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Anal. Chem. 1985, 57, 2110-2116
Performance Improvements of Gas-Diffusion Ion-Selective and Enzyme Electrodes George G. Guilbault,* Jerzy P. Czarnecki, and Mohammad A. Nabi Rahni Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148
The subsequent steps of measurements with gas ion selective and enzyme electrodes for ammonia and urea assays are discussed and experimentally examined. Improvements of procedure and lnstrumentatlon have been investigated, including a new electrode-sample cell unit, allowing easy change of the solutions, and in some cases higher sensltivlty and a faster base line recovery.
Enzyme electrodes have been in analytical use for over 20 years (1-3) and in recent years have been extensively studied ( 4 ) . These devices combine convenient electrochemical principles of operation with the selectivity of enzymes. Use of a gas-diffusion hydrophobic membrane makes an enzyme electrode even more selective. However, gas-sensing enzyme electrodes are often slow in response and require long times to reach base line recovery (5),although essential improvements in the immobilization technique have made them faster and more stable (6-8). Assays using a gas-sensing enzyme electrode consist of the following steps: (1) immobilized enzyme is exposed to a sample; (2) the enzymatic reaction produces a pH-changing gas; (3) the gas diffuses both back to the sample and toward the pH sensor (through the enzyme and the gas-permeable membrane, and the layer of the sample solution between them); (4)the gas dissolves in the internal filling solution, changing its pH; (5) the pH jump is measured; and (6) the initial state of the electrode is recovered, enabling the next assay. All of these six steps influence the electrode performance, e.g., its sensitivity, detection limit, linear dynamic range, analysis time, and the time of base line recovery. Depending on which of these analytical parameters are essential in a given case, each of the six steps can be tailored to specific needs. Step 1. We can select the mode of exposure of the enzyme to the sample (e.g., open or closed container, with or without stirring, flow-through cell, etc.): the easier the mass transfer, the faster the equilibrium. Step 2. For a given specific activity of the enzyme (units per milligram), to obtain a higher total activity of enzyme on the electrode, the thickness of the enzyme layer has to be increased. This results in sometimes slower (instead of faster) response, and, when the sample holder is an open beaker, the height of the response itself is sometimes smaller, due to the escape of some of the gaseous products. To minimize the impact of this step, a thin layer of highly active enzyme should be used. Step 3. Presently, enzymes are often immobilized on the electrodes, on a support of pig intestine or collagen. In order to get to the pH sensor, the gas has to diffuse through the enzyme gel, through the intestine or collagen layer, and through the hydrophobic membrane. The layer of the sample-solution between the intestine (or collagen) and the hydrophobic membrane, no matter how close the membranes are to each other initially, is generally a thick gap which hinders the gas diffusion. This phenomenon occurs to a lesser degree with a nonenzyme, e.g., ammonia, ISE,and is due to a swelling of the intestine or collagen membrane. The bidi0003-2700/85/0357-2110$01.50/0
rectional diffusion (toward the pH sensor and back to the sample depends on the partition coefficient (p) of the gas between the internal fillhg solution of the electrode and the sample (9). This coefficient depends mainly on the difference of the pH of the two solutions. Let us note that only when p > 1can one expect an improvement of the detection limit and of the sensitivity by decreasing the volume of the internal filling solution (in order to accumulate more gas in the solution). Otherwise, the small volume (thin layer) of the filling solution makes the response (and the recovery) faster but not higher in magnitude. However, some alternative principles can improve the process, e.g., those described by Meyerhoff, where ammonia is trapped and concentrated in buffer (10). The diffusion in step 3 depends highly on the permeability of the membranes. Different types of membranes have been examined and reported in many analytical papers (e.g., ref 7 and ll),but further investigation is required. Step 4. The dissolving of the gas in the internal filling solution, resulting in the pH jump, has also been studied, and it has been found that the lower the detection limit of the electrode, the shorter the linear range of sensitivity. For an ammonia ISE,using an NH4Cl filling solution, at a concentration of lo-' M, the Nernstian range covers 5 decades (1X lo-' M to 5.0 M), while at a concentration of M the linear range is only between 1 x M and 5 x lov4M (9). It is obvious that the lower the initial concentration of NH&l filling solution, the higher (but less linear) the initial pH jump in the vicinity of the limit of detection. Step 5. General purpose glass electrodes are used in gas membrane electrodes to sense pH changes in enzymatic analyses, since it is easy to stretch the membranes against the large, flat or slightly curved electrode tip. This results in almost inevitable trouble in the recovery of the base line, since the reference junction is usually distant from the pH-sensing glass. The gas penetrates deep into the film of the internal filling solution toward the reference junction, reaching its equilibrium concentration rather slowly. But it is even slower for the dissolved gas to diffuse out of the long and narrow space completely, and on its way out of the reference junction, the gas passes through the electrode tip, preventing it from restoring the initial (or at least acceptable) value of pH. As a result, rapid assays are possible only for samples of subsequently increasing concentration of the analfle. It has been both theoretically and experimentally shown by Ross et al. (12)that the recovery time from the f i i itself is always slower than the response time due to the differences in the nature .of the concentration gradients. Theoretically, this is the shortest possible recovery time. The recoveries discussed in this paper are practically even much slower. So, the problems of the ammonia diffusing deep toward the reference junction refer to a decrease in the recovery time in order to get it as close to the theoretical limit, as possible. If one could easily change the internal filling solution after every assay, faster base line recovery might be expected. In the present work such an electrode has been designed, tested, and compared with standard probes. Changing the internal filling solution after every assay can overcome the theoretical limitation, since there is no need for diffusion to 0 1985 American Chemlcai Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
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Flgure 2. Comparison of typical response curves for ammonia assays and base line recovery with (1) “Double Injection Electrode” and (2) regular (Radelkis)electrode: Celgard 2400 (pobpropylene)membrane: ammonia concentration 3 X lo-* M; base line recovery in buffer (0.2 M Tris pH 8.5) with Radelkis electrode (bottom curve) or with Double Injection Electrode (after injection of fresh filling solution into the electrode (top curve); B, base Ilne; BR, base line recovery; S,response to “3.
Figure 1. (a) “Double-injection”enzyme electrode, allowing easy, fast change of both sample and filling solution: (1) microelectrode; (2) opening for Internal filling solution; (3) electrode chamber; (4) sample chamber; (5) empty space; (6) droplet (film of internal filling solution); (7)O-ring; (8)sample change; (9)membranes; (10) sample (1 mL); (11) magnetic stirring spin bar (2 X 7 mm). (b) Modified jacket for regular ammonla electrode.
occur. Furthermore, the other steps of an enzymatic assay (diffusion of ammonia, special filling solution, etc.) have been investigated.
EXPERIMENTAL SECTION Apparatus. All the pH measurements have been made with a Corning pH-meter, Model 110, coupled to an Omniscribe recorder through a dc offset module (Schlumberger). The electrodes used were an ammonia sensitive electrode set, Type 015833055 (Radelkis, sold in the U.S. by Universal Sensors, P.O. Box 736, New Orleans, LA 70148), MI-410 micro-combination pH probes (Microelectrodes,Inc.), and a Corning pH-combination electrode. The hydrophobic membranes studied for their permeability were polypropylene (Celgard),“Rubber Teflon COz” (Radelkis), silicone rubber (Radelkis),and “pOz”(Corning). Their commercial names and the pore sizes (when specified by the manufacturer) are given in Table 111. Some of the membranes were laminated upon supporting layers, such as polypropylene unwoven or screen of different porosity. This porosity of the support is not specified by the manufacturer but, since the main purpose of this part of the experiment is purely practical, this specific knowledge is not required. There is one important difference between our work and those described by Arnold et al. (11): the laminated support on the membrane used by them was facing the external (sample) solution, whereas our supporting layer was placed between the hydrophobic membrane and the electrode glass surface. Their approach is quite proper for ammonia assay, but since we were concerned with both the ammonia and urea producing ammonia assays, where an extra membrane (e.g., intestine) is added, it was more appropriate to have the support reversed to reduce the escape of ammonia. The Radelkis “Fil-NH3-1”or 0.15 M NH4Clsaturated with AgCl were used as the electrode internal filing solutions. The internal filling M amsolution for the “Double Injection Electrode” was monium chloride. Initially, lo9 M HC1 is injected into the electrode which is transformed into loF3M NH4Clby the diffusing ammonia.
Figure l a shows the principle of the new design of an enzyme electrode, which allows a fast change of both the filling and the sample solutions. Both the electrode jacket and the cell are made of Plexiglas, lined inside with Teflon to reduce carry-over. Both solutions are changed by injections with syringes. After the upper electrode compartment was filled with filling solution, the solution was blown out of the lower electrode compartment with air. Only minute amounts (several microliters) of the solution remained in the compartment after such a treatment, and this is mainly in the form of a film covering the tiny 1.2 mm 0.d. tip of the electrode, extending 1.5 mm up to its reference junction. The electrode tip is the only wettable part inside the electrode compartment. The volume of the sample chamber was about 1 mL, small enough to avoid unnecessary dilution of the evolving gas, which would result in slow attainment of equilibrium, and still large enough to produce a high concentration in the filling solution. The principle of changing the internal filling solution was also applied in an alternative design shown in Figure lb. A regular pH electrode was mounted into a modified jacket, allowing a change of the internal filling solution after every assay. Reagents. Chymotrypsin, urease, bovine serum albumin, and glutaraldehyde were obtained from Sigma Chemical Co., St. Louis, MO. The other chemicals used were reagent grade. Procedure. With both the standard (Radelkis) and injection type electrodes, the immobilization procedure used was as follows: The electrode jacket was covered first with the hydrophobic membrane and then with the pig intestine membrane. “Plastic Rubber” (Duro) was used as sealant and the membranes were kept in place and stretched with an O-ring. A drop of chymotrypsin solution (0.5 mg in 0.5 mL of H,O) was put upon the intestine. After 10 min the intestine was rinsed with water and wiped with tissue paper. Twenty microliters of a 0.2 M Tris buffer (pH 8.5) solution containing 15 mg of bovine serum albumin and 5 mg of urease was applied upon the intestine and was left for 5 min to d a w the solution to penetrate (partially)into the intestine. Then 1 MLof a 6.25% aqueous solution of glutaraldehyde was added and the mixture was stirred and spread uniformly with a piece of nylon string, until the polymer formed. The electrode was allowed to dry at room temperature for 3 h and then was placed in an open jar containing water at about +5 OC. The membrane was kept about 1 in. above the water, in order to prevent drying, which usually results in peeling of the enzyme gel off of the support.
RESULTS AND DISCUSSION The performance of this new electrode unit has been tested both on ammonia as well as on ammonia-releasing urea assays. In the first series of experiments, ammonia assays were performed both with the new injection electrode and with a regular Radelkis ammonia electrode. The results are shown in Figure 2 (polypropylene membrane); results obtained with the silicone membrane are comparable. Typical urea assays
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Table I. Contribution of Some Factors in the Total Time of Base Line Recovery after Ammonia and Urea Assays with Flat Electrodes (Radelkis) '30 relative increase in recovery timen ammonia assays urea assays 50%
factor
80%
50%
80%
recovery recovery recovery recovery
removal of NH8 off the surface of the membranes into the solution (no stirring vs. stirring) 120 370 30 280 hinderance in the enzyme gel (with gel vs. no gel) (no gel) (no gel) 50 50 "The percentage values are for a relative increase for the factors given in parentheses above. They have been obtained using a complete base line recovery at regular assay conditions as a reference. For example with no stirring, the recovery time for removal of NHBoff the surface of the membranes into solution is 370% longer than that in a stirred solution for 80% recovery to a complete base line. Table 11. Response Time, Response Signal, and Time of 50% Base Line Recovery for Ammonia and Urea Analyses Using an Injection Electrode with and without Intestine Laminated on a Polypropylene Membrane with Different Adhesives" ammonia analyses no enzyme gel
ammonia analyses with enzyme gel
urea analyses enzyme gel
55 mV 1.7 rnin >20 min .5 mV 15 rnin 15 rnin
44 mV 9 min 1 min fil.ex 55 mV 7 min 15 rnin fil.ex
62 mV 2 min >20 min 23 mV 5 min 15 rnin
54 mV 2.5 rnin 1 min fil.ex 45 mV 3.5 min 5 min
92 mV 6 rnin 12 min
76 mV 6 rnin 8 rnin
Cyanoacrylic Adhesive filled
h(mV)
100 mV 1 min
t80%
nonfi11ed
20 min 87 mV 1min 20 min
h(min) ~80%
recw4.
Rubber-Type Adhesive 1O%b 50%b
No Adhesive 105 mV 4.5 min 10 min
h(mV), height of 100% response signal; tm, time of attaining 90% of the response height (rnin); recm%,time of 50% recovery of the base line (min); filled, refers to filling of the membrane pores prior to applying the adhesive in order to prevent the adhesive from blocking the pores (soluble filling); fiLex, refers to the base line recovery with changing of the filling solution. *Content of adhesive.
A
23L
curve
5
11-2
a
2-3 b
Md
'10 mm
time
Figure 3. Comparison of the base line recovery after assays of high (2 X lo-' M) and law (2 X M) urea concentrations: Radelkis ammonia electrode covered with Celgard 2400 (polypropylene) and urease immobilized on pig intestine; curve A, the mV sensitivity expanded 1OX over that of curve 9; initial urea concentration marked on the curves; BR base line recovery.
with flat electrodes are shown in Figure 3. Some of the possible reasons for the slow recovery times found with regular electrodes have been investigated, and the results are shown in Table I. These results have been obtained by calculating the percent increase in the recovery time. Two of the possible reasons for the delayed recovery were studied; i.e., the removal of the ammonia from the electrode surface into the solution and diffusion through the enzyme gel. The percentages were calculated as the ratios of the recovery time (1) with and without stirring and (2) with and without the enzyme gel.
Figure 4. Comparison of characteristic ammonia assay curves with two ammnia (Radelkis) electrodes, one kept in a loose jacket slightly touching the membrane vs. an electrode kept in a tighter jacket. Regions are (a) base line, (b) linear increase, (c) nonlinear increase, (d) inclined plateau, (e) steady plateau, and (1)base line recovery. I n cvve A, the electrode was kept in a loose jacket, sli@tIy touching the membrane. I n curve 9, the electrode was kept in a tighter jacket, pressing hard against the membrane. The internal filling solution at steady state is NH,Ci.
In the injection electrode described here, most of the sources of slow base line recovery have been reduced, except for the accumulation of the gas in and between the membranes in the double membrane enzyme electrodes. T o improve this problem, we attempted the lamination of intestine onto the porous polypropylene membrane, either with adhesive or with heat embossing (both of these methods are recommended by the manufacturer of the polypropylene membranes). Heat embossing turned out to be unsuitable (no reliable bond with intestine is formed). Hence, two types of adhesives have been examined: a cyanoakyl and a rubber type. The performance of such laminated membranes has been studied and the result
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Table 111. Comparison of Ammonia Permeability of Various Membranes@
membrane 1. K-442 (2400 between polypropylene nonwoven) 2. K-443 (2400 on polypropylene screen) 3. 2400 (0.02 ,urn) 4. 2402 (two-ply 2400) 5. 2500 (0.04 wm) 6. 4400 (same as 3 on nonwoven) 7. 4510 (same as 5 on nonwoven) 8. Teflon “COz” “POZ.* 10. silicon + net 11. intestine 12. collagen 13. “Teflon rubber” “NH,membrane“
initial slope jump pH/s pH 0.41 0.16 1.09 1.04 1.01 0.67 0.87 0.00 0.00
5.78 5.46 5.32 5.14 5.78 4.95 5.46 0.01
time of time of base line inclined steady recovery plateau, plateau, pH units min min after 1 min
3.2 3.2
4.2 5.7 11.7 8.3 4.8 8.0 4.8
3.3 3.3 3.2 0.8
1.14 0.77 3.64 2.05 2.96 1.59 2.82 0.01
notes very strong membrane very strong membrane
strong membrane membrane stronger than 2400 strong membrane very strong membrane very strong membrane
0.00
0.00
0.03
4.32
1.6
3.3
0.90
0.21 0.20 1.49
5.38 1.66 5.52
0.8 3.2 3.2
8.0 4.0 8.9
3.46 1.50 4.77
very weak membrane; firm plateau 1.5 min delayed start noisy; rather weak membrane
‘1-7 Celgard polypropylene, 8 and 13 Radelkis membranes, 9 Corning membrane, 11 and 12 without any hydrophobic membranes. Conditions: The membranes were sealed on the electrode jackets with rubber type adhesive and O-ring. “Fil-NH3-1”Radelkis filling M; recovery in 0.15 M “&I. solution (0.3 mL) in the jackets. Ammonia concentration: 3.0 X
4l 3
I
i I
E
Figure 5. Comparison of the performance of different filling solutions in ammonia detectbn for urea assays: (A) distilled water, flat electrode (highest theoretical sensitivlly); (B) “Fil-”,-l” (Radelkis), fiat electrode; (C) 0.05 M NH,Ci, fiat electrode; (D) 0.05 M KCI, flat electrode; (E) 0.001 M HCI, injection electrode; (- - -) the range of unstable signals.
are shown in Table 11, as compared to no adhesive used. The ammonia permeability of various membranes has been measured, using a Radelkis ammonia electrode and a jacket filled with NH4C1. The numerical results are shown in Table 111. Some of the typical response curves shown in Figure 4 illustrate their common features and the influence of electrode/membrane configuration on the recovery of the base line. With regular pH electrodes, put in electrode jackets and covered with hydrophobic membranes, the internal filling solution has to interact reversibly with the gas being assayed. With the new double injection electrode design, which allows quick changing of the filling solution, it is possible to use also irreversibly acting f i i n g solutions (one-assay filling solutions). Thus, there is no need to compromise between sensitivity and linearity or on the time of base line recovery. The sensitivities of some filling solutions toward ammonia have been experimentally determined and compared with the highest theoretical sensitivity (see Figure 5). In practical assays,’achieving the highest sensitivity has turned out to be impractical, especially for the double-membrane enzyme electrodes, since the residual ammonia concentration remaining after the preceding assay is high enough to give a large response with such over the sensitive filling solutions (e.g., distilled water or KC1). These observations have led to the idea of a new principle of filling solution: the diffused gas (ammonia)
10
tmejminj
Figure 6. Ammonia assays wlth 0.001 M HCI as internal filllng solution, injection electrode. The concentrations of ammonia samples are marked on the diagrams.
chemically reacts with or “titrates” the filling solutions, e.g., diluted HCl, thus changing its pH more than that in the case of regular filling solutions (like NH,Cl). The results of the experiments on the new type of the filling solution are shown in Figures 4,5, and 6. We believe this is the first time that a tip-type electrode has been used as the pH sensor for an ammonia ion selective assay. The results of Figure 2 indicate that the total response height is about the same for both flat and tip-type electrodes, using regular filling solutions (e.g., NH,Cl). This is expected, since for a partition coefficient p = 1, the equilibrium concentration of ammonia in the internal filling solution does not practically depend on its volume, which is usually much smaller than that of the ammonia-releasing sample. However, the injection electrode has proven superior over the regular electrodes in two aspects: (1)in the use of the new, supersensitive “titrating” filling solution; and (2) in the time of base line recovery (with all types of filling solutions). Figure 5 illustrates the improvement in the detectability of urea enzyme assays, when diluted HCI is used as internal filling solution. As little as lo4 M NH, is determinable. This is not very important in urea-in-blood analyses, but it may be very important in other applications. There are two important reasons for this improvement in the detection limit: (1)when ammonia titrates diluted HC1, the pH changes from 2-3 to 9-10; (2) the partition coefficient of ammonia between acidic solutions and solutions of pH 7-9 is almost infinity, compared to a the partition coefficient of 1between two solutions, both at pH 7-9. This forces all, or almost all, of the evolved ammonia to transfer into the acidic filling solution, remarkably increasing the sensitivity and improving the lower detection
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limit. The ability to greatly improve the base line recovery (in some cases) has been illustrated in Figure 2. The presence of the hundreds of microliters of the filling solution, held in conventional electrodes, squeezed deeply toward the reference junction, is eliminated with the injection electrode. Curve E of Figure 5 represents a typical titration curve differing completely from those obtained upon dissolving ammonia in regular filling solutions. Although in the HC1 curve there is a linear range (dashed section) with very high sensitivity (slope), this range is analytically unless, being extremely unstable. Still the total jump between the initial and the final branch of the curve is more than 5 times higher than that for the regular (“Fil-NH,-l”) filling solution (curve B). Let us remember that the partition coefficient p is much greater than 1 with HC1, so that actual sensitivity is even greater, due to the almost complete transfer of the ammonia into the filling solution, Figure 6 shows that the level of maximum signal using HC1 as a f i solution does not depend on ammonia concentration as much as it does with regular filling solutions; it is only the matter of slower or faster response. Such type of response (lack of proportionality, high and steep total jump of almost the same height for either high or low concentration of the analyte) suggests possible application of this electrode in “ON-OFF” type of detection devices, e.g., for sensitive triggering of valves, alarms, or switches, where the 1-V signals obtained are preferable over the usual tens of millivolt range observed. This means that the titration-type filling solution in an ISE makes a new alternative over those now used. It should be explained that the use of the injection electrode was necessary for obtaining the results shown in Figure 5 in the case of HC1. Even if it was not for the reference junction (Ag/AgCl) of the Radelkis electrode (not permitting HCl as f i g solution), flat electrodes operate on much larger volumes (at least 20-50 pL) than the film-droplet (1-2 pL) of a microelectrode tip. With regular type filling solutions, the pH response for a given concentration of ammonia does not depend on the type of the pH electrode used. In fact, it is the same with either flat or tip electrodes, but it does depend on the volume of the filling solution. Hence, large volumes would be unfavorable. The repeatability of the volume and shape of the filling solution is difficult to attain from filling to filling. This occurs especially in the case of the injection electrode, where the droplet is kept in place only by capillary forces. However, the thin, large film of a “flat” electrode is not reproducible either. Diffusion, osmosis, changing wettability and drying processes, as well as shrinking and expanding of the membrane make the film unstable. If the partition coefficient is close to 1,this instability results only in slower or faster saturation of the solution with the gas. With the highly selective partition of ammonia between a sample-solution of pH about 7 , and the filling HC1 of pH about 3, most of the ammonia is transferred from the sample to the droplet of HC1, until it has been completely titrated to the same pH as that of the sample. Then the partition coefficient drops sharply down to a value close to 1. In this way the partition coefficient drops sharply down to a value close to 1. In this way the partition coefficient, changing during the assays, produces a square-shaped curve (Figure 6), equalizing all the assays to almost the saturation level. This makes the unfavorable influence of the fillingsolution volume less significant. Typically saturation curves repeat the same pattern, as shown in Figure 4, curves A and B. The initial increase is linear (section 2-3), followed by a nonlinear increase (3-4), an inclined (almost linear) plateau (4-9, and then a steady plateau (H), Le., full saturation. A possible explanation of such a typical pattern is given in Figure 7. The film of the filling solution is not uniform, neither in
‘W 1’
time
Figure 7. Suggested explanation of the typical ammonia-saturation patterns obtained with “flat” electrodes showing three concurrent processes (these three processes overlap, producing typical plots llke those in Figure 4, to which the symbols 1, 2, 3, 4, 5 refer): (a) electrode glass, (b) electrode jacket, (c) gap between the glass and the jacket fllled with filling solution, (d) O-ring, (e) hydrophobic membrane: regions 2-3, saturation of the film of the filling solution; 2-4, saturation of the wedge-shaped part of the filling solution; 2-5, saturation into the depth of the gap between the electrode glass and the jacket, toward the reference junctlon.
its shape nor in access to the diffusing ammonia. There is the thinnest, uniform section of the film near the center of the electrode tip, a wedge-shaped ring around it, and a thinwalled cylinder between the electrode glass and the jacket. Ammonia simultaneously diffuses into all three parts, and saturation is subsequently attained. All three concurrent processes produce a curve, like the A or B curves of Figure 4, and the sections of the curve refer to the parts of the filling solution film (see Figure 7 ) . Experimental data show that the faster the response (due to the thinner film of the solution), the poorer the repeatability, when the jacket or the solution is changed. These differences can be as large as 100%. The most probable explanation is in the tolerance of the mechanical fitting of the electrode and the jacket. Let us consider an example: if the opening in the jacket is 5 mm with a tolerance f 0.5 mm, then the outer diameter of the electrodes can differ from 4.90 to 5.00 mm. Actual electrode jacket sets are machined to exact specifications, with the intention to make the gap as narrow as possible, but, instead, have gaps differing from 0.00 mm to 0.15 mm. If a larger gap was assumed (e.g., 0.2 mm), the actual gaps would be 0.1 mm to 0.3 mm larger, but more uniform (differing by a factor of 3, instead of a factor larger than 15). The conclusion is that it is better to design electrode jackets with a looser fit to the electrode glass; since even with the same jacket, it is easier to assemble the electrode in the same way each time. Porous membranes stretch against electrode tips mostly irreversibly, producing irreproducible conditions of diffusion, especially in the wedge-shaped part of the solution (area 2-4 of Figure 7). We suppose that the main source of the delay in the base line recovery in ammonia assays is the cylindrical part of the solution in the inside of the jacket (region 2-5 in Figure 7 ) . Ammonia dissolved there can only very slowly diffuse out, lagging for many minutes, especially in more concentrated samples (Figure 3). Table I and Figure 2 also explain some reasons of the delay. With enzymatic analyses, requiring two membranes with another film of sample solution between the membranes, the delay is especially prolonged. The results of Mascini and Guilbault (7) (comparatively fast base line recovery) were obtained with enzyme immobilized directly on a Teflon membrane without intestine/collagen, and thus without the additional layer of the sample solution between the membranes. The intestine support is only used
ANALYTICAL CHEMISTRY, VOL. 57, NO. 11, SEPTEMBER 1985
n
(A I
Flgure 8. Improvement of base line recovery with the modified jacket (curve A), compared with a regular jacket (curve B); lo-* M NH, has been assayed.
IO 20 tlrne(m1T
30
Flgure 9. Typical response curves for ammonia assays using the modified jacket electrode (Note: lower concentrations can readily be
measured after higher ones). because of the poor adhesion of enzyme gel to Teflon. The main limitation in the total time of assays is the base line recovery. According to our investigations this consists mainly of two major components: retention of the gas (1) in the cylindrical part of the filling solution and (2) between the two membranes. In the case of ammonia assays there is only the first component, and thus, the “Double Injection Electrode” has reduced the base line recovery of ammonia assays from minutes to seconds (Figure 2); 1 min is sufficient for a full cycle of ammonia analysis and recovery. This superiority of the injection electrode over regular noninjection electrodes is much less dramatic with urea analyses, where the second of the two sources of the base line recovery (lag retention of the gas in and between the membranes) is in effect. This is even a greater effect due to the lack of any mechanical support to stretch the membranes tight. The experiments with laminated membranes (Table 11) show that lamination offers a possible solution. The alternative design is to change the filling solution, and the results are described in Figures la, 8, and 9. Only the thinnest section of the solution film remains unchanged, while the filling solution of the wedgedshaped and cylindrical sections of the jacket (the main source of lag in recovery) is periodically changed. The design of the electrode jacket allows easy, rapid changing of the internal filling solution between measurements, without disassembling the electrode. This reduces the time of base line recovery and allows more analyses per hour. As it is illustrated in Figure 8, 90% base line recovery takes about 30 min for curve B, whereas, it is about 1 min for curve A. Rapid analyses of lower-concentrated samples can follow higher-concentrated ones, without long waiting periods. Such electrodes are now commercially available from Universal Sensors, Inc. The examination of the permeability of different membranes was intended to determine the following: (1)whether there are significant differences in ammonia permeability between different pore-sized membranes, and, if one- or two-ply membranes are better; (2) if the advantages of reinforcement (offering better mechanical support and resistance,
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so thinner films of filling solution can be achieved) prevail over the disadvantages of the larger distance between the glass surface and the sample; (3) to what extent the permeability is different for different materials (polypropylene, Teflon, silicone); and (4) whether the limiting step of the diffusion is in the intestine/collagen or in the hydrophobic membrane. The results of the examination are given in Table 111. It can be concluded that the best of the membrane materials studied is double-ply polypropylene (one-ply has unidirectional textural flaws, causing its cracking when stretched against the electrode tip). Both intestine or collagen alone are almost as permeable for ammonia as polypropylene, and this gives evidence that they alone do not cause that observed delay in the base line recovery of enzyme electrode. The delay is rather due to the solution gap. Paradoxically, thicker membranes (Table 111)have a faster response. A probable explanation is that thicker membranes are stronger, allowing thinner films of the filling solution. Hence a larger part of the electrode tip is covered with the thin film section of the solution layer. When the membrane is mechanically stronger and pressed closer against the glass, the fiim of the filling solution is made thinner, thus giving rise to a faster response. This is in good agreement with Arnold’s (11) finding that this influence is much stronger upon the response time than on the recovery time; the latter is many times slower due to factors other than just the film thickness. Another factor influencing the final analytical performance (i.e., the internal filling solution) has already been partially discussed with the respect to the new “titration” principle of filling solution. Figure 5 shows a comparison of potential electrode performance with regular filling solution (“FilNH3-1”) and with several other, more sensitively responding solutions (KC1, HC1). Added for comparison is pure water, the solution with the highest possible sensitivity. The results shown in Figure 5 confirm the assumption that concentrated common-ion buffers (like “Fil-NH3-1”applied in ammonia or urea assays) are not very sensitive but are selective and offer linearity over a wide concentration range. They can be recommended for assay without changing of the filling solution. However, fast analyses are limited to samples of subsequently increasing concentration, otherwise prolonged base line recovery is required. The more sensitive filling solutions (e.g., dilute NH4Cl) has a lower limit of detection and also higher sensitivity (slope), but the linear range is small at higher concentrations. When the filling solution is based on use of a common ion buffer (common with the analyte), the solution is selective toward that ion. Such buffers reduce the sensitivity toward any changes in pH involving the influence of the common ion, but they reduce the sensitivity toward other ions to a much greater extent. However, permeable hydrophobic membranes prevent most other substances (except for a limited group of gases) from interfering with ammonia analyses. Thus, in some analytical problems, where sensitivity and detectability are more important than selectivity, use of more sensitive “one assay” filling solutions (e.g., KC1, diluted NH4C1,HC1) with the “double injection electrode” described here can offer another alternative. Such a system allows fast, trouble-free changing of the filling solution after every assay. This can make an assay and, sometimes more important, base line recovery, as fast as those in Figure 2, e.g., about 1min. Initially, ammonia reacts with HC1 filling solution, producing ammonium chloride which ultimately leads to ammonia/ammonium chloride equilibrium. The resulting pH inside both the regular and the injection type electrodes is almost the same, but the total signal jump for the injection electrode is higher, since it starts from a lower pH value. The initial concentration of ammonia to neutralize HCl does not sig-
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Anal. Chem. 1985. 57. 2116-2120
nificantly deplete the analyte, provided that the sample volume is large enough. Although, the final steady-state values are the same for both the electrodes, the main advantage of using HC1 instead of NH&l is much faster recovery. Changing the internal filling solution can be made using both Double Injection (microelectrode) and modified jacket electrodes. The limitation of the Double Injection sensor is in its dynamic properties, whereas the modified jacket electrode cannot use filling solutions antagonistic to the reference junction. In the case of enzyme analyses, interfering gases, if any, can be removed from the sample prior to the analysis by any of the degassing techniques, such as those developed in liquid chromatography.
CONCLUSION In order to improve the analytical performance of ammonia and urea ISEs, five out of the six steps of an individual assay have been studied. The “Double Injection Electrode” described here, built on a tip-microelectrode, improves the changing and holding of the sample, dissolution of the gas in the filling solution, pH measurement, and especially base line recovery, making the ammonia assay extremely fast (full cycle
with base line recovery less than 1 min). A new “titration” principle of a gas-ISE filling solution has been proposed and found to be useful for some analytical problems. Registry No. Ammonia, 7664-41-7; urea, 57-13-6.
LITERATURE CITED Clark, L. C.; Lyons, C. Ann. N . Y . Acad. Scl. 1962, 102, 29. Updike, S.; Hicks, G. Nature (London) 1967, 2 1 4 , 986. Guilbault, G. G.; Das, J. Anal. Blochem. 1970, 3 3 , 341. Guilbault, G. G. “Handbook of Enzymlc Analysis”; Marcel Dekker: New York, 1977. Riley, M. In ”Ion Selective Electrode Methodology”;CRC Press: Boca Raton, FL, 1979;Vol. 11, Chapter 1. Guilbault, 0. G.; Tarp, M. Anal. Cbim. Acta 1974, 7 3 , 335. Mascini, M.; Gullbault, G. G. Anal. Chem. 1977, 49, 795. Havas, J.; Guilbault, G. G. Anal. Chem. 1982, 5 4 , 1999. Hansen, E. H.; Larsen, N. R. Anal. Chim. Acta 1975, 7 8 , 459. Meyerhoff, M. E. Anal. Chem. 1980, 52, 1532-1534. Arnold, M. A. Anal. Chlm. Acta lg83, 154, 33-39. Ross, J. W.; Riseman, J. H.; Krueger, J. A. Pure Appl. Chem. 1973, 3 6 , 473-487.
RECEIVED for review November 5, 1984. Accepted May 20, 1985. The authors kindly appreciate the financial support of the Environmental Protection Agency (Grant No. R808532).
Chronopotentiometry at a Dropping Mercury Electrode: Effects of Electrode Sphericity for an Electrode Process with a Preceding Chemical Reaction Jesus Galvez* and Maria L. Alcaraz Laboratory of Physical Chemistry, Faculty of Science, Murcia 30001, Spain
Tomas Perez and Manuel H. Cordoba Laboratory of Analytical Chemistry, Faculty of Science, Murcia 30001, Spain
A theoretical study of the kinetic response for the CE mechanism in DME chronopotentiometry by using a perturbation function of the form Z ( t ) = ZoPis presented. Equations for the potential-time curves and for the transition times have been derived by taking into account the sphericity of the electrode. An experimental verification of the theory has been carried out with the Cd2+/EDTA system by using the function Z ( t ) = l o t .
Table I. Notation and Definitions
heterogeneous rate constants of the forward and reverse charge-transfer reaction apparent heterogeneous rate constant for charge transfer at Eo rate constants of the chemical reaction equilibrium constant of the chemical reaction ( = k 2 / k l ) constant of proportionality of electrode area time-dependent electrode area (=Aot2l3) rate of flow of mercury (3m/47rd)‘i3
In a previous paper (I),we developed the theory concerning the use of the perturbation function I ( t ) = IOtW+1/6 (w 2 0) for a single-transfer reaction. We adopted the expanding sphere electrode model (ES) and showed that the effect exerted by the sphericity of the electrode on the transition times may be so great that the more simple model of expanding plane electrode (EP) for the DME is not valid. On the other hand, the theory for the CE mechanism in DME chronopotentiometry has been also derived (Z), although the E P model for the DME was used. Hence, the aim of the present paper is to extend the corresponding theory for this mechanism by adopting the ES model and to make clear the influence exerted by the curvature of the electrode on the kinetic response. The theory has been tested by obtaining the rate constant values for the system Cd2+/EDTA from measurements of poten-
spherical correction parameter (=(12Di/ (7$2))’/2t’/6) (12Di/(7$2))’/2 (Dc/OD)
time-dependent electrode potential E(t)- E o
time-dependent Faradaic current (=IOt”+1/6) constant applied rate of Faradaic current increase (eq 1) kinetic transition time for the ES model transition time for the ES model when no kinetic effects are involved Euler gamma function other definitions are conventional
tial-time curves ( E / t ) and transition times.
THEORY Notation and definitions are given in Table I. A CE mechanism is described by the scheme
0003-2700/85/0357-2116$01.50/00 1985 American
Chemical Society
