INSTRUMENTATION: WATER AND WASTEWATER ANALYSIS 569
(e) Enzyme and microbial electrodes 45,54 – 56
Enzyme and microbial electrodes can be an amperomet-
ric, membrane, or a conductometric electrode convered by
a film or membrane containing the immobilized enzyme or
microbial organisms. A permeable membrane intervenes
between the enzyme or microbial film and the electrode (see
Figure 23). In the course of measurement the analyte diffuses
through the immobilized enzyme undergoing an enzymatic
reaction. One of the participants of the reaction, product or
reactant, is sensed by the electrode. These devices provide a
unique combination of two selection processes—specificity of
enzyme or microbial reactions and of the selective electrode.
Measurements for two electrochemical modes, ampero-
metric and potentiometric, are made as follows: In the poten-
tiometric mode the log of the concentration of the detected
species is a linear function of the measured electrode potential
(Nernst equation). In the amperometric mode the concentra-
tion of the detected species is proportional to the electrode
current. The potential of the amperometric electrode chosen
corresponds to the redox potential of the detected species.
Through the stoichiometry of the enzyme or bacterial reac-
tion, the concentration of the analyte is easily calculated from
the concentration of the detected species.
However, the compatibility between conditions for enzy-
matic and microbial action and the optimal electrode sens-
ing environmenta in the form of pH, solvent, ionic strength,
temperature, etc. must be taken into account. At times the
compromises made in the conditions do not serve well the
measuring objective of the enzyme or microbial electrode
ensemble. This point will be illustrated.(i) Enzyme electrodes 45,54,56
Enzymes are immobilized in several ways, e.g., absorp-
tion on a porous inorganic structure or polymer gel mem-
brane or copolymerization with a compatible monomer to
form a thin membrane. The immobilized enzyme film is
viable for from about two to four weeks. The modular con-
struction of the electrode permits easy replacement of the
immobilized enzyme membrane.(x) Potentiometric type
A potentiometric enzyme electrode may be formed
from a gas-sensing electrode assembly containing an ion-
or molecular-sensitive electrode. The immobilized enzyme
membrane is inserted in the space allotted for the thin film
of internal solution (see Figure 23). An example of this elec-
trode is the measurement of urea where reaction with the
enzyme urease is illustrated as follows:ureaH O^22 urease CO 2NH^4
→ . (28)At an alkaline pH the forms NH 3 which is detected by an
ammonia, gas-sensing electrode. Alternatively, a pH/glass
electrode can be used to determine the change in pH due
to the hydrolysis of the NH^4 ion. Potentiometric enzyme
electrodes are also available for the analysis of L-lysine and
L-tryptophane.Incompatibility of enzyme or bacteria with the selective
electrode can occur because of the differences in pH for opti-
mal enzyme, microbial, and selective electrode activity. For
example in the determination of urea, the enzyme, urease,
converts urea to ammonium ion, NH^4 , and bicarbonate ion,
HCO 3. This reaction optimally occurs at a pH of about 7. An
ammonia gas-sensing electrode is the electrode used and the
NH^4 ion, enzymatically formed, requires a pH of 8 or more to
change NH^4 to ammonia, NH 3 , which is sensed by the elec-
trode. Thus none of the components are operable, optimally,
at each other’s best pH. An alternative selective electrode in
this enzyme electrode is a glass electrode which detects NH^4.
However, Na^ ^ and K^ ^ , are also sensed thereby obviating some
of its selectivity.
An automatic analyzer is available for the analysis of
urea that addresses the problem of optimal pH conditions
(see Part Two Section 5, b,(3), ( c ),( iii )).(y) Amperometric type
The glucose electrode consists of an amperometric probe
covered with immobilized glucose oxidase. The enzymatic
reaction is as follows:β -D-glucose O 2 H 2 O glucose oxidase → H 2 O 2
D-glucono- ∆ -lactone. (29)Either oxygen or peroxide may be detected as a measure of
the glucose content. The decrease in oxygen or the increase
in peroxide is proportional to the glucose concentration.
Therefore, an oxygen or peroxide amperometric electrode
can be used. Each electrode has a specific advantage; the
oxygen-based electrode doesn’t respond to other reaction
substituents but the peroxide does respond to other oxidiz-
able components of low molecular weight. However, the
oxygen electrode has a lower sensitivity than the peroxide
probe, since it has a high background signal.
The oxygen electrode (see Section III,B, 2, b, (2), (a)) is
used in a number of enzyme electrodes because of its selec-
tivity.^56 Amperometric enzyme electrodes are also avail-
able for the analysis of alcohols, L-amino acids, ascorbate,
L-glutamate, lactate, lactose/galactose, oxalate, salicylate,
sucrose, and uric acid.^56
Enzyme electrodes have been incorporated into an auto-
matic or semi-automatic analyzer (see Part Two Section
5, b,(3), ( c ),( iii )).(ii) Microbial electrodes 52,53
Microbial electrodes are similar in construction and oper-
ation to enzyme electrodes. In the microbial electrodes a poly-
meric membrane containing immobilized organisms is placed
between the electrode and the analyte solution. The nature of
the analyte dictates the species of organism. Several examples
of analytes, immobilized organisms and electrodes that com-
prise a number of microbial electrodes are listed in Table 9.(x) Potentiometric type
Glutamic acid and glutamate can be detected by a micro-
bial electrode utilizing a CO 2 , gas-sensing electrode. 57,58 TheC009_005_r03.indd 569C009_005_r03.indd 569 11/23/2005 11:12:25 AM11/23/2005 11:12:25