862
ANALYTICAL CHEMISTRY
Bessey ( 1 7 ) are used with the Beckman spectrophotometer (1.25 microgram of Fast Green, final dilution t o 0.25 ml.) the limit should be less than 0.005 microgram of chlorine; however, this assumes that both the oxidation reaction and microdiffusion remain quantitative in this ultramicro range. I n fact, preliminary trials show that, while 0.1 microgram of sodium chloride can be determined with these microcells, reagent blanks are high and the oxidation-diffusion reaction is slow and probably not quantitative. A modification of the Conway cell, allowing the use of volumes of the order of 0.01 ml., will be necessary for accurate analysis of quantities of the order of 0.1 microgram of chlorine. Specificity of Analytical Reactions for Organic Chlorine Compounds. It is not possible to identify an unknown chlorinated compound by a single chloride analysis, but two or more dechlorination reactions will make possible a calculation of reaction rates, and a partial identification. For example, the ratio of “chloride formed a t 95’ in 30 to 40 minutes” t o “chloride formed at 25’ in 60 minutes” can be calculated from Table I. It is near 1 for D D T and lindane, 2 for T D E , 5 for toxaphene, 10 for chlordan, and 15 for methoxychlor. Some chlorinated insecticides, such as aldrin and dieldrin, do not react with sodium n-propoxide, and \vi11 not interfere with analyses for other reactive insecticides. A mixture of 2 insecticides can be analyzed if their reaction rates with sodium n-propoxide differ. For example, a mixture of z micrograms of chlordan and y micrograms of D D T will yield 0.16 y) = A micrograms of sodium chloride in the 95”, (0.45 z 0.16 y) = B micrograms of 30-minute reaction, and (0.045 z sodium chloride in the 2 5 O , 60-minute reaction. Both z and y can be calculated by solving the simultaneous equations. The factors are derived by calculation from the data in Table I. Applicability to Insecticide Residues. Method C can be used to determine residues of the order of 1 to 10 p.p.m. in fresh plant leaves, and 10 t o 100 ).p.ni. in oils and fats. If preliminary treatment of the extracts TX ith concentrated sulfuric acid is included in the procedure, the sensitivity is increased by a factor of 10. Further purification of the extracts by chromatographv would not only increase t h e smsitivity. but might also make
+
+
possible the separation of a mixture of insect’icides, or of an insecticide from its partial breakdown products. LITERATURE CITED
(1) Calam, C. T., Clutterbuck, P. W., Oxford, A. E., and Raistrick, H., Biochem. J . , 41,458-63 (1947). (2) Carter, R. H., Nelson, R. H., and Gersdorff, W. A., Advances in Chem. Series, No. 1, 271-3 (1950). (3) Clutterbuck, P. IT., hlukhopadhyay, S. L., Oxford, il.E., and Raistrick, H., Biochem. J . , 34, 664 (1940). (4) Conway, E. J., ” hlicro-Diffusion Analysis and Volumetric Error,” 3rd ed., Chap. XXIV, XXIII, London, Crosby Lockwood, 1950. ( 5 ) Cristol, S.,J . Am. Chem. Soc., 67, 1494-8 (1945); 69, 338-42 (1947). (6) Davidom, B., J . Assoc. Ofic. Agr. Chemists, 33, 130-2 (1950). (7) Dudley, H. C., IND.ESG. CHEM.,ANAL.ED., 11, 259-61 (1939); Public Health Repts., 56 (19), 1021-7 (1941). (8) Gordon, H. T., AKAL.CHEM.,23, 1853 (1951). (9) Gunther, F. A , , IWD.ENG.CHEM.,h . 4 ~ ED., . 17, 149-50 (1945). (IO) Gunther, F. A., Harris, W.D., Blinn, R. C., Kolbeaen, M. J., Simon, H. S.,and Barkley, J. H., ANAL.CHEW,23, 1835 (1951). (11) Haslam, J., and Squirrell, D. C. IT.,Biochem. J . , 48, 48-50 (1951). (12) Judah, J. D., Ibid., 45,60-5 (1949). (13) King, E. J., and Bain, D. S., Ibid., 48, 51-3 (1951). (14) Kirk, P. L., “Quantitative Ultramicroanalysis,” New York. John Wiley & Sons, 1950. (15) Koenig, N. H., Kuderna, J. G., and Danish, A. A , , ANAL. CHEM.,submitted for publication. (16) LaClair, J. B., IND. ENG.CHEM.,ASAL. ED., 18, 763-6 (1946); ANAL.CHEM.,20, 241-5 (1948). (17) Lowry, 0. H., and Bessey, C. -i., J . Biol. Chem., 16, 633 (1946). (18) March, R. B., Ph.D. thesis, University of Illinois, 1949. (19) Peel, E. IV., Clark, R. H., and Xagner, E. C., IND.ENG. CHEDI.,ANAL.ED., 15, 149-51 (1943). (20) Raistrick, H., A’ature, 163, 553-4 (1949). (21) Schechter, M. S.,Pogorelskin, bI. .1.,and Haller, H. L.. ISD. ENG.CHEM.,ANAL. ED.. 19, 51-3 (1947). (22) Scott, B. A., J . SOC.Chem. Ind. (London).67, 1-2 (1948). (23) Lmhoefer, R. R., ISD. ESG. CHEW,-4s.~~. ED., 15, 383-4 (1943). (24) yswanathan, R., Biochem. .J.. 48,239-40 (1951). (25) Ton Oettingen, W.,and Sharpleas. N., J . Pharmacol. Erptl. Therap., 88, 400 (1946). RECEIVED for review August 22. 1931.
.\ccepted Fehrilary 13, 1952.
Fluorometric Determinations of Traces of Fluoride H O R i R T H. WILLARD AND CHARLES A. HORTOK’ Zniversity of Michigan, Ann irbor, Mich.
I
S 1938 Goto (6) outlined three methods for the qualitative spot-test detection of fluoride using fluorescence. Onemethod ,depended on the bleaching by fluoride of the strong yellow-green fluorescence of the zirconium-niorin complex in hydrochloric acid solution. The other fluorescence tests were based on the restoration of the violet fluorescence of salicyclic acid in acetate solution, which had been bleached by ferric or titanium ions, ,owing t o formation of fluoride complexes of these two metallic ions. Later, Bourstyn ( 1 ) proposed a quantitative method based on the effect of fluoride on the fluorescence of the aluminumPontachrome Blue-Black R sl-stems used in methods for aluminum published by him and by Reissler and White ( I I ) , but he ,did not investigate the method suggested. Okac ( 9 ) developed a viwal volumetric method for large amounts of fluoride, using aluminum chloride as the titrant and nioiin as the fluorescent indicator, but did not give the effects ,of variables in detail. l l o r e recently Feigl and Heisig ( 3 ) have published a test for fluoride depending on the quenching of the fluorescence of paper treated with aluminum 8-hydroxyquinolate. 1 Present addre-. -Ridge, T c n n
K-25 Plant, Carbide and Carbon Chemicals Co.. Oak
Many other fluorometric methods are known in which fluoride has a deleterious effect, but, none has been investigated as an approach t o the quantitative determination of traces of fluoride. This paper reports an investigation of several fluorometric systems as possible methods for the determination of traces of fluoride with greater sensitivity than is possible with the common colorinietric methods. Two satisfactory systems are discussed in detail and results on the unsatisfactory systems are reported briefly. REAGENTS AIVD APPARATUS
Water and alcohol were redistilled. -411 chemicals used were reagent grade. Commercial grade organic indicators were used unless otherwise noted. Fluoride solutions were stored in maxlined bottles. A Klett fluorometer was employed for most of the work. Corning, Lumetron, and Wratt,en filters were used in the fluorometer. ALUMIKUM-OXIKE SYSTEM
Gentry and Sherrington (4)suggested a fluorometric method for aluminum using the fluorescence of aluminum oxinate (8hydroxyquinolate) in chloroform solution. Preliminary experi-
V O L U M E 2 4 , NO. 5, M A Y 1 9 5 2
862
~~
This work was undertaken to develop a precise new instrumental method for determining extremely small amounts of fluoride. The two methods developed depend on the competitive complexing of aluminum by fluoride and organic reagents which fluoresce with aluminum. One method depends on extraction in chloroform of unreacted aluminum as the fluorescent oxinate at pH 4.7. The other is based on the decrease in fluorescence of an aluminum-morin complex measured in 50% alcohol at pH 4.9 and estimates down to 0.2 microgram of fluoride with excellent precision. Methods for determination of such minute amounts of fluoride are important in present-day water analysis and in biochemical and toxicological investigations.
merits showed that the fluorescence of chloroform extracts containing aluminum oxinate followed the modified Beer’s law applying for fluorescence for estractions a t p H 4 to 5 or about 9. The intensity of fluorescence varied indirectly with the oxine (8quinolinol) concentration and the extent of the linear range varied directlj- with the oxine concentration, Maximum fluorescence for 1 micromole of aluminum or less in 50 ml. of chloroform was obtained with an osine concentration of 0.025% and by making two %-nil. extractions a t pH 4.7. The fluorescence has a maximum intensity a t 520 to 570 mp and is best measured using Corning 5850 primary and Corning 3385 secondary filters. The presence of fluoride in the aqueous phase before estraction ties up some of the aluminum as a complex ion, reducing the amount present as oxinate and extractable with chloroform. Sensitivity to traces of fluoride was favored when using very small amounts of alunlinum and the minimum quantity of osine for masiinuni fluorescence and minimum color in the extract. Figure 1 illustrates the type of curve and range obtained under varied conditions. The method will detect 0.5 to 20 niicrogranis of fluoride using 1 micromole of aluminum. The necessity of extraction adversely affected the precision of this method. The usual ions which react with aluminum or oxine or precipitate with fluoride at pH 4.7 interfered seriously in this method. ALUMINURI-XlORIK SYSTEM
The investigation of the aluninum-morin system was sugqcsted by Okac’s (9) work, and the work of White and L o w ( 1 2 ) , Kavanagh (8), and other3 using this reagent for aluminum was also helpful. Preliminary work showed that the fluorescence of 100
90
80 W
$ 70
this system follows Beer’s law to over 60 micrograms of aluminum using 40% methanol containing 4% of saturated technical morin,. buffered a t p H 3.0 with ammonium acetate and sulfuric acid. Maximum fluorescence was observed a t 500 mp, in agreement with data given by Goto (5, 7 ) . Traces of fluoride markedly affected the fluorescence of a small amount of aluminum. Several variables must be controlled carefully for reproducible results and standards should be run with each set of unknowns. A Corning 5850 primary and a Lumetron B-530 secondary filter and a nonfluorescent 10-ml. test-tube cell were used in a N e t t fluoronietei. EFFECT OF VARIABLES ON ALUMINUM-MORIN SYSTEM
Type and Concentration of Morin. The most satisfactory morin was pure sublimed morin, recrystallized from acetic acid, supplied by T. Y. Toribara. S e s t most satisfactory was moriri supplied by T. Schuchardt, Ltd., 1,~opoldstrase4, Munchen 23, Germany. Eastman Kodak Co. technical morin, even after. purification by elementary procedures, is decidedly inferior. Quercetin, supplied by S. B. Penick and Co., is intermediate in quality as an alternate indicator. The optimum concentra,tion of niorin depends on its purity and on the amount of aluminum used as a basis in the method. The. minimum amount for masiniurn fluorescence with the fixed amount of aluminum should be used. Fcr one lot of Schuchardt morin, 1.3 mg. per 100 micrograms of aluminum gave maximum fluorescence in a total volume of 100 nil. of 50% alcohol a t p H 4.7. Type and Concentration of Alcohol. Methanol, absoluteethyl alcohol, or 95y0 ethyl alcohol (denatured with methanol) were equally satisfactory in enhancing the fluorescence of the complex. Isopropyl alcohol was not as satisfactory, and higher alcohols were not sufficiently miscible with the buffered aqueous solution used. The fluorescence increased linearly with increasing alcohol content. The concent,ration was fixed a t 5070, however, because higher concentrations introduced other complications. Concentration of Aluminum. A low aluminum content such as 27 or 54 micrograms per 100 ml. was found desirable as a basis for the I o ~ e s t ranges of fluoride, because with such small amounts it required less fluoride t o form the, AlF++ and AlFS+ ions which predominate in the region of greatest sensitivity for this method.
v)
pH and BufEer Used. Figure 2 shows the. variation of fluorescent intensity for a given amount of aluminum a t various p H values in 50y0 alcohol. Greatest sensitivity occurs a t p H 3.4, but in this region fluorescence is also. very sensitive to slight p H variations. Although light intensity is slightly less a t p H 4.9, it is much less sensitive to slight pH variations. The latter pH is recommended where more than 0.2 p.p.ni. of fluoride is expected. -4 high buffer concentration was found necessary to control the p H Rithin narrow limits.
K W
560 Y
k t E W z
50
40
30
20 10 MICROGRAMS OF FLUORIDE
Figure 1.
Variation in Sensitivity to Fluoride of Aluminum-Oxine System under Varied Conditions
-4fter the aqueous solution had been adjusted to p H 4 using a glass electrode p H meter, 8 nil. of a buffer n.ere added which was 1 Jl in acetic and monochloroacetic acids and 1 J l in the corre-
a64
ANALYTICAL CHEMISTRY
Table I. Fe(II1) Cu(I1) Ca ZrO++ Sn(I1) Cr(1II) .41
Table 11. Xa hln(I1)
28
Interfering Ions in Aluminum-Morin Fluorometric Method for Traces of Fluoride Si
so
Pb Th Bi Sr Ga
co Be Ba UO* AsOr--ab+++
25
CrOd-NO2 S-Citrate Tartrate
+
LE
sod-c10 -
R.E.
Ions Tolerated in Moderate Amounts in Aluminum-Morin Method K Li C1CIOaclodNOI-
Br-
I-
Acetate
10
COa-Chloroacetate
7 4
sponding sodium salts, for every 100 ml. of final 50% alcohol solution. The pH should be maintained a t 4.90 + 0.08, measured in 50% alcohol. If pH 3.4 is used, the value should be controlled within f0.04unit, and a 2 t o 1 mixture of dichloroacetate and monochloroacetate buffers used, adjusted to give pH 3.40 in 50% alcohol. Acetate and mono- and dichloroacetate buffers gave the most satisfactory results; phthalate and formate buffers were not as satisfactory; citrate and bisulfate buffers were unsatisfactory. 21
I
Figure 3.
Effect of Fluoride on AluminumMorin System
would not apply in this medium, the trend of consecutive formation of aluminum fluoride complexes is similar for the experimental conditions presented here. The equilibrium constants under these conditions have not been established. The empirical shape of the curve and great dependence on various factors make it advisable to run some standards along with each group of unknown samples.
IS
*
15
Table 111. Precision of Method Using Pure Sodium Fluoride Solutions
;le
(Using 54 of aluminum with morin in 100 ml. of final solution) FFFFFFFTaken. Found, Taken, Found. Taken, Found, Taken, Fcund,
z w
F-
c 9
z
6
Y
'1
Y
Y
0.20
0.22 0.15 0.20 0.25 0.18 0.45 0.52 0.50 0.55 0.78 0.70 0.72 1 00 i 05 0.98 1 03 1.00 1.25 1.15 1.32
1.50
1.52 1.48 1.50 2.03 2.00 1.98 3.00 3.03 2.95 3.08 4.05
3 2
3
4
5 PH
6
7
8
Figure 2. Variation of Aluminum-Morin Fluorescence with pH in 50% Ethyl Alcohol
Interferences. -411 ions interfere which complex or precipitate aluminum, which form strongly colored or fluorescent complexes wkh the indicator or react with it, and which precipitate fluoride in the pH range used. The ions that interfere are listed in Table I and ions which do not int,erfere in moderate amounts are given in Table 11. Only the radicals indicated (Table I) were tested; however, other forms of the same elements probably would interfere. Excessive amounts of silicate interfere, but the amounts present in hydrofluosilicic acid do not. Thus, to avoid the numerous interferences, it is advisable to separate the fluoride by Willard and Winter's (15) method for liquid samples or as hydrogen fluoride by pyrohydrolysis (10) for heavy metal fluorides. Calibration and Results. .4 typical calibration plot of fluorescence versus fluoride concentration is shown in Figure 3. It is evident that the decrease in fluorescence does not follow Beer's law. This is because more than one fluoride complex is formed by aluminum ions. As the fluoride concentration increases the AlF++, ,11F,+, AlF,, AIF4-, and perhaps hlF6-- and AIF,--complexes form consecutively. In this series, since more and more fluoride is required to tie up a certain amount of aluminum, the rapidity of the decrease in fluoresence lessens with consequent IOBS in sensitivity and precision for large amounts of fluoride. Fsrtunat,ely, the sensitivity is highest in the lowest ranges of fluoride where other previous methods have less accuracy. Brosset and Orring ( 2 ) have previously established the equilibrium constante for the formation of aluminum fluoride complexes in acidic aqueous solution. rllt'hough their constants
0,50
0.73 1.00
1.23
2.00 3.00
4.00
Y
6 00
7.00 8.00
d.Y>
5.00
3.90 4.15 5.10 4.90 5.02
10.00 12.50
Y
6.2 5.6 6.3 5.7 6.0 7 4 6 7 7.1 7.7 8.5 7.9 7.5 8.1 10.3 9.6 10.0 12.6 12.1 12.9
Y
15.0
17.5
20.0 22.5 25.0 35.0
Y
15.5 14.9 14.5 15.3 17.0 16.4 17.5 16.7 17.6 19.5 20.7 20.2 22 24 21 23 27 33 37 36
Some results with pure sodium fluoride solutions are given in Table 111 for the procedure below and in Table IV for 27 micrograms of aluminum as a basis. I n the first case the reproducibility is about f 0 . 2 microgram for 0.1 to 5 micrograms, h 0 . 4 microgram for 5 to 15 micrograms, d ~ 0 . 6microgram for 15 to 20 micrograms, and j=l microgram up to about 60 micrograms of fluoride. The precision on other samples was about half of that for pure sodium fluoride samples. PROCEDURE
Apparatus. h Klett fluorometer equipped with standard thickness Corning 5850 and Lumetron B-530 (maximum transmittance a t 530 mfi) primary and secondary filters, respectively; a 10-ml. nonfluorescent test-tube cell; and the reflecting mirror sumlied with the Klett were used. The lamphouse was cooled wvit6 an air stream. Reagents. ALUMINUMSTOCKSOLUTION. Dissolve 2.3744 grams of reagent grade potassium aluminum sulfate, KAl(SOa)r 12H20, in 1 liter of redistilled water. Dilute 200 ml. to 1 liter with redistilled water, giving a solution containing 27 micrograms of aluminum per ml.
865
V O L U M E 2 4 , NO, 5, M A Y 1 9 5 2 WATER, Use redistilled water throughout. ALCOHOL.Use redistilled, nonfluorescent, 95% reagent grade ethyl alcohol. MORIS SOLUTION.Dissolve 0.25 gram of the purest possible morin (2’,3,4’,5,7-pentahydroxyflavone) in 250 ml. of alcohol. T. Schuchardt, Ltd., Munich, provides a satisfactory product. Technical grade morin is unsatisfactory.
Table IV.
Precision of hIethod Using Pure Sodium Fluoride Solutions
(Cslng 27 y of aluminum wlth morin in 100 ml. of final solution) FFFFFFFTnLrn, Found, Taken, Found, Taken, Found Taken, Found, Y Y Y Y 5 Y Y Y
F-
0.35
0.34 0.37 0.33
2.50
2.56 2.48 2.46 2.53
7.00
7.1 6 7 7.2
15.0
15.2 14.7 15.8
050
051 0 00 0 53
310
310 3.05 3.18
800
7 8 8 4 8 1
180
170 18 3 18 7 17 2
0 io
0 68 0 71 0 66
4.00
4 12 4 03 3 9‘4
9.50
9.2 9 6 9.1
20 0
19 20 22
1.10
1.11 1.08 1.10
4.50
4.46 4.58 4.55
11.00
11.3 11.5 10.9
25.0
28 23 20
1.50
1.51 1.47 1.54
5.00
5.1 4.8
fluorescence. This is masked in stronger solutions by the deep yelloiv color of the oxinate solutions. Less than 200 micrograms of flugride did not appreciably affect the fluorescence of the oxinate formed from 50 micrograms of zirconium in either 601vent. Thorium-Oxine Systems. The strong color of this complex makes it necessary t o use solutions containing less than O.Olyo oxine in chloroform or 70 to 95% alcohol, to obtain a fluorescence that follows Beer’s law over a small range of thorium ion concentration. The fluorescence is rather weak even a t the optimum p H of 4.7. The change in fluorescence of 25 or 50 micrograms of thorium on addition of 20 to 150 micrograms of fluoride was slight and other systems showed much greater sensitivity. Thorium-1-Amino-4-hydroxyanthraquinone System. The procedure recommended by White and Lowe (13) was used to Etudy the effect of fluoride. Erratic results were obtained in the presence of fluoride, probably due to the effect of the thorium fluoride formed on the stability of the fluorescent colloidal lake. Attempts to stabilize the colloid by addition of various protective colloids or bv addition of alcohol all failed. I
5 3
4 1
’
3 0
p H BUFFER(for final p H 4.90 in 50% alcohol). RIix 94.48 grams of monochloroacetic acid, 57.5 ml. of glacial acetic acid, and 40.5 grams of sodium hydroxide slowly into about 250 ml. of water and make up t o 500 ml. SODIUM FLUORIDE SOLUTION.Dissolve 0.4524 gram of sodium fluoride in 1 liter of water. Dilute 10 ml. t o 200 ml. as needed. Solution contains 10 micrograms of fluoride per ml. Store in polyethylene bottles. BLANKSOLUTION. Mix 2 ml. of morin solution, 48 nil. of alcohol, and 8 ml. of buffer solution and dilute t o 100 ml. with water. REAGENT STOCKhhXTURE. Mix 2 volumes of morin solution, 48 volumes of alcohol, 8 volumes of buffer, and 2 volumes of dilute aluminum solution. PROCEDURE. After separating fluoride from any interferences, adjust the p H to 4 using a p H meter and make up to a definite volume. Pipet an aliquot of 35 ml. or less containing not over 20 micrograms of fluoride into a 100-ml. volumetric flask. Add exactly 60.0 ml. of reagent stock mixture. Mix well and adjust to 100 ml. with water. Mix again and allow to stand 20 minutes. At about the same time prepare a similar flask using distilled t a t e r jnatead of fluoride, and three or four standards in a similar fashion. Rinse out the 10-ml. test-tube cell with the aluminum standard, then fill to 0.25 inch (1.25 cm.) of the top. Wipe off the exterior of the cell with an alcohol-moistened cloth and place in the fluorometer. Note the position of the small dot on the cell and always insert in the same position. Adjust the fluorescent reading to 260 on the potentiometer by adjustment of the slit diaphragm. Rinse out the cell twice with 50% alcohol. Wash the cell with the blank solution, fill, and read fluorescence. Rinse, fill, and read fluorescence for each of the standards and unknowns. Plot the standards on a curve and read the fluoride content of the unknowns. The blank gives a measure of fluoride in the reagents and other variables causing low readings. UNSATISFACTORY SYSTEMS
Aluminum-Pontachrome Blue-Black R System. The procedure given by Weissler and White(l1)for the determination of aluminum Tvas employed. The effect of fluoride was studied by the addition of known amounts of sodium fluoride to a fixed amount of aluminum. The fluorescence unexpectedly increased as fluoride was increased t o 40 micrograms, using 20 micrograms of alumfnuni as a standard, then leveled off, and finally decreased slowly, as shown in Figure 4. This irregular behavior eliminated this method from further consideration. Zirconium-Oxine System. Dilute solutions of zirconium oxinate in alcohol or chloroform show a fairly strong yellow
I
I
I
I
1
I
I
I
I
I20
180
240
300
360
60 MICROGRAMS OF FLUORIDE
Figure 4.
Effect on Aluminum-Pontachrome Fluorescence pH 5.0
Boron-Benzoin System. One trial of the effect of fluoride on Weissler and White’s (14) fluorometric method for boron using benzoin showed that this system is not sensitive to traces of fluoride. ACKSOWLEDGbIENT
The authors appreciate the supply of pure sublimed morin provided by Taft Y. Toribara, University of Rochester. This work was done under contract with the Technical Command, Chemical Corps. LITERATURE CITED
Bourstyn, H., Bull. soc. chim. (France), 8, 540 (1941). Brosset, C., and Orring, J., Collection Czeckoslou. Chem. Communs., 10, 177-81 (1938). Feigl, F., and Heisig, G. B., AnaI. Chim. Acta, 3, 361-4 (1949). Gentry, C. H. R., and Sherrington, L. G., Analyst, 71, 432-8 (1946). Goto, H., J . Chem. Soc. J a p a n , 59, 457-64 (1938). Goto, H., Toholzu I m p . Cniu. Sci. Repts., Ser. ( l ) , 29, 269-71 (1940). Ibid., pp. 461-9. Kavanagh, F., IND.EXG.CHEM.,ANAL.ED.,13, 108-11 (1941). Okac, A., Collection Czechoslov. Chem. Communs., 10, 177-81 (1938). Rodden, C. J., Ed., “Analytical Chemistry of the ,\Ianhattan Project,” pp. 729-31. Sew York, John Wiley & Sons, 1950. Weissler, 8., and White, C. E., IND.EXG.CHEM.,ANAL.ED., 18, 530-4 (1946). White, C. E., and Lon-e, C. S.,Ibid., 12, 229-31 (1940). Ibid., 13, 809 (1941). White, C. E., Keissler, A . , and Busker, D., Ibid., 19, 802-5 (1947). Willard, H. H., and Winter, 0. B., Ibid., 5 , 7-10 (1933). RECEIVED for review December 21, 1951. Accepted March 6, 1932. Presented a t t h e Southwide Chemical Conference, Atlanta, Ga., October 16 t o 18, 1950. Abstracted from a doctoral thesis presented b y Charles A . Horton t o t h e University of Xichigan.
