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Anal. Chem. 1990, 62, 1526-1528 Nelson, H.; Bend. D.; Erickson, R.; Mattson, V.; Lindberg, J. U S . EPA Report EPA/600/3-86/023, U.S. EPA: Duluth, MN, 1986. Gulens, U.; Leeson, P. K.; Segutn, L. Anal. Chim. Acta 1984, 756, 19-31. Stumm, W.; Morgan, J. J. Aquatic Chemistry: An Introduction Emphaslzing Chemical €quillbria in Natural Waters ; Wiley-Interscience: New York, 1970; p 268. Cabaniss, S. E. Envlron. Sci. Technol. 1987, 27, 209-210. Skogerboe, R. K.;Wilson, S. A.; Osteryoung, J. G.; Florence, T. M.; Batiey, G. E. Anal. Chem. 1980, 52, 1980-1962. Perrin, D. D.; Dempsey, B. Buffers for pH and Metal Ion Control; John Wiley 8 Sons: New York, 1974; 176 pages. Good, N. E.; Winget, G. D.; Winter, W.; Connolly, T. N.; Izawa, S.; Singh, K. M. M. 6iochemstry 1968, 3 , 467-477. Power, J. F.; LeSage, R.; Sharma, D. K.; Langford, C. H. Environ. Technol. Lett. 1988, 7 , 425-430. Shapho, A.; Diamond, P.; Warner, I.M. J . fhys. Chem. Patonay, 0.; 1988, 9 0 , 1963-1966. Underdown, A. W.; Langford, C. H.; Gamble, D. S. Environ. Sci. Techno/. 1985, 79, 132-136. Teasdale, R. D. J . Soil Sci. 1987, 3 8 , 433-442. Sekerka, I.; Lechner, J. F. Anal. Lett. 1978, A l l , 415-427. Fltch, A.; Stevenson, F. J.; Chen, Y. Org. Geochem. 1988, 9 , 109-1 16. Berger, P.: EwaM, M.; Liu, D.; Weber, J. H. Mar. Chem. 1984, 74, 289-295. Holm, T. R.; Barcelona, M. J. I n froceedlngs of the eound Water OeOChemktty Conference 7988; Water Well Journal Publlcation Co.: Dublin, OH, 1988; pp 245-287. Newell, A. D. An investNtbn of copper-organic complexes in the fatuxent Rlver. Matyknd; UNC, Department of Environmental Science 8 Engineerlng: Chapel HIII, NC, 1983. Cabaniss. S. E.; Shuman. M. S. Geochim. Cosmochim. Acta 1988, 52, 195-200.
Sir: While some of Ryan and Ventry’s criticisms ( I ) are correct, they have missed the principal point of our paper (2). They ignore the motivation for fluorescence quenching (FQ) measurements, which is to describe metal speciation in solutions containing humic and fulvic acids. The authors also incorrectly assert that FQ measurements alone can be used to calculate equilibrium K values for copper-fulvic acid binding withoug having to calculate free (or inorganic) copper concentration ( I ) . FQ titration data can be represented as FREL (relative fluorescence) which has a low relative standard deviation ( < 5 % ) for all data points. Ryan and Ventry argue that binding parameters CL (complexation capacity) and equilibrium K
can be determined from a model (3)that ”does not involve a calculation of [Cu2+]”( I ) . It would be indeed surprising if binding parameters determined without calculating [Cu2+] could accurately describe metal binding (predict [CuL] and [Cu2+]in solution). If this contention were true, the findings of the paper in question (2) and earlier papers ( 4 , 5) would be irrelevant. In fact, their argument is incorrect because (1)any method of determining K must include a term for [Cu2+],and the method used in ref 3 is no exception and (2) binding constants generated by the method used in ref 3 do not necessarily describe metal binding well. Although they correctly describe the fluorescence intensity as a function of added metal, this is not the purpose of the measurements. Determining KUsing FQ. Since the definition of K (eq 1) uses [Cu2+],either [Cu2+]or some mathematically equivalent term must be used to calculate or curve-fit K . [Cu2+] calculated by
(25) Perdue. E. M.; Lytle, C. R. Symposium on Terrestrlal and Aquatic Humic Materials, Chapel Hill, NC, 1981. (26) Bates. D. M.; Watts, D. 0. Nonlinear Regvesslon Anabsis and Its Applicatbns; John Wiley & Sons: New York, 1988; 365 pages. (27) Hering, J. G.; Morel, F. M. M. €nnvhon. Sci. Technol. 1988, 22, 1469-1478. (28) Plankey, B. J.; Patterson, H. H. Environ. Sci. Technol. 1988, 22, 1454- 1459.
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Current address: Chemistry Department, University of Lowell, Lowell, MA 01854.
David K. Ryan*J Lisa S. Ventry Edgerton Research Laboratory New England Aquarium Boston, Massachusetts 02110 RECEIVED for review August 14, 1989. Accepted March 12, 1990. This work was supported in part by Grant R-812101 from the U.S. Environmental Protection Agency, Office of Research and Development, and by a Donner Research Fellowship to D. K. Ryan from the University of Massachusetts, Boston, Environmental Sciences Program. L. S. Ventry was at the New England Aquarium through the Doctoral Internship Program, Department of Chemistry, Northeastern University, Boston, MA.
[Cu2+1 = CUT - [CUL]
(2) has high relative error if most of the total copper is present in bound form ( 2 , 4 ) . In the approach developed by Ryan and Weber (3,6) and adopted by others (7-9),a solution of ligand is titrated with copper and [Cu2+] is calculated by mass balance from CUTand [CuL] using eq 2. K is defined as in 1above. Combining maw balance in total ligand concentration CL = [L] + [CUL] (3) with eq 1, the fraction of bound ligand, X (X = [CuL]/CL) is shown to be a function of K and [Cuz+] K[Cu2+]
X=
1
+ K[CU2+]
(4)
Equations 4 and 2 are combined to give X as a function of K , CL, CUT,and X K(CUT - XCL) X = 1 + K(CUT - XCL) (5) Equation 5, in which [Cu2+]has been replaced by CUT- XCL), is written as a quadratic in X and the values of K and CL are found by nonlinear regression. (See eqs 3-9 of ref 3.) This method clearly relies upon a calculation of [Cu2+], contrary to the claim of Ryan and Ventry (3) quoted above; this is obvious on comparing eqs 4 and 5. Rearranging eq 5 into a quadratic does not remove the dependence on calculated [Cu2+],and neither does using FREL (proportional to (1- X)) instead of X. Describing Metal Binding. It is important to recall the motivation for these experiments. We study the binding of metals like Cu(I1) to dissolved organic material like fulvic acid because “(t)he transport, toxicity, and removal of trace metal ions in aquatic systems is intimately related to their speciation” (7). Consequently, we wish to be able to predict metal speciation in natural waters as a function of total metal,
0003-2700/90/0362-1526$02.50/00 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 14, JULY 15, 1990
Table I. pCu and X Predicted by Two Methods CuT, p M 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 15.00 20.00 25.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
calculated pCu ref 4” FQb 10.50 9.74 9.26 8.99 8.79 8.63 8.49 8.37 8.25 8.13 6.94 6.46 6.21 6.03 5.90 5.78 5.67 5.58 5.50 5.17 4.96 4.81 4.69 4.52 4.40 4.30 4.22 4.16 4.10 4.05
7.72 7.42 7.24 7.11 7.01 6.93 6.86 6.80 6.74 6.69 6.36 6.15 5.99 5.86 5.75 5.65 5.56 5.48 5.41 5.12 4.92 4.78 4.67 4.51 4.39 4.29 4.22 4.15 4.09 4.04
calculated X ref 4 FQ 0.0094 0.0187 0.0281 0.0374 0.0467 0.0560 0.0653 0.0746 0.0838 0.0930 0.1767 0.2487 0.3171 0.3811 0.4443 0.5005 0.5494 0.5970 0.6408 0.7722 0.8468 0.8915 0.9113 0.9380 0.9549 0.9584 0.9650 0.9688 0.9732 0.9806
0.0086 0.0171 0.0256 0.0341 0.0425 0.0510 0.0594 0.0678 0.0759 0.0841 0.1652 0.2422 0.3146 0.3825 0.4462 0.5032 0.5544 0.6012 0.6457 0.7762 0.8460 0.8842 0.9058 0.9312 0.9490 0.9582 0.9709 0.9729 0.9804 0.9852
Calculated by using TITRATOR (21) with binding parameters from ref 4 given in the text. bCalculated by using TITRATOR (21) with log K = 5.658, CL = 9.46 pM.
organic ligand concentration, pH, ionic strength, and other variables. FQ ought to be useful for measuring metal binding by pure solutions of weak complexing agents. Results from Cu(1I) quenching of tyrosine and salicyclic acid confirm this (3, 10). However, humic materials like FA appear to contain a mixture of strong and weak-binding ligands (4, 11). Strong-binding ligands complex most of the metal at low (Le., environmental) CuT:CLratios. Under these conditions, binding parameters detemined solely from FQ data can underrepresent the strong-binding sites (see Table 2 in ref 4), leading to large overestimates of [Cu2+]. This is particularly true if a simplistic two-parameter model is used, since in that case the binding parameters represent only the weak-binding ligands. For example, consider the results of Fish and Morel (4). A copper titration of 5 mg of C/L of Grassy Pond FA was carried out a t pH 6.0 in 1 mM NaC104. The [Cu2+]data were well described by postulating three binding components with log K and CL values of 10.52 and 0.18 pM, 8.28 and 1.34 pM, and 5.63 and 9.15 pM. We constructed a synthetic data set from these binding parameters by calculating pCu and X as a function of CUT (Table I). This data set was curve-fit by the method of ref 3 using nonlinear regression (12) on the quadratic form of eq 5, with X as the dependent variable and CuT as the independent variable. The resulting binding parameters (log K = 5.658, CL = 9.46 MM)were used to calculate pCu and X. Table I shows that the parameters fitted to X by the method of ref 3 predict X well on an absolute basis. These same parameters fit the pCu data very poorly. At low CUT (
