Spectrophotometric Determination of Copper and Zinc in Animal Tissues J. T. McCALL and G. K. DAVIS Department of Animal Husbandry and Nutrition, Florida Agricultural Experiment Station, Gainesville, Flu.
T. W. STEARNS Deparfment of Chemistry, University of Florida, Gainesville, Flu. A method for the determination of copper and/or zinc in animal tissues is based on the difference between the absorbance values of a solution containing both copper- and zinc-Zincon complexes and of the same solution after destroying the zinc-Zincon complex with Versenate. Equal absorbance values for equal concentrations of the two complexes at 61 0 mp made it possible to use one calibration curve for both elements. The absorbances of these metal complexes follow Beer’s law over the concentration range from 0.1 to 2.4 p.p.m. The complexes were stable for 1 hour. Interfering ions are removed by precipitation as the hydroxides in the presence of an excess of ammonium hydroxide. The method is rapid and accurate, and both elements are determined in the same solution by essentially the same procedure.
T
increased emphasis on trace mineral elements in animal nutrition, especially copper and zinc, developed a need for more rapid and accurate methods of analysis for these elements. Although there are many chromogenic reagents suitable for the determination of traces of copper in animal tissues.. only diphenylthiocarbazone and di-2-naphthylthiocarbazone have been widely accepted for determining zinc. Both reagents require several time-consuming extractions at carefully controlled p H values. Rush and Yoe ( 9 ) described a method for the colorimetric determination of copper and zinc using Zincon (2-carboxy - 2’ - hydroxy - 5’ - sulfoformazylbenzene) as the chromogenic reagent. Copper forms a blue complex n i t h this reagent 15-hich is stable over the pH range from 8.5 to 9.5. This difference in stability of the tn-o metal complexes a t different p H values was the basis of their method for determining both copper and zinc in the presence of each other. The copper concentration was determined b y measuring the absorbance of a solution containing both copper and zinc complexes a t p H 5.2, where the abHE
sorbance resulted entirely from the copper complex. The concentration of both metals m-as found by measuring the absorbance of a similar solution at p H 9.0, where the absorbance was due to both the copper and zinc complexes. The difference between the absorbances of the two solutions gave the zinc concentration. The authors could not obtain satisfactory results using this procedure, because at p H 5.2 there \vas a precipitate of the reagent in most samples. This made it impossible t o measure the absorbance due to the copper complex accurately. The maximum color development of both the copper and zinc complexes from p H 8.5 t o 9.5 suggested determining both elements in the presence of each other by selectively destroying the complex of one of the elements viithout affecting the color intensity of the other. EXPERIMENTAL
Destmction of Zinc-Zincon Complex. T h e addition of a f e n drops of a disodium (ethylenedinitri1o)tetraacetate solution (Versenate) t o a test solution containing coppera n d zinc-Zincon complexes completely destroyed all traces of color resulting from t h e zinc complex b u t did not affect the color intensity of the copper complex. I n the presence of excess Zincon reagent, 3 drops of a 4% Versenate solution was enough t o chelate 100 y of zinc in 50 ml. of solution a t p H 8.5 t o 9.5. Absorbance Curves. T h e zinc complex shoived a maximum absorbance a t 620 nip, and the maximum for the copper complex occurred a t 595 mp, Both complexes had the same absorbance value for equal concentrations of the ions a t 610 mp. All subsequent measurements viere made at this wave length, nhich greatly simplified the subtraction of absorbance values, and only one standard calibration curve \vas required for the calculation of both elements. Addition of Versenate to solutions of the copper complex, the zinc complex, and a reagent blank had no effect on
the absorbance curves of the copper complex or the reagent blank. The values of the zinc solution and the reagent blank, after adding Versenate t o each, were the same. There was a slight absorbance of the reagent blank above 600 mp 1% hich decreased after addition of the Versenate. This effect was attributed t o a very small contamination, presumably zinc, in the Zincon reagent. Stability and Sensitivity. Both copper a n d zinc formed a blue color immediately upon addition of Zincon t o qolutions containing these ions a t pH S.5 to 9.5. Xo change v a s found in the absorbance values of either complex after 1 hour in diffused light. There was a slight decrease in absorbance with both complexes after 24 hours relative to the amount of metal complex in the solution. Solutions containing 4.0 y, or less, with respect to the metal ions, decreased about 1% in absorbance in 24 hours. The more concentrated solutions, containing a total of 10.0 y, decreased about 4% in absorbance over the same period. ilddition of the Versenate solution had no effect on the stability of the copper complex or the reagent solution. Both the copper- and zinc-Zincon complexes had molar extinction coefficients of about 19,000 a t 610 mp. Both complexes follow Beer’s law over the concentration range from 0.1 to 2.4 p.p.m. ( 2 ) . Interfering Ions. Cobalt, manganese. nickel, aluminum, molybdenum, beryllium, bismuth, cadmium, and iron n ill interfere in the determination of copper and zinc using Zincon, but only iron is normally present in animal tiisues in concentrations large enough to cause appreciable error. Elements such as calcium, magnesium, and phosphorus, present in animal tissues in relatively high concentrations, n ill form a precipitate a t the p H required for the determination and must be removed prior t o the determination. Precipitation of the iron, magnesium, calcium, and phosphate from a basic solution, in the presence of excess ammonium hydroxide, will eliminate the interference caused b y these ions n i t h no loss VOL. 30, NO. 8, AUGUST 1958
1345
of copper or zinc. Table I lists the recovery of copper and zinc from solutions which hare had some interfering ions removed by this precipitation method. Iron, manganese, and maguesium nere present a t levels of 100 y and calcium and phosphorus were present a t levels of 500 y before prc’cipitation. The recovery of c o p p r and zinc in the presence of the niaxinium amounts of these ions that may be present in a sample solution, with no separation, is given in Table 11.
Table 1.
APPARATUS A N D REAGENTS
All absorbance measurements were made with a Beckman Model DIT spectrophotometer, using matched 1.00-cm. Corex cells, and were repeated rrith a Bauschand Lomb Spectronic-20 colorimeter, using matched 1.80-cm. tubes. Any colorimeter or spectrophotometer having a band pass of 20 mp or less at a wave length of 610 mM may be used. A Beckman p H meter, Model G, was used for all pH measurements. Reagent grade chemicals were used in all solutions. Standard copper solution. Dissolve 0.3928 gram of copper sulfate heptahydrate in distilled water, add enough hydrochloric acid to make the final solution about O,lArl and dilute to 1000 ml. From this stock solution, prepare a dilute standard containing 1.0 y of copper per nil. Standard zinc solution. Dissolve 0.1000 gram of uowdered zinc in a slight excess -of coicentrated hydrochloric acid, and dilute to 1000 ml. From this stock solution, prepare a dilute standard containing 1.0 y of zinc per ml. Buffer solution, p H 9.0 Prepare a Clark and Lubs buffer by adding 21.3 ml. of 0,2-Jr sodium hydroxide to 50 ml. of 0.2.2’ boric acid in 0 . 2 s potassium chloride and diluting to 200 ml.
Recoveries of Copper and Zinc
(Interfering ions removed by precipitation) Present, y Found, y cLl Zn c11 Zn 0.0
0 0 20
8 0
6 4 2 0 10
0 0 0 0 0
4 6 8 10 0
0 8 6 4 2 0 10
0 0 0 0 0
0 1 1 0 1 0 0
Table II.
Added, y Cu Zn 0.0 0.0
Fe ..
5.0
50 50
5.0
.. ..
0 2 4 6 8 10 0
Recovery
..
of Copper and Zinc
(Interfering ions not removed) Present, y Mn Ca Mg K . .
, .
..
2
100
20
..
..
2 ..
100
2
100
..
50
0 1 0 2 0 0 0
..
..
..
..
20 20
Table 111. Determination of Copper and Zinc in Liver Digests
Zincon Method, P.P.hI. Zinc Copper 137.0 62.0
Sample 1 2 3 4 5 6
136.0 134.0 154.0 i43.0 138.0
68.0 74.0 75.0
62.0
78.0
Carbamate RIethod ( I ) , Copper, P.P.M. 139.0 138.0 138.0 125.0 145.0 137.0
io0
..
..
cu 0.0 0.0
..
..
5.0 5.0
..
..
5.0 4.9
126
100
5,o 5,0
i26
..
..
..
Recovered, y
P
..
z I1
0.0 0.0 5.0 4.9 50 5.0 5.0 5.0
Zincon reagent solution. Dissolve 0.130 gram finely polvdered Zincon (LaRIotte Chemical Products Co., Chestertown, hxd.) in about 2 nil. of 1.ON sodium hydroxide, and dilute to 100 ml. Versenate solution. Dissolve 4.0 grams of disodium (ethylenedinitri1o)tetraacetate in distilled water, and dilute to 100 ml. About 3 drops of this solution will complex 100 y of zinc. RECOMMENDED PROCEDURE
The sample t o be analyzed may be
Recovery of Copper and Zinc Added to Liver Sample 6
Table IV.
Added, Cu 8 0 6 0 4 0
20 00 10 0
1346
Recovered,
y
Zn 20 4 0 6 0 80 10 0
00
cu
80 6 1 4 0 21 0 0 10 1
ANALYTICAL CHEMISTRY
y
Zn 20 39 61 82 10 1
00
cu
% Recovery
100 0 100 8 98 8 105 0 0 0 101 0
Zn
100 0 97 101 101 100
5
T 9
5
dissolved by any appropriate means. For animal tissues, wet digestion with concentrated nitric acid and 60% perchloric acid is recommended. The final solution of the tissue digest must be colorless. Place an aliquot of the sample solution, containing a combined total copper and zinc concentration of about 10 y, in a centrifuge tube with a conical tip. Add concentrated ammonium hydroxide until the solution is basic, then add an excess of approximately 2 nil. Add about 1ml. of 6N sodium hydroxide to facilitate removal of ammonium liydroxide in a later step. Mix thoroug centrifuge the solution a t a rela centrifugal force of 3000 for about 5 minutes, and decant the clear supernitant liquid into a beaker. Wash the precipitate with about 5 ml. of 3 5 ammonium hydroxide, centrifuge again, and combine the supernatant liquid with that from the first centrifugation. The precipitate may be removed by filtration just as effectively, but more time is required for a large number of samples. Heat the solution until the strong odor of ammonia is gone. Add concentrated hydrochloric acid dropwise until 1 drop makes the solution acid. If the concentration of interfering ions is equal to or less than the amounts listed in Table 11, or if there is no precipitate when the p H is raised to 9.0, the separation step may be omitted and the procedure started with the addition of the buffer to an aliquot of the sample solution. Add 10 ml. of the buffer solution and adjust the p H to 9.0 with either 11sodium hydroxide or 1 N hydrochloric acid. Add 3 ml. of the Zincon reagent. transfer to a 50-ml. volumetric flask. and dilute to volume m-ith distilled water. For reagent blank, use distilled water instead of the aliquot of sample solution. Carry standard copper and zinc solutions, containing up to 10 y, with a combined concentration of 10 y, through the same procedure, and use the absorbance values of these solutions at 610 mb to plot a calibration curve. RIeasure the absorbance of each saniple solution a t 610 mp with the reagent blank in the reference cell. Add 3 drops of the Versenate solution to each of the sample solutions including the reagent blank, and again measure the absorbance a t 610 mpl using the reagent blank containing Versenate in the reference cell. The first value represents the absorbance due to both the copper- and zincZincon complexes. The second value. after addition of the Versenate, is due to the copper complex alone. The amount of copper in a solution may be calculated by comparing this value directly to the calibration curve. The difference b e h e e n the absorbance values before and after addition of the Versenate is due to the zinc complex, and the zinc content may be calculated b y comparing this d u e to the calibration curve.
DISCUSSION
A11 test analyses comparing the proposed procedure t o the carbaniate method, or testing the recovery of added amounts of copper and zinc, were made x i t h liver samples. A comparison of the analysis of liver digests using the carbamate method and the Zincon method is given in Table 111. Good agreement was found in all except sample 4. All of this sample was used, so the analysis could not be repeated. Different amounts of copper and zinc were added to a liver digest solution,
and the recovery of the added copper and zinc v a s measured b y analysis (Table IV). K h e n 2 y of either ion vias added, the maximum error of recovery !vas 5%; n-hen 10 y was added, the maximum error of recovery was 5%; when 10 y was added, the niaximum error \vas 1%. Other tissues, not including hone, were analyzed for copper and zinc content, and the results nere 11ell within the range of copper and zinc values r q ~ o r t e din the literature for these tissues. The most important advantage of this niethod is that both elements are de-
termined by essentially the same procedure, The only reagent which must be added in an exact volume is the Zincon reagent, and there is no extraction required t o concentrate the complex or remove interfering ions. LITERATURE CITED
(1) Cherig, K. L., Bray, R. H., A N ~ L . CHEJI.25,655 (1953). (2) Rush, R. U., Yoe, J. H., Ibid., 26, 1395 (1954).
RECEIVED for review Sovember 5 , 1057. .Iccepted March 18,1958.
Potentiometric Titrations with (Et hylened initrilo)te traaceta te Use of Masking Agents to Improve Selectivity JAMES
S. FRITZ,
MARLENE J. RICHARD, and SHIRLEY K. KARRAKER
Institute for Atomic Research and Department o f Chemistry, Iowa State College, Ames, Iowa
b Potentiometric titration of metal ions with EDTA using a mercury metal indicator electrode i s a useful but rather nonselective method. Complexing agents mask certain interfering metal ions to allow interesting and selective analytical determinations. With citrate as a masking agent, cadmium(ll), copper(ll), mercury(ll), lead(ll), or zinc(It) can b e titrated in the presence of beryllium(ll), chromium(lll), iron(lll), molybdenum(VI), niobium(V), antimony(Ill), tin(lV), tantalum(V), thorium(lV), titanium(lV), uranyl(ll), tungsten(Vl), and zirconium(1V). In some cases tartrate can b e substituted successfully for citrate. Rare earths and some bivalent metal ions can be titrated in the presence of aluminum or uranium, using 2,4-pentanedione or sulfosalicylate as a masking agent.
Porterfield (2) used a mercury iiietal electrode of different design in coiijunction with coulometric titrations of metal ions with electrically generated EDTA. The use of a mercury metal electrode for the determination of stability constants for certain metal chelates also has been studied (4). Reilley and Schniid (3) studied the principles and scope of potentiometric E D T A titrations using a mercury indicator electrode. Potentiometric complexometric titrations using a mercury electrode are based on the fact that the potential of the mercury electrode depends on the concentration of free mercuric ions in solution. Direct titration of a metal ion with E D T A can be carried out if a sniall amount of mercuric-EDTA complex is first added to the solution to be titrated. The following equilibrium is established : ;\I+"
titrations of nietal ions n i t h (ethylenedinitrilo)t e t r a a c e t a t e (EDTA) have been carried out using visual indicators to detect the end point. Khile visual indicator methods are very convenient, it is often desirable to perform a coniplexometric titration 11ithout the complicating presence of a visual indicator. Siggia, Eichlin, and Rheinhart (5) showed that E D T A and certain other chelating agents can be titrated potentiometrically with any of several metal ions. These titrations were carried out in a pyridine-water solution using a mercury-coated platinum indicator electrode. Reilley and OST
+ H g Y 2 $ ;\IY-4+n + Hg-2
-1
I
r0l .\
I 200 z_
ml o f 005 M EDTA
Figure 1.
Direct titration of thorium
(1)
where Y represents a n anion of EDTA. A nietal ion that forms a strong E D T A complex n ill force this equilibrium far to the right, especially a t the start of the titration nhere the ratio of (lI+") to (HgtOt,l)is often of the order of 500. If the metal-EDTA complex is very strong, the metal may all be titrated before the free mercuric ions react Tvith the titrant. The (Hg-?) remains virtually constant until the stoichiometric point is reached, and then there is a sudden drop in potential caused by the complexation of mercuric ions by EDTA. This is illustrated by the titration curve of thorium (Figure 1). The
1001
40
Figure 2.
I
45
1
,
50 55 ml of 005 M EDTA
I
6.0
Direct titration of cerium(ll1)
true elid point is the point of intersection of the horizontal and nearly vertical parts of the curve. A metal ion that forms a n EDTA complex of about the same strength as the mercuric-EDTS complex r d l give a titration curve similar t o that of cerium (Figure 2). In the earlier stages of the titration, the mass effect resulting VOL. 30, NO. 8, AUGUST 1958
1347
