Cu(I1) and the same acid and azide mentioned before. Optical measurements were taken a t 400 mp, which is in the xicinity of the wivelength of the maximum absorption of the copper azide complex, 380 mp. Iron Determination. -4mixture of 5 ml. of 0.008X Fe(III), 2.5 ml. of O.1N HCl, and 0.2 ml. of 1J1 azide indicator, diluted to 25 ml. with distilled water, gave an intenqe red color. It is important to emphasize here that the amount of azide should not exceed the above limit, otherwis? the titration mixture would become turbid because of the formation of ;he basic ferric azide. Even in cases where more acid was added to prevent 1 he formation of the basic salt, increased azide concentration would render the end point obscure. The addition of 0.02V E D T I to the above mixture masks the red color and the end point is reached when the solution turns from orange-red to faint yellow. The pH of these titrations should be below 3. Back-titrations were carried out by adding exceis EDTA to the iron solution and backtitration with 0.008A11 Fe(II1). The end point in this case i.j sharper and is obtained when the solution turns from faint yellow to orange-red. Spectrophotometric titrations were also carried out using 1 ml. of 0.008X Fe(II1) and the color mas measured at 450 mp) which is the wavelength of maximum absorption for the ferril: azide complex.
Zinc Determination. A known excess of EDTA, 17.5 ml. of 0.02MJwas added to 2 ml. of 0.1148X Zn solution, and 2 ml. of 1N sodium azide was added. The mixture was titrated against 0.02M Cu(I1) solution. I s the copper ion was introduced, it reacted with the EDTA, forming the blue chelate. The blue color increased gradually, and when the end point was reached, the solution turned suddenly to green, the color of the copper azide complex. Aluminum and Sickel Determination. -1mixture containing 15 ml. of 0.01X hl(III), 17.5 ml. of 0.02Jf EDTA, and 1 ml. of 1-ll azide indicator, was titrated against 0.02-1.1 copper solution. The end point was detected as above. In a similar fashion, the known excess of EDT.1 added to the nickel solution and sodium azide indicator was backtitrated against the standard copper solution.
tity of the titrant at the end point. The zinc titrations could be carried out in solutions of pH ranging from 2 to 5, with no effect on the result,. The amount of azide indicator could also be varied widely. In fact, the larger the amount of indicator, the more obvious was the end point. However, it is adviqable not to increase the acid concentration, as volatile hydrazoic acid is irritating to the mucous membranes of the nobe and eyes. Attempts were made to determine Pd(II), Pt(IV), Cr(III), and U(V1) by titrating the excess EDTA against copper, but the color produced bj- these ions with the azide masked the end point.
RESULTS AND DISCUSSION
Chem. 8,346 (1958). (3) Schwarzenback, G. S., Analyst 80, 713 (1955). (4)Sherif, F. G., Awad, Aida, A m / . Chim. Acta 26, 235 (1962). (5) Sherir. F. G., Oraby, W. M., Sadek, H., J . Inorq. iYucl. Chem. 24, 1373
The data are depicted in Figure 1 and compared with LTolumetric results in Table I. In the determination of copper, changes of pH within a range of 4 to 6 were permissible in both forward and back E D T d titrations. Slight variations in either the acid or the azide concentration were found to have no effect on the sharpness and/or quan-
LITERATURE CITED
(1) Elshamy, H. K., Sherif, F. G., E g y p t . J . Chem. 1, 257 (1958), (2) Saini, G., Ostacoli, G., J . Inorg. Nucl.
(1962).
( 6 ) Welcher, F. J., "The Analytical Uses of Ethylenediaminetetraacetic Acid," p. 140, Van Kostrand, Princeton, 1957.
RECEIVEDfor review May 16, 1963. Acrepted September 4, 1963.
Extraction of Submicrogram Amounts of Molybdenum with Cu pfe rro n-Chlorof o rm Using Molybde num-99 W. B. HEALY' and W. J. McCABE Soil Bureau, and Institute of Nuclear Sciences, Department o f Scientific and Industrial Research, Wellington, N. Z.
b Extraction of Mo a t 0.01 - to 0.1 -pg. levels with cupferron-chloroform has been followed using Mogg. The extraction is most efficient, giving a distribution ratio of over 200 between the chloroform and ciqueous phases, Over 90% of Mo can be separated in one extraction when 'only 0.1 pg. is present in 1 liter of aqueous phase. Phases must b e separated within an hour of extraction, ovherwise breakdown of cupferron results in return of Mo to the aqueous phase. Bone samples in preparatioii for extraction can be ashed a t temperatures up to 850" C. without loss of Mo.
iatry," 11. 169, 1987, Wiley, New York, N. Y.). I n this laboratory, analysis of various materials, including teeth, bone, soft animal tissues, urine, and water, for submicrogram amounts of molybdenum has necessitated a preliminary extraction of molybdenum from acid solutions into an organic phase. This paper reports the use of Mog9to check on the effectiveness of cupferron in complexing molybdenum and facilitating its extraction into a chloroform phase when molybdenum is present in the range 0.013 to 0.13 pg. EXPERIMENTAL
T
cupferron (ammonium salt of N-nitrosophenylhydroxylamine) t o extract a number of elements, including molvbdenum. from acid and neutral solutims into organic phases has been recently reviewed (Morrison, G. H., Freiser, Henry, "Solvent Extraction in Abialytical ChemHE USE OF
and Materials. CUPFreshly prepared 6% aqueous solution of analytical grade cupferron (ammonium salt of N nitrosophenylhydroxylamine) , Mo9'. Solution of sodium molybdate containing 1.3 pg. of molybdenum for each microcurie of MoS9. Apparatus
FERRON.
CHLOROFORM. C.P. COUNTINGAPPARATUS. A Philips (iModel PW.4032) Scalar was used with a Geiger-Muller Liquid Counting Tube (20th Century Electronics Type M.6H). Four solutions were used for these extraction studies: (1) 2N HC1. (2) T550 Solution. Twenty grams of ovendried human teeth were ashed in a platinum crucible a t 550" C. for about 6 hours. The ash was dissolved in concentrated HC1, diluted, and filtered through a ?io. 542 paper into a 250-ml. flask. The re4due and filter paper mas re-ashed for 4 hours a t 580" C,, dissolved in a few milliliters of concentrated HC1 and added to the volumetric flask, which was made up to volume. The final solution was approximately 2 N in HC1. (3) B550 Solution. Seventeen grams of oven-dried bone (femur of sheep) were ashed at 550" C. for 6 hours, dissolved in concentrated HC1, Present address, Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tenn.
VOL. 35, NO. 13, DECEMBER 1963
21 17
Extraction of Mo (0.013 and 0.1 3 p g . ) from 2 N HCI and from Extracts of Teeth and Bone
Table I.
0.013 pg. MO ChloroAqueous form &Io exphase, phase, tracted, c.p.m. 7% c.p.m. 3 330 Si
21%’ HCl
II. Breakdown of Mo-Cupferron Complex (0.13 pg. Mo) Aqueous _Postextraction time phase, h Min. c.p.m. ... 11 15 ... 35 21 1 .. 3 1 25 22 1 55 110 2 25 700 .~ 2 45 780 4 35 920
Table
Fresh Cupferron Added 25 28 5 50 27 5
Table Ill. Extraction of 0.1 3 pg. MoPhases Separated within 5 Minutes of Extraction
Sample 2NHC1 T550 B550 B65O
First extraction CHCla phase, c.p.m. 3170 3410 3310 3360
Second extrac- &Iog8in tion first CHCls extracphase, tion, c.p.m. % 27 99 60 98 58 98 56 98
Table IV. Extraction of 0.13 p g . Mo from 1 -Liter Water Samples
Sample Distilled water City water
First extraction CHClo phase,
Second phase,
first extraction,
1790
110 45
94 98
c.p.m.
1960
extrac-
tion
CHC13 c.p.m.
M099
(j&
Table V. Effect of Ashing Temperatures on M099 Content Bone Ash, c.p.m.
Ashed
550” C.
1010 560 1090
21 18
Ashed 650°C. 1010 5 70 1070
Ashed
750” C.
1040 5io 1110
ANALYTICAL CHEMISTRY
in
of
Ashed 850” C. 1040 540 1110
0.13 pg. MO ChloroAqueous form Mo exphase, phase, tracted, c.p.m. c.p.m. % 240 1800 50
and made up to 250-ml. volume as for T550 solution. (4) Bcj50 Solution. Fifteen grams of oven-dried bone were ashed a t 650” C. for 6 hours, dissolved in concentrated HCl, and made up to 250-ml. volume as for T550 solution. Extraction Procedure. Aliquots (50 ml.) of each of the four solutions were placed in separatory funnels, followed by 1 ml. of 6y0 cupferron solution and 10 ml. of CHCls. This was shaken for 1 minute and the two phases were allowed t o separate. The CHCls phase was drawn off and discarded. This was repeated three times so that the solutions were Mofree, or nearly so, prior to addition of Mog9. The appropriate amount of Mog9was added plus 1 ml. of 6% cupferron, followed by 11 ml. (to ensure sufficient volume to fill liquid counter) of CHC13. The separatory funnel was shaken for 1 minute and allowed to stand. Where several extractions mere made a further 1 ml. of cupferron and 11 ml. of CHCL were added in each case. Where the CHCla phase did not separate cleanly, it was transferred to a centrifuge tube and centrifuged. The clear CHCla phase was then removed by pipet and placed in the counting tube. RESULTS
Two standards are included in the results t o serve as reference points for extraction efficiency. I n the “aqueous” standard, the activities of the two Mo levels used, 0.013 and 0.13 pg. (0.01 and 0.1 pc. respectively) were measured in the liquid counter in 11 ml. of H20. I n the “extracted” standards, the Mog9activities were measured in the CHC13 phase made up of two successive extractions of the 2147 HCl aqueous phase t o which Mog9had been added. Fresh cupferron reagent was added a t each extraction, and the final volume of the CHCl, phase made up t o 11 ml. Prior experiments had shown that no hIog9activity could be detected in a third extraction. The lower activity in the “extracted” standards would appear t o reflect the greater beta absorption in the cI3c1~ phase. Extraction efficiencies were calculated by expressing the Moggactivity in the CHCls phase of a sample, as a percentage of Mog9 activity in an “extracted” standard (CHC13 phase).
The results of extraction of Mo from the four solutions is given in Table I. All count rates have been corrected for background and decay. While transfer of Rlo to the CHC13 phase in one extraction was promising, the results were somewhat variable. To lessen the possibility of contamination, the lower activity aqueous phases were counted first, followed by the CHCls phases. There was, therefore, some time elapse between the measurement of activity in aqueous and organic phase of a particular sample. The 0.13-pg. Mo series had the greatest time elapse between extraction and counting, and these show lower extraction efficiencies. This suggested that the &Io-cupferron complex might be unstable and break down with time, the Mo returning to the aqueous phase. The fact that in some samples the sum of activities in the two phases fell short of the known activity --eg., in T550, 0.13 pg. Mo, 25% of Mog9activity could not be accounted for--supported this possibility. When a 0.13-pg. level of Mo (0.1 pc. RIog9)and the extraction procedure are used, the activity of the aqueous phase mas followed over several hours. After initial separation of the two phases, a sample of the aqueous phase was removed, centrifuged, and counted. Following counting, the sample was returned to the separatory funnel and reshaken for 1 minute. This operation was repeated several times and the postextraction activities are given in Table 11. After 5 hours, when the bulk of the &lo had returned to the aqueous phase, a further 1 ml. of 6% cupferron was added and the operation was repeated. It can be seen that fresh cupferron resulted in extraction of &Io back t o the chloroform phase. It appears that the Mo-cupferron complex breaks down rapidly after about 1.5 hours, probably as a result of free cupferron breaking down and falling below a certain minimum concentration. Extraction of the 0.13-pg. Mo series was repeated and in all cases counts were made within 5 minutes of extraction. the CHC& phase only being counted. Results are given in Table 111. It would appear that better than 98% of the >lo is removed in the first extraction, indicating a distribution coefficient of over 200.
Extraction of Mo from Water. One liter of water was made approximately 0 . 2 5 5 by the addition of 5 ml. of concentrated HC1, 0.13 pg. of Mo was added followed by 5 ml. of 6% cupferron and 25 ml. of CHC13. Following shaking for 1 minute (not too rigorous, otherwise phases take a long time to separate), the chloroform phase was counted within 5 to 10 minutes. Results for distilled and city water samples are given in Table IV. Results show that 94 to 98oJ, of the
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, pH 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
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