Anal. Chem. 1988, 60, 2418-2421
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assayed for Na and K simultaneously in 0.1-0.2-nL aliquots, usually without dilution, within the normal concentration range of tubular fluid samples. Bicarbonate ion has been shown to spuriously enhance the Na signal; a method of avoiding this error is described. Registry No. Na, 7440-23-5; K, 7440-09-7; HC03, 71-52-3; &Boa, 10043-35-3;NaC1,7647-14-5;NaHC03,144-55-8;Na$04, 7757-82-6;Na2HP04,7558-79-4 Na2C03,497-19-8; KCl, 7447-40-7; KHCO3, 298-14-6;K2S04,7778-80-5;KzCO3,584-08-7;K2HP04, 7758-11-4.
(4) Levine, D. Z.; Byers, M. K.; McLeod, R. A.; Luisello, J. A. J. Clin. Invest. 1979, 63. 59-66. (5) Levine, D. 2 . ; Roinel, N.; De Rouffignac, C. Kidney Int. 1982, 22, 634-649. (6) Paschen, K.; Fuchs, C. Clin. Chim. Acta 1971, 35, 401-408. (7) Solera, J. J.; Crislino, L. C.; Conky, M. K.; Kahn, H. L. Anal. Chem. 1983 55, 204-208. (8) Krasowski, J. A.; Copeland, R. R. Anal. Chem. 1979, 51, 1843-1649. (9) Knott, A. R. At. Absorpt. News/. 1975, 14(5). (IO) Subramanian, K. S.;Chakrabarti, C. L. Frog. Anal. At. Spectrosc. 1978, 2, 287-308. (11) Parekh, N.; P o p , G.; Galaske, R.; Galaske, W.; Steinhausen, M. Pfluegers Arch. 1973, 343, 1-9.
LITERATURE CITED
RECEIVED for review August 12,1987. Accepted June 6,1988. A portion of this work was presented at the 29th Annual Conference of the Spectroscopy Society of Canada (1982). This work was supported by theMedical Research Council, Grant MT3836.
Kuntrlger, A.; Antonetti, S.;Couette, c.; Coureau, c.; Amiei, c. Anal. Chem. 1974, 46, 449-454. Good, D. W.; Wright, F. S.A m . J. Physid. 1979, 236(2), F192-F205. Vurek, G. G.; Bowman, R. L. Science (Washington, D . C . ) 1985, 749, 448-450.
Fluorescence Quenching Measurements of Copper-Fulvic Acid Binding Stephen E. Cabaniss' and Mark S . Shuman*
Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599- 7400
Current methodologies for calibration and analysis of fluorescence quenching (Fa)data cannot be recommended for studies of Cu binding by dissolved organic matter. Parameter estknates based solely on FQ data and a sknple 1:l model are unreliable due to strong covarlance among the three fltting parameters and large errors in computed [Cuz+] If total copper >> [Cu2+]. Addltional errors a r k because the relationship between quenchlng (0) and bound Cu concentration [CuL] Is not always linear as commonly asswned. Catlbratlng the fluorescence measurements Independently wHh a technlque more dlrectly related to metal concentratlon (as., potentlometry)Is recommended. Atthough fluorescence quenchlng is fast enough for use In klnetic studies, its error properties compare unfavorably with those of the CU-PAR spectrophotometric method In most cases.
Weber and co-workers ( I , 2) introduced fluorescence quenching (FQ) as a method for observing free ligand in copper equilibria with DOM (dissolved organic matter)
+
cu2+ L + CUL where L is a fluorescing ligand such as fulvic acid (FA). The concentration of bound copper, [CuL], is assumed to be proportional to the fluorescence quenched, Q (see Glossary for definitions). The method was originally validated with model ligands (I,2 ) and compared to ion-selective electrode (ISE) measurements (I). Binding parameters estimated from FQ titrations were compared with those estimated from other analytical techniques (2). Several researchers investigated the underlying assumptions, error propagation, and potential usefulness of the method (2-4). Some authors reported dif-
* A u t h o r t o w h o m correspondence should be addressed.
C u r r e n t address: C h e m i s t r y Department, U n i v e r s i t y of N o r t h Carolina, Chapel Hill, N C 27599-3290.
ficulties in obtaining reasonable parameter estimates (2,5, 6). The present study investigates the causes of these diffi-
culties; it employs FQ and Cu-ISE measurements to evaluate the FQ titration methodology and the usefulness of FQ for kinetic studies of Cu2+binding to a standard fulvic acid (FA). Fluorescence Quenching Titration Methodology. Saar and Weber (I)added Cu2+to soil FA solution at pH 3-6 and found that Q was proportional to [CuL] calculated from ISE measurements. Ryan and Weber (2) describe a FQ titration methodology with which to estimate average binding parameters LT and K,, (defined in the Glossary). They tested the method on the model ligand tyrosine and on a soil FA at three pH values and concluded that it could reliably determine these parameters. This method explicitly assumes that (1)the fluorescent fraction of DOM is representative of all the sample DOM, (2) the average parameters Kcu and LT adequately represent the data, and (3) 1:l stoichiometry applies. It also implicitly assumes that Q is proportional to [CuL], that is Q = AfCuL] (1) where A is a constant with a value dependent on instrument setup as well as sample characteristics. Ryan and Weber used a nonlinear regression algorithm with CUTas the independent variable and fluorescence intensity, I , as the dependent variable to estimate the parameters LT,KcU,and Imin.This approach estimates A , since (2) A = (I,,, - Imin)/& Fluorescence quenching has several attractive characteristics for environmental studies. It is fast, sensitive to low concentrations of DOM, and requires no supporting electrolyte. FQ complements metal detection techniques since it observes free ligand directly and the same instrumentation may be used for light scattering measurements (2). Fish and Morel (3) and Cabaniss and Shuman ( 4 ) noted the high relative error in Q at low [CuL]and the corresponding low error, relative to metal detection techniques, at high [CuL]. Cabaniss and Shuman
0003-2700/88/0360-2418$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988
(4)suggested that fluorescence could be calibrated by a metal detection technique and used in stopped-flow studies of CuDOM complexation kinetics. Data Analysis Problems. The fluorescence titration method outlined above has produced unreasonable binding parameters in several instances. For example, Ryan and Weber (2) note that although titrations of FA at pH 5 were reproducible and changes in fluorescence were substantial, the nonlinear regression “did not give reasonable results”. Estimates of LT from four replicate titrations were “highly scattered and often ...negative”. Newell (6) examined Cu2+ binding by whole and sizefractionated Patuxent River DOM using FQ titrations and algal bioassays (7). Fluorescence intensity was measured in a flow-through cell at pH 8.0 using an excitation wavelength of 350 nm and emission wavelength of 435 nm. The FQ titrations gave unreasonably low LT estimates ( 2 mg of C/L and pH 7.00; departure from linearity is small at DOC 5 but substantial at DOC 10. Fluorescence decreases gradually as CUT increases a t all three pH values (Figure 1). Since copper is nearly 100%
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ANALYTICAL CHEMISTRY, VOL. 60, NO. 21, NOVEMBER 1, 1988 200 ~
1
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CUTuM Figure 1. Fluorescence intensity versus C y at pH 5.14 (A),pH 7.00 (0),and pH 8.44 (0). Solution is 5 mg of C/L Suwannee River FA, 0.10 M NaCIO,. 200 I
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00
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CUT u M Flgure 2. Fluorescence intensity versus C y at 1 (0),2 (A), and 5 (0) mg of C/L Suwannee River FA. Solution is pH 7.00, 0.10 M NaCIO,.
bound in organic complexes at both pH 7.00 and pH 8.44 (13), the similarity of the titration curves is expected. High CUT causes precipitation at pH 7.00 and pH 8.44, and maximum CUTfor these titrations was
