EPA-600/4-76-034
October 1976
Environmental Monitoring Series
BIOLOGICAL ELECTRODES SPECIFIC FOR
PHOSPHATE AND NITRITE IONS
^
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, Georgia 30601
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL MONITORING series.
This series describes research conducted to develop new or improved methods
and instrumentation for the identification and quantification of environmental
pollutants at the lowest conceivably significant concentrations. It also includes
studies to determine the ambient concentrations of pollutants in the environment
and/or the variance of pollutants as a function of time or meteorological factors.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/4-76-034
October 1976
BIOLOGICAL ELECTRODES SPECIFIC FOR
PHOSPHATE AND NITRITE IONS
by
George G. Guilbault
University of New Orleans
New Orleans, Louisiana 70122
Number R-800359
Project Officer
Thomas B. Hoover
Analytical Chemistry Branch
Environmental Research Laboratory
Athens, Georgia 30601
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
ATHENS, GEORGIA 30601
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DISCLAIMER
This report has been reviewed by the Athens Environmental
Research Laboratory, U.S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the
contents necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation
for use.
11
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ABSTRACT
Biological electrodes have been developed for the selective
analysis of phosphate and nitrite ions at micro-levels. A
phosphate electrode was constructed by using two enzymes,
alkaline phosphatase and glucose oxidase, which were immobilized
and mounted on the surface of the platinum electrode. The
competitive inhibition of phosphate ion on alkaline phosphatase
slows down the hydrolysis of glucose-6-phosphate to glucose,
which is subsequently used by glucose oxidase as a substrate in
the presence of molecular oxygen. The phosphate ion
concentration in the reaction mixture was found to be
proportional to the oxygen consumption.
The assay of nitrite was accomplished with the use of nitrite
reductase which catalyzes the reduction of nitrite into ammonia.
Nitrite in the range of 10-*M-10~2M could be accurately
determined with either soluble or immobilized enzyme using an
air-gap electrode as sensor.
Preparations of benzidine phosphate and hexammino cobalt nitrate
in silicone rubber for liquid ion exchange and triphenyltin
electrode have been attempted and results obtained were
unsatisfactory due to their poor selectivity.
This report was submitted in fulfillment of R 800359-02 by
University of New Orleans under the sponsorship of the
Environmental Protection Agency. Work was completed as date of
12-31-75.
111
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ACKNOWLEDGMENTS
Three post doctoral associates. Dr. M. Nanjo, Dr. T. J. Rohm and
Dr. S. S. Kuan, were employed on the project. One graduate
student, Mr. C. H. Kiang, was employed, doing basic research
associated with his Ph.D. degree to be received next year.
This support from EPA is gratefully acknowledged, as is the kind
assistance and advice of Dr. Thomas Hoover, of the Environmental
Research Laboratory in Athens, Georgia.
IV
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CONTENTS
Sections Page
I. Conclusions 1
II. Recommendations 2
III. Introduction 3
IV. Results and Discussion 4
V. References 7
v
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SECTION I
CONCLUSIONS
Attempts to employ an insoluble salt or ligand-association
complex for the selective assay of phosphate ion have been made.
Among the salts tested, good sensitivity was achieved with the
phosphonium and triphenyltin salts, but both lacked sufficient
selectivity, therefore, the use of these substances in electrodes
for standard routine assay of phosphate is not recommended.
However, the use of these electrodes as an aid to double check
the phosphate ion concentrations at micro-levels is recommended.
On the other hand, the use of extremely selective phosphate and
nitrite enzyme electrodes for the detection of POj, 3~ and NO2~ is
highly suggested for routine analysis due to their good
sensitivity and selectivity. These electrodes are highly
selective for these ions.
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SECTION II
RECOMMENDATIONS
It is recommended that further work be done on these electrodes,
to insure their widespread use in environmental sampling for
nitrite and phosphate. Prototypes have been developed, but
additional work must be done to evaluate these electrodes, and
perfect them for experimental use in everyday situations.
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SECTION III
INTRODUCTION
The objective of this research project was to develop selective-
ion electrodes for phosphate and nitrite ions that can be used
for the continuous monitoring of these ions at microlevels.
Because of the great sensitivity of enzyme systems (as little as
10~l2g of some substances is determinable) such systems have
proven useful for the assay of various substances. Enzyme
systems can be used to build in selective electrodes for
substances for which selective conventional electrodes cannot be
made. Phosphate and nitrate ions are pollutants commonly present
in water, being discharged from detergents, fertilizers and other
sources. We took two approaches to this problem. The first was
the development of an electrode that employed an insoluble salt
as the sensing element. The second approach involved the use of
enzyme electrodes in which the enzyme catalyzed a reaction
involving phosphate or nitrite ion and the reaction was followed
electrochemically.
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SECTION IV
RESULTS AND DISCUSSION
The preparation of a solid-state electrode for orthophosphate has
been reported (1) . This electrode was prepared by mixing a
glutaraldehyde-thiourea polymer with silver ion and dibasic
phosphate to obtain an insoluble complex (PCPX-1) . This material
was mixed with silver sulfide and pressed into a pellet which is
then used as the sensing membrane of an electrode. This
electrode responds to dibasic phosphate concentration over the
range 10-* to 10-* M. Sulfate ion interfers with the performance
of the electrode and the device loses sensitivity after 48 hours.
To improve the stability and selectivity of the solid-state
electrode, we investigated the use of derivatives of thiourea
(i.e., thiocarbanilide) ; 1,3-didecyl-2-thiourea; 2,2'-
diethylthiocarbanalide; 1 r3-diethyl-2-thiourea and 1,1,3,3-
tetramethyl-2-thiourea) for the preparation of silver complexes
similar to PCPX-1. All of these compounds yielded silver sulfide
and various organic products when treated with silver nitrate.
Electrodes prepared from these products in the same manner as the
original solid-state electrode have essentially the same
properties as the PCPX-1 electrode. Since no improvement was
made, this phase of investigation was not pursued.
The use of ligand-association complexes of 2,2*-bipyridine; 1,10-
phenanthroline and related compounds with transition metals as
exchange materials for the preparation of liquid ion- exchange
electrodes has been fully investigated. Liquid-type electrodes
prepared from these complexes were sensitive to C10^~, I~, Br~,
NO3- and Cl~, generally in the order cited. A limited response
to orthophosphate was achieved by converting the
trisbathophenanthroline iron (II) complex to the orthophosphate
form and using the resulting product to construct a liquid ion-
exchange electrode. During the study of methods for the
preparation of liquid ion-exchange electrodes, we found that
incorporation of the exchange solution in poly (vinyl chloride) ,
PVC, improved the performance of the electrode. We reported
these findings in Reference 2 and gave a summary of the results
of studies of solid-state and ligand-association complex
electrodes at the ACS National Meeting in Dallas, April, 1973.
During the period covered by this grant, the investigator
prepared a report for IUPAC (3) , and reviewed the field of ion-
selective electrodes
We studied the possibilities of using the reaction of benzidine
and various complex salts of cobalt (III) as membrane materials.
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These compounds are selective for phosphates, but were found to
lack the mechanical and electrical properties necessary for the
preparation of ion-selective electrode.
The next area investigated involved the use of quarternary
ammonium salt as exchange materials for the preparation of a
phosphate selective electrod^ (5) . The quaternary ammonium salt,
General Mills Aliquat 3365 (methyltricapryl-ammonium chloride) ,
was used to prepare a divalent phosphate electrode (6) and
electrodes for a variety of anions have been reported (7) using
the same salt after conversion of the anion to that appropriate
for the type of electrode constructed. We prepared similar
electrodes which showed linear response to orthophosphate over
the range 10-* to 10~* M at pH 4.3. These electrodes employed
quaternary ammonium phosphate salts as exchange materials. The
stability of such electrodes is excellent, but the selectivity is
poor with selectivity coefficients greater that unity for common
ions (C1~, OAc~, Br~) . However, the nature of the organic groups
which are attached to the central nitrogen atom greatly
influences the selectivity of such electrodes and we were able to
construct electrodes with varying selectivities by changing the
type of quaternary ammonium salt used. The selectivity is
closely related to extraction coefficients as has been recently
reported (8, 9) .
In conclusion, precipitate-type electrodes (benzidine sulfate or
phosphate and hexammino- cobalt (III) nitrate in silicone rubber) ,
and liquid ion-exchanger electrodes based on quaternary, ammonium
and phosphonium and triphenyltin salts were tested for the assay
of phosphate ions. Good sensitivity was achieved with the
phosphonium and triphenyltin salts, but both lacked sufficient
selectivity for routing assays (5) .
An enzyme electrode which senses oxygen consumption for the assay
of phosphate ion (10~3-10~* M) was constructed by using two
enzymes together:
alkaline phosphatase
glucose-6-phosphate - > glucose + phosphate
glucose oxidase
glucose — • - > gluconic acid + H2O2
The competitive inhibition by phosphate ion added caused a
smaller and slower oxygen consumption which could be detected by
a platinum disc electrode at -0.6 V vs. SCE amperometrically.
This dual enzyme electrode was also found useful for the assay of
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oxyacids other than phosphate, such as arsenic, tungstate,
molybdate and borate (10). The electrode is completely specific
for phosphate tinder controlled conditions, and is the best
electrode thus far developed for phosphate ion.
The enzyme, nitrite reductase, was extracted and purified from
spinach leaves; the freeze-dried preparation is completely stable
for at least 4 months if kept in a freezer. The enzyme catalyzes
the reduction of nitrite to ammonia in the presence of reduced
methyl viologen as electron donor. An assay of nitrite can be
based on the measurement of the ammonia formation, with an air-
gap electrode as sensor. Nitrite in the 10~* M-5 x 10~2 M range
can be accurately determined with either soluble or immobilized
enzyme, but the latter is stable for at least three weeks, is
less susceptible to interferences during assay, and can be used
repeatedly for about a hundred runs. These advantages make the
method very simple, valuable and economical for the routine
analysis of nitrite ion (11) .
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SECTION V
REFERENCES
1. Shu, F. R. and G. G. Guilbault, An Ion-Selective Electrode
for Dibasic Phosphate Ion. Anal. Lett. J>(8) , 559-565
(1972).
2. Rhom, T. J. and G. G. Guilbault, New Methods for the
Preparation of Perchlorate Ion-Selective Electrodes. Anal.
Chem. 46» 590-592 (1974).
3. Guilbault, G. G. , Recommendations for Nomenclature of Ion-
Selective Electrodes. IUPAC Report, 1975.
4. Guilbault, G. G. and T. J. Rhom, Ion-Selective Electrodes
and Enzyme Electrodes in Environmental and Clinical Studies.
Intern. J. Environ. Anal. Chem. j», 51-64 (1975).
5. Nanjo, M. , Timothy J. Rhom and G. G. Guilbault, An
Investigation of Polyphenyl-Onium Bases and Other Materials
for Phosphate Ion-Selective Electrodes. Anal. Chim. Acta.
17, 19-27 (1975).
6. Nag el berg, I., L. I. Braddock, and G. J. Barbero. Divalent
Phosphate Electrode. Science 166 (3911), 1403-1404, December
T969.
7. Coetzee, C. J. and H. Freiser. Liquid- Liquid Membrane
Electrodes Based on Ion Association Extraction Systems.
Anal. Chem. 11(8), 1128-1130, July 1969.
8. Reinsf elder, R. E. and F. A. Schultz. Anion Selectivity
Studies on Liquid Membrane Electrodes. Anal. Chim. Acta
(Netherlands) 65, 425-435 (1973) .
9. Back, S. and J. Sandblom. Role of Solvent Extraction
Parameters in Governing the Potential Selectivity of Liquid
Membrane Electrodes. Anal. Chem. 45_(9)» 1680-1684, August
1973.
10. Guilbault, G. G. and M. Nan jo, A Phosphate-Selective
Electrode Based on Immobilized Alkaline Phosphatase and
Glucose Oxidase. Anal. Chim. Acta 7_8, 69-80 (1975). "
11. Kiang, Chi h- Hen, S. S. Kuan, and G. G. Guilbault, A Novel
Enzyme Electrode Method for the Determination of Nitrite
Based on Nitrite Reductase. Anal. Chim. Acta 80, 209-214
(1975). —
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1. REPORT NO. ,
EPA-600/4-76-034
2.
4. TITLE AND SUBTITLE
3. RECIPIENT'S ACCESSIOt*NO.
5. REPORT DATE
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
Biological Electrodes Specific for Phosphate and
Nitrite Ions
October 1976 (Issuing date")
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
G. G. Guilbault
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG \NIZATION NAME AND ADDRESS
University of New Orleans
Lake Front
New Orleans, Louisiana 70122
10. PROGRAM ELEMENT NO.
1BAQ27
11. CONTRACT/GRANT NO.
R 800359
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Athens, Georgia 30601
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Biological electrodes have been developed for the selective analysis of
phosphate and nitrate ions at micro-levels. A phosphate electrode was constructed
by using two enzymes, alkaline phosphatase and glucose oxidase which were
immobilized and mounted on the surface of the Platinum electrode. The competitive
inhibition of phosphate ion on alkaline phosphatase slows down the hydrolysis of
glucose-6-phosphate to glucose which is subsequently used by glucose oxidase as a
substrate in the presence of molecular oxygen. The phosphate ion concentration in
the reaction mixture was found to be proportional to the oxygen consumption.
The assay of nitrite was accomplished with the use of nitrite reductase which
catalyzes the reduction of nitrite into ammonia. Nitrite in the range of 10~^M-
10 M could be accurately determined with either soluble or immobilized enzyme using
an air-gap electrode as sensor.
Preparations of benzidine phosphate and hexammino cobalt nitrate in silicone
rubber for liquid ion exchange and triphenyltin electrode have been attempted and
results obtained were unsatisfactory due to their poor selectivity.
This report was submitted in fulfillment of R 800359-02 by University of New
Orleans under the sponsorship of the Environmental Protection Agency. Work was
completed as date of 12-31-75.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
*chemical analysis
*electrodes
*ion-exchangers
alkaline phosphatases
enzymes
nitrites
perchlorates
*ion-selective
phosphate
glucose oxidase
biological electrode
nitrite reductase
05A
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)'
UNCLASSIFIED
21. NO. OF PAGES
14
20. SECURITY CLASS.(Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
*USGPO: 1976 - 757-056/5422 Region 5-11
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