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Anal. Chem. 1983, 55, 298-304
Calibration of Copper Ion Selective Electrode Response to pcu 19 Alex Avdeef,’ Jerome Zabronsky, and Hans H. Stuting Department of Chemistry, Syracuse Universiiy, Syracuse, New York 13210
Aikailmetrlc titrations (pH 3-1 1) of aqueous solutions containing 1 mM Cu2+ and 11-16 mM ethylenedlamlne (0.2 M ionlc strength, KNO,; 25 “C), uslng the Beckman 39612 cuprlc Ion selective electrode and the 39501 comblnatlon glass electrode, have indlcated that the copper electrode conslstentiy responds ilneariy (99.2% Nernstlan slope) in the range pCu 3-19. Individual readings, gathered by a mlcrocomputerlred titrator, stabillred in 1-7 mln.
Biologically significant copper-binding molecules, such as amino acids, small peptides, and enzymes such as superoxide dismutase, form very stable metal complexes: the equilibrium concentrations of free (uncomplexed) Cu2+ions can be as low as lo4 to M. Cupric ion selective electrodes (CuISEs), whose emf response can be related to the concentration of free Cu2+ ions, can be valuable probes in the determination of reaction stoichiometries and metal binding strengths ( I , 2). However, the usefulness of a CuISE in the study of such metal-binding reactions can be severely limited (3-8),unless the electrode is f i t calibrated with metal-ion buffers (9) which can regulate metal ion concentrations at extremely low levels (1 ppb) limit of since it inevitably leads to a Nernstian response (12-15). This is not surprising; attempts M are to dilute unbuffered stock solutions to [Cu2+]< extremely unlikely to be successful due to trace impurities from various sources, including the electrode itself (16). The literature of CuISEs is replete with clues that the electrodes can be used to measure emf corresponding to copper concentrations at the picomolar level and that such electrode responses perhaps ought not be viewed as “anomalousn (7). Wada and Fernando (3) calibrated CuISE using NHs as a metal-ion buffer and obtained pCu (pCu = -log [Cu2+]) measurements as high as 11. Pick, Toth, and Pungor (4)also used the Cu2+/NH3system to produce calibration buffers working up to pCu 12. Hansen, Lamm, and Ruzicka (5) used Cu2+/EDTA (ethylenediaminetetraacetic) and Cu2+/NTA (nitrilotriacetic acid) buffers to obtain calibration curves (emf vs. pCu) which were linear up to pCu 12. Blum and Fog (6), using similar buffers, were able to extend the calibration curves to pCu 15,although the curves were not entirely linear. More recently, van der Linden and Heijne (7) studied metal-ion buffering with EDTA and trien (triethylenetetraamine) and were able to observe CuISE emf changes of 500 mV, suggesting very high pCu values. (Conversion to a pCu scale was not reported.) As a part of our continuing study of methods of electrode calibration, we began to explore the metal-ion buffering effected by ethylenediamine (en). Propitious circumstances to the critical examination of the CuISE response in the subpicomolar (“anomalous”) region include: (a) accurate knowledge of the relevant equilibrium constants and (b) accurate knowledge of total reactant concentrations. In the present study we addressed the above two issues (a) by
carefully determining the metal binding constants by performing nearly 70 pH-metric titrations, covering a wide range of total reactant concentrations and ionic strengths and (b) by devising a regression method to determine minor systematic errors in the total reactant concentrations in each of the alkalimetric pH titrations. In a subsequent series of pCumetric titrations, the pCu values (calculated from the precise equilibrium constants) correlated with the measured emf values in a remarkably linear fashion in the entire pCu range 3-19. Furthermore, the curves possessed >99% ideal Nernstian slope (theoretically 29.58 mV/pCu at 25 “C). Our study contributes to a significant extension of the previously reported linear dynamic range of CuISEs.
EXPERIMENTAL SECTION Stock Solutions. A standardized acidified (”0,) copper nitrate (J. Matthey, “puratronic”) stock solution was prepared. Ethylenediamine dihydrochloride was obtained by neutralizing the corresponding base (Aldrich) and purified by two recrystallizations. A standardized stock solution was prepared. The KOH titrant solution (J. T. Baker, “Dilut-it”) was prepared as previously described (17). Potassium nitrate (J.T. Baker, analyt) was used as an auxiliary electrolyte. All stock solutions were prepared and stored under moisture-saturated nitrogen in an inert atmosphere box (Vacuum Atmospheres). CuISE Pretreatment. The Beckman 39612 solid-state (CuS/Ag2S)CuISE was stored in distilled water (exposed to air) while not in use. Before each titration, the shiny surface of the pellet was buffed gently (30 s) with Orion 948201 polishing strips (1419). The electrode was then rinsed vigorously with distilled water and placed into 0.025 M H2S04for 10 min, which serves to clean the electrode (18). Before use, the electrode was again vigorously rinsed with and soaked in highly purified distilled water. We did not find it advantageous to use EDTA (5) or ascorbic acid (20) in the pretreatment procedure. Automatic Titrator. The computerized automatic titrator used in this study, an upgraded version of an earlier design (21), consisted of a Cromemco 22 microcomputer (Z80A processor) equipped with a floppy disk device (CP/M operating system). The data collection program used, T2MlB (22),was coded in FORTRAN. Interfaced to the computer were a Corning 130 pH meter, a modified Beckman 97200 electrode switch box (connecting the combination glass electrode (Beckman 39501) and the CuISE to the meter), a magnetic stirrer, and a stepper-motordriven Gilmont precision glass-piston buret assembly of our own design. Titration Method. Two series of titrations were performed. Table I summarizes the total compositions of the six solutions (first series) prepared for alkalimetric titrations using both the CuISE and the glass electrode. The solutions contained 11-16 mM em2HC1, variable amounta of Cu(NO& and HNO, (4mM), and enough KNOBto elevate the total ionic strength to 0.2 M. These solutions were prepared in the inert box by dispensing aliquota of the stock solutions with a Gilmont precision glass-piston micrometer syringe. Each of the solutions was titrated with KOH (from pH 3 to pH 11)outside of the box, using the computerized titrator. Premoistened N2was slowly passed over the solutions in order to exclude O2and COP The temperature was maintained at 25 k 0.1 OC. After each addition of titrant, the magnetic stirring was ceased, and a series of pH readings (1every 6 s) was commenced. When the average change in pH was C0.005 min-’, the pH value was recorded automatically. The computer then connected the meter
0003-2700/83/0355-0298$01.50/00 1983 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 2, FEBRUARY 1983
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Table I. Composition of Solutions Titrated and Derived Calibration Parameters total concns,a M expt [Cu(NO,),] no. (*2%) 1 2 3 4 5 6
[en+2HCl] (*00.5%) 0.011 14 0.015 02 0.015 07 0.014 92 0.015 56 01.01527
0.000 85 0.000 79 0.000 79 0.000 82 0.000 90 2.1 x 10-7e
[HNO,] (+6%) 0.000 77 0.000 77 0,000 75 0.000 80 0.000 7 1 0
comb. glass electrode
-
A
SH
E"', (mV)
SM,mV/pCu
0.10 (2)d 0.07 ( 3 ) 0.07 (3) 0.10 (2) 0.12 ( 3 ) 0.08 (3)
1.001 (3) 1.006 (4) 1.006 (3) 1.006 (2) 1.001 (4) 1.005 ( 4 )
306.1 (8) 308.1 (12) 309.7 (15) 306.6 (6) 317.2 (14) 305.4 (20)
-29.38 -29.35 -29.21 -29.38 -29.41 -26.47
M
0.24 0.22 0.22 0.22 0.19 0.19
CuISE (vs. satd Ag/AgCl (7) (9) (10) (5) (11) (11)
GOFC
2.2 2.5 1.0 1.3 2.0
1.3
a The relative errors were determined from least-squares refinement of total concentrations. Average total ionic strength (KNO, supporting electrolyte), Goodness-of-fit, eq 7 . Estimated standard deviation in the least significant digit of the preceding quantity. e Estimated "background" concentration of Cuz+,originating from various sources of impurities (see text).
to the CuISE and a series of emf measurements was performed. The emf was recorded when the average change in potential was
