CONCLUSION
The combination of coulostatic and potentiostatic instrumentation has been shown to be effective in improving the risetime of potential step experiments, particularly for large applied steps and for solutions which have poor conductivity. The circuits presented have a simplified design and may be conveniently connected to an existing potentiostat. Although for the circuit given, a coulostat-assisted pulse may be applied only for one polarity, alteration of the basic design to handle bipolar pulses is a logical extension of this work. The application of this device should be especially effective in the study of the kinetics of fast reactions in nonaqueous
solvents. The 3-microsecond risetime for a 3-volt step as well as the allowable 2000 Hz repetition rate make it an attractive candidate for use in IRS spectroelectrochemical studies. This instrumentation allows the shortest measurement time to be improved by at least an order of magnitude over presently attainable values.
RECEIVED for review May 4, 1972. Accepted July 10, 1972. The authors wish to thank the National Science Foundation (Grant No. GP-27744) and the Air Force Office of Scientific Research (Grant No. AFOSR-72-2238) for financial support.
Automated Determination of Permanganate and Dichromate Using a Porous Catalytic Oxygen Electrode B. Fleet and A. Y. W. Ho Department of Chemistry, Imperial College, London, S . W.7
J. Tenygl Polarographic Institute, Czechoslovak Academy of Science, Prague I , Czechoslovakia A novel approach to the automated determination of permanganate and dichromate is described. Both methods depend on the stoichiometric reactions of the analyte with hydrogen peroxide in acid medium to liberate oxygen which is then measured by a new type of coulometric oxygen analyzer. Synthetic solutions resembling those normally found in the final titrimetric step for the standard permanganate value and the C.O.D. methods were analyzed using the present procedure. Additives which were normally used in the official standard C.O.D. method (Ag+ and Hg2+)and permanganate method (phosphoric acid and urea) did not affect the analytical usefulness of the present procedure. For highly precise work, however, the standards would have to be treated in an identical manner to the samples. The present procedure is not only useful as an automated finish for standard (manual) methods but can also be readily adapted to completely automated procedures for determining oxygen demand (both the permanganate value and the C.O.D.). It thus offers an attractive alternative to the existing automated permanganate and dichromate methods based on measuring the optical densities of solutions, especially when the sample is colored or turbid.
THEULTIMATE PURIFICATION of an effluent from organic matter must involve a process of oxidation, and the assessment of the amount of oxygen required for this purpose has been the subject of much research over many years. Although no single procedure has been found satisfactory for all effluents and in all circumstances, three methods have been found useful to assess the extent of organic pollution in natural waters, sewage, and effluents by estimating the amount of oxygen utilized in the oxidation of the sample (1-3). The (1) “Standard Methods for the Examinations of Water and Wastewater,” American Public Health Association, 12th ed., New York, N.Y., 1965. (2) “Official, Standardised and Recommended Methods of Analysis,” Society for Analytical Chemistry, Savile Row, London, 1963, p 175. (3) Association of British Chemical Manufacturers-Society for Analytical Chemistry, Joint Committee, Determination of Oxygen Demand, Atialyst, 82, 683-708 (1957). 2156
first of these involves the measurement of the oxygen absorbed from acid permanganate solution (permanganate value), while the second method is based on the oxygen uptake from boiling acid potassium dichromate solution (dichromate value of Chemical Oxygen Demand, C.O.D.). These two methods are essentially chemical in nature while the third method, the Biochemical Oxygen Demand (B.O.D.) is a biochemical procedure. In general, the B.O.D. determination which measures the amount of organic matter oxidized as a result of activities of aerobic bacteria under prescribed conditions (5 days at 20 “C) is the best available single test for assessing organic pollution especially in natural waters and domestic sewage (3, 4). The test has the advantage of simulating as far as possible the natural process of oxidation of organic pollutants that actually occurs in a river or stream. With industrial wastes, however, great caution must be observed. As the B.O.D. test is so dependent on the activities of bacteria, the presence of any toxic substances or bactericides could affect markedly the B.O.D. value and hence render it useless as an indicator of organic pollution. Such bactericides include heavy metals such as lead, copper, mercury, and chromium and also phenols, formaldehyde, free chlorine, cyanide, etc. which are toxic even when present in very low concentrations. Under such circumstances, the permanganate value and the dichromate value (C.O.D.) are more reliable as indicators of organic pollution. These chemical methods have the additional advantage of being relatively quick and simple and are useful for routine control work, once the B.O.D. to C.O.D. ratio has been established for a particular waste (5). The determination of permanganate value and dichromate value (C.O.D.) has been described in detail in a number of standard and authorized methods (1-3). Such standard (4) G. McGowan, C. C. Frye, and G. B. Kershaw, “Royal Cornmission on Sewage Disposal, 8th Report,” Vol. 2, Appendix Pt. 2, Sect. 2, H.M.S.O., London, 1913, pp 93-99. ( 5 ) L. Klein, “River Pollution I-Chemical Analysis,” Butterworths,
London, 1968, p 31.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
procedures normally consist of a final titrimetric finish to determine the amount of permanganate (or dichromate) that has been consumed. With the awareness of the importance of water pollution control and the introduction of strict legislation regarding the quality of trade effluents, it is inevitable that the demand for such analyses will continue to increase. Consequently, the development of continuous or automated methods will prove to be of great value. Procedures for automating the permanganate value method (6) and the C.O.D. methods (7) have been described, both of which depend on measuring the decrease in absorbance of the reagent. Problems arise when the sewage or effluent being monitored is colored or turbid and a procedure which does not depend on measuring the optical densities of solutions has to be developed. Tenygl, Fleet, and West (8) have recently described the use of a new type of porous catalytic silver electrode in a 3electrode-system oxygen analyzer. The electrode is robust and designed to work in a flowing stream. The response is almost independent of temperature and pressure of the sample. Coulometric efficiency approaching 100% can be attained by correct manipulation of the flow rate. It was thought that by introducing a sample of acid permanganate (or dichromate) at a constant rate for a fixed interval (interspersed with water wash) into a reaction chamber or mixing coil, it might be possible to determine the concentration of the sample using the above mentioned oxygen analyzer provided that the sample reacts stoichiometrically with another reagent (which is supplied in excess) to liberate oxygen. Under such circumstances, the analytical system as a whole resembles an automated coulometric titrator. The integrated response (quantity of electricity generated) is therefore proportional to the concentration of the sample. The only common reagent which reacts with potassium permanganate and dichromate in acid solutions to give oxygen is hydrogen peroxide. In this study, automated methods are described for determining permanganate and dichromate based on their reaction with hydrogen peroxide to liberate oxygen which in turn is being measured using the coulometric oxygen analyzer. An investigation of the effects of some inorganic substances (e.g., chloride ion, silver and mercuric ions) and urea on these automated procedures has also been made. Such substances may have been the normal constituents of water and waste water or they may have been added in the standard procedures to aid the digestion process and/or to remove interferences. EXPERIMENTAL
Apparatus. SAMPLE HANDLING AND PRESENTATION. AutoAnalyzer modules (Technicon Corp., Tarrytown, N.Y.) consisting primarily of a sampler and a proportioning pump with associated pump tubes and glass fittings were used. Samples were presented at a rate of 10/hr and 20/hr for the permanganate and the dichromate methods, respectively. A ratio of sample to water wash of 1:2 was used in both methods. COULOMETRIC OXYGENANALYZER.The three-electrode oxygen analyzer has been described by Tenygl, Fleet, and West (6) A. Henrikesen, “Automation in Analytical Chemistry-Technicon Symposia 1965,” Mediad Inc., New York, N.Y., 1966, p 301.
(7) M. H. Adelrnan, “Automation in Analytical ChemistryTechnicon Symposia 1966,” Vol. 1, Mediad Inc., New York, N.Y., 1967, p 552. (8) J. Tenygl, B. Fleet, and T. S.West, Narure, in press.
Figure 1. Flow diagram for the automated permanganate and dichromate methods. Tube dimensions in brackets refer to the dichromate procedure
(8). The circuit diagrams for the potentiostat and the integrator used have also been presented (9). RECORDERS.Two Servoscribe recorders (Smiths Industries Ltd.) were used to measure both the integrated and the non-integrated signal. Flow Diagram. The Aow chart for the automated permanganate and dichromate methods is shown in Figure 1. Sample, hydrogen peroxide solution, and nitrogen are mixed; the oxygen liberated in the reaction is then carried along in the nitrogen stream and presented to the sensor after the liquid phase has been removed by passing the gas-liquid mixture through two consecutive phase separators. The second phase separator was introduced to ensure that no liquid phase would reach the sensor. Different manifolds have been tried but the one presented gave most satisfactory and reproducible results. Materials and Reagents. All chemicals used in connection with this study were of A.R. grade. 0.063N POTASSIUM PERMANGANATE STOCKSOLUTION. Two grams of potassium permanganate was dissolved in 750 ml of hot distilled water. The solution was heated to 90-95 “C for 3 hours, cooled, and diluted with distilled water to 1 liter and stored in the dark for 3 days. The supernatant liquid was then filtered through glass wool and the filtrate stored in a dark glass bottle. The solution was standardized by adding sulfuric acid and potassium iodide and titrating the liberated ion with sodium thiosulfate using starch mucilage to mark the end point. The thiosulfate was, in turn, standardized with potassium iodate. These standardization procedures were recommended in the Official, Standardised and Recommended Methods of Analysis in connection with the determination of permanganate value (2). The strength of this stock permanganate was found to be 0.063N (0.997 x 0.063N). 0.2N POTASSIUM DICHROMATE STOCK SOLUTION.Dried A.R. grade potassium dichromate, 4.904 grams, was dissolved and made up to 500 ml with distilled water. STOCK HYDROGEN PEROXIDE SOLUTION. Phosphoric acid, 5 ml, was added to 25 ml of “100 volume” hydrogen peroxide solution and the solution made up to 1 liter. The solution was stored in the dark in a dark glass bottle. HYDROGEN PEROXIDE WORKINGSOLUTION.Fifty milliliters of 1ON sulfuric acid was added to 50 ml of stock hydrogen peroxide solution and the solution diluted to 200 ml with distilled water. This solution should be freshly prepared. PURIFIED NITROGEN SUPPLY. Oxygen-free-nitrogen (British Oxygen Company) was further purified by passing through (9) B. Fleet, J. Tenygl, and A. Ho, Taianra, 19, 317 (1972).
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A
B
0.05
0.10
0.15
CONCENTRATION
025
0.20 of
0.30
7
7
7
7
0.35
KMnOq ( X N/80)
Figure 2. Calibration graph for permanganate determination an electrode assembly similar to the coulometric oxygen analyzer immediately before use.
Figure 3. ( A ) Non-integrated signals obtained for the permanganate method ( B ) Integrated signals obtained for the same series of standards Sample concentration ( X 0.125N KMnOa): (1) 0.05; (2) 0.10; (3) 0.15; (4) 0.20; (5) 0.25; (6) 0.30; and (7) 0.35
RESULTS AND DISCUSSION
Theory. Hydrogen peroxide can react either as a reducing agent or a n oxidizing agent, both in acid and in alkaline solutions. Its reactions with permanganate and dichromate in acid solutions are described below: 2 Mn04-
+ 5 H202+ 5 H+ =
+ 8 H 2 0 + 5 O2 + H20r = Cr207+ H,O 2 Mnz-
2 CrOa Chromic acid anhydride
Cr.0;
perchromic anhydride
(Ref IO)
(Ref 11)
+ 4 H202 = CrLO:i+ 4 H,O + 4 O2
The reaction between acid permanganate and hydrogen peroxide has been used in conventional titrimetric procedures for determining hydrogen peroxide but the reverse does not apply-. This is p:irtly due to tht: fact that there are already many methods for titrimetric determination of permanganate. But a mort: important reason is that hydrogen peroxide is unstable and hence is unsatisfactory even as a secondary standard. Hydrogen peroxide solutions can, however, be stabilized by the addition of a small amount of phosphoric acid which removes heavy metal ions and thus minimizes the extent of catalytic auto-decomposition. Such solutions are then sufficiently stablized to be of use in our procedures (IO) A. 1. Vogel, “Quantitative Inorganic Analysis,” Longmans, Green, New York, N.Y., 1968, p 295 (11) J. W. Mellor. “Comprehensive Treatise in Inorganic and Theoretical Chemistry,” Vol. 1 I , Longmans, Green, New York, N.Y., 1966, p 3.55. 2158
in which hydrogen peroxide is always supplied in excess. Under such circumstances, the exact strength of the hydrogen peroxide solution need not be known and a wider margin of variation can be tolerated. To prevent the hydrogen peroxide from undue exposure to direct sunlight, the reaction mixing coil, the reservoir supplying hydrogen peroxide, and the transmission tubings should all be covered with black cloth. This is a further safeguard against auto-decomposition of hydrogen peroxide. The reaction between hydrogen perioxide and acid dichromate is, in fact, more complicated than that represented in the equations above. Several intermediate products have been suggested and these have been summarized by Mellor in his comprehensive treatise (II), but these intermediates, most of which are oxides and acid anhydrides of chromium in its various oxidation states, are readily decomposed with the liberation of oxygen and formation of chromic oxide or sulfate. We have found that under the conditions employed in our procedure (large excess of hydrogen peroxide and concentrated sulfuric acid medium), the amount of oxygen liberated in proportional to the amount of dichromate present. Permanganate Method. PREPARATION OF CALIBRATION CURVE.The results obtained using dilute Permanganate standards are shown in Table I. Samples contained from 1.0 to 7.0 ml. of stock permanganate solution, together with 10 ml of 10N sulfuric acid and diluted to 100 ml. The concentration standards were chosen to cover the range from 0.05 to 0.35 X 0.125N which is the range commonly encountered in the final titration step of the permanganate
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
Table I. Integrated Signals (Recorder Units) at Varying Concentrations of Permanganate Corrected Permanganate concn Signal Blank signal 23.0 14.8 8.2 0.05 X 0.125N 29.3 14.8 14.5 0.10 X 0.12SN 35.8 14.8 21 .o 0.15 X 0.125N 0.20 X 0.125N 41.5 14.8 26.1 0.25 X 0.125N 48.2 14.8 33.4 54.0 14.8 39.2 0.30 X 0.125N 60.0 14.8 45.2 0.35 X 0.125N
value test. A rectilinear relationship between the integrated signal and concentration over the range studied is obtained (Figure 2). REPRODUCIBILITY. The reproducibility of the method and the extent of cross contamination between samples is shown in Figure 3. There was no significant difference in both the nonintegrated ( 3 4 and integrated (3B) signals obtained with the same solution whether the determination has been carried out in an increasing order of concentrations or in the reverse order. It is thus evident that there is very little influence from cross-contamination of samples. The reproducibility is demonstrated by the results obtained for 10 replicate determinations of a 0.35 X 0.125N permanganate standard (Table 11). INTERFERENCES. Two main classes of interferences can be differentiated when this method is used for assessing the permanganate value of sewage and industrial wastes. The first of these is due to the presence of oxidizable inorganic substances such as ferrous salts, nitrites, sulfides, thiosulfates, and thiocyanates (12). These would reduce the permanganate and give permanganate values which are no longer reliable as indicators of the degree of organic pollution. This limitation, however, is inherent of the permanganate method itself and is not created by our modified procedure. In some instances, the limitation can be overcome by performing a 3-minute permanganate test and subtracting it from the 4hour value. The other main class of interference is encountered from oxidizing agents ( e . g . , dichromate ion, ceric ion, etc.) which react with hydrogen peroxide in acid medium to liberate oxygen. When these interfering substances are present, the permanganate value would be lowered. Once again, this drawback is not a peculiar feature of the present procedure since all redox methods for determining permanganate would be affected by the presence of these interfering oxidants. In such cases, it is possible to carry out a blank correction with the sample and hydrogen peroxide in acid solution. In addition to the above mentioned main classes of interferences, the effect of two common occurring anions on the results obtained with the present procedure must be considered. The first of these is chloride ion which constitutes a serious interference in both the permanganate and dichromate methods. According to Roberts (13), the interference is due to the action of liberated chlorine on certain organic substances which are not normally oxidized by acid permanganate and thus high permanganate values are obtained. This interference is particularly serious if iron salts are present, for example in mine wastes. Roberts (13) also observed (12) L. Klein, “River Pollution I-Chemical Analysis,” Butterworths, London, 1968, p 120. (13) R. F. Roberts, Amdyst ( L m d o ~ i )80, , 517 (1955).
Table 11. Reproducibility of the Automated Permanganate Method. Results of Consecutive Replicate Analyses Integrated Statistically signal useful (corrected) x i - 31 ( x , - X)* information -0.5 45.2 0.25 45.4 -0.3 0.09 Mean, E = 45.1 45.2 -0.5 0.25 46.8 +1.1 1.21 std deva = 0.68 45.7 0 0 45.2 -0.5 0.25 re1 std devb = 1 . 4 8 z 46.2 0.25 +O. 5 44.8 -0.9 0.81 45.4 -0.3 0.09 47.4 +I .7 2.89 a
std dev
iqxi
-
=
re1 std dev
X)2
/I-1
st dev
=
~
).
l/Z
mean x 100%.
Table 111. Effect of Using Dilute Phosphoric Acid in the Permanganate Determination Sample A B C Strength of 0.25 X 0.125N 0.25 X 0.125N 0.25 X 0.125N KMnOl Acid used sulfuric phosphoric phosphoric NaCl (ppm) Nil Nil 500 (in final solution)
Number of replicates Mean, X, Variance, s t 2
8
8
8
47.6
46.9 0.48
46.6 0.31
0.18
that errors due to the presence of chloride ion are greatly reduced by substituting dilute phosphoric acid for the dilute sulfuric acid used in the test, We, therefore, examined the influence of phosphoric acid on the present method. The results shown in Table I11 were obtained by performing replicate analyses of 3 permanganate solutions, A, B, and C, all of 0.25 X 0.125N, but in different acid media, with one containing added sodium chloride. The mean values of the analytical signals obtained for these samples were different although they contained the same concentration of premanganate. The Student-t distribution was then used to test the significance of these differences. By using the formula (14): -
-
- xz
XI
t =
(“-
+ ____ (nz - 1) nl +
1 ) ~ ~ 2
i
q 1 : 2
’:1-’r
1
+-
a t value of 2.5 for the sets of data, A and B, was found. Similarly, a t value of 1.6 was obtained for the sets B and C. Since table-t value (14) at 0.05 level of significance and 14 degrees of freedom is 1.76, it is evident that when dilute phosphoric acid is substituted’for dilute sulfuric acid, there is a slight but statistically significant decrease in the analytical signal. For accurate work, therefore, a separate calibration curve would have to be constructed based on a series of known standards prepared in a phosphoric acid medium. The presence of chloride, on the other hand, does not cause a statistically significant change in the reaction between per(14) J. E. Freund, “Modern Elementary Statistics,” Prentice Hall, Englewood Cliffs, N.J.. 1960, p 270.
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Table IV. Use of Mercuric Ion to Mask the Interfering Chloride Ion in the Dichromate Method Sample D E Concn of dichromate 0.01N 0.01N Acid medium 12N H2SOa 12N HzSOa Chemicals added Ag+, Hg2+ Ag+, Hg2+ Concn of chloride Nil 200 ppm in final s o h Number of replicates 6 6 Mean, X i 34.86 34.78 Variance, si2 0.11 0.053
5C
-
-z
40
k
a Y K 0
30 K “
w
:
a
g 20
z
0
3 10
0.4
0.2
0.6
QB
CONCENTRATION of
1.0
K2Cr20,
1.2
1.4
1.6
L8
( X N/100)
Figure 4. Calibration graph for dichromate determination __ Dichromate solution alone
- - - Dichromate + Ag+ and Hg2+
manganate and hydrogen peroxide in a phosphoric acid medium. The other important common interference is from nitrite ion. The A.B.C.M.4.A.C. Joint Committee (2, 3) has recommended that the nitrite be destroyed by acidifying the sample and the blank, adding 1 gram of urea to each and allowing the solutions to stand for 5 minutes before adding the permanganate solution. We observed that the presence of urea in permanganate standards does not affect the analytical response with our method as the means obtained for 2 sets of 7 replicate determinations of samples X and Y (identical except for the presence of urea) were exactly equal. The use of urea to decompose nitrite, however, results in the liberation of carbon dioxide which destroys the surface of the silver electrode. Hence, this method cannot be used in the present procedure unless a carbon dioxide absorption device is introduced between the phase separator and the sensor. A more satisfactory method of removing nitrite is by the addition of sulfamic acid where the products of the reaction H+ NO,-0.SOz.NHz = N2 HS04HzO (15) d o not affect the electrode. Dichromate Method. PREPARATION OF CALIBRATION CURVE.The results obtained using dichromate standards are shown in Figure 4. Samples contained from 1.0 to 9.0 ml of stock potassium dichromate solution together with 20 rnl of distilled water and 65 ml of 18N H2S04and the solution diluted to 100 ml. A rectilinear relationship between the integrated signal and concentration over the range studied (0.2 to 1.8 X 1N) is obtained. REPRODUCIBILITY. A typical recording of non-integrated (A) and integrated (B) signals obtained for the standards used in the preparation of the calibration curve is shown in Figure 5. It is evident that no cross-contamination occurs at the sample rate used. Consecutive replicate analyses were performed on a 0.018N dichromate standard solution. The reproducibility was satisfactory with the mean of 13 determinations equal to 60.8 (recorder units), the standard deviation = 0.69, and the relative standard deviation = l .13 %. INTERFERENCES. The discussion of interferences in the automated permanganate method is also relevant to the dichromate method. Because silver sulfate has now been used in the standard method for C.O.D. to aid digestion of some organic substances ( I ) , and mercuric ion has been used to complex the chloride ion (16), the effect of silver and mercuric ions on the analytical signals obtained by this automated procedure was investigated. As seen in Figure 4, the pres-
+
B
6
Figure 5. ( A ) Non-integrated signals obtained for the dichromate method ( B ) Integrated signals obtained for the same series of standards Sample concentration (X 1.0 N K2Cr20,): (1) 0.20; (2) 0.40; (3) 0.60; (4) 1.0; (5) 1.4; and (6) 1.8 2160
+
+
+
(15) A. I. Vogel, “Quantitative Inorganic Analysis,” Longmans, Green, New York, N.Y., 1968, p 605. (16) H. A. Dobbs and R. T. Williams, ANAL. CHEM.,35, 1064 (1963).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 13, NOVEMBER 1972
ence of these two ions at concentrations similar to those normally found in the final titration step of the C.O.D. determination (1100 ppm Ag+ and 1200 ppm HgZ+)does not interfere and a linear calibration curve is obtained. The effectiveness of mercuric ion as a masking agent for chloride ion was examined by adding 1 ml of a 2% wjv sodium chloride solution to 10 ml of a 277, w/v mercuric nitrate solution. To this was added 5 ml of stock dichromate solution and 65 ml of 18N sulfuric acid (containing 2.5 grams of silver sulfate per liter) and the solution was diluted to 100 mi. The results obtained with this solution and a standard made up in exactly the same way but with the omission of chloride are summarized in Table IV. The t value for the two sets of data, D and E, was 0.5. Since this is much lower than the table-t value, at 0.05 level of significance and 10 degrees of freedom, of 1.812, then it is apparent that under these conditions, sodium chloride at concentrations up IO 200 ppm in the final solution does not interfere. By varying the amount of mercuric ion and the
volume of effluent sample used, higher concentrations of chloride can be tolerated. CONCLUSION
The permanganate and dichromate methods described here can be applied not only as an automated finish to standard procedure for permanganate and dichromate values of sewage and effluents but can also be readily adapted to a completely automated procedure for determining the oxygen demand (permanganate and dichromate value) itself. The application of such a completely automated method in the actual determination of the oxygen demand of sewage and effluents will be reported in a later communication. RECEIVED for review August 30, 1971. Accepted June 20, 1972. One of us (A. Y . W. Ho) thanks the Association of Commonwealth Universities for the award of a research grant. The authors also gratefully acknowledge the financial assistance of the Technicon Corporation.
Enzyme Electrodes Based on the Use of a Carbon Dioxide Sensor. Urea and L-Tyrosine Electrodes G. G. Guilbault and F. R. Shul Department of’ Chemistrj, Louisiana State University in New Orteam, New Orleans, La. 70122 The electrode properties of a CO? electrode are evaluated. Although the response time of a CO?electrode varies with the substrate concentration, the slopes of calibration curves agree with the Nernstian prediction, ;.e., 59 mV/dec and 30 mV/dec for NaHC03 and NH&l internal solutions, respectively. The linear concentration range i s between 10-4 and 10-1M. Urea and L-tyrosine electrodes are prepared by coupling a layer of enzyme with a COS electrode. Both electrodes show properties inherited from the COz sensor. The selectivity of these electrodes is superb. Only acetic acid shows a slight interfering effect on electrode response from pH 5-7.
DURINGTHE PAST FEW YEARS, ion-selective membrane electrodes have attracted more and more attention because of their convenience as well as their accuracy in assay. Among the many applications of ion-selective electrodes, their use in the analysis of amino acids has become increasingly important. With a layer of urease immobilized on the sensor part of a cation electrode, an electrode that shows Nernstian characteristics in potentiometric measurement of urea as a result of the formation of ammonium ion at the electrode surface has been described (1-3). By the same token, glutamine and many other amino acids have been determined with cation
sensitive electrodes coupled with an enzyme layer ( 4 , 5). Although the sensitivity and the response time of these enzyme probe electrodes are quite satisfactory, the cation interference inherited from the sensor electrode is always a major disadvantage of utilizing these electrodes in analysis. Thus, in the presence of alkali metal ions, additional pretreatment of the sample solutions is necessary in order to eliminate the cation interference effect (6). More recently, Llenado and Rechnitz have reported a new kind of enzyme electrode made by coupling the enzyme, pglucosidase, to a non-glass membrane electrode, namely, the cyanide electrode (7, 8). When this electrode is exposed to an amygdalin solution, the cyanide ion, produced in the enzymatic hydrolysis of amygdalin, gives rise to the potentiometric response of the electrode system. In addition to the applications mentioned above, specific ion electrodes can also be used to measure the activity of enzymes (9). In an attempt to develop enzyme electrodes which are virtually free of any interference in amino acid assay, we have investigated the possibility of utilizing a new sensor, namely, the COSelectrode, for the preparation of enzyme electrodes. (4) G. G. Guilbault and F. R. Shu, Alia/. Chim. Acta., 56, 333
(1971). Present address, Department of Chemistry, National Tsing Hua University, Sinchu, Taiwan, Republic of China.
(5) G. G. Guilbault and E. Hrabankova, ANAL. CHEM., 42, 1779 ( 1970).
(6) G. G. Guilbault and E. Hrabankova, A/ru!. Cliim. Acta, 52,
(1) G. G. Guilbault, R. K. Smith, and J. G. Montalvo, Jr., ANAL. CHEM., 41, 600 (1969). (2) J. Montalvo, Jr., and G. G. Guilbault, ihid., p 1897. (3) G. G. Guilbault and J. G. Montalvo, Jr., J.-Amer. Chem. Soc., 92, 2533 (1970).
287 (1970). (7) R. A. Llenado and G. A. Rechnitz, ANAL. CHEM.,43, 1457 (1971). (8) G. A. Rechnitz and R. A. Llenado, ibid.,p 283. (9) G. G. Guilbault, W . F. Gutknecht, S. S. Kuan, and R. Cochran, A d . Biochern. 46, 200 (1972).
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