6th Annual Summer Symposinm-Less Familiar Elements
Analytical Chemistry of Micro Quantities of Beryllium TAFT Y. TORIBARA AND RUTH E. SHERMAN Division of Pharmacology and Toxicology, Department of Radiation Biology, School of Medicine and Dentistry, University of Rochester, Rochester, N . Y . Because of the very toxic nature of beryllium, submicrogram quantities of the element are of biological importance. Colorimetric, fluorimetric, and spectrographic methods used to determine small quantities of beryllium are subject to interferences by other elements, often necessitating a separation scheme to isolate the element before measurement. The fluorimetric method based on the interaction of beryllium with purified morin is the most sensitive chemical determination tested and compares favorably in sensitivity with the spectrographic method. The separation of beryllium from bone proved to be most difficult because of the large quantity of calcium phosphate. Using a combination of steps consisting of precipitation, electrolysis with a mercury cathode, absorption on an ion exchange resin, and complexing with acetylacetone, it was possible to isolate completely the smallest measurable quantity of beryllium from all biological samples. The radioisotope beryllium-7 was employed in the determination of the efficiency of each step of the separation scheme.
T
HE problems in the determination of beryllium may be divided into three categories: methods of estimation, removal of organic material, and removal of interfering substances. I n the subsequent discussion, the various procedures available in each category are presented without regard to any special requirements. These will vary with the needs of the analyst. The authors’ interest is in the detection of beryllium in biological material and, because beryllium is so very toxic (16), special emphasis is given the analysis of minute samples of the cation by very sensitive methods of detection. I n discussing the sensitivities of the various methods, it is important to bear in mind what the figures mean in terms of total beryllium taken for analysis. Thus, a sample for colorimetric or fluorimetric analysis is usually made up to a minimum volume of 10 ml. by the time the reagent, buffer, and stabilizer are added. I n the spectrographic method, the crater on the arc may hold anywhere from 0.1 to 0.5 ml., but this quantity is taken from a sample which has been concentrated to a specified volume (usually 1 ml.),
it has been demonstrated positively that complexes are formed by the interaction bettyeen the beryllium and the dye ( 2 3 ) . Depending upon the ratio of dye to beryllium, the complex may exhibit molecular ratios of 1 dye to 1 beryllium or 2 dye to 1 beryllium. Under the conditions employed in the colorimetric determination, the colored compound is the 2 dye to 1 beryllium complex. -4lkannin is prepared from an extraction of alkanet root, while naphthazarin is a synthetic product (21). Based on the extensive study made on these two dyes, a very reliable colorimetric method has been developed with a sensitivity as great as that of any other colorimetric method yet reported. The most sensitive chemical test for beryllium is the fluorescence developed by its interaction with morin at pH’s greater than 11, and considerable interest (10, 18, 15, 24) has been shown in this reagent for estimating small quantities of the element. Commercial morin is of variable and doubtful purity, but a purification scheme has been developed (6). With the pure product the sensitivity of the method is increased nearly tenfold ( 2 4 ) . A serious difficulty universally encountered is the rapid decrease in fluorescence after the reagents are mixed. An alkaline stannite has been used (10, 12) to stabilize the fluorescence. 4 s originally described, this stannite solution must be freshly prepared each day. A more permanent stabilizing solution may be prepared by mixing stannite and hydrazine dihydrochloride and storing under nitrogen. A number of reducing agents and bivalent cations have been investigated as stabilizers without success. The mechanism of fading and stabilization remains obscure. Most of the commercial fluorimeters use a mercury vapor lamp giving very intense ultraviolet radiation as the exciting source for the fluorescence. It was felt that this radiation aggravated the problem of fading. After some preliminary studies on the excitation frequencies, i t was found that an automobile headlight lamp excited sufficient fluorescence to energize a system consisting of two barrier-layer type photocells and a Leeds and Northrup No. 2430-D (0.00045 pa. per mm.) galvanometer. The light
Table I.
Accuracy in the Range 1 to 10 Micrograms
Reagent Quiniaarin-2-sulfonic acid ( 7 ) Aurintricarboxylic acid (11) Alkannin (Sp) Naphthaaarin (82) Eriochrome cyanin R (20)
Error, y 0.1 0.2 0.06 0.06 0.05
METHODS OF ESTIMATION
Table 11.
There are three general methods for the determination of microgram quantities of beryllium: colorimetric, fluorimetric, and spectrographic. Many colorimetric methods are reported, for almost any dye which may be used for aluminum may be adapted for use with beryllium. A number of these colorimetric procedures have been investigated in this laboratory, and Table I shows the accuracy obtainable in several of these methods. The minimum useful range for colorimetric methods is in the 1- to 10-microgram region, although much smaller quantities may be detected. The dyes are nonspecific and the interferences in the alkannin method, shown in Table 11, are typical. The reagents alkannin and naphthazarin both have the same chromophoric group and react similarity with beryllium. The conditions for color formation have been studied thoroughly ( 2 2 ) , and
Interferences in Alkannin Method (22) (4.667 Be)
Ion Ca Zn .4I -Mg cu Fe Li
1Mg. 0
0.1 M g . 0
Ppt.
Ppt.
+ + Ppt.
Ppt. 0
+
+ Ppt. +0 0 0
0 0
0. 30interference. P p t . Formation of visible precipitate. Increase in colorimetric reading. Decrease in colorimetric reading.
+. -.
1594
0.01 JIg. 0
+ t+ + 0
V O L U M E 25, NO. 11, NOVEMBER 1 9 5 3
1595
entered through the bottom and was measured from two sides of a quartz cell 25 mni. square and 50 mm. high containing 10 ml. of bolution. Figure 1 shows a working curve obtained under the most sensitive conditions (limitations are imposed by the size of the blank). On the working curve the blank reading has been adjusted to a minimum on the galvanometer. The actual blank reading obtained against distilled water was 45 mm., while a
steps which are applicable to the Qeparation of beryllium from bone: 1. 2. 3. 4. 5. 6.
,4sh to remove organic matter. Dissolve and remove calcium or phosphate. Collect beryllium. Remove cations by mercury cathode or oxine extraction. Separate beryllium from alkali and alkaline earth metals. Collect beryllium for measurement. ASHIYG
Table 111. Comparison of Sensitivities Coloriinetric ( $ 8 ) Fluorimetric Spectrographic On arc Spark ( 1 7 ) ( 2 ml.)
0 1 gdnic
JIicrogram 30% 0.004 + 20% 0.2
+
0 , 0 0 2 i. 507, ( 8 ) 0.0006 (18) 0.004 j= 50%
Table 1%'. Recovery of Berjllium-7 Added to Yarious Salts Salt HC1. 1 nil.
SHL1, 1
gram
Be Carrier Added, y 20
co
20 20
Ignited a t 750O for 17 hours
103
Evaporated with infrared lamp and ignited a t 500' for 1 hour
100.3
20 20 20
SA9HP04 1 gram XaC1, 1 gram 5 S HCI
Recovery,
Treatment Evaporated t o dryness n i t h infrared Ignited a t 750' for 17 hours Ignited a t 750° for 17 hours after driving off XHdC1 carefully Evaporated t o dryness with infrared Ignited a t 750' for 17 hours
Carrierfree
102
102 99 2
material may he removed by wet or dry ashing. Dry ashing seems to lend itself to routine work more readily and is uqually favored. It has been reported ( 6 )that the direct ashingof urine a t temperatures a. loiv as 500" C. resulted in losses as high as 8iyOof the beryllium when a platinum dish was used. -4nother source ( 1 4 ) reported losses up to 90% when the sample was treated first a t a low temperature follotved by a few- minutes in a muffle a t 900" C. Such losses were not observed by Underwood and Seuman ( 2 2 ) in their standardization of a solution of beryllium metal dissolved in hydrochloric acid. They found that exactly the same results were obtained in evaporating such a solution to dryness and igniting to the oxide whether sulfuric acid had been added or omitted.
99.7
98.4
w n p l e containing 0 . 0 1 ~of beryllium in the 10-ml. final volume gave a reading of 32 mm. above the blank. The spectrographic method (2, 6 , 14, 17, 18) will determine the qniallest quantities of beryllium. but transposed into quantities which must be actually present in a sample, the fluorimetric method is comparable. Table I11 shows a comparison of the sensitivities of the three methods of analysis. The quantities for the colorimetric and fluorimetric methods are actual amounts present in the final 10-ml. volume. I n the arc spectrographic method, the quantities shown must actually be in the arc. This quantity is usually taken from a minimum final volume of 1 nil. ( 6 , l d j 18), although a method has been developed ( 2 ) where the entire sample is evaporated to dlyness under carefully regulated conditions and scraped into the crater. -411 three methods-colorimetric, fluorimetric, and spectrographic-are subject to interferences, and it is necessary to employ some kind of qeparation scheme. I n dust samples ( 1 4 ) a separation scheme is necessary for the spectrographic method in order to obtain a more uniform matrix for excitation rather than to eliminate spectral interferences. The separation scheme also serves to extend the usefulness of the quantitative methods by concentrating the element. Since the interferences are many for the colorimetric and fluorimetric methods, the separation schemes essentially isolate the beryllium. The fluorimetric method using morin has the advantage over many of the colorimetric methods, in that aluminum does not interfere and need not be separated. Biological materials such a? urine, blood, soft tissues, and bone may, in some cases, contain micro quantities of beryllium. Determination of beryllium in such samples has been the subject of a number of papers (6,Q. 10, 12, 14,17, d i ) , each of which contains a slightly different separation scheme. Many of the methods ( 6 , 14, 17, 24) are applicable only to the determination in urine or dust samples. Because of the large quantity of calcium phosphate present, bone poses much more of a problem than does other biological material. A study has been made to determine the completeness of each step in a separation scheme (13), and this will serve as a basis for the present discussion. -411 the separation schemes involve all or part of the folloming
lo0
% 0.004
0.008
0.012
0.016
0.020
0
MICROGRAMS B E R Y L L I U M in IO m l
Figure 1.
Fluorimetric Response of Beryllium-Morin Solutions
Ashing conditions have been carefully studied (19), with the conclusion that no losses by volatilization occur when an aqueous solution of beryllium is evaporated and ignited a t temperatures as high as 750" C. Microgram quantities of beryllium to which the radioisotope beryllium-7 was added were mixed with 1-gram quantities of the main salt constituents of urine. These solutions were evaporated to dryness in platinum dishes under an infrared lamp and then placed in a muffle furnace a t 750" C. The samples were removed from the platinum by evaporating with hydrofluoric acid to fumes of sulfuric acid and diluting with water. Measurement of the activity with a dipping tube counter gave the results in Table IV. Longer heating a t 750' C. with periodic measurements showed no loss of beryllium activity over an 8-day period. Samples of urine were ignited under conditions for which losses had been previously reported ( 6 ) , with the results shown in Table V. Samples n-hich had been ignited a t 7.50" C. could be removed from the platinum dish by the hydrofluoric evaporation, but the beryllium could not be absorbed on a cation exchange column nor extracted into benzene n ith acetylacetone. -4fter the salts had been heated qtrongly with concentrated sulfuric acid for several minutes it was possible to absorb the beryllium from solution with a ration exchange resin and to extract it with acetylacetone. It is believed that losses previouqly attributed to volatilization n-ere poor recoveries caused by conversion of the beryllium t o a ielatively insoluhle oxide. Samples of bonr which had been
ANALYTICAL CHEMISTRY
1596 Table V.
Recovery of Beryllium from Urine
(50-ml. urine samples dry-ashed in platinum dishes) Temperature, O C.
500
Average
Be Added, Recovery, Method of Solution Concd. heated strongly
Concd. HCI 750'' H F and HzSOi e r a p . t o fumes of SUIfunc 7500 H F and H2S04 evap. t o fumes of SUIfuric a Samples could not be extracted with acetylacetone. 500
Y
%
4.03
95
4.03 4.03
96 92
8.06
102
ashed a t 600' C. gave no trouble. To preclude the possibility that the beryllium might behave differently after having been ingested in an animal, the radioisotope beryllium-7 with and without carrier was injected into rabbits. Complete recoveries were obtained after ashing the urine, soft tissues, and bones of such animals. For small samples of urine (50 ml.), tissues, and filter papers containing dust, a wetrashing procedure using nitric and sulfuric acids ( 2 , 6, 1 4 ) or nitric and perchloric acids (14, 17) is often used. For large quantities of urine, perchloric acid should not be used because of the large quantities of potassium present. For large quantities a semidry technique using nitric acid only has been described (19). I n this procedure 500 ml. of urine are evaporated almost to dryness after the addition of 25 ml. of concentrated nitric acid. Toward the end of the evaporation the sam le starts to foam and puffs of smoke appear. The reaction is se6-sustaining and may be spectacular, varying from a small amount of scintillation to a strong blue continuous flame with much volatile matter evolved. The volatile material does not carry any beryllium with it. Any remaining organic matter is oxidized by further evaporations with 10-ml. portions of concentrated nitric acid. This is a rapid procedure, as a sample may be completely ashed in 1.5 hours. ISOLATION OF BERYLLIUM
When the beryllium content is extremely low, it is necessary to take a large sample. Following the removal of organic matter, the beryllium is separated from the bulk salts by some gathering procedure. Most commonly some collecting precipitate is formed in the presence of a suitable cation by raising the pH. In the case of a bone solution, raising the pH precipitates a large quantity of calcium phoqphate, which carries down most of the beryllium. The bulk of either the calcium ion or the phosphate ion must be removed prior to the gathering step. Precipitates formed in strong acid solution do not carry any beryllium with them. Calcium is best removed as the sulfate from acid solution (10, 19) to which alcohol has been added to decrease the solubility. Calcium has also been immobilized by complexing with ethylenediaminetetraacetate ( 2 ) , although this scheme would not be suitable if a collecting precipitate was to be used, as the collecting cation would be complesed and form no precipitate. Phosphate has been removed from acid solution as the bismuth salt ( 1 2 ) ; the excess bismuth ion must be removed as the sulfide. Phosphate may be removed quantitatively from an acid solution by passing the solution through a column of Dowex 50 resin converted to the bismuth form. However, part of the beryllium is also retained by the column, and the procedure is worthless as a separation. Among the collecting precipitates used are calcium phosphate (2, 6, 19), ferric phosphate ( I O , 12), aluminum phosphate and aluminum hydroxide (IO, 24), and manganous phosphate ( 1 7 ) . It is reported that the slightest noticeable precipitate of calcium phosphate ( 2 ) or manganous phosphate ( 1 7 ) collects the beryllium quantitatively. This is true, provided there is sufficient beryllium present. When 0.1 microgram of beryllium was added to 10 grams of bone ash (19), a maximum of 70% of the beryllium was gathered by calcium phosphate. With 1.3 micrograms of
beryllium added to 10 grams of bone ash, 95% of the beryllium was collected under the same conditions. The most quantitative collecting procedure of those tried is to use a cation exchange column in the hydrogen form. A bone ash sample is dissolved in dilute hydrochloric acid, and most of the calcium is removed by precipitation as the sulfate. The filtrate is diluted to give an acidity of approximately 0.5 S and passed through a Dowex 50 column in the hydrogen form. If sufficient resin is used to absorb all polyvalent cations preqent. quantities of beryllium as small as 10-10 gram (carrier-free radioisotope) are absorbed completely. The cations may then be stripped from the column with 5 N hydrochloric acid. The effect of complexing anions depends upon the pH, as shown in Table VI. The lower pH is approximately that of a 1-liter dilution of 10 grams of bone ash, from which the calcium has been removed by precipitation with sulfuric acid. The higher pH is that at which oxine and acetylacetone extractions are usually carried out. At pH 5.0 tetrasodium ethylenediaminetetraacetate is a very poor complexer of beryllium, while phosphate is a very effective one. It is probably the HPO,-- ion which is effective in complexing the beryllium, as there is no interference to absorption of beryllium (in the presence of phosphate) a t pH 1.8. The mercury cathode is very convenient for removing many cations (10, 12, IS, 19). Oxine (8-quinolinol) is used to precipitate iron, aluminum (14, 241, and other metals from acetatebuffered solutions. A simpler technique is to extract the metal oxinates with chloroform ( I S ) . This reagent is especially useful for the colorimetric methods in which aluminum interferes. As a h a 1 step in the separation scheme it is necessary to collect the beryllium in the desired volume for analysis. Aluminum. which does not interfere in the fluorimetric method of analysip using morin, is the most used gathering agent (10, 12, 24). A more rapid and absolutely quantitative method for collecting the smallest measurable amounts of beryllium (carrier-free isotope) is an extraction of the acetylacetonate into benzene (19). Beiyllium forms a well-characterized and very stable compound with acetylacetone ( 1 ) . This compound was first applied to the separation of milligram quantities (a),then to microgram quantitieq ( I S ) , and finally to the preparation of carrier-free beryllium-7 ( 3 ) . I n carrier-free quantities evaporation of a benzene solution of the acetylacetonate to dryness resulted in significant losses of activity. Thenoyltrifluoroacetone forms a similar compound with beryllium (4), which is nonvolatile and has been used for the preparation of carrier-free beryllium-7. The acetylacetonate i q readily decomposed by acid, as evidenced by the complete backextraction of beryllium into the aqueous phase \Then the benzene
Table VI.
Absorption of Carrier-Free Beryllium-? by Dowex 50 ("4) Be Absorbed
PH 1.8 1.8 2.0 5.0 5.0 5.0
%
Additions per Liter 0.057 mole Hap04 0.057 mole NalHPOd a n d HCI 4 ml. acetylacetone 0.057 mole Hap04 and acetate buffer 4 ml. acetylacetone 2 .O grams tetrasodium ethylenediaminetecraacetate
105 100 79 2.8 15.:
97
Table VII. Extraction of Carrier-Free Beryllium-? in the Presence of 6.1 Grams of Phosphate at pH 4.5 Extraction
Minutes Stirred with Bcetylacetone
1. l a t 2nd a n d 3rd
40 5
:;}95
2.
1st
3.
1st
4.
1st
30 25 40 30 90 45
80 1 7 ) ~ ~ 82 3 13.lj95 4 86 5 13.5} loo
2nd 2nd 2nd
Extracted, 5%
V O L U M E 25, N O . 11, N O V E M B E R 1 9 5 3 solution is shaken with 5 N hydrochloric acid for a few minutes. The thenoyltrifluoroacetonate is very stable, and the backextraction of beryllium into the aqueous phase is complete only ,tfter 80 hours when concentrated hydrochloric acid is used ( 4 ) . The acetylacetonate lends itself more readily to a rapid separation -( heme. Conditions for using acetylacetone vary. For separation in 111 esenre of other complexing anions, it is desirable to use as low a pH as possible. If the p H is too low, beryllium acetylacetonate \ r i l l not be formed. At the higher pH’s the greater ionization oi the acetylacetone favors its solubility in the aqueous phase and increases the difficulty of extraction of beryllium acetylacetonate into the organic phase. One procedure (8)recommends a pH betneen 3 and 4, another (IS) recommends 4.5, while a p H of 6 IT as used in the preparation of carrier-free beryllium-7 ( 3 ) . All these procedures used a benzene solution of acetylacetone to extract the beryllium from the aqueous phase. In the preparation of the radioberyllium, an extraction time of 2 hours was used. -4more thorough study of the extraction procedure (19) showed that a p H between 4 and 5 was more suitable than the lower range. Further it was shown that stirring the aqueous phase with acetylacetone for 5 minutes before addition of the benzene was more effective than using a benzene solution of acetylacetone. Stirring trith a propeller tilted to force the liquid downward gave much more reproducible results than did manual shaking, and a total extraction time of 15 minutes was sufficient. The extraction time is a function of the anions present in 9o1ution. With a small quantity of phosphate (50 to 100 mg. as PO4) present, complete separation of beryllium is obtained with one 15-minute extraction (5 minutes u i t h acetylacetone plus 10 more minutes after adding benzene). Table VI1 shows some result? obtained when none of the phosphate from 10 grams of bone ash was removed. The back-extraction of the beryllium into hydrochloric acid takes a small quantity of acetylacetone, which is sufficient to interfere in the final measurement. The acetylacetone may be removed by evaporating the arid solution and igniting gently (below 600” C., or heating with a burner just enough to burn the organic material). The beryllium is then taken up in a small amount of hydrochloric acid. The acetylacetone step removes the alkaline earths and the alkalies. Iron and aluminum also form complex acetylacetonates, which are soluble in benzene. Iron is removable in a mercury cathode. iiluminum need not be removed if the morin method is used. It may be removed with oxine. DI SCU s SIOY The choice of a method depends upon the quantity of beiyllium and other ions present in the sample. If the quantity of pample is not limited, a separation scheme may iqolate sufficient quantities of beryllium to allow any method to be employed. Of the colorimetric methods tried, the alkannin or naphthazarin method ( 2 2 ) has been found most reliable. The fluorimetric method using purified morin is more sensitive and aluminum does not interfere. As fluorimetry approaches the spectrographic method in sensitivity, its use should become more general. Purified morin may be obtained easily in several steps, starting with morin obtained from T. Schuchardt, Ltd., Leopoldstrasse 4, Munchen 23, Germany. The determination of beryllium may be carried out on a large qample of urine with the semidry-ashing procedure (19). As the volume has been reduced greatly during ashing, it is unnecessary to use a preliminary gathering step Electrolysis to remove most of the cations, followed by the use of acetylacetone to complete the separation, makes a relatively simple procedure. Seven 500-ml. samples of urine each containing 1.3 micrograms of beryllium and radioisotope beryllium-7 were carried through this separation scheme with recoverages ranging from 97.5 to 101.7% (average 99.9%). Two samples containing carrier-free radioisotope beryllium-7 gave recoveries of 96.6 and 100.3%.
1597 For bone, dry ashing a t 600” C. is most rapid. After calcium removal as the sulfate, the most quantitative gathering procedure is to pass the acid solution through a Dow-ex 50 column in the acid form. If the bone contains more than 0.17 of beryllium per gram of ash, the metal may be gathered with a calcium phosphate precipitate obtained by raising the pH of the solution, from which most of the calcium has been removed as the sulfate. -4fter solution in sulfuric acid, the separation of the beryllium is completed by electrolysis using a mercury cathode followed by the acetylacetone extraction. Recoveries between 95 and 98% have been obtained for quantities of beryllium as small as the rarrier-free radioisotope (less than gram). For soft tissues such as the liver, kidney, and spleen, wet ashing n i t h nitric acid and finishing with perchloric acid are very satisfactory. Using the same separation procedure as for ashed urine, complete recoveries of carrier-free radioisotope have been obtained. C O l CLUSION
Adequate methods are available for the determination of cstremely small quantities of beryllium, although all suffer from interferences. Rapid procedures have been developed to isolate the beryllium. A little more work remains to be done to develop the ultimate sensitivity of the fluorimetric method using morin, the limitation being imposed by the size of the blank of the purified reagent. The ideal reagent would be specific for beryllium, one which in itself did not fluoresce but produced an appreciable stable fluorescence in its interaction with beryllium. The ultimate sensitivity for such a reagent would be limited only by the electronic devices available for meaiuring the light emitted. LITERATURE C I T E I )
Arch, A,, and Young, R. C., “Inorganic Syntheses,” \-oL 11, ed. by UT.C. Fernelius, p. 17, New York, McGraw-Hill Book Co., 1946. Barnes, E. C., Piros, W.E., Bryson, T. C., and Wiener, G. W., ANAL.CHEM..21. 1281 (1949). Bolomey, R. ii., and Broido, A., dtomic Energy Commisbion, ONRL 196 (December 1948). Bolomey, R. A., and Wish, L., J . Am. Chem. SOC.,7 2 , 4483 .
I
(1950).
Bonner, J. F., J r . , Atomic Energy Commission, R e p t . UR-111 (April 1950). Cholak, J., and Hubbard, D. M . , . i l s t ~ CHEM., . 20, 73 (1948). Cucci, M. W., Neuman, W. F., and Mulryan, B. J., Ibid., 21, 1358 (1949). ~~
I
Flagg, J. F., unpublished work. Hyslop, F., Palmes, E. D., .klford, W. C., .Monaco, -1.R., and Fairhall, L. T., Natl. Inst. Health, Bull. 181 (1943). Klemperer, F. W.,and Martin, A. P., ANAL.CHEM.,22, 828 (1950).
Kosel, G. E., and Neuman, W.F.. Ibid., 2 2 , 936 (1950). Laitinen, H. A., and Kivalo, P., Ibid.,24, 1467 (1952). Neuman, W. F., and Kosel, G. E., Atomic Energy Commission, Rept. UR-35 (June 1948). Peterson, G. E., Welford, G. d.,and Harley, J. H., ~ A L CHEM.,22,1197 (1950). Sandell, E. B., IXD. ENG.CHEX,ANAL.ED., 12, 674 (1940). Scott, J. K., “Pneumoconiosis,” ed. by A. J. Vorwald, p . 369, New York, P. B. Hoeber, Inc., 1950. Smith, R. G.,Boyle, A. J., Frederick, W. G., and Zak, B., ANaL. CHEM.,24,406 (1952). Steadman, L. T., personal communication. Toribara, T. Y . , and Chen. P. S., J r . , ANAL. CHEM.,24, 539 (1952).
Toribara, T. Y . , and Cucci, M. W., unpublished work. Toribara, T. Y . , and Underwood, A. L., ANAL.CHEM.,21, 1362 (1949).
Underwood, A. L., and Neuman, W. F., Ibid., 21, 1348 (1949). Underwood, A. L., Toribara, T. Y . , and Neuman, W. F., J. A m . Chem. SOC.,72,5597 (1950).
Welford, G., and Harley, J., Am. Ind. Hug. Assoc. Quart., 13, 4 (1952). RECEIVED for review J u l y 13, 1 9 3 . Accepted August 24, 1953. Based on work performed under contract with t h e United States Atomic Energy Commission a t t h e University of Rochester Atomic Energy Project, Rochester, N. Y .
.
