589
Anal. Chem. 1984, 56,589-591
Table 111. Results of Analytical Photometric Titrations of Calmagite with Cu(II)= calmagite, re1 std dev, source % PPt company A 56.3 1.1 B 51.0 4.0 54.4 2.8 C 70.8 1.5 D purified ( 2 0 ) 91.8 2.4 a For each titration 5 pmol of calmagite was taken and 2 g of titrant was required. Results are the mean of six determinations in each set.
-
-
Beyond chemical considerations, the linearity of the curves reflects the low level of drift in the instrumentation and also the benefits of signal averaging.
CONCLUSIONS The computer-controlled gravimetric titrator described here is well suited to (a) analytical applications where high precision is required and (b) the study of complexation equilibria in solution. In addition, the system can be programmed to support other applications such as the high-speed Gran titrations proposed by Yamaguchi and Kusugama (17) and the “standard-less” titrations proposed by Barry, Meites, and Campbell (18). Further refinements in the titrator-control software, particularly in the areas of computer optimization of the operational parameters involved in data collection, are under way. The titrator-control applications discussed here can be implemented on virtually any computer or microprocessor.
The amount of data collected is small, the necessary calculations are simple, and the timing and execution-speed requirements are minimal.
ACKNOWLEDGMENT This work was supported by the Natural Sciences Engineering and Research Council of Canada and by the University of Alberta. LITERATURE CITED Meltes, L. CRC Crit. Rev. Anal. Chem. 1979, 8 , 1. Guevremont, R.; Kratochvll, B. Anal. Chem. 1978, 5 0 , 1945. Moran, B. W. Trans. Am. Nucl. SOC. 1978, 30, 270. Tamberg, T. Fresenlus’ Z . Anal. Chem. 1978, 291, 124. Slanlna, J.; Bakker, F.; Lautenbag, C.; Llngerak, W. A.; Sier, T. Mikrochlm. Acta 1978, 519. Punaor. E.:Feher. 2.:Naoy, -. G.; Toth. K. CRC Crlt. Rev. Anal. Chem. 1983, 14, 175. Gans, P. A&. Mol. Relaxation Interact. Processes 1980, 18, 139. McBryde, W. A. E. Talanta 1974, 2 7 , 979. Leggett, D. J. Am. Lab. (FairfleM, Conn.) 1982, 14 (l),29. Alcock, R. M.; Hartley, F. R.; Rogers, D. E. J . Chem. SOC., Delton Trans. 1978, 115. Kratochvll, B.; Makra, C. Am. Lab. (Fairflekf, Conn.) 1983, 15 (1). 22. Lufi, L. Talanta 1980, 2 7 , 221. Chrlstlansen, T. F.; Busch. J. E.; Krogh, S. C. Anal. Chem. 1976, 4 8 ,
1051. Leggett. D. J. Anal. Chem. 1978, 5 0 , 718. Ebel, S.; Reyer, B. Fresenius’ Z . Anal. Chem. 1982, 312, 346. Kratochvll, B.; Maitra, C. Can. J . Chem. 1982, 6 0 , 2387. Yamaguchl, S.;Kusuyama, T. Fresenlus’ Z . Anal. Chem. 1979, 295,
256. Barry, D. M.; Meltes, L.; Campbell, B. H. Anal. Chim. Acta 1974, 6 9 ,
143. Martin, C. R.; Frelser, H. Anal. Chem. 1979, 5 1 , 803. Kratochvll, B.; Nolan, J.; Cantwell, F. F.; Fulton, R. B. Can. J . Chem. 1981, 5 9 , 2539.
RECEIVED for review August 9, 1983. Accepted October 20, 1983.
Welght Tltrimetry with Equivalence Point Detection by Differential Electrolytic Potentiometry Pablo Cofr6* and Georgina Copia
Facultad de Quimica, Pcntificia Universidad Catdlica de Chile, Casilla 114-0, Santiago, Chile Weight titrations have been known for a long time. They have recently been reviewed (1))and their advantage of higher precision over volumetric titrations has been pointed out by different authors (1-3). However, we could get no real advantage from this technique, if we did not have the titration end point located with the same high precision. Differential electrolytic potentiometry (DEP) has been thoroughly studied as an end-point location technique (4). When applied to argentimetry ( 5 ) ) a precision of 0.04% is obtained. In this work, we make an appraisal of the practical precision and accuracy obtained by the classical gravimetric method, precipitating silver as AgCl, and the weight titration of silver with KBr, using DEP. Results obtained are really promising and have encouraged us to apply the method to real samples. This will be the subject of a future paper. EXPERIMENTAL SECTION Apparatus. A disposable syringe was used as a weighing buret with a siliconized capillary tube attached to the needle. Drop weight delivered was 2-5 mg. 0003-2700/84/0356-0589$01.50/0
Weighing of the syringe was done with a Sartorius Model 1602 MP electronic analytical balance. Weighing of 1L bottles for solution preparation was done with a Sartorius Model 1404 MP8 top-loading balance. Twin silver wire electrodes (0.5 cm2)were made as described elsewhere (6). The constant current source was assembled as described in the literature (7). The potential difference between indicator electrodes was monitored by means of a Metrohm Model E510 pH meter attached to a Metrohm Model Labograph E478 strip chart recorder. Reagents. All chemicals were reagent grade. Procedure. Twin silver electrodes were activated by immersion in 1:l (v/v) nitric acidwater for a few seconds and then cathodiied for 1min, with a 3-V dry cell in 1:lOO (v/v) nitric acid-water with a platinum anode. Electrodes were immersed in the sample solution only when titration was completed to 95-98%. The titration was stopped when the first decrease in potential difference was observed. Titration curves were obtained by using the pipet-dilution method (8).
RESULTS AND DISCUSSION Electrode Response. This was investigated by voltammetry, and results found are shown in Figure 1. Curves A 0 1984 American Chemical Society
590
ANALYTICAL CHEMISTRY, VOL. 56,NO. 3, MARCH 1984
F
101.11
,
I
I I I
,
ia,pAU -100
-200
,
,
-300 -400 -500 -600
1
-700
,I -800
E, mV vs H g / H g 2 S 0 4 SAT Figure 1. Voitammograms in 0.1 M HNOBwith a 0.5 cm2 silver wire electrode: (curve A) before equivalence point, [As+] = 2 X lo-' M; (curve B) after equlvalence point, [Br-] = 2 X lo-' M; (curve C) supporting electrolyte 0.1 M HNO,, electrode without adsorbed nitrogen oxides; (curve D) supporting electrolyte 0.1 M HNO,, electrode with adsorbed nitrogen oxides (first scan); (curve E) same as D, second scan: (curve F) solution saturated with oxygen. Scan speed was 10 mV s-' for curves A, B, C, and F and 100 mV s-' for curves D and E.
and B show the current-potential relation of silver indicator electrodes before and after the equivalence point. Zero-current potential shifts from -50 to -350 mV when titration proceeds through the end-point region. Any substance that might be reduced within this range should interfere with the indicator electrodes. The effect of dissolved oxygen was investigated. There is a reduction current, curve F, at the end of the potential range previously mentioned; therefore oxygen does not interfere with the titration since its effect starta after the end point has been reached. Electrode pretreatment was then investigated. Figure 1 shows voltammograms of the supporting electrolyte, 0.1 M nitric acid, when different electrode pretreatments were applied. We soon discovered that the usual electrode pretreatment by immersion in 1:l (v/v) nitric acid-water could produce interference with the normal electrode response (curves D and E). There is a reduction peak current which tends to vanish as succesive voltammograms (D before E) are done on the same electrode. This was attributed to the presence of nitrogen oxides, NO or NO2,produced by reaction of silver with nitric acid, which might get adsorbed on the silver surface. The reduction peak is observed within the potential span determined. This means that as the electrode potential shifts toward more negative values when titration goes through the endpoint region, the presence of adsorbed nitrogen oxides, should interfere with the cathode, and distort the titration curve obtained. Curve C corresponds to an electrode treated as described in the Experimental Section. There is no reduction peak at all. Nitrogen oxides present on the electrode surface have been reduced by the cathodization procedure described. DEP Titration Curves. Reported DEP determination of halides with silver (5)made us choose KBr as the most suitable titrant for the determination of silver. In order to determine the best conditions to perform titrations later, we studied the influence of the polarizing current density on the titration curves. Results obtained for current densities in the range 0.5-10 pA/cm2 are shown in Figure 2. When higher current densities are used, higher peaks are obtained, which make potential measurements, E A , easier. Although the peaks obtained are broader, which might result in a loss of precision, the titration curve width measured at one-half of the peak height, when 0.2 M KBr solution is used, is in the range of 10-25 FL and we could clearly determine
VOLUME OF 0,18M KBr Flgure 2. Effect of polarizing current density on DEP titration curves:
I (HA/cm2), (A) 0.5; (B) 1; (C) 2;
(D) 3; (E) 5; (F) 10. [Ag']
=2X
lo-' M. Titrant was 0.18 M KBr.
the end point with a precision of &2 pL. From these curves we chose a current density of 2 FA/cm2 as working condition for future titrations. Peak heights, when plotted against the logarithm of the current density, give a straight line as predicted by theory (9). Smaller and irregular peaks are obtained in the presence of nitrogen oxides. When these are eliminated from solution by boiling, a well-defined peak is obtained. The amount of sample titrated has a marked effect on the titration curve obtained. There is a peak height reduction and peak width increase when larger samples are used. When larger samples are titrated, the amount of AgBr precipitate has a more pronounced adsorption effect on the ions present in the solution. When larger samples are used, electrode surface blocking is more pronounced. For this reason, final results were always obtained by dipping indicator electrodes in solution only after 95-98% of the titration had been performed. Weight Titrations. In order to asses the quality of results, a set of ten identical samples (0.5 g each) of silver foil were analyzed by gravimetry as AgCl and weight titrimetry as AgBr. Two different analysts tested the new method. Gravimetry gave a silver content of 99.94390 with S = 0.047% standard deviation. Weight titrimetry with DEP gave 99.954% with S = 0.040% and 100.049% with S = 0.04590 for the two analysts. Positive bias of results obtained by the second analyst was attributed to an incompletely dried KBr used to prepare the titrant solution. However, this did not affect the precision of results. By comparison of both techniques, we conclude that weight titrimetry could be a good alternative for gravimetry. Results obtained are as accurate and as precise as those given by classical gravimetry, if not better. One aspect that is not shown by these results is the time involved in analysis. It takes only 30 min for an experienced analyst to complete determination of one sample, by this method. This is a real advantage over gravimetry when a small number of samples is analyzed. Another advantage of this method is the possibility of automation. Our analytical balance has a BCD output which, together with the BCD output of the milivoltmeter, could be monitored, and control of a syringe pump by means of a microprocesor could be accomplished.
Registry No. Ag, 7440-22-4; nitrogen oxide, 11104-93-1.
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Anal. Chem. 1984, 56,591-593
LITERATURE CITED (1) Kratochvil, B.; Makra, C. Am. Lab. (Falrfkkf, Conn.) 1983, 15(1), 22, 24. 26. 28. 29. (2) Biler,'E. A.; Swift, E. H. J . Chem. Educ. 1972, 49, 425. (3) Thoburn. J. M. J . Chem. Educ. 1959, 38,616. (4) Bishop, E.; Cofr6, P. Anakst ondo don) 1978, 703, 162. and references therein. (5) Bishop, E.; Dhaneshwar, R. G. Analyst (London) 1982, 87, 207. (6) Cofr6, P. J . Chem. Educ. 1983, 60, 421.
(7) Hartshorn, L. 0.;Blshop, E. Analyst (London) 1971, 96, 885. (8) Bishop, E. Anal. Chim. Acta 1959, 20, 315. (9) Bishop, E. PrOC. SAC COnf. 1965, 416-429.
RECEIVED for review September 21,1983.
Accepted November 21, 1983. This work was supported by the Direccidn de Investigacidn de la Pontificia Universidad Catdlica de Chile, under Grant No. 4/82.
Air-Dried Enzyme Electrodes Gilbert Bardeletti a n d Pierre R. Coulet*
Laboratoire de Biologie et Technologie des Membranes d u CNRS, Universitt? Claude Bernard, Lyon I , 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cgdex, France Performance and characteristics of immobilized enzyme electrodes appear strongly dependent on the type of enzyme, the support, the immobilization procedure, and the conditions of utilization, namely, industrial or clinical (1-3). Their use still appears generally confined to the laboratory and is very often impaired by the conditions of storage. In our laboratory collagen films were chosen for enzyme immobilization and a mild method of general use has been previously developed for the covalent binding of numerous enzymes ( 4 , 5 ) . Beside polymembrane bioreactors, enzyme electrodes have been designed with either mono-, bi-, or multienzyme active membranes (6, 7). The aim of the work reported here was to test the performance of an air-dried enzyme electrode, to widen its field of usefulness, and to make it more easy to handle. EXPERIMENTAL SECTION Apparatus. The polarograph supplied by Solea-Tacussel, Villeurbanne, France, was similar to that used with our previously described glucose electrode (6). The sensor consisted of a silver cathode covered with silver chloride and a platinum anode on which the enzymic collagen membrane is maintained in close contact by a screw cap. The potential of the platinum anode was fixed at +650 mV vs. Ag/AgCl/Cl- and the anodic current was proportional to the amount of oxidized peroxide generated during assays at the enzymic membrane level. Reagents. Glucoamylase (GA, EC 3.2.1.3, 1,4-a-D-glUCan glucohydrolase) lyophilized powder, 50 U/mg, was purchased from Merck. Glucose oxidase (GOD, EC 1.1.3.4) grade I lyophilized powder, from Aspergillus niger, 210 U/mg, peroxidase (POD, EC 1.11.1.7) grade I lyophilized powder, from horseradish, 250 U/mg, and ABTS (2,2'-azinqbis(3-ethyl-6-benzothiazolesqlfonate))were supplied by Boehringer, France. The 80-100 pm thick collagen films (F61 and F70 type) were provided by the Centre Technique du Cuir, Lyon, France. All other reagents were pf the highest grade commercially available. Procedure. Enzyme Immobilization. Collagen films were activated by using the acyl-azide process previously developed in our laboratory. For glucoamylase (GA) and glucose oxidase (GOD) two coupling procedures, random and asymmetric were used ( 4 , 5 ) . Disks of the chosen area adapted to the electrode tip were cut out of these films and mounfed on the sensor. Electrodes were filled with 0.2 M acetate buffer and 0.1 M KCl pH 5.5. Measurements. Enzyme activities for free or immobilized GOD and GA were determined at 30 "C following the H202formation using either the POD-ABTS reagent (8) or electrochemical detection (9). For substrate determination, the enzyme electrode is immersed in 20 mL of 0.2 M acetate buffer and 0.1 M KCl, pH 5.5, at 30 "C. The electrode could be used either with glucose alone or with maltose according to the reactions leading to H2OP maltose
+ H20
2P-D-glucose
(1)
(GA)
0003-2700/84/0356-059 1$01.50/0
P-D-glucose + HzO + 0
D-gluconic acid
2
+ H202
(GOD)
(2)
(3) After equilibration the sample is added and then the current increases and reaches a new stable level from which the steadystate response time can be deduced (Figure 1). For further assays, the sample can be added successively after obtaining each plateau provided the final concentration in the reaction vessel is kept below M.
RESULTS AND DISCUSSION Among the characteristics of our immobilized enzyme electrode, the activity of the surface-bound enzyme related to the sensitivity and the thickness of the inner space between the membrane and the transducer are two important factors. In the work presented here the search for new conditions of storage, namely, to keep the enzyme electrode dry in the air a t room temperature, led us to focus on the influence of the thickness of the inner space on the electrode response. Prior to the air-drying procedure, a control experiment is performed. When glucose or maltose is added to the buffer solution into which the enzyme electrode is dipped, a current vs. time plot can be recorded and reaches a maximum value Io after a few minutes corresponding to the steady-state response time to (Figure la). Then the enzyme electrode is air-dried and kept at room temperature. To study its behavior after this treatment and a chosen period of storage, the enzymic sensor is reimmersed in buffer and tested after 1h of equilibration. When the electrode is kept plugged to the potentiostat a 10 min equilibration is sufficient to obtain a low and stable base line current allowing the best detection. After this dry storage an identical measurement is performed with the same setting of the device and the transient signal recorded (Figure lb). Comparing the curves, it can be seen that for the same variation of substrate concentration, I,, is lower than Io, showing that the probe sensitivity I / C is affected, but the time necesary to reach the plateau is strongly lowered, thus improving likewise the response time t,. These two factors were then more carefully studied for either glucose or maltose determinations as a function of dry storage. Furthermore the influence of asymmetric and random immobilization on the performances of the probe was investigated. Sensitivity. As already reported for enzyme collagen membrane electrodes the sensor responds to glucose or maltose within 4 to 5 concentration decades (5, 6). To estimate the evolution of sensitivity with dry storage, we have determined the value of I / C for the electrode which 0 1984 American Chemical Society
