Mo in the aqueous phase is removed to the chloroform phase in the first extraction. It could wclll be that the efficiency is even higher, for the aqueous phase still contained fine droplets and surface lenses of CHCl, difficult t o settle out. The activity leves for the second extraction are probably somewhat high on this account. Ignition Temperatiires for Bone Samples. Bone and t1:eth samples are not easily ashed free of organic matter at temperatures (500" to 550" C.) normally used. "Ireover, when the ash is dissolved in acid in preparation for extraction with cupferron 3 portion of the organic matter appears to dissolve also, imparting a brown color to the solution. Even if residual carbon is re-ashed and dis.solved, this does not, of course, prevent organic matter from being taken up when the bulk of the sample is jirst dissolved in acid. Traces of organif: matter prevent clean separation of the chloroform and aqueous phases after shaking and so interfere seriously with 1\10 extraction. The bone samples (B€50) used in the extraction studies were free of this interference because of the higher (650" C.) ashing temperature, and phases separated cleanly and quickly. Other studies being c m i e d out in this laboratory involved injection of blo99 into rats. When thck animals were sacrificed, it was possible to obtain a number of femurs containing quite considerable amounts of hI09~,and as this h l had~ been ~ taken ~ up by metabolic processes it was most likely in a form normal to bone. The femurs were cleaned of soft tissue and three groups of femurs were ashed in platinum basins
for 18 hours a t 550" C. The three samples were then ground and counted in metal planchets. Four counts were taken for each sample, the planchet being rotated 90" between counts. Mean counts are recorded, but the four separate counts agreed well. After counts were made a t 550" C., the samples were returned to the platinum basin and ashed a t 650" C. for 2 hours. The samples were transferred to the planchets and recounted. This operation was repeated a t temperatures of 750" C. and 850"C. Results are given in Table V, counts being corrected for decay. These showed that at tcmperatures up to 850" C. no X o g 9 mas lost from the bone samples. DISCUSSION
Submicrogram determinations may be affected by sample variation in cations and anions apparently without effect a t higher microgram levels Quantitative separation of the elements concerned from the sample matrix minimizes such effects and may be essential for accurate determinations by helping to reproduce standard curve conditions. Extraction of an element, usually in the form of a complex by a solvent immiscible with water, is an especially valuable separation technique, not only because of the selectivity possible with the choice of complexing agents, p H optima, and organic solvents available, but also because the extraction may be repeated several times if the distribution ratio is not particularly favorable.
The Ago-cupferron complex appears to have a distribution coefficient of over 200 between the chloroform and aqueous phases. Cupferron is, therefore, a most effective reagent for complexing Mo in dilute acid solutions, and ensuring its extraction into the CHCia phase. The Mo-cupferron residue left after evaporation of CHCl, is readily digested with HN03-HClOa. Care must be taken that the CHCls phase is removed within perhaps 1 hour of extraction, otherwise the Xo-cupferron complex breaks down rapidly with return of M o t o the aqueous phase. Two or three extractions ensure quantitative removal of Mol for a single extraction will remove about 95% of Mo from the aqueou3 phase in amounts of the order of 0.01 pg. I n work not reported here, even 0.001-pg. amounts of MO would alqo appear t o be extracted quantitatively. -1 similar efficiency can be seen in the extraction of 0.1 pg. of Rlo from 1 liter of aqueous phase into 25 ml. of C"C1,. Ashing of bones or teeth a t 650' C. ensures that all organic matter is removed. With material ashed at 550" C. the presence of traces of organic matter inhibits the separation of aqueous and chloroform phases. S o losses of I f 0 9 9 appear to take place when bone is ashed a t temperatures up t o 850" C. RECEIVEDfor review April 23, 1963. Accepted September 9, 1863. This work was supported in part by C. S. Public Health Grant D-965.
Spectrophotometric Estimation of Copper(I) Using Rubeanic Acid AGNES PAUL Alagappa Chettiar College o f Technology, University of Madras, Madras, India
b The possibility of the spectrophotometric estimation of copper(1) as a soluble chloro complex of rubeanic acid i s discussed. This molecular formula of the complex formed i s determined b y the method of continuous variation and the probable structure of the complex i s indicated. The method compares favcrably with similar methods, and i s applicable in a concentration range of 2 to 12 p.p.m. of copper(I).
T
HE SPECTROPHOTOJIETRIC STUDY O f
copper(1) has bee11 carried out in the past using several organic reagents. To mention but a few instances, copper (I) in ammoniacal solution has been estimated by complexing with 2,2'dipyridine (14),and separately with
2 ,2 ',2"-tripyridine and 2,2'-biquinoline (10). Other reagents used for the purpose have been described (5, 6-9, 19, 11, 21, 27). Some of these reagents are claimed to be specific for copper(1) with minimum interference from other ions, but their cost and availablity have not always been favorable. Rubeanic acid (dithiooxamide) can be synthesized easily to a high degree of purity (14) and it works well in the spectrophotometric study of copper(1). The suitability of the reagent for the colorimetric estimation of copper(I1) has already been established by previous workers (1, 2, 4, 10, 16, 16, 15). Its application to the spectrophotometric study of copper(1) has not been investigated. The present investigation shows that the reaction of copper(1) in the
form of its chloro complex with ruheanic acid is instantaneous, resulting, therefore, in a rapid method. EXPERIMENTAL
Reagents. C U P R O ~ SCHLORIDE. Cuprous chloride used during this investigation was prrpared in a pure state according to the method given by Palmer (1.2). Commercial samples of cuprous chloride are usually partially oxidized to the cupric state and they have to be freshly reduced by sulfur dioxide and dried before they can ,be used for such investigations. RUBEANIC ACID. Three-tenths gram of rubeanic acid was dissolved in 100 ml. of 95 to 96% ethyl alcohol (this strength is not very critical). The solution had a tinge of orange, and 0.5 f 0.01 ml. was used per estimation. VOL. 3 5 , NO. 13, DECEMBER 1 9 6 3
2119
BUFFER. A phosphate buffer was prepared by adding 4 ml. of 1N sodium hydroxide to a solution containing 71.6 grams of Na2HP04.12Hz0 and making up a total volume of 1 liter with distilled water. This buffer showed a p H of 9.4 to 9.5. STABILIZER. Five milliliters of a 20% aqueous solution of hydroxylamine hydrochloride was added per estimation to stabilize the cuprous complex. Apparatus. The absorbance measurements were made with the Beckman B spectrophotometer equipped with a set of 4 Beckman borosilicate glass rectangular cells. Procedure. ilbout 10 mg. of freshly prepared cuprous chloride was accurately weighed and dissolved in 10 ml. of a saturated solution of sodium chloride. From this stock solution different aliquots were pipetted out into standard 25-nil. flasks. One milliliter of phosphate buffer and 5 ml. of 20% hydroxylamine hydrochloride were added to each aliquot, and the volume wab made up in each case to the 25-ml. mark with distilled water. Finally, 0.5 i 0.01 ml. of 0.3% rubeanic acid was added with stirring. The absorbance of each samTable 1.
Quantitative Determination of Copper(l1 Vol. of Wt. of copper
cuprous chloride, ml.
in mg./liter, av.,of 5
0 5
0 8
1 0 1 5 1 7 2.0
experiments
Std. dev.
2 638 4 E58 5 0170 7 5080
&(I 1339
8 ,5600 12.8380
f 0 1549 f O 1013 A 0 2116 f 0 1808 10.1030
Table II. Interfering Cations and the Colors of Their Respective Rubeanate Complexes
Cations Silver Cobalt Bismuth Copper(11) Mercury( I ) Nickel
Iron( 111) Iron( 11)
Aluminum Palladium Platinum
Color of rubeanate complex Yellow, turning t o greenish black Yellow-brown Tan
Grass-green Brown turning black Blue-red Faint orange Dull green Yellow t o greenish black tinge Brown Rose
ple was measured with the spectrophotometer at various wavelengths. Reagent blank correction was applied in all cases. Readings obtained indicated that the 400-mp region showed maximum change in absorbance for unit change in concentration of the copper(1) ion. However, the rubeanic acid reagent also showed appreciable absorbance at this range and therefore the 425-mp region was the next best for copper(1) estimation, as the absorbance due to the reagent was negligible in this region. DISCUSSION
The molar absorptivity values for six samples of the copper(1)-rubeanate complex were 743.0, 749.0. 743.2, 755.3. 776.4, and 745.9 which show that the absorbance obeys Beer's law. -1series of quantitative determinations of copper(1) nere carried out taking known weights of cuprous chloride in saturated sodium chloride solution, determining their respective absorbancez on the spectrophotometer, and referring the values to the working graph. The estimated values \\-ere in good agreement with the weighed values. Standard deviations are indicated in Table I. Cations shonn in Table I1 tan interfere n-ith the estimation if present in large amounts. Their concentrations can be reduced conciderably by suitable means, such as electrodeposition. before estimating the copper(1) ions. However, if they are present as trace impurities, their interference can be obviated by the addition of suitable buffers and/or sequestering agentc (R6,2&'). Approximate limits of tolerance of some of the diverse ions usually found with copper are shown in Table 111. Cations of silver require, for masking. K C X or thiourea which also form complexes with cuprous ions, and hence are not tolerated in this estimation. Silver ions are completely eliminated as inqoluble AgCl nhen the cuprous ions are complexed with saturated sodium chloride solution to give the soluble chloro cuprous complex for the estimation. Anions such as periodate, nitrite, thiocyanate, and ferricyanide give yellow coloration with the reagent. or react with hydrovylamine hydrochloride. Hence, their presence alco is not tolerated. Though the broun cuprous-rubeanate
Table Ill. Tolerance Limits of Diverse Ions
Metal Co(I1)
CoCI2.6H20
Ni(I1)
Sic12 6H20
Fe(II1) Fe(I1)
FeC13 6H20 Fe(I1) ammonium sulfate
2 120
Sdded as
ANALYTICAL CHEMISTRY
Complexing agent Tolerance limit Malonic acid or 20 p.p.ni. EDTA (sodium salt) Malonic acid or 12 p.p.ni. EDTA (sodium salt) Fluoride or phosphate 1000 p . p m EDTA (sodium salt) 4 p.p.m. . .
complex is formed over a wide range of p H (from 4 to 9.5) the color stability was maximum in the alkaline range. Stabilizers are necessary in various colorimetric studies of copper(1) (6, 13, 18, 2 4 ) . I n this laboratory Metol (monomethyl pura amino phenol sulphate), quinol, hydrazine hydrate, and dimethl 1 formamide were tried as stabilizers without much success. The two former reagents are very susceptible even to small changes in hydrogen ion concentration, and their behavior becomes unpredictable due to rapid tautomeric changes in their constitution. Hydroxylamine hydrochloride was found to be the best stabilizing agent for this estimation; the brown color of the cuprous rubeanate complex was stable for more than 15 minutes in the alkaline medium. Composition of the Cuprous-Rubem a t e Complex. The composition of the complex was determined by t h e method of continuous variation proposed by Job (11) and adopted by Vosburgh and Cooper ($6). TThen the different mole ratios of copper(1) to reagent n ere plotted against their respective absorbances for different wavelength< (Figure l ) , i t was observed that there was no variation in the location of the maxima, from which it was inferred that only one product was formed, or that the complex was homogenous The position of maxima of the continuous T ariation method showed the ratio of copper(1) to reagent in the complex to he 3 : 2 Hon ever, the gravimetric 2nd volumetric analyses of the ,iolid comple.; indicated the ratio of coppei (1) to rpngent to be 1 : 1. Therefore, in the solid state the complex exists in a monomeric state with one ligand per copper(1) atom. X-ray studies have indicated that the dithiooxamide (rubeanic acid) molecule is planar with a center of symmetry. Barcelo (see 17) observed that the infrared spectra of the metal complexes of dithiooxaniide or S , N ' , disubstituted dithioovamide molecule showed strong absorbance of the Tu'-C=S group, but the characteristic absorption of the S-H bond was not found in any of the spectra. which agreed with the formula based on the trans-keto form. Correlating the above two observations. and the results of the gravimetric and volumetric analyses, the structure of copper(1)-rubeanate complex in the solid state may be represented as: S=-CNH
\
bu
7
HzN-C=S Hoxel-er, the continuous yariation method, which is a specific analytical method for trace quantities of very weak Complexes, indicated the ratio of copper
t
8
10
Mole Fraction of [Cu Clrl- S o h . 6 4
2
0 (X10%4)
0.8
working graph be prepared using the conditions adopted for sequestration. ACKNOWLEDGMENT
Illy grateful thanks are due to P. B. Janardhan for his guidance and keen interest in this work. I also express my indebtedness to Madras University for the facilities made available to me.
0.6
Y
x
B
i0.4
, “Experimental Inorganic Chemistry, pp. 129-130, Cambridge Cniversity Press, 1954. (13) Pflaum, R.T., Popov, A. I., Goodspeed, N. G., ANAL.C m v . 27, 253 (1955). (14) Pheline, J. M.,Castro, R., C‘ongr. Group Avan. Methodes Anal. Spectrog. Prod. ;Met. ( P a r i s ) 8 , 47-59, 177-187 (1947). (15) Ray, P., 2. Anal. Chem. 79,O-i (1929). (16) Ray, P., Ray, R. &I.,Quart. J . I n d . Chem. Soczely 3, 118 (1926). (17) Richard, N., et al., Chemical ReI
0
Figure 1.
P
4
-- M o l e Fraction of
6 Rubeanic Acid
8
10 ( X10 %4)
-+
Identification of rubeanic acid-cuprous complex by method of continuous variation
(I) to reagent to be 3 : f ‘ . Therefore, in solution, in the presence of a high concentration of sodium chloride, there is a possibility of the form:rtion of a polynuclear complex when the two amido groups of two monomeric complexes by their proximity are bridged through a copper(1) atom belonging t o the (CuC12)Ka complex. The structural formula of the brown polynuclear complex in solution can be r2presented as:
Ka
Applications. The method is of analytical interest. It does not claim greater advantage t h a n t h e estimation of copper(I1) by t h e same reagent. However, i t (:an work as a n alternate method to t h e latter. T h e application of this mei;hod is limited b y t h e limited occurrence of copper in its unusual univalency. Although not confirmed, i t m a y be of value i n t h e estimation of copper in :uprite (CutO), in the evaluation of cup::ous oxide t o be used in ceramics, red glaze of porcelain, red glass, and electroplating, and in the estimation of cuprous oxide used in fungicide and in antifouling paints (ship bottom paints). It should also be ap-
plicable to the evaluation of cuprous chloride used in catalysis, preservatives, fungicides, and in wet-cell batteries (cuprous chloride and magnesium). I n all the above instances where cuprous oxide or cuprous chloride is handled, the extraction may be done with saturated sodium chloride while bubbling sulfur dioxide through it, when (CuCl2)Ka complex is formed. The spectrophotometric method discussed in this paper can then be applied directly after elimination of free sulfur dioxide. The method has not been of quantitative value when the extraction involved acid digestion. Interferences. T h e ions t h a t are usually met in aqsociation with cuprous oxide or chloride are cobalt, nickel, iron(III), and magnesium. Of these, t h e last is not a n interfering ion. If t h e interfering ions are present in large proportion, their prior elimination by electrodpposition is necessary. Iron(II1) complexes with t h e phosphate buffer used in t h e experiment and does not interfere under the conditions of the experiment as has been mentioned earlier. Sickel and cobalt, if present in small amounts, can be sequestered with malonic acid, ethylenediamine (26), or EDTA, and thus their interference can be eliminatpd. However, the addition of these sequestering agents, especially malonic acid, considerably reduces the stability of the cuprous-rubeanate complex. Larger amounts of the hydroxylamine hydrochloride stabilizer (about 15 to 20 ml. per estimation) have to be used when EDTA is used in the experiment, and it is also recommended that a separate
/
%
search Dept., Mallinckrodt Chemical Works, St. Louis, & I o , p. 3.50 “Further Studies on the Metal Complexes of N,LT’-Disubstituted Ditliiooxamides.”
Proc. 6th Intern. Conf. on Coorcltnatton Compounds, Detroit 1961, Stanley
Krishner, ed.
(18) Robert, L. M7., Owens, 11. I,. Jr., Slate, J. L., ANAL.CHEZI. 27, 1614-16 (1955). (19) Schilt, -4.-4.,Smith, G . F.,.lnaZ. Chim. Acta. 15, 567 (1956). (20) Smith, G. F., ANAL. C m x . 26, 1534-8 (1954). (21) Smith, G. F., McCurdy, W.€I., Jr., Ibid., 24, 371 (1952). ( 2 2 ) Smith, G. F., Wilkins, D. H., Ibid. 25, 510 (1953). (23) Tananaev, I. V., Levitman, d. Ya., Z h . Analat. K h i m . 4, 212-19 (1049). (24) Tartarine, G., Gazz. Chim. Ital. 63, 597-600 (1947). (25) Vosburgh, W. C., Cooper, C.; R., J. Am. Chein. SOC.6 3 , 437 (1941). ASAL. (26) West, P. IT.,I N D . ENG.CHEZ~., ED. 17, 740 (1945). (27) Wilkins, D. H., Schiff, A. A., Smith, G. F., ANAL.CHEX 27, 1574 (1955); (28) Willard, W. H., Diehl, H., Advanced Quantitative Analyses, p. 285, Van Xostrand, Sew York, 1943.
RECEIVED for review September 26, 1962. Accepted August 7, 1963. VOL. 35, NO. 13, DECEMBER 1963
2121
