Spectrophotometric Determination of Copper in Fertilizer with Neocuproine E. R. Larsen
The Inspectorate of Fertilizers, Lottenborgvej 24, DK-2800 Lyngby, Denmark
In the determination of copper in Danish soils, EDTA has been used as an extracting agent, and the extracted amount of copper was measured colorimetricaly using diethyldithiocarbamate by Henriksen, (1). This method has been applied to fertilizers containing less than 5% copper (Anon). A method, in which the copper(1) specific reagent 2,9dimethyl-1,lO-phenanthroline (neocuproine) was used, was described by Smith and MacCurdy, Jr., ( 2 ) and modified by Gahler ( 3 ) . This study was made to evolve a method using neocuproine in determining copper in fertilizer extracted with EDTA and to examine whether it would be possible to omit the time-consuming extraction of the colored complex, formed of colored copper(1)-neocuproine, into a water-immiscible liquid.
Table I. Colorimetric D e t e r m i n a t i o n of Copper in Samples with K n o w n C o n t e n t Absorbance
1 2
3 4
5
RESULTS AND DISCUSSION To prove the validity of the procedure used, the experiments shown in Table I were carried out. From the principles given on complex formation by Aa. H e n r i k s e n . N a t u r e , 178, 499 (1956). ( 2 ) G . F. Smith and W . H. McCurdy J r . , Anal. Chem., 24, I. 371 (1952).
(1)
(3) A . R.
Gahler, A n a l . Chem., 26, 577 (1954).
0 0 Water 100 pg Cu, 10 ml buffer solution B, 0 . 2 2 5 0 . 2 3 5 5 ml copper reagent 100 pg Cu, 10 ml buffer solution B, 0 . 2 2 5 0 . 2 3 0 12 ml extraction solution, and 5 ml copper reagent 0.010 100 pg Cu, 10 ml buffer solution B, 0 12 ml extraction solution, and 5 ml 50 % 2-propanol 10 ml buffer solution B, 12 ml ex- 0 0.015 traction solution, and 5 ml copper
reagent
EXPERIMENTAL Apparatus. Absorbance measurements were made in 1-cm cells with a Bausch & Lomb Spectronic 20 colorimeter. The reagents used were analytical grade. Demineralized water was used in preparation of the solutions. Reagents. Extraction solution. Dissolve in about 500 ml water, 14.0 g sodium hydroxide, 45.0 g tris(hydroxymethy1)aminomethane, and 50.0 g ethylenediaminetetraacetic acid. Add water to lo00 ml. 50% 2-Propanol. Dilute 250 ml of 2-propanol with water to 500 ml. Copper Reagent. Dissolve 500 mg neocuproine, C14H12N2 in 200 ml50% 2-propanol. Buffer Solution A . Add to about 150 ml water, 5.0 g zinc oxide, 14.0 g nitrilotriacetic acid and 18.0 g tris( hydroxymethy1)aminomethane; dilute with water to 200 ml. Buffer Solution B. Add shortly before use 1.0 g hydroxylamine hydrochloride per 100 ml buffer solution A. Procedure. Weigh out 4.9-5.1 g of the fertilizer sample to be analyzed. Boil it half an hour with 400 ml extraction solution. After cooling, transfer the solution to a 500-ml volumetric flask; add water to the mark. Filter and dilute, if necessary, to a copper content of 3 to 50 pg per ml. Transfer two aliquots, containing from 45 to 250 pg copper, to 50-ml volumetric flasks. Note! No more than 12 ml extraction solution, i . e . , 0.6 g EDTA must be transferred. Add so much extraction solution that, each flask contains a total of 12 ml. Add 10 ml buffer solution B to each flask and 5 ml copper reagent to one and 5 ml 50% 2-propanol to the other. Dilute with water to the mark and mix well. After 10 minutes ,and within 24 hours, measure the absorbance a t the wavelength of 450 nm using as reference the sample without neocuproine. Determine the copper concentration by means of an absorbance us. copper concentration curve. Prepare the standard solutions by adding the above-mentioned reagents to different volumes with known copper content and measure the absorbance using as reference a solution containing all the reagents used except the copper reagent, substitute for this the addition of 5 ml50% 2-propanol.
Content in 50 ml
No.
The After 15 following minutes day
6
As No. 3 but w i t h 12.8 mg iron
0.270 0.300
added as Fe(I1) 7 8
As No. 4 but w i t h 12.8 mg iron 0.040 0 . 0 7 0 added as Fe(I1) As No. 3 but with 150 mg ammo- 0 . 2 4 0 0 . 2 4 0 nium nitrate
Ringbom ( 4 ) , it follows that the maximum value of the conditional stability constant for the Cu(1)-neocuproine complex should be reached in a solution having a p H value about 7 , since the pKa value of neocuproine is 5.8 ( S i l l h et al., 5 ) . However, the Cu(1)-EDTA complex does not form (6). The &-value for the Cu(1)-neocuproine is 19 ( S i l l h et al., 5 ) . From this was predicted that it should be possible, in a solution containing EDTA and with a pH value between 6 and 7, to obtain a color reaction for copper with neocuproine. In fact a reaction appeared, but the color developed slowly and incompletely and, with a greater concentration of EDTA, the color did not appear. To overcome this difficulty, zinc oxide equivalent to the maximum amount of EDTA was added to buffer solution A. As mentioned above, the Cu(1)-EDTA complex is unstable, thus, although the stability constant of the Cu(I1)EDTA complex is greater than that of Zn(I1)-EDTA, Zn(I1) will replace copper from EDTA when hydroxylamine is added to the solution. Cu(1) then easily reacts with neocuproine. In the procedure given by Smith and McCurdy, Jr. ( 2 ) and by Gahler ( 3 ) ,the colored copper-neocuproine complex was extracted into a water-immiscible liquid from a n aqueous solution containing citrate. Strictly speaking, the great selectivity for the neocuproine reagent toward copper showed by Gahler (3) is only valid when using Gahler's method. As far as the author understands, the citrate ( 4 ) A . Ringbom, "Complexation in Analytical Chemistry," Interscience,
NewYork. N . Y . , 1963. (5) L . G . Sillen and A . E. Martell, The Chemical Society, London, Spec. Pub/. No. 17. 1964. (6) T S. West, "Complexometry with EDTA and Related Reagents," BDH Chemicals Ltd., Poole, 1969. A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 8 , J U L Y 1974
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prescribed in Gahler's method prevents complex building cations other than Cu(1) from reacting with neocuproine. From the values of the stability constants where the 'ligands are EDTA, citrate, nitrilotriacetate, and neocuproine (see S i l l h et al., 5 and 7), it follows that in the procedure given here, complex building cations other than Cu(1) are prevented from reacting with neocuproine by nitrilotriacetate or EDTA, by EDTA with respect to cations which have greater stability constants with EDTA than that of Zn(I1) and EDTA. So, with the procedure given here, it is reasonable to expect great selectivity toward copper. The pK, Value of tris(hydroxymethy1)aminomethane is about 8 and of hydroxylamine about 6. Thus, it is easy to maintain the solution a t a desirable pH value between 7.0 and 7.5 for the color reaction to take place. Smith and McCurdy, Jr. ( 2 ) found that halides and, in particular, nitrate precipitated the copper-neocuproine (7) L. G. Sillen, E. Hogfeldt, A. E. Marteil, and R. M. Smith, The Chemical Society, London, Spec Pub/.No. 25, 1971.
complex in an aqueous solution. In this study, it was found that the solubility of the copper-neocuproine complex was improved by adding a water-miscible alcohol. For this reason, neocuproine was added in a solution of 5wo 2-propanol. Table I shows that, with the procedure given here, it is possible to determine at least 0.1% copper in ammonium nitrate. With EDTA, some cations give colored solutions (West, 6). The cations that with EDTA or nitrilotriacetic acid give colored solutions absorbing light of the wavelength 450 nm, would interfere, if, in the measuring of absorbance, a solution containing an aliquot of the sample to be analyzed and all reagents used except neocuproine was not used in the reference cell. An example is given in Table I with a sample containing iron. Received for review June 1, 1973. Accepted January 21, 1974.
Model for Competitive Binding Assays-The Shape and Location of the Inhibition Curves Donald J. Laurence and Graeme Wilkinson institute of Cancer Research, Chester Beatty Research Institute, Fulham Road, London S W 3 6JB, England
The increasing application of competitive binding assays has required the development of mathematical models to represent the binding curves. Berson and Yalow ( I ) and Ekins, Newman, and O'Riordan (2) have discussed conditions for obtaining the maximum sensitivity from an assay. Feldman and Rodbard ( 3 ) and Feldman, Rodbard, and Levine ( 4 ) have presented graphical findings for a number of important special cases. The information obtained from the curves can come from their location on the concentration axis and from their shape. Studies of tumor-derived antigens ( 5 ) have posed the question of identity between substances in low concentration in body and tissue culture fluids and the materials purified from the tumor itself. In these cases, when the identity of the analyzed product is in question, the location of the curves is less informative than is their shape. Location depends both on the concentration and the affinity for protein of the test substance. If the substance cannot be assayed by an alternative method, it is not possible to separate these two aspects of the problem. The present theory was developed to determine conditions that are suitable for detecting affinity differences between competitors of unknown concentration. The conditions selected are not the same as those that would nor-
mally be chosen in order to establish a sensitive assay. When a test of identity with a sensitive assay appears to confirm the identity of competitors of different origins, it should be considered whether the test is capable of giving an alternative conclusion. The curves we have considered are plots of degree of inhibition as a function of logarithm of the ligand concentration. The degree of inhibition I is a normalized linear function of the ratio of bound to total ligand that increases from 0 to 1 as ligand concentration increases. On a log concentration scale, changes in absolute unitage of ligand, or affinity variations, cause a scale shift but no change in shape of the curves provided the maximum bound fraction ro is kept constant. It was found that the equations are of a simple analytical form if .the ligand concentration and shape parameters are expressed in terms of I and ro. As indicators of shape, the symmetry of the curves, the maximum slope, and the span are taken. The Inhibition Curve. For an equilibrium between ligand and protein with a single association constant K and no interaction between binding sites
(1) S. A. Berson and R. S. Yalow in "The Hormones," vol 4, G. Pincus. K. V. Thimann, and E. E. Astwood, Ed., Academic Press, New York, N . Y . , 1964, pp 567-72. (2) R. P. Ekins, G E. Newman and J. L. H. O'Riordan in "Radioisotopes in Medicine,'in Vitro Studies," R. L. Hayes, F. Goswitz, and B. E. P. Murphy, Ed., U . S . Atomic Energy Commission, Oak Ridge, Tenn.. 1968, pp 59-100. ( 3 ) H. Feldman and D. Rodbard in "Principles of Competitive Protein Binding Assays," W. D. Odell and W. H . Daughaday, Ed., J. E. Lippincott. Philadelphia, Pa., 1971, pp 158-216. ( 4 ) H. Feldman, D. Rodbard, and D. Levine, Anal. Biochern., 45, 530 (1972). ( 5 ) D. J. R . Laurence and A. M. Neville. Brit. J. Cancer, 26, 335 (1972).
where B and F are concentrations of bound and free ligand, respectively, and Q is the concentration of free protein binding sites. If the ligand or protein are multivalent, the equivalent concentrations should be considered. Taking the total concentration of ligand and protein sites as p and q, respectively, and defining
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ANALYTICAL CHEMISTRY, VOL. 46, NO. 8 , JULY 1974
BIF
=
KQ
Y = ( K q ) - ' , r = B j p . and m = q / p then:
r2
- d1
+ m(1 + y ) ] + m=O
(2)
