Anal. Chem. 1996, 68, 3665-3669
Diffusional Titration of Metal Ions in Microliter Samples with Potentiometric Indication Huijun Xie and Miklo´s Gratzl*
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106
Calcium, magnesium, copper, and zinc ions were determined in 1- and 20-µL samples in the millimolar and micromolar concentration ranges with diffusional microtitration using EDTA as the reagent and a microfabricated gold/mercury amalgam electrode for potentiometric end point indication. Typically, a reproducibility of about 3% was obtained in 20-µL samples with nitrogen jet stirring. A spatially averaging indicator electrode designed to compensate for minor inhomogeneities was tested in 1-µL samples without stirring, yielding 4% reproducibility. Linear calibration graphs in 20-µL copper, zinc, and calcium samples and in 1-µL copper samples were obtained over broad concentration ranges. Based on the pH dependence of stability of EDTA-metal complexes, copper could be determined selectively in low-pH medium in the presence of magnesium, calcium, and other metal ions. With proper pH adjustment, the hardness of tap water could also be obtained in 20-µL samples using a combination of standard additions and dilutions. The setup developed is extremely simple yet practical for metal ion determinations in microliter samples. Diffusional microtitration employs, along with surface tension and capillary forces, a reproducible concentration gradient within a suitable membrane to deliver reagents into extremely small samples via diffusion.1-6 To titrate microscopic (femto-, pico-, and nanoliter) droplets, the diffusion membrane is placed in the tip of a pulled glass capillary filled with a buffer containing the reagent. With this simple device, called a diffusional microburet (DMB), acid/base3,4 and complexometric6 determinations have been performed under a microscope. The methodologies developed in these studies were intended for research applications with physicochemical and biological problems in mind. Another set of studies1,2,5 involved microliter-size samples (typically, from 1 to 20 µL), with the objective of developing practical analytical applications for still very small but macroscopic samples. This approach required a setup different from the one developed for the microscopic range: a planar diffusion membrane was used to separate the reagent reservoir from the sample droplet. Visual1 and potentiometric2,5 end point indications have been used so far in these experiments. The combination of available microfabrication technologies with the principles of diffusional microtitration resulted in a (1) Gratzl, M. Anal. Chem. 1988, 60, 484-488. (2) Gratzl, M. Anal. Chem. 1988, 60, 2147-2152. (3) Gratzl, M.; Yi, Ch. Anal. Chem. 1993, 65, 2085-2088. (4) Yi, Ch.; Gratzl, M. Anal. Chem. 1994, 66, 1976-1982. (5) Diefes, R.; Hui, K.; Dudik, L.; Liu, C.-C.; Gratzl, M. Sens. Actuators B 1996, SNB 030/2, 133-136. (6) Yi, Ch.; Huang, D.; Gratzl, M. Anal. Chem. 1996, 68, 1580-1584. S0003-2700(96)00262-4 CCC: $12.00
© 1996 American Chemical Society
compact device where the sample assumed an ideal semispherical shape, and the electrochemical sensor used for end point detection was directly deposited onto the inert sample holder around the diffusion membrane.5 Recently, a similar integrated arrangement was shown to be equivalent to a rotating electrode system for 20-µL semispherical samples when a mild gas jet directed tangentially toward the sample provided for its rapid stirring.7 Thus, the principles of using diffusion and other natural phenomena for microvolume sample analyses1-7 are by no means limited to titration techniques alone: electrochemical stripping analysis, and the combination of stripping and reagent delivery (necessitated by potentiometric stripping analysis), can be performed now in very small samples and without the need for any moving mechanical parts.7 In this work, the above integrated approach was combined with an indirect scheme to determine metal ions complexometrically in 1- and 20-µL samples, using a microfabricated gold/mercury amalgam electrode for end point indication. Surface tension and adhesion forces kept the microliter-size sample droplets semispherical and in place. A jet of humidified nitrogen was used to stir the 20-µL samples during reagent delivery. In 1-µL samples, diffusion within the sample was effective enough to ensure an already good degree of homogenization. The remaining minor inhomogeneities have been compensated for by using a special electrode design for spatial concentration averaging.8 Therefore, no moving mechanical parts were needed to analyze either the 1or the 20-µL samples. Ca2+, Mg2+, Cu2+, and Zn2+ were determined in the millimolar and micromolar concentration ranges using EDTA as the reagent. Hardness of tap water samples was also analyzed by this method, and the results are compared with those of conventional techniques. EXPERIMENTAL SECTION Apparatus. The setup used in this work for complexometric determinations in microliter samples consists of a reagent reservoir with a saturated calomel reference electrode and a sensor assembly (Figure 1). The latter was made of a Pyrex glass substrate (thickness, 0.23 mm) with a hole in its center for the diffusion membrane (diameter, 0.30 mm for 1-µL samples and 0.57 mm for 20-µL ones). A hydrophobic silicone elastomer ring was deposited on top of the Pyrex so that it automatically centers and keeps in place the sample droplet around the membrane. The ring was made in two sizes, one (i.d., 1.56 mm) for 1-µL and another (i.d., 4.24 mm) for 20-µL samples, such that the corresponding sample droplet automatically assumed a semispherical (7) Cserey, A.; Gratzl, M. Anal. Chem., submitted. (8) Gratzl, M.; et al. Unpublished work, Case Western Reserve University, 1993.
Analytical Chemistry, Vol. 68, No. 20, October 15, 1996 3665
Figure 1. Schematic diagram of the device and sensor used in this work for performing complexometric diffusional microtitrations. (A) Side view of the device. (B) Top view of the microfabricated sensor assembly, consisting of the Pyrex glass sample holder, microfabricated sensor and diffusion membrane. The example shown is used for titrating 1-µL samples. The shape of the gold/mercury amalgam electrode is designed to compensate for minor concentration inhomogeneities within the semispherical sample during titrations.8 Since the 20-µL samples were stirred to achieve homogeneity, the electrode shape in that case was immaterial. (The nitrogen jet stirrer is not shown in Figure 1B since the 1-µL samples did not need stirring.) The inner diameter of the hydrophobic silicone elastomer ring was 1.56 mm for 1-µL droplets and 4.24 mm for 20-µL samples, corresponding to the different circular bases of the respective semispherical volumes. Also, a larger hole was used for the diffusion membrane in the 20-µL case (diameter, 0.57 mm, compared to 0.30 mm for 1-µL samples).
shape when applied (Figure 1B shows the sensor assembly for 1-µL samples). Between the silicone ring and the hole in the center, a gold base sensor was deposited in a special spiral shape. This shape was intended to compensate for minor concentration inhomogeneities within the sample.8 This method was used only for dealing with inhomogeneities in 1-µL samples, where diffusion within the droplet was efficient enough to achieve a good degree of homogeneity during diffusional titration.1 The shape of the sensor can be arbitrary for the 20-µL sample assembly, where a mild jet of humidified nitrogen was used to mechanically stir the sample.7 (The actual sensor shape for 20-µL samples was similar to the one shown in Figure 1B, due to convenience in microfabrication.) Reagents. Copper and zinc samples were made by dissolving CuSO4‚5H2O and Zn(NO3)2‚6H2O, respectively, in pH 5 acetate buffer. The calcium and magnesium samples were made from Ca(NO3)2‚4H2O and MgSO4‚7H2O, respectively, with pH 10 boric acid buffer. The tap water samples were buffered to pH 10 with the same buffer and used in a series of standard dilutions and additions, as described later. All chemicals were of analytical grade from Fisher, Sigma, and Aldrich. Procedures. A gold base electrode of ∼3000 Å in thickness was made by thin-film sputtering on Pyrex. Details of the procedure are the same as those described in ref 5 for Pt sputtering, except in this work 50 Å of chromium was first deposited to ensure a strong adhesion of the gold layer to the Pyrex. A gold/mercury amalgam electrode was then obtained by electrodeposition of Hg on the surface of the microfabricated gold sensor from 5 × 10-4 M Hg(II)-EDTA in 0.1 M perchloric acid. A BAS 100-W potentiostat was used for the plating with a Ag/AgCl reference electrode (filled with 4 M KCl saturated with AgCl) and a platinum wire as a counter electrode. The potential for the reduction of Hg(II) to Hg(0) was set at 300 mV, a value where the reduction current density was about 0.6 µA/mm2. After 3666
Analytical Chemistry, Vol. 68, No. 20, October 15, 1996
about 40 min of plating, the nominal thickness of the mercury film on the gold (calculated from the total charge, the density of Hg, and the sensor dimensions) was about 800-1000 Å, which formed an amalgam with the underlying gold. A diffusion membrane of Sepharose gel (0.2-0.3 mm thick) was then placed into the hole in the center of the Pyrex holder.5 This completed the preparation of the sensor assembly. The potential difference between the microfabricated gold/ mercury amalgam electrode and the reference was monitored during titrations by a Radiometer PHM 84 pH meter whose output was connected to a computer for data acquisition. As soon as a sample droplet is applied with a 1- (Rainin) or 20-µL (Eppendorf) micropipet onto the Pyrex assembly, the potentiometric cell becomes complete. At the same time, the reagent begins to diffuse through the Sepharose gel membrane into the sample. The amount of the applied sample can be identified on the basis of the time needed to reach the end point.1-6 RESULTS AND DISCUSSION Shapes of Complexometric Microtitration Curves. A gold/ mercury amalgam electrode immersed into a solution containing bivalent metal ions that can form an EDTA complex and a small added amount of Hg(II)-EDTA exhibits the following potential (somewhat modified from ref 9, p 120):
E ) E°Hg +
(
)
KMeY Me2+ S S log [HgY2-] + log 2 KHgY 2 MeY2-
(1)
where Y represents EDTA, Me2+ denotes the metal ion to be titrated, and S is the Nernstian slope (59.16 mV at room temperature). The other symbols have their usual meanings. (9) Flashka H. A. EDTA Titrations; Pergamon Press: Oxford, UK, 1964.
Figure 2. Diffusional microtitration of a 600 µM copper sample of 20-µL volume with 0.05 M EDTA in pH 5 sodium acetate buffer. The titration curve (A) and its smoothed first derivative (B) are shown. The end point time, 7.81 min, corresponds to the minimum in curve B.
Since the intrinsic stability of Hg(II)-EDTA is extremely high (log K ) 21.8, from ref 9), the potential varies during a titration practically only with the last term of eq 1, which undergoes the greatest change at the equivalence point. Therefore, the titration curve will exhibit an inflection which can be indicated and used as the end point.9 For the complexometric titrations performed in this work, this end point coincides with the chemical equivalence point (in other words, there is no end point error) if sample dilution is negligible.10 With diffusional reagent delivery, this is precisely the case.1-6 Copper ions can be titrated in solutions as acidic as pH 5 because of the high stability of Cu2+-EDTA (intrinsic stability, log K ) 18.8; apparent stability at pH 5, log Ka ) 18.4, from ref 9). Figure 2A displays a diffusional titration of 600 µM Cu2+ in a 20-µL sample under such circumstances with nitrogen jet stirring.7 Since reagent delivery is monotonic (and, after an initial transient, linear) with time,2 the potential was recorded as a function of time. As eq 1 predicts, the potential decreases as the titration progresses. The end point (coinciding with the inflection point in Figure 2A) can be detected as a sharp minimum in the first derivative (Figure 2B). Reproducibility. It was found earlier that sample contamination and cross-contamination can be a severe problem in such determinations, due to sample and/or reagent adsorption onto the surface of the material used for sample holder/sensor substrate.5 The reason for this is the large contact surface-tovolume ratios involved (7.1 cm-1 for 20-µL and 19.2 cm-1 for 1-µL samples). In redox determinations using a ceramic substrate, (10) Meites, L.; Meites, T. Anal. Chim. Acta 1967, 37, 1-11.
Figure 3. Calibration for 20-µL calcium samples of 200-600 µM Ca(NO3)2 (with pH 10 boric acid buffer). Reagent, 0.05 M EDTA sodium salt and the same buffer. The figure shows also the correspondence between individual titration curves and the end points on the calibration curve. (A) Titration curves for different calcium amounts. All are shown in the same coordinate system. To visualize the relationship between the recorded curves and the resulting calibration, the end point times are plotted along the horizontal instead of the usual vertical axis. (B) Calibration curve. The end points were obtained from the smoothed first derivatives of the titration curves shown in panel A. From the graph, t° ) 2.49 min, and S° ) 1.94 nmol.
errors in the order of 25% and higher have been observed for 20-µL samples, due to high adsorptive retention by the ceramic material. When Pyrex was chosen as a substrate, however, exactly the same determinations yielded very good reproducibilities in the order of
