x i h e of K R , fits the synthetic values of 11- - ATon i t h a mean deviation of only 0.0067,. Actually, the precision of thc interferometer readings is so good t h a t the factor which limits the accuracy in these measurements is the q n t h e t i c value of N - IV,.
LITERATURE CITED
( 1 1 ..\dams. L. H.. J . Ani. Chem. Sac. 37. 1181’ ( 1915j.
(2) Adams, L. H., J . Wash. i l c a d . Sci. 5 , 267 (1915). (3) Bacarella, 8. L., Finch, d., Grun-
Kald, E., J . Phys. Chevz. 60, 573 (1956). (4) Barth, W.,2. wiss. Phot. 24, 145 (1926). ( 5 ) Bauer, K., “Physical,,llethods of Organic Chemistry, A. Keissberger, 2nd ed., p. 253, Interscience, Kew Tork, 1949. (6) Candler, C., “Modern Interferometers,” Hilger & Watts, London, 1951. ( 7 ) Evans, R. M., “Introduction to Color,” Wiley, Sew York, 1948. (8) Faust, R C., Marrinan, H. J., B?zt. J . A p p l . Phys. 6 , 351 (1955). (9) Karagunis, G., Hawkinson, A,, Danikohler, G., Z . p h y s i k . Chem. A151, 433 (1930). (10) Kruis, -1., Zbid., B34, 13 (1936 ,
(11) Macy, R., J . Srn. Chem. SOC.49,30TO (\ -1927). - - . I
(12) Marshall, H. P., Grunwald, E., Ibid., 76, 2000 (1954). (13) Williams, W. E., “Applications of
Interferometry,” 4th ed., AIethuen, London. 1950. (14) Tates, H.‘ W., -Optical Works RIanager, Hilger & Watts, Ltd., London, private communication.
RECEIVED for reviex May 5, 1956. .4ccepted September 25,1956. Work carried out under contract between the Office of Naval Research and Florida State University. Reproduction in whole or in part is permitted for any purpose of the L-nited Ststes Government.
Separation and Determination of Microgram Amounts of Molybdenum GLENN R. WATERBURY and CLARK E. BRICKERI University of California, Los Alarnos Scientific Laboratory, los Alarnos,
,The separation and determination of microgram amounts of molybdenum in plutonium alloys and other samples in the presence of iron are accomplished by extracting the molybdenum into hexone (4-methyl-2-pentanone) from a 6M hydrochloric acid-0.4M hydrofluoric acid medium. This i s followed b y back-extraction into water, removal of iron by precipitation from the aqueous extract as hydrated iron(ll1) oxide, and colorimetric estimation of the molybdenum in the aqueous extract using chloranilic acid for the color reagent. For 32 determinations o f 19 to 96 y of molybdenum in sobtions containing various foreign ions, an average value for the molybdenum found of 99.870, with a standard deviation of 1.670, was obtained. Of 29 foreign metals investigated, only tin, tungsten, and bismuth interfered seriously with t h e determination.
B
*E of the need foi an analytical method for the determination of 0.01 to lYG molybdenum in plutonium and plutonium alloys, possible methods foi the determination of molybdenum were imestigated. For these loir concentiations especially TT here only a smdl aniount of sample is uvnilnble, :t quantitative separation and a sensitive nietliod for the deterniination are necessary. Although the precipitation E C it
Present address, Chemistry Department, Princeton University, Princeton, N . J.
N.M.
and recovery of microgiam amounts of molybdenum using a-benzoinoxime have been reported (16), extraction separations are usually more readily adaptable t o microgram amounts, and in these cases colorimetric methods offer the sensitivity required. Consequently, the present investigition has been limited to extiaction methods for separation couplrd n-ith coloriniptric methods for the estimation of molybdenum. Since the first mention of the ether extraction of molybdenum from a hydrochloric acid solution by Pechard in 1892 (16), methods for the separation of molybdenum by extraction have been discussed by many authors. In general, these methods involve the extraction of molybdenum cupfeirate by chloroform (1. 7 , 20) or ethyl nitrate ($I), the extraction of molybdenuni thiocyanate by various organic solvents (8,14, 19. %), the extraction of molybdenum dithiol by h t y l or amyl acetate (9, 28). and the extraction of molybdenum from mineral acids into organic media (3, 15, 19, 24). Of these methods, the evtraction of molybdenum from mineral acids n i t h nn organic solvent utilizes the most simple system, and the investigation perfoimed 11)Seliclou- and Diamond (15) indicates that this separation can lie highlv efficient, depending on the solvent and acidities used. From their study, the extraction of molybdenum from G to 7 M hydrochloric acid by hexone (-1methyl-2-pentanone) was shon n to be about 96% efficient; the efficiency of the L I S U ~ Idiethyl ctlier extrartion iq les..
Tiibutyl phosphate extracts niolylxlenum more efficiently than does hexone. but the number of other elements coextracted is larger. The colorimetric determination of molybdenum hss been reported using thiosulfate (5), tannic acid (W3), hydrogen peroxide (279, hydrogen sulfide (1, d), potassium ethyl xanthate (I?), phenylhydrazine ( I O ) , dithiol ( 2 ) , and thiocyanate-tiniII) chloride (11, 12). Of these methods, the thiocyanate-tin(11) chloride color procedure is the most widely used. Although molybdate is f i b t e r l as an interference in the d r t w
lii
STIRRER CHUCK
DIA’rlETEi HOLE
L
J
15rnrn.
Figure 1. Hollow stirrer extractor with test tube VOL. 2 9 , NO. I , JANUARY 1957
129
6-mm TUEING,
Figure 2. Liquid transfer apparatus in test tube with stopcock
niination of zirconium using chloranilic acid (6, I S ) , no procedure based upon the molybdenum-chloranilic acid color was found in the literature. I n the present work the separation of molybdenum from 6M hydrochloiic acid solution by extraction into hexone and the colorimetric estimation of molybdenum using chloranilic acid are described. Although this method as developed specifically for the dettwiiination of molybdenum in plutonium alloys, the procedure applies equally n-ell to most samples that can he dissolved to yield a final solution GJI in hydrochloric acid. APPARATUS AND REAGENTS
DISHES,platinum, n ith lip. HEAT LAMP, infrared, n-ith socket, switch, extension cord, and ring stand attachments. HOTPLATE, electric. SPECTROPHOTOMETER, Beckiiiaii Model DU, with Corex cells of 1-cm. light path. EXTRACTOR, h o l h tube type, RS shown in Figure 1. LIQUID TRASSFER APPAR.4TUS, a5 shown in Figure 2 . PIPET,polystyrene plastic, 5 nil. n it11 0.5-ml. graduations. HYDROCHLORIC ACID, concentrated, reagent grade. HYDROFLUORIC ACID, concentrated, reagent grade. HYDROFLUORIC ACID, 4M. Dilute 16.26 grams of concentrated hvdrofluoric acid 100 ml. with water. "Store this solution in a polyethylene bottle. PERCHLORIC ACID, 70 t o 72y0,reagent grade. PERCHLORIC ACID, 2M. Dilute 166.6 nil. of 70 to 72y0 perchloric acid to 1 liter with water. 130
ANALYTICAL CHEMISTRY
CHLORASILIC ACID, Eastman piwctical grade. Purify this reagcnt as follows ( 2 6 ) : Dissolve 8 grams of the chloranilic acid in 1 liter of boiling water and filter while hot. Extract the filtrate twice a t about 50" C. with 200-ml. portions of benzene; discard the organic Iayerq. Cool the aqueous phase in ice water. Filter the red crystals that separate and nash with three 10-m1. portions of water. Dry these crystals at 115" C. Prepare the reagent solution by dissolving 90 mg. of the purified crystals in 100.0 ml. of water. HEXONE(4 - METHYL - 2 - PESTAKONE), Castman white label, or methyl isobutyl hetone, Matheson, Coleman and Bell. HEXONE, equilibrated with 6-34 hydrochloric acid and 0.464 hydrofluoric acid. Add approximately 100 ml. of hexone to a solution containing 20 ml. of water, 25 ml. of concentrated hydrochloric acid, and 5 ml. of 4M hydrofluoric acid. Stir this mixture for 10 t o 15 minutes, and then allow the two phases to separate completely before using the upper layer for extractions of molybdenum. Alnays use freshly equilibrated hexone, and discard any of this solvent remaining a t the end of each day. SCDIUMHYDROXIDE, 0.2-11. Dissolve 4 grams of sodium hydroxide pellets in 500 ml. water. Store this solution in a polyethylene bottle. STASDARDMOLYBDEKUJI SOLUTIOX. Dissolve a weighed amount of pure molybdenum metal in aqua regia and evaporate the resulting solution to dryness. Add a felv milliliters of water and about 1 ml. of concentrated ammonia to the residue anti again evaporate to diyncss Transfer the residue with n-ater to a volumetric flask and dilute to volume. If a more dilute molybtlenum solution is needed, dilute a measured volume of the original solution to a linou-n volume n-ith n ater. DISSOLUTION OF SAMPLE
Plutonium alloys are dissolved by tieating a known weight of alloy in a centrifuge tube with water, followed by the dropwise addition of concentrated hydrochloric acid. The acid is added s l o ~ l yto prex-ent the rapid evolution of hydrogen or loss of sample by frothing or spraying. After the evolution of hydrogen ceases upon further addition of hydrochloric acid, the centrifuge tube and its contents are warmed to ensure the complete reaction of any metal. The reaction mixture is centrifuged, the supernatant liquid is transferred to a volumetric flask, and any residue is placed in a platinum dish. For simple plutonium-molybdenum alloys, no residue will remain, and it is necessary only to dilute the hydrochloric acid solution to volume before taking an aliquot for analysis. If a residue is found, it is treated with hydrofluoric, nitric, and sulfuric acids, and evaporated until strong fumes of sulfur trioxide appear. The resulting mixture, if completely soluble in water, is added to the solution.in the volumetric flask. If the residue is not completely soluble in mater, the mixture is
centrifuged; tlie soluble portion is added t o the volumetric flask, and any residue is placed in a gold crucible. This residue is fused with a mixture of 0.1 to 0.5 grain of sodium hydroxide and 0.2 to 1.0 gram of sodium nitrate, depending on the amount of residue present. The fused melt is dissolved in water, acidified rvith hydrochloric acid, and added to the solution in the volumetric flask. The combined solutions in the volumetric flask are diluted to volume with water. A n aliquot from the solution of the dissolved sample is taken for analysis by the recommended procedure. If the aliquot needed for the analysis is larger than 4 ml., if the acid concentration in the dissolved sample is not known accurately, or if there is an appreciable amount of nitrate present, the aliquot is treated n-ith a few drops of concentrated sulfuric acid in a platinum dish and evaporated to dryness. The residue is then dissolved in 4 ml. of water plus 5 ml. of concentrated hydrochloric acid and 1 ml. of 4M hydrofluoric acid and transferred to a 25 X 150 mm. test tube. This solution is extracted and analyzed for molybdenum as described in the recommended procedure.
If the sample cannot be dissolved by the procedure given, it will probably require an unusual treatment, and no effort is made here to give directions for dissolving all types of samples. Honever. any disolution procedure may be used, provided the resulting solution can be conxerted to chloride, perchlorate, or sulfate prior to the application of the reconimended procedure. RECOMMENDED PROCEDURE
Transfer tlie dissolved sample containing 100 y or less of molybdenum to a 25 X 150 mm. test tube. Add sufficient u-ater t o make the volume of tlie sample 4 ml., and then add 5 ml. of concentrated hydrochloric acid and 1 ml. of 4 M hydrofluoric acid. Add 10 ml. of equilibrated hexone, and stir the mixture for 5 minute? with the hollow stirrer. After the layers have separated completely, use the liquid transfer apparatus to remove the organic layer as quantitatively as possible without sucking off any of tlie aqueous phase. Add about 5 ml. of hexone, and without stirring remove the organic layer again as completely as possible. Extract the aqueous layer for 5 minutes each TTith two additional 5-ml. portions of equilibrated hexone, and remove the organic layer as completely as possible each time. Add 5 to 8 ml. of water to the combined organic phase in the test tube of the liquid transfer apparatus, and stir the mixture for 3 to 5 minutes with the hollow stirrer. After the layers have separated, drain off the bottom layer into a platinum dish. Extract the organic phase two more times with 5-ml. portions of 17-ater, and combine the aqueous extracts in the platinum dish. Evaporate the combined aqueous extracts to dryness under a heat lamp. Add 5 or 6 drops of concentrated acid and about 1 ml. of water to the residue
aiid evaporate to dryness again under the heat lamp. Moisten the residue with 2 drops of concentrated hydrochloric acid, and t,hen transfer the residue quantitatively with water and 5 ml. of 0.2.11 sodium hydroxide to a 25-m1. volumetric flask. Warm the solution in the volumetric flask under the heat lamp for about 30 minutes t o coagulate any hydrous oxides that may have precipitated in the alkaline solution. Cool the coagulated solution and dilute t o ~~olullle. Filter the diluted solution through a dry Kliatmaii S o . 42, or equivalent grade, filter paper into a dry container. Transfer 20.0 ml. of the filtrate t o a plntinuni dish and evaporate to dryness under tlie heat lamp. For samples containing more than 100 y of molybdenum, take a smaller aliquot of this filtrate. Dissolve the residue in about 3 ml. of water, and acidify the solution by adding hydrochloric acid cautiously to minimize loss by spraying. Evaporate the acidified solution to dryness again under the heat lamp. Transfer the residue quantitatively with 2-26 perchloric acid to a 10-ml. volumetric flask and dilute to T-olume with this acid. Remove at least 3 ml. of this solution to a n absorption cell, and measure the absorbance (L41) a t 350 mp against 2 M perchloric acid. Add 3.00 ml. of chloranilic acid solution to the solution remaining in the 10-ml. volumetric flask and dilute to volume n-ith the solution in the absorption cell. hleasure the absorbance (A2) of this solution against, a solution containing 3.00 ml. of chloranilic acid solution and sufficient 21Tf perchloric acid to make 10 nil. Calculate the absorbance due to the molybdenum (AM,) by multiplying the second measured absorbance by 10/7 and subtracting t,he first measured absorbance. I.:str:wt and carry through the entire procedure 10 ml. of 6 V hydrochloric acid wliich is also 0.4M in hydrofluoric acid. Subtract the absorbance ( A b l a n k ) of this l h n k from the abqorbance of the saiiiple to obtain the corrected absorbance (-4Y ~ ~ for ~ ~the~ mol!-ljtienmn . ) in the saiiiple. Prepare a calibration curve for molybdenum by evaporating knon-n amounts of the standard molybdenum solution plus 2 t o 3 drops of concentrated hydrochloric acid to dryness in platinum dishes. Transfer the molybtlenum residues quantitatively with 2-11 perchloric acid to lo-ml. volumetric flasks containing 3.00 nil. of chloranilic acid solution and dilute to volume with the 2-11 acid. Measure the absorbance of these solutions :igainst a solution prepwed by diluting 3.00 ml. of chloranilic acid solut'ioii t o 10 nil. rrith 2d1 perchloric acid. To cnlculate the amount of molybdenum. divide the corrected absorbance AM^^^^^ ) for the molybdenuni from the sariiple by the absorbance per microgram of molybdenum obtained from the calibration curve. Because only 20 ml. of the filtrate from the 25-m1. volunietric flask were taken for t,he molybdenum analysis. multiply t,he micrograms of molyhdenum found by 1.25 to obtain the tot:+] amount of molybdenum in the
Table I.
Extraction Efficiency Data for Molybdenum
;\lo Taken, Mg. 0.040
Efficiency, Cxtracta11t Diethyl ether
67 /C
82
81 80 Hexoiic
1.00
Hesoiie
1.50
Hesone
0 .?I0
Aqueous back-
94 97 '3 9 93 95 '33 9 *?I '33 100
1,OO
Aqueous back-
08
0.50
Hesoiie
extraction extraction
Table II. Effect of Hydrofluoric Acid on Extraction of Molybdenum 110 Hydrofluoric Absorbance Taken, Acid, at 360 -/ 31 lllp 65 28 0 0 0 723 0-1 0 729 t57 66 0 0 0 613 0 4 0 629
sample. If an aliquot smaller than 20 ml. was taken, multiply the micrograms of molybdenum found from the cor~ ~ the ~ . ) rected absorbance ( A Y ~ ~ by appropriate factor to obtain the total amount of molybdenum in the sample. EXPERIMENTAL RESULTS AND DISCUSSION
Extraction of Molybdenum. I n the extensive study by XelidoIT- and Diamond (f?) of the extraction of molybtienum f i om iliineral arid wlutions, *everal experimental variahles. including oiganic soh ent, acid, acid strength, and temperature, were investigated. They reported that hexone was 96 to 97% efficient in extracting molybdenum fiom G V hydrochloric acid at room temperatuie. A verification of theii results as obtnined using the hollon -stirrer estractor described in this report n ith a 3- to 5-minute eytraction peiiod. X single evtraction of 10 nil. of solution G.11 in hydrochloric acid and 0.4M in hydrofluoric acid v i t h an eqiial volume of heyone n a s 93 to 97% efficient (Table I \ , I\ liereas an efficiency of 537, n a s found nhen diethyl ether n as the extractant. Bis(2-chloroethyl) ether and n-butyl ether were much less efficient as extractants. When the chloranilic acid color method for molybdenum IT as applied directly to the residue obtained after evapointion of the organic layer, er-
ratic results were frequently obtained because of slight charring of the organic fraction upon evaporation. By back extraction of the molybdenum into n-ater and evaporation of the aqueous layer, the inconsistent results were eliminated. One back extraction of the liexone layer using n-ater is 98 to 100% efficient in extracting t'he niolybdenum (Table I). Hy making three hexone extractions and three aqueous back extractions, an over-all efficiency of about 1007, n-as obtained (Table V1j. Because of the estensive invcstigatmion by Selidow and Diamond and the w r y satisfactory rstraction efficiency obtained with hexone, no further study of extractants, acid strength, or other esperinientid variables pertaining to the extraction was made. Xlt,hough tlie pi'esence of hydi,ofluoric acid in the aqueous extraction medium is not always necmsary, some samples that contain tantalum, tungsten, and other metals were analyzed for molybdenum, and it was essential to havc hydrofluoric acid in these solutions to prevent precipitation of the foreign elements. Because hytirofluoI,ic acitl did not interfere n-ith the estraction of molybdenum (Table 11) and hnd :i beneficial effect with samples containing some foreign metals, the use of 0.431 hydrofluoric acid and 636 hydrochloric acid for all samples is reconimendrtl. Colorimetric Determination of Molybdenum Using Chloranilic Acid. Althoiigh molybdate is listed as an interference in the determination of zirconium using chloranilic acid (6, 13). the iise of chloranilic acid as n color reagent for molybdenum was not foiiiitl in the literature. The niolybdenmn-chloranilate color is developed simply and is very sensitive. Whereas most other color methods described fur molybdenum involve extraction, reduction, or other operations in the rolor development, t,hc molybdenumchloranilate color is developed directly and immediately upon adding a measured amount of the reagent t o the molybdenum solution. Only the acidity need be controlled to gk-e a reproducible color thnt is stable for sevrral hours. To select t,he proper waye length for measuring the molybdeiium-chlorariilate complex in 1.4M perchloric acid, tlie absorbance of a solution 1.29 X 10-3N in chloranilic acid and the absorlmice of a similar solution contniiiing 65 y of molybdenum in 10 nil. and the same chloranilic acid conrrntration were determined using 1.5-21 perchloric acid as a reference solution; these curves are shown in Figure 3. Because of the strong absorbance of the chloranilic acid reagent belovi 350 mp, the concentration of the reagent would need to he controlled very stringently if measurements of the molybdeniim-
vot. 29,
NO. I , JANUARY 1957
0
131
chloranilic acid complex were made a t less than 350 mp. Although the absorbance of the molybdenum-chloranilic acid color is less at 350 mp than a t lower wave lengths, this slightly reduced sensitivity a t 360 mp is offset by making it unnecessary to reproduce the chloranilic acid concentration exactly from sample to sample. By measuring the ahsorbance of the molybdenum-chloranilic acid color a t 350 mp, good linearity with molybdenum concentration and excellent reproducibility from day t o day were obtained. The molar absorptivity of this complex is 1.04 x 104. The effect of chloranilic acid concentration on the molybdenum-chloranilic acid complex was studied by measuring the absorbance of solutions 0.5 x to 2.0 X 10-3M in chloranilic acid, 4.5 X 10-4M in molybdenum, and 1.4W in perchloric acid using reagent blank solutions as reference liquids. The absorbance is highly dependent upon the chloranilic acid concentration (Figure 4),and no limiting value of the abqorbance is reached in the concentration range studied. The limited solubility of chloranilic acid in I .4M acid does not permit the use of more concentrated solutions. I n addition. the absorbance of the molybdenum-chlorinate complev is less dependent on chloranilic acid for concentrations above 1 X 10-4J1 than for loiver strengths. T o achieve good sensitivity for molybdenum. low depeiidence of the absorbance on chloranilic acid concentration, and no danger of precipitation, a chloranilic acid concentration of 1.3 X is recommended. The continuous variation method n as used to determine the nature of the iiiolybdenum-chloranilic acid complex. S i n e solutions in 1.4M perchloric acid n ere prepared in IT hich the molybdenum concentration and the chloranilic acid concentration varied from 0.0 to -1.0 X 10-4M, but the total concentration of these two substances \vas constant a t 4.0 X 10-4M. The absorbances of these solutions were measured using reagent blank solutions for reference a t 350 mp. A plot of the absorbances as a function of molybdenum and chloranilic acid concentrations (Figure 5 ) passed thiough one maximum w l u e n hen the molybdenum and chloranilic acid concentrations were each equal to 2.0 X These results indicate a 1 to 1 iatio of molybdenum to chloranilic acid in the colored complex. The effect of acid strength on the color 11as deteimined by measuring the absorbance of solutions 0 to 3 . 5 X in perchloric acid and 1.29 X 10-3X in chloranilic acid containing 38.4 y of molybdenum in 10 ml. These data (Table 111) show that the ahsorbance of the complex increases 1~1th
132
ANALYTICAL CHEMISTRY
1.7
I
I
I
I
1.5
-
13
MOLYBDENUM CYCORANILATE
I 1 W 0
Z
2
09
U
0 u)
$
07
05
.,
03
0 .I 320
I
I
I
I
I
I
I
I
I
I
340
360
380
400
420
440
460
480
500
520
WAVE LENGTH, MILLIMICRONS
Figure 3.
Absorption curves for chloranilic acid and molybdenum-chloranilate
07
I
I
I
I
I
I
I
I
1
I
-
0.6 -
CHLORANILIC ACID CONCENTRATION,
Figure 4. Absorbance acid concentration
Mx
of molybdenum-chloranilate as
decreasing acid concentPation n ith a mayimum absorbance a t about 0.0iM acidity. A somenhat similar effect was found by Frost-Jones and Yardley (6) in their study of the sirconiumchloranilic acid color. Hon ever, the lower acidities also enhance the interference caused by other ions. The 1.451 acid strength recommended in this procedure is a compromise to minimize interferences while maintaining a relatively high sensitivity. Using the recommended acid and chloranilic acid concentrations, the color obtained is reproducible from day
io4
a function of chloranilic
to day nnd stable for several hoiur. Hon ever, a small increase in absorbance with time of standing is observed. I s shown in Table IV, an average increase of 2.5y0over the original reading was found for six solutions containing 53 to 207 y of molybdenum that nere alloned to stand for 15 hours. The absorbance of the molybtlenumchloranilate complex is affected by temperature changes. Three solutions containing 2.70 nig. of chloranilic acid and approximately 43 y of molybdenum in 10 ml. were allowed t o remain in the spectrophotometer cell compartment,
0.800 -
I
0.700
A'
Table IV. Effect of Time on Molybdenum-Chloranilic Acid Color
Molybdenum Taken, Y
53 4 63 2 94 0 128 1
0.600
160 1
207 0
w 0.500 0
-4bsohmce
-4bsoib-
After
Inimediately
15 hours
0 580 0 687
0 590 0 710 1 C38 1 425 1 800 2 31
1 022 1 392 1 740
2 25
ance Increase, % 17
Av.
z a
3 3 15
2 4 3 4
2 7 2.5
m
5
0.400
v)
m
a
0.30 0
0.2 0 0
0.100 ILOR
llLl
CENTRATI N,
M
x
io4
I 1
0 4.0
1.0
2.0
3.0
2.0
3.0 1.0
MOLYBDENUM CONCENTRATION, M Figure
x
0.0 lo4
5. Plot of continuous variation data for molybdenum-chloranilate
and readings n-ere taken a t known intervals. In addition, the three solutions were warmed to 60" to 70" C. and then read. A decrease in absorbance of about 1Sy0 was observed n hen the solutions were warmed to GO" t o 70" C.; the absorbance decreased by only 27, after being warmed by standing 12 minutes in the unthermostated cell compartment of the spectrophotometer. Obviously, for reproducible results. absorbance readings should be made within 1 hour after preparing the solutions, and the solutions should not be permitted t o be narmed above room temperature. K h e n the color is developed using the conditions described, the absorbance is reproducible within 1% (Table V), and a n absorbance of 0.0109 is obtained per microgram of molybdenum in 10 ml. of solution. Although the absorbances in Table V were measured on different days using different solutions, approximately the same absorbance per microgram of molybdenum n-as obtained. However, it is recommended that known molybdenum samples or standards be analyzed a t the same time unknonn samples are determined to eliminate possible discrepancies caused by variation in the solutions, temperature, or other experimental conditions.
Table 111. Effect of Acid Strength on Molybdenum-Chloranilic Acid Color
.4cidity, -11 3 5 2 8 2 1 14 0 7
Absorb-
ance 0 134 0 207 0 318 0 460 0 583
Acidity, AbsorbJi ance 0 0 0 0 0
X5 I4
07 035 000
0.609 0 618 0 634 0 624 0 517
Interferences. Xlthoi:gli the chloiaiiilic acid color method for niolybdenum is sensitive, it is not highly selective. Thamer and T'oigt (26) and FrostJones and Yardley (6) reported that hafnium, uranium(IS-), thorium, tin(I1) and tin (IV) , titanium ( IT'), iron (111) , tungsten(VI), antimony(III), andmolybdenum(V1) formed colored complexes n i t h chloranilic acid in addition to zirconium. I n a study of the effect of 29 elements on the color reaction, some of the above interferences nere verified, and the number of metals investigated n as expanded. Measured amounts of solutions of the foreign elements and of molybdenum were evaporated to dryness in platinum dishes and dissolved in 2 M perchloric acid. The solutions n ere transferred
to 10-nil. volunictric flasks, and 3 n i l . of chloranilic acid solution and suficient 2-11 perchloric acid t o make 10 ml. were added. The absorbances of these solutions were measured a t 350 inp against reagent blanks. The residues remaining from the evaporation of some of the foreign ion solutions n-ere not coiiipletely soluble in 231 perchloiic acid, and negative int,erferences resulted; other foreign nietals enhanced the color. Iron, zirconium. uranyl, bismuth! tin, dichromate, vanadium, and tungsten caused serious interference, and copper, titanium, cadniiuni, manganese( II), magnesium, chromium (1111, nickel, strontium, lead, xrsenic, thalliuni, silver, aluminurn, 111ercury, zinc, cerium(II1) and ceiiuni(IY), rutlieniuni, neodymium, and co1)alt caused no intrrference n.hen prcsent in equal amounts hy weight to the molybdenum. Jl-lien t,he ratio by w i g h t of the foreign inctal to molybdenum ~ v s sincreased to 23 to 1! interference to varying degree resulted also from t,it,anium, silwr. lead; copper, aluniinum, mercuyy. zinc, arsenic, tantalum, and thxlliuni. S o effect on the color was caused by chloride or sulfate ions. The effect of the foreign metals that caused some interference in the color reaction n-:is determined in the entire procedure. lleasured amounts of solutions containing the foreign elcment and molybdenum n-ere e v a p o r a t d to dryness in platinum dishes, and the residues were dissolved in 10 nil. of GJ1 li~-clrochloricacid containing 0.431 hydrofi uoric acid. Tlie rcsulting solutions were taken through the reeommended procetlure. The extraction 011eration elini-inated the interferelice caused hy wveral of the elements n-ith the color reaction. S o intrrfereiice rcsultcd for weight ratios of the foilon-ing nietals to niolybtlenuni of 100 to 1 : iron. zinc, zirconium, 1 1 1 ~ tonium, silver, copper. lead, arsenic, mercury, t~lialliuni, aluminum, chrumiuni, uranium, and titanium. Tlie int,erference caused by large amounts of vanadiuni n m eliminated Lvheu the ratio of vxiwrlium to molybdenum \\-:IS VOL. 29, NO. 1, JANUARY 1957
e
133
reduced to 10 to 1, but even small amounts of bismuth, tin, and tuiiosten interfered with the procedure. Initially, attempts were made to determine molybdenum by extracting a 6-44 hydrochloric acid solution of the sample with hexone, evaporating the extract to dryness, and determining the molybdenum in the residue colorimetrically n i t h chloranilic acid. The interference of several ions, notably iron(III), made modifications necessary in this simple extraction procedure Several unsuccessful attempts Li-ere made to eliminate the iron interference, including reduction of the iron with sulfurous acid, complexing the iron !\ itli fluoride or phosphate, nieasurement of the color a t two wave lengths and correcting for the iron interference, selective back extraction of the iron, and back extraction of the iron and molybdenum with sodium hydroxide solution follo~ved by filtration of the iron. Of these unsuccessful methods, the back extraction with sodium hydroxide and subsequent precipitation of the hydrated iron(II1) oxide was the most promising. Analytical results for 10 to 80 y of molybdenum in the presence of 0.04 to 1.0 mg. of iron nere from 7 to 18% high, presumably because of incomplete precipitation of the iron in the presence of fluoride ion.
T a b l e V. R e p r o d u c i b i l i t y of Molybdenum-Chloranilic Acid Color
l\folybdenum Taken, ”i
19 22 22 33
38 44
57 RG
rlbsorb-
ance 0 204 0 212 0 243 0 239 0 244 0 241 0 245 0 242 0.418 0 419 0.628
Absorbance,’ y
Rlo
0 0106 0 0110 0 0109 0 0107 0 0109 0 0108 0 0110 0 0108 0 0109 0 0109 0.0109 0.628 0 0109 AV. 0 0109 Std. dev. 0 0001
By back-eutracting the organic phase n it11 water, the iron and molybdenum
ale quantitatively removed from the orqanic phase. The fluoride ion may be eliminated then by evaporation of the aqueous extract to dryness, followed by 3, hydrochloric acid treatment and a second evaporation to dryness as described in the recommended procedure. The precipitation of iron is complete when the fluoride-free solution of the residue is treated nith dilute 134
ANALYTICAL CHEMISTRY
sodium hydroxide. Tlie basic solution is cooled, diluted to volume, and mixed prior to filtration of the iron to eliminate the need for mashing the hydrated iron oxide precipitate and the possibility of peptizing the precipitate.
Table VI. Analytical Results for Molybdenum in Known Solutions 310 Foreign 310 Taken, hletal Added, Found, cc ,c blg. Y
19.22 38.44
38.44 57. 66 76,88 96,lO 96,lO 38.44
102.4 99.0 101.4 101.2 99.3 101 0 98 6 99.7 98.9 97.6 98.4 97.1 97.7 98.7 97.2 99.1 101.2 102,9 102.4 101.6 100.0 101.4 99.4 100.4 101.7 98.4 98 5 99.3
Pu, 17 PU, 33 Pu, 50 cu, 4 38.44 As, 4 Zn, 4 v, 0 5 Cr, 0 . 5 Ti, 4 38 44 U, 40 u, 20 u, 12 Bg, Pb, Hg; 4 nig. each 99.4 TI, Al; 4 mg. each 100.2 40.00 PT a 98.3 PT“ 100.3 Av. 99.8 Av. Dev. 1.3 Std. Dev. 1.6 ‘Solutions containing 0.1 mg each of Zr, Ce, Nd and Ta; 1.0 mg. of Fei 0.2 nlg. of Ru and 3.0 nig. of Co.
Following the evaporation of the aliquot of the filtrate to dryness, the varying amount of residual base in the residue must be neutralized to prevent variations in the acidity of the final eolution, As stated under the discussion of the color reaction, the acidity must be carefully controlled in the development of the molybdenum-chloranilic acid color. Consequently, the residue is treated with a small amount of water and hydrochloric acid and evaporated to dryness. As stated, the hydrochloric acid shoidd he added carefully to avoid loss by spray. A slight turbidity or discoloration occasionally remains in the final 2115 perchloric acid solution prior to the addition of the chloranilic acid. By measuring the absorbance
of this 2.11 perchloric acid solution be fore and after the addition of the chloranilic acid solution, as described in the recommended procedure, a correction for the absorbance of the turbidity or discoloration may be mad?. Reliability. Because no standard samples of plutonium-molybdenum nllops were available, the reliability of the method was lmsed upon analytical results obtained for knoirn solutions of molybdenum and foreign metals. The k n o n n solutions uere made 6M in hydrochloric acid and 0.4M in hydiofluoric acid, and the molybdenum w 5 extracted and determined colorimetricnlly using the recommended procedure. The absorbances obtained for molybdenum follon ing the entire e.;traction-colorimetric procedure were compared with the absorbances of known amounts of molybdenum which were taken through the color development procedure only (Table V), and the per cent molybdenum found was computed from these relative nbsorbances. Data for 32 representative determinations of molybdenum (Table VI) show an average for the molybdenum found of 99.8% with a standard deviation of 1.6%. The recommended procedure has been used for the analysis of samples of plutonium alloys and for solutions containing a wide variety of metallic ions. The reproducibility of this method is within 3YG for multiplicate determinations of samples containing over 10 to 15 7 of molybdenum. A single determination requires about 6 or 7 hours. but 10 determinations may be performed simultaneously in about 2 days. LITERATURE CITED
(1) Bertrand. D.. Bull. .
I
SOC.
chim. France
6,1676’(1939). (2) Bickford, c. F.i Jones, w.s.?Keen% J. S.,J . Am. Pharm. ASSOC., Sci. Ed. 37,255 (1948). (3) Blair, 4.A., J . Am. Chem. SOC.30, 1229 (1908). (4) . . Burton, J. I.. Ind. Ena. Chem. 19. 406 (i927). ’ (5) Falciola, P., Ann. chim. app2. 17, 261 (1927). (6) Frbst-Jones, R. E. U., Yardley, J. T., Analyst 77, 468 (1952). (7) Gibson, hx.3 Atomic Energy Research Establishment, Harm-ell, England, Rept. No. AERE C/M 64 (May 1950). (8) Grimaldi, F. s., Wells, R. C., IND. ESG. CHEM.,AKAL.ED. 15, 315 (1943). (9) Hamenee, J. H., Analyst 65, 152 (1940). (10) Hauptmann, H., Balconi, bl., 2. anorg. u . allgem. Chem. 214, 38 (1933). (11) Hiskey, c. F,, ~ ~ ~y. ~ l v .~J, . ~ Am. Chem. SOC.62, 1565 (1940). (12) Hurd, L. C., Allen, H. 0.f I N D . ExG. CHEX.,ANAL.ED.7, 396 (1935). Reagents (13) Johnson, Tv.c,, for Metals,” p. 29, Chemical Publishing Co., Sew York, 1955.
h
~
S.,Beckwith, R. S., J. SOC. Chem. Ind. (London) 67, 374
( 1 4 ) Kapron, AI., Hehman, P.
(20) Piper, C.
(1945). (15) Selidow, I., Diamond, R. M., J . Phys. Chem. 5 9 , 710-18 (1955). (16) Sichols, RI. L., Rogers, L. H., IXD. E ~ G CHEX, . ANAL. ED. 16, 137 (1044). (1;) Pavelka, F., Loghi, A . , Xikrochernie z w . Mikrochinz. Acta 31, 138 (1943). (18)Pechard, E., Conipt. rend. 114, 173 (1 892). (19) Per&, D. D., S e w ZeaZand J . Sci. Technol. 28A, 183 (1946).
(1948). (21) Robinson, W. O., Soil Sci. 66, 317 (1948). (22) Sandell, E. R., “Colorimetric De-
I,., 1x0. ENG. CHEXl., ANAL. ED. 17, 5 i 3
termination of Traces of Metals,” Vol. 111, 2d ed., pp. 453-68, Interscience, New York, 1950. (23) Steiner, O., 2. anal. Chem. 81, 389 (1930). (24) Swift, E. H., J . .4m. Chenz. SOC.46, 2375 (1924).
(25) Thamer, 13. J., Voigt, A. F., Ibicl., 73, 3197 (1951). (26) Thamer, B.J., Voigt, A . F., J . Phys. Chem. 56, 227 (1952). (27) Weissler. A , . IND. ESG. CIHXI.,. ~ A L . ED. 17. 645 (1945). (28) Wells, J. ’E., Pemberton, R., d n d y s l 72, 185 (1947).
RECEIVEDfor review October 8, 1056. . Accepted October 25, 1956. Work done under the auspices of the U. S. -1toniic Energy Commission.
Factors Influencing Validity and Confidence Limits
of Pantothenic Acid Estimation MicrobiologicaI Assay with Lactobacillus Casei MIRIAM F. CLARKE Department of Physiological Chemisfry, The Woman’s Medical College of Pennsylvania, Philadelphia, Pa.
Earlier work had demonstrated a linear relationship between the logarithm of pantothenate per tube and the amount of acid formed upon incubation with 1. casei, provided glucose was increased in the medium to a level that would eliminate i t as a limiting factor. Now a change in the media from peptone to enzymehydrolyzed casein yields a straight line but with a much greater slope. Factors affecting the slope of this logarithmic dose-response curve were examined critically: glucose content of the medium, size and method of preparation of the inoculum. Most favorable conditions for valid assays were found when 3% glucose and heavier inoculum (from enriched medium) were employed and when dosage corresponded to 0.125 to 0.25 y o f calcium pantothenate. Under these conditions, the slope of the doseresponse curves was between 13 and 18, and assays of 2 X 2 or 2 X 3 design, using 16 or 18 tubes, yielded confidence limits of 98 to 102%;.
S
on the original microbiological assay (19), in nhich L. casei n-as eniployed for the quantitative determination of pantothenic acid, suggested that the medium could be improved by the addition of one or more growth-stimulating substances present in rice polishings concentrate ( 6 ) as well as by a n increase in glucose and buffer content (5, 27). Y o r e recent n ork has s h o m that the (hired improvement in the medium for TUDICS
this organism could be attained by incipasing the peptone, or replacing the peptone, yeast extract, and acid-hgdrolyzed casein by a charcoal-treated enzymatic hydrolyzate of casein together with the usual vitamin, purine, and pyrimidine components, and n i t h appropriate increases in glucose and buffer (4, 16, 23). As the casein preparation mnde n-ith enzymes offered such marked improvement over the nitrogen sources in the earlier media, i t became unnecessary, for practical assay purposes, to resort to concentrates of natural products to provide the necessary adjuvants to the medium. Moreover, use of the enzyme-hydrolyzed caqein in media containing pantothenate hut lacking one of several other vitamins had provided n basis for satisfactory assays (23). K h e n used for riboflavin assayq, this medium n i t h added glucose had alloned a long, steep doseresponse curve and a small experimental error which compared very favorably n i t h other media used for riboflavin
(4). Preliminary experiments with the Roberts-Snell medium n ith graded doses of pantothenate indicated that the charcoal treatment of the casein preparation varied somen h a t in the degree of removal of pantothenic acid or of other substances capable of enhancing bacterial growth. High titrations were occasionally encountered in response to zero dose (blanks). Low “ceiling” or maximum acid output sometimes occurred \then preparations n-ere employed n hich yielded satisfactorily low blank titrations. The
size of tlic inoculum and the methotl used in its prepamtion had n. considerable effect upon the results obtained. The culture itself seemed to be providing significant amounts of growth stimulating substances but, of course, increases of pantothenate from this source rvere undesirable. Experiments n-ere therefore first directed ton-arc1 modifications of the inoculum. Clianges in the amount of glucose n-ere tried to see whether the higher maximum output of acid allo1Ted by 3 7 , glucose ~ o u l dgive as satisfacbory results with pantothenic acid assays as had heen the case with riboflavin. 1Iodifications n-ere sought which would yield a long dose-response curve of proved linearity and steep slope, and a reasonably m a l l experimental error. PROCEDURE
The Roberts and Snell (63) medium was the basis for these experiments. It contained, in the 10 ml. in each test tube: enzymatic casein preparation (23), 4 ml. (==200 mg. of casein, prepared from vitamin test casein, Xutritional Biochemicals Corp.); salt solution C (23), 0.2 ml.; glucose, 200 mg.; cystine. 1 mg.; sodium acetate, 200 mg. KHzPOl and K2HP04,25 mg. each ; adenine sulfate, guanine hydrochloride, and uracil, 100 y each; pyridoxine, 10 y ; nicotinic acid, thiamine, and riboflavin, 5 y each; p-aminobenzoic acid, 1 y ; biotin, 0.04, and folic acid, 0.02 y. The only modification of this medium which n-as investigated v-as the increase of glucose from 2 to 30/,, as indicated under results. Media nere prepared one day before their actual use in an assay. ant1 stored ~
r;
VOL. 29, NO. 1, JANUARY 1957
135
