tional Ag/AgI electrode in equilibrium systems also (see Figure 4). The following observations are in order: The solubility limit is somewhat higher for the Ag/AgI electrode; the response of this electrode to changes in [I-] was more sluggish, perhaps because equilibrium established itself more slowly on the finely divided AgI(s); and in an acidified iodide solution through which oxygen is bubbled, the potential of the Ag/AgI electrode is shifted to more positive values-i.e., in the direction that would increase the difference between the potentials of the two electrodes in Figure 3. There seems
little doubt that the presence of oxidizing agents produces a mixed potential at a n Ag/AgI electrode. ACKNOWLEDGMENT.
The many helpful suggestions of Karl H. Pearson are gratefully acknowledged. RECEIVED for review June 30, 1969. Accepted August 27, 1969. This work was supported by the Robert A. Welch Foundation under Grant A-254.
Sensitized Cation Selective Electrode J. Montalvo, Jr., and G . G . Guilbault Department of Chemistry, Louisiana State University in New Orleans, New Orleans, La. 70122
IN A RECENT STUDY (I), the results of a thorough investigation of the response, selectivity and use of the cation electrode for determination of NH4+was reported. The application of this electrode to a study of the kinetics of deaminase enzyme systems (urease, asparaginase, glutaminase, amino acid oxidase, and amine oxidases) was likewise reported. In a more recent study (2), urea was determined by immobilizing the enzyme urease in a layer of acrylamide polymer on the glass surface of a cation electrode sensitive to ammonium ion. The substrate urea diffuses to the enzyme electrode and reacts with the immobilized enzyme to produce ammonium ion at the surface of the electrode. In this paper, the use of a film of immobilized urease on a glass electrode is reported
for the determination of cations. EXPERIMENTAL
Preparation of the Coated Electrode. The gel solution was prepared by dissolving 3.0 grams of acrylamide monomer and 0.58 gram of N,N’-methylene-bisacrylamide (Eastman) in 25 ml of 0.1M tris buffer, pH 7.0. To catalyze the photopolymerization, 2.7 mg of riboflavin and 2.7 mg of potassium persulfate were added. The solution was stored in the dark at room temperature until used and remade every two days. One milliliter of the polymer solution was pipetted into a small centrifuge tube containing a weighed amount of enzyme, 175 mg urease/cc of gel solution. The enzyme suspension was stirred for two minutes and was allowed to dissolve for 20 min at room temperature. The mixture was chilled in the refrigerator at 2 “ C for 10 min and centrifuged for 20 min. The clear supernatent was carefully transferred to a test tube with a 21-mrn i.d. A monovalent cation electrode (Beckman Instrument Co., Catalog Number 39137) was washed well with distilled water, wiped dry with tissue paper, and mounted upside down. A strip of nylon net, 350 p thick, (nylon stocking, J. C. Penney and Co.), 2” X 2”, was placed over the glass bulb of the electrode. A rubber “0” ring was used to anchor the netting over the rigid glass bulb. This “0’ring was placed about inch below the sensing part of the glass bulb and held the netting in place while a second “0” ring was (1) G. G. Guilbault, R. Smith, and J. Montalvo, Jr., ANAL.CHEM., 41, 600 (1969). (2) G. G. Guilbault and J. Montalvo, Jr., J . Amer. Chem. SOC., 91, 2164 (1969).
placed just below the edge of the sensing glass bulb. The first “0” ring was cut away and the netting over the glass bulb carefully inspected for any folds which can be removed by pulling on the netting below the remaining “0” ring. The netting was then cut flush with the “0” ring. The resulting electrode was dipped into the enzyme gel solution making sure all of the pores of the netting are filled with solution. The electrode was then removed from the solution and the glass wall above the “0” ring wiped free of solution. Excess liquid does not cling to the netting because of the surface active enzyme preparation. The netting gives mechanical rigidity to the enzyme gel layer and also is used to control its thickness. The electrode was placed in a water-jacketed tube. Oxygen inhibits the polymerization and was removed by bubbling with NP through the tube for 15 min. The polymerization reaction which requires light, was irradiated with a G. E. BBA photoflood lamp equipped with a reflector. The controlled temperature in the photopolymerization was 28 “C (measured with a mercury bulb thermometer in place of the electrode). After one hour of polymerization, the electrode was equilibrated in tris buffer for one day before use. Apparatus. A Beckman Zeromatic Model I1 pH meter and a standard fiberjunction, saturated calomel reference electrode (SCE) were used. Millivolt measurements were made by operating the pH meter in the h 7 0 0 mV range and were recorded on an Electroscan 30 (chart speed of the recorder varied from 20 to 100 seciinch). In those studies involving Ag+ ion a salt bridge filled with tris-HNOs buffer was used to prevent contamination of the SCE. All measurements were carried out in a thermostated cell at 25 f 0.01 OC. Chemicals. Tris(hydroxymethy1amino-methane)buffer, was used to maintain a constant pH 7.0, and a constant ionic strength, 0.1M. The enzyme urease was obtained from the California, Corp. for Biochemical Research, Grade B (activity 375 Sumner units per gram of enzyme). Procedure for Determination of Cations. Potentiometric measiirements of the steady state response for the construction of calibration curves and the study of the effect of various parameters (film thickness, gel concentration, etc.) on the steady state response for the coated electrode were carried out in the conventional manner. All solutions were magnetically stirred with a bar made of Teflon (Du Pont). An aliquot of the cation to be determined (NH4+, Na+, etc.) is pipetted into a 100-ml beaker containing 50-ml of buffer. The steady state potential is read, and the ion concentration of cation is determined from a calibration plot of potential us. log of cation concentration. After determination of an ion, the film of immobilized enzyme was washed free of the ion by placing both the coated electrode and the reference
ANALYTlCAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
1897
I
W
I
I
-2
I
-3
Log [NH,CL],
li
I
-4
M 30
Figure 1. Calibration plots of potential (us. SCE) us. log NH4+ ion for the uncoated ( A ) and the coated ( B ) electrodes A 0
0
wI
I
+
98% r e s p o n s e
Enzyme electrode Cation electrode TIME ( S e c )
Figure 2. Dynamic response curves for the enzyme coated ( A ) and uncoated ( B )electrodes to NH4+ion
electrode in the automatic electrode washer. The potential is monitored until a low level of ion concentration is obtained in the enzyme gel layer. The rate of flow of tris buffer through the electrode washer was 4.35 ml/min. RESULTS AND DISCUSSION
Response of Electrode to Ammonium Ion. The experimental curve obtained for the response of the uncoated electrode is shown in Figure 1, Curve A. The response is not completely Nernstian, a slope of about 56 mV/decade change in concentration is obtained. The response is linear with concentration in the range 10-l to 10-3M NH4+ ion. Below 10-3M NH4+, however, the response was nonlinear with concentration for the solutions studied. Various pretreatments of the electrode did not extend the linear range to lower concentrations, although a faster, better response was obtained regardless of the NH4+ concentration if the electrode was soaked in triply distilled water for several days before use. More drift was observed if the electrode is soaked in NH4+ solution before use. Hydrolysis of the NH4+ ion in solution was considered (1) in calculations of the activity reported in Figure 1, Curve A . At a pH of 7, the ratio of [NHd+]/[NHJ is about 180; hence almost all of the NH4+is in the cationic form. Curve B in Figure 1 was obtained when the electrode is covered with a film of immobilized urease, 350 I.( thick and containing 175 mg urease/ml of gel solution. The coated electrode has a response that is 31 mV more positive than the uncoated electrode, and hence allows a more sensitive measurement. The slope of Curve B is identical to Curve A . Besides being more sensitive to NH4+ion, the response of the coated electrode is linear with NH4+ion down to about 5 X lO-jM, whereas the uncoated electrode is linear only to 10-3M NH4+. The increased response and more linear response at low NH4+ concentrations is due to the fact that the film of immobilized urease is acting as a cation exchanger. When*the electrode is covered only with the gel layer, which contains no enzyme, the potential response of the electrode us. NH4+ion concentration is the same as when the electrode is uncoated. This is because the polymer is neutral and possesses no net fixed charged groups at pH 7. On the other hand, when the urease preparation is added to the gel, fixed negative charges from ionized groups of the enzyme (iso-electronic point, pH 1898
9.5
x ~O-~MNH,CI
5.5) give to the gel cation exchange properties. This lends to an increase in positive potential response for a given concentration of NH4+ ions, so Curve B lies above A in Figure 1. Dynamic Response Characteristics. The response characteristics of the coated electrode were evaluated by exposing the electrode to a rapid change in ammonium ion concentration and recording the potential us. time curve. A typical response curve to 9.5 X 10-3M NH4Cl is shown in Figure 2. The time interval for 98% of the steady state response for the uncoated electrode is 23 sec for 9.5 X 10-3M NH4Cl. The time required for the diffusion process to reach the steady state depends primarily on the film thickness for the enzyme coated cation electrode. This suggests that diffusion into the enzyme gel layer is the slow step in reaching the steady state. The time interval for 98% of the steady state for the coated electrode for 9.5 X 10-3M NH4Cl is 23.5 sec for a 60 I.( enzyme gel layer, and 42 sec with a 350-11 coating, Curve A, Figure 2. With 175 mg urease/ml gel solution the steady state response potential to NH4+ was essentially independent of the enzyme gel film thickness in the range 60-350 11. The response was likewise independent of the gel composition when varying the per cent gel from 5 to 17.6 at constant ratio of monomer/ crosslinking agent or variation of the per cent crosslinking agent from 5 to 19 at constant gel composition. Increase of the enzyme concentration in the gel from 175 to 230 mg/ml gel did not significantly increase the response. After determination of an NH4+ ion concentration, the electrodes are removed from solution and the enzyme gel layer is flushed out in a automatic electrode washer. Figure 3 shows typical wash curves for lo-* and 10-3M NH4C1with a 350-11 enzyme gel layer thickness. The washout time decreases with decrease in enzyme gel layer thickness, increase in flow rate of buffer through the electrode washer, and decrease in NHa+ion concentration. The high response rate of the electrode to NH4+ ion and the high washout rate of NH4+ion from the coating indicates that continuous monitoring of ammonium ion activity in aqueous solutions is possible. Excellent reproducible voltages (d~0.5 mV average) over a 19-day period at 10-3M
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
W V v)
in
>
> E
W’
a
0
I
I
I
50
100
150
Time
1
(Sec)
Figure 3. Wash curves for the enzyme coated electrode. Wash rate 4.35 ml/min; 350 v thick netting A : 10-’M NHdCI B: 1 0 - 3 NHXI ~
NH4+ ion were obtained. This means that after the initial calibration curve is obtained, the electrode could be used for a long period of time without recalibration. Essentially all other instrumental methods for NH4+ ion and other cation determinations, such as flame photometry, require frequent recalibration. Response of Other Cations. The response characteristics of the coated electrode to other monovalent cations investigated, Ag+, K+, H+, Na+, and Li+, was identical in all respects to the NH4+ion studies. Increased response was always obtained relative to the uncoated electrode. The readings were highly reproducible from day to day. The response of the uncoated electrode was found to be Ag+ > K+ > H+ > Na+ > Li+ >> Mg2+,Ca3+. In order to establish the selectivity of the enzyme electrode, the selectivity ratios, K, defined by Eisenman’s simplified equation for glass electrodes (3):
where determined with respect to NH4+. These results indicate almost the same selectivity order for the coated and uncoated electrodes. The coated electrode showed essentially no response to Mg2+ and Ca2+. This is similar to that reported for the uncoated electrode ( I ) . Because there is no significant change of ionic specificity of the electric potential when one surface of the cation electrode is coated with a relatively thick enzyme gel layer, it is difFi(3) G. A. Rechnitz, Chem. Eng. News, June 12, 1967,p 146.
Table I. Cation Selectivity Ratios Relative to Na+ for the Sensitized Cation Electrode Selectivity ratio (Khfl + / M x a+) Cation Found ( 1 ) Calculated (2)0 &+ 6.62 x 6.63 x H+ 9.80 X 9.81 X K+ 1.00x 10-1 1.00x 10-1 NH4+ 2.36 X 10-l 2.35 x 10-I Li+ 1.56 X 10’ 1.56 X 10‘ Calculated from K L +INa+ = KL +/NH$+ / K N a+,WH~+.
cult to distinguish the coated and uncoated electrodes. While the other surface of the coated electrode remains as good and sound a glass electrode as ever, the enzyme film makes the glass electrode more sensitive to cations, but does not seriously affect the selectivity order. This could mean that the selectivity order of the enzyme film is the same as that of the cation electrode or that the high ionic permeability and leakage through the enzyme gel film offsets any distinct selectivity of the enzyme exchanger. Eisenman ( 4 ) showed that the sensitivity of a cation, Lf, relative to a M+ cation is interrelated to the sensitivity of the L+ and M+ cation relative to a third cation, N+, by
(3) for a glass electrode. Cation selectivity ratios relative to Na+ for the coated electrode are shown in Table I. The ratios in column (1) were determined using Equation 2 with M2+being the Na+ ion. The calculated values in column (2) were obtained with Equation 3, where N+ is NH4+ ion. The excellent agreement of the found and calculated selectivity ratios for the coated electrode shows that Equation 3 can be used to calculate selectivity ratios relative to any ion to which the coated electrode is responsive. This work has given sufficient information about the practical applications of the enzyme coated electrode to show that further study and improvement of this type of electrode is needed. The insensitivity and freedom from interference from alkaline earth ions, the ease of preparation and handling, the increased sensitivity, and the more linear response to low cation concentrations should offer more opportunities to extend the usefulness of this electrode for rapid cation determination. RECEIVED for review April 23, 1969. Accepted August 28, 1969. This work was supported by the National Science Foundation, Grant No. GB 12669. (4) G.Eisenman, Ed., “Glass Electrodes for Hydrogen and Other Cations,” Marcel Dekker, New York, 1967,p 270.
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
1899
