution was mixed, let stand 15 minutes, and measured in the fluorometer.
HE QUALITATIVE
A typical calibration curve is illustrated in Figure 1. In solutions from 0.02 to 0.2 pmole of tin in 10 ml. all points fall on a straight line. The solutions should be mixed in the order given above, because of solubility factors. Flavonol is extremely soluble in dimethylformamide and only slightly soluble in water. The acid is necessary to prevent precipitation of tin and to assure proper acidic conditions for the complex formation. The amount of acid added is in accordance with the acidity of the original tin
APPARATUS
The American Instrument Go. Polyfluorometer equipped with a H100A4 mercury lamp and a primary filter, Corning No. 5860, was used for quantitative measurements. A Wratten C-5 filter mas used as a secondary filter and the instrument was standardized against a solution of quinine sulfate, 0.37 in 25 ml. of O,lAr sulfuric acid.
Figure 1. Relation of tin(1V) concentration to fluorescence intensity with flavonol in 33% dimethylformamide, 3 ml. of 1N sulfuric acid, 2.0 ml. of 0.05% flavonol in 25 ml. of solution
REAGENTS
TINSTOCKSOLUTION.A stock solution of 50 pmoles of tin per ml. in 18N sulfuric acid was prepared by dissolving 0.5935 gram of granular tin in 50 ml. of hot 18N sulfuric acid. The solution was heated to sulfuric acid fumes, cooled, and diluted t o 50 ml. with water. It was transferred t o a 100-ml. volumetric flask and diluted to the mark with 18N sulfuric acid. For routine work a solution of 1.0 pmole of tin per ml. in 3N sulfuric acid was prepared 1486
ANALYTICAL CHEMISTRY
16vi
w a 0
i 14-
1
I
I
I
I
I
Figure 2. Relation of acid concentration to fluorescence intensity of tin(lV)-flavonol complex in 33% dimethylformamide
solution. At acid concentrations less than those specified, tin is likely to precipitate. The fluorescence is greatly decreased if more than 8.0 ml. of 1.O.V sulfuric acid is present in 25 ml. of the mixture. For a maximum fluorescence, the flavonol concentration should be a t least in a 4 to 1 molar ratio to the tin. Quantitative measurements on tin chloride solutions were not satisfactory unless the tin was converted to a sulfate, and most of the chloride removed. To accomplish this the sample was made 18A’ with sulfuric acid and heated until sulfuric acid fumes appeared. The solution was then diluted so that the acidity nas about 3.Y.
1
30,
A
0
0.2
0.4
06
0.8
I
MOLE F R A C T I O N OF S n l l V )
DISCUSSION
Figure 3. Fluorescence emission spectrum of tin(IV)-flavonol complex in 33’70 dimethylformamide
300
The p H for optimum fluorescence of the tin-flavonol chelate was investigated with both hydrochloric and sulfuric acids t o control the acidity. At comparable p H values, the sulfuric acid solutions always gave higher fluorescent readings than the hydrochloric acid solutions. The decrease of fluorescence in chloride solutions is probably due t o a decrease in tin ion concentration by the formation of a chloride complex. The maximum fluorescence was at p H 1.25 to 1.45, or a sulfuric acid solution of 0.05 to 0.10N in water solution and 0.12N sulfuric acid in 33% dimethylformamide solution. Solvents. Three solvents were tried as a medium for this test: mater, 30yoethyl alcohol, and 33% dimethylformamide. Although the fluorescent intensity in the three solutions varied only slightly, a dimethylformamide solution was the most satisfactory because of the greater solubility of flavonol and of the reaction Acidity.
400 W A V E L E N G T H Imp)
Figure 4. Effect of wave length of exciting light on fluorescence intensity of tin(IV)-flavonol complex in 33% dimethylformarnide
0 250
I
I
I
I
I
215
300
325
350
375
400
425
450
WAVE LENGTH ( r n 4
Figure 5. Absorption spectrum of tin(lV)-flavonol complex in 33% dimethylformamide 0.002% flavonol with 4 moles of tin(1V) for each mole of flavonal. Readings a t every 5 mp
Figure 6. Combining ratio of tin(lV) to flavonol
33 yo dimethylformamide A . Max. amt. of tin(IV), 2 pmoles in 25 ml. B . Max. amt. of tin(IV), 1 pmole in 25 ml.
products in this medium. The fluorescent readings were stable for approximately 3 hours in a 33% dimethylformamide solution, whereas a precipitate formed in a water or 3070 ethyl alcohol solution after about 10 minutes. For quantities of tin in the order of 0.15 pmole, a 10% dimethylformamide solution is sufficient. Smaller amounts of dimethylformamide result in higher fluorescence intensity readings. When stability is of minor importance, as in the ordinary procedure of qualitative analysis, water solutions are satisfactory. The establishment of a final solution with a pH 1.25 to 1.45 cannot be precisely determined when 33% dimethylformamide is used as a solvent; hence an experiment was conducted to find the optimum quantity of sulfuric acid to use with this solvent. A series of solutions of constant tin content in 33% dimethylformamide was tested mith varying amounts of 1 .ON sulfuric acid (Figure 2). This experiment showed that from 2.5 to 3.5 ml. of 1.ON sulfuric acid per 25 ml. of final solution gave reasonably constant fluorescence intensity readings. Valence State of Tin. Tin(I1) was compared with tin(1V) to determine if flavonol chelated equally well with either form of tin. The tin(I1) solution was prepared from metallic tin in 6‘4’ hydrochloric acid. The amount of tin(I1) present was determined by titration with potassium permanganate. The fluorescence of this solution on the addition of flavonol was dependent upon the tin(1V) present and was unaffected by tin(I1). Fluorescence Emission Spectrum. T h e spectrum of the tin-flavonol VOL. 29, NO. 10, OCTOBER 1957
0
1487
pH is greater than 4 or in 33% dimethylformamide if the solution is less than 0.03N in sulfuric acid. A t the acidity of the tin test less than 20 pmoles of these elements in 10 ml. does not interfere. QUALITATIVE PROCEDURE
The test may be used for the determination of tin in the qualitative scheme of analysis.
Figure 7. Variation of fluorescence intensity of tin(lV)-flavonol complex with varying amounts of flavonol
,4. 0.2 Hmole of tin(IV), 3 ml. of IN sulfuric acid in 25 ml. of 3370 dimethylformamide B . Same as -4except no tin(1V) present
fluorescence under excitation with a 365 mp source is approximately from 420 to 520 mp (Figure 3). The small inflection a t 365 to 370 mp on the graph is caused by reflected mercufy lines a t this point and is not pertinent to the fluorescent spectra. Under the acidic conditions of this test, flavonol alone produces a green fluorescence. A VCTratten C-5 filter was selected as a secondary filter for the fluorometer t o transmit the blue fluorescence of the tin-flavonol chelate and to prevent the passage of the green fluorescence of flavonol. Excitation Spectrum. The excitation spectrum for the tin-flavonol complex shows a peak at 405 mp (Figure 4). A high pressure mercury vapor lamp (G.E. H100A4) proved satisfactory for the irradiation of the sample. Absorption Spectrum. The absorption curve for a 0.002% flavonol solution which contained 2 moles of tin for each mole of flavonol is shown in Figure 5 . The peaks on the curve are a t 310, 346, and 405 mp. The relatively low absorption peak a t 405 mp is the point of maximum excitation. In concentrations of over 0.002% flavonol the absorption a t 405 mp is much more pronounced. Ratio of Tin to Flavonol. The method of Vosburgh and Cooper, as applied by Freeman and White (d), was used to determine the ratio of tin to flavonol in the chelate (Figure 6). The molecular ratio was 1 t o 1. Varying concentrations of flavonol with constant tin concentrations were tested to determine the amount of flavonol required for maximum fluorescence (Figure 7). The tests indicate 1488
ANALYTICAL CHEMISTRY
that a ratio of 3 to 8 moles of flavonol for 1 mole of tin should be present for maximum fluorescence. This ratio of flavonol to tin is considerably in excess of the stoichiometric quantity, but it is probably necessary to force most of the tin into the complex. Sensitivity. On the fluorometer the test was sensitive to 0.002 pmole of tin in 10 ml., or about 0.027 per ml. Visually about 0.27 per ml. is required for good distinction from the blank. Interferences. More than 20 pmoles of fluoride or phosphate will quench the fluorescence of a 0.1 pmole tin sample in 25 ml. Zirconium will fluoresce under the same conditions as tin and hence is a positive interference, Copper(I1) , chromium(III), cobalt (II), nickel(II), and iron(II1) do not interfere unless present in high enough concentrations to color the solutions. Arsenic(II1 or V), antimony(II1 or V), mercury(I1), lead(I1) , bismuth(II1) , cadmium(II), zinc(II), manganese(II), niobium(V), titanium(1V) , tantalum(V), and tungsten(V1) do not interfere under the conditions of the test. hfolybdenum(V1) reduces the intensity of the tin fluorescence markedly. Hydrogen peroxide partially prevents the interference of molybdenum but not sufficiently for quantitative results. Any element which precipitates in 33% dimethylformamide, which is 0.12N with sulfuric acid, will interfere with the test. Tungsten does not precipitate under the conditions of the tin test, but if the solution is made 0.3N with acid it precipitates and produces a fluorescence. Traces of aluminum(II1) and thorium(1V) produce a fluorescence with flavonol in water solutions if the
In the absence of fluoride, phosphate, and zirconium ions, the test for tin may be made directly from the solution after Group I (lead, silver, and mercury) has been removed. If fluoride, phosphate, or zirconium ions are present, the antimony and tin must be precipitated as in the Group I1 procedure. At this point in the usual scheme of analysis, antimony and tin sulfides are dissolved in concentrated hydrochloric acid. This solution is evaporated to about half its volume and becomes about 6 N in hydrochloric acid. In either of the above cases, 5 to 10 drops of the solution is used for the test. After the addition of 5 drops of 1 . O S sulfuric acid and 5 drops of 0.01% flavonol solution, the mixture is diluted to a convenient volume of 5 or 10 ml. A blank, in which 5 t o 10 drops of 6 N hydrochloric acid are substituted for the test solution, should also be prepared. On observation under ultraviolet light, a rich blue fluorescence of the sample confirms the presence of tin in contrast to the bright green fluorescence of flavonol itself in the absence of tin. This test may be performed on paper but it is not so sensitive as in solution, The procedure on paper is as follows. A piece of spot test paper is moistened with 0.01% flavonol and dried. A drop of tin(1V) or zirconium solution in 0.LY sulfuric acid dropped on the flavonol spot will produce a bright blue fluorescence when exposed to 360 m p radiation. The flavonol alone has a green fluorescence. ACKNOWLEDGMENT
The authors wish to express appreciation to ilrthur D. , Little, Inc., for partial financial support of this project. LITERATURE CITED
(1) Alford, K. C., Shapiro, L., White, C . E., ANAL. CHEM. 23, 1149 (1951). (2) Freeman, D., White, C. E., J. Am. Chem. Soc. 78,2688 (1956). (~. 3 ) Grimaldi, F. S., White. C. E., A N ~ L . CHEM.’25, 1886 (1953). (4)Liska, K., Chem. Listy. 49, 1656 (1955). (5) Luke, C. L., ANAL.CHEM.28, 1276 (1956). (6) Schubert, L., Ph.D. thesis, University of Maryland, College Park, Md., June 1954.
RECEIVEDfor review March 5, 1957. Accepted May 15, 1957.
