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Polarographic Oxygen Electrode]
JOHN KANWISHER
Woods Hole Oceanographic Iristitution, Woods Hole, Massachusetts
ABSTRACT
A method has been developed for the continuous monitoring of oxygen tension in solu-
tions and gases. The oxygen is reduced at a platinum surface which is covered with a
plastic membrane. The silver-silver oxide reference electrode is also included under the
membrane. Drift is less than 10 per cent for a period of days, and temperature compcnsa-
tion is possible. A number of uses to which it has been put arc described.
The intimate involvement of oxygen in oxide reference electrode. Molecular oxy-
nature makes its measurement a problem of gen is rcduccd at the platinum surface, and a
recurring interest. In many investigations current flows. The magnitude of this
prcscnt techniques of oxygen detcrmi- current depends upon the amount of oxygen
nation limit the amount and type of data reaching the platinum surface. Thus a
that can be taken. A method of con- bare platinum electrode in sea water makes
tinuously monitoring oxygen tension in an acceptable polarograph for a short time.
liyuids and gases will bc described in this The sensitivity oji such an electrode de-
paper. It is based on the polarographic creases with time, apparently due to metals
reduction of molecular oxygen at a plati- plating out on the platinum. The sensi-
num electrode. Many of the previous tivity cannot be regained by reversing the
shortcomings of polarographic systems have current. In complex biological media such
been overcome. The variety of tasks to as blood, red cells or other material usually
which it has been applied include the dc- collect on the surface and reduce the sensi-
termination of biological oxygen demand in tivity by partially blocking the access of
domestic sewerage, continuous flow res- oxygen. Dropping mercury electrodes
pirometry of a variety of marine and fresh- partly overcome the difficulty by continu-
water invertebrates and fishes, determina- ally renewing the metallic surface, but they
tion of respiration and photosynthesis in are inconvenient and may bc poisonous to
algae, analyzing respiratory gases for oxygen, living systems. Such chemical and physical
determination of oxygen tension in l-ml factors have prevented polarographic
samples of water and biological fluids, and methods of oxygen determination from being
vertical profiles of oxygen in fresh and widely used.
salt water. Interest has been sufficient to In an effort to overcome the shortcomings
indicate that the technique should be of a bare platinum electrode in biological
published separately as a research method. fluids, Davies and Brink (1942) covered the
An explanation of how the electrode func- metal with agar and also a membrane of
tions will be followed by a description of collodion, and Clark and colleagues (1953)
some of the above uses to indicate its later investigated a variety of other plastic
potentialities. A more complete descrip- films. A recent symposium has sum-
tion of the theory and chemical behavior of marized the present state of the art (Fedcra-
the electrode will be prcscntcd elsewhere. tion Proceeding 11957). Interest in such
systems has been mostly among biologists
I. TII-EORY OF THE OXYGEN ELECTRODE for application to specific problems. The
The present system consists of a platinum present work was undertaken to develop a
electrode held at - .8 volts to a silver-silver generally useful technique for biological
1 Contribution No. 1014 from the Woods Hole
and oceanographic work.
Oceanographic Institution. Preliminary experiments with uncovered
210
POLAROGRAPEIIC OXYGEN ELECTRODE
electrodes led to the conclusion that a
practical platinum polarographic system
must have two features. The platinum
should operate in a simple medium to prc-
vent, changes in electrode sensitivity due to
chemical interference, and there should
also be a standardized diffusion arrange-
ment, between the platinum surface and the POLYETHYLENE OR
medium in which oxygen is being measured. TEFLON MEMBRANE
Polyethylene and teflon have the necessary
properties to realize both of thcsc con- RETAINING
RING
ditions. Molecular oxygen can diffuse OUT
through them, while at, the same time they
are impermeable to ions. The platinum
electrode can opcra.tc behind a thin film of
polyethylene in a pure solution of KOH.
Under this condition its sensitivity does
not change. The plastic represents a
considerable but fortunately unchanging
diffusion barrier.
The physical arrangement of the electrode TO CELL ABOVE
system is shown in Figure 1. The silver-
silver oxide reference clcctrodc is con- REGULATED FLOW IN
centric around the platinum. A film of
polyethylene is stretched tightly over this
and held by a retaining ring. The film is
put on the electrode while the entire as-
sembly is held under the surface of a so- GLASS BEAD
MIXING
lution of 0.5 N KOH. A small amount of CHAMBER
this solution is trapped under the plastic.
FIG. 1. A. Exploded view of the oxygen elec-
It forms only a very thin layer between the trodc showing how the membrane is stretched
plastic and the platinum. tightly over the end surface and held this way by
The initial reaction of the oxygen af the an elastic retaining ring. B. Cell with which the
platinum forms OH ions. electrode can be introduced into a stream of solu-
tion or gas. C. An arrangement for continuous
flow respiromctry of small fishes or other aquatic
4e- + O2 + 2HZ0 ---) 40H- (1) animals.
A piece of indicator paper laid across the
platinum under the plastic shows increased system will last for many months of con-
pH a few seconds after a current due to O2 tinuous operation.
starts to flow. The 011 ions then diffuse The diffusion situation of O2 in the clec-
to the reference electrode and react with the trode is as follows. When the electrode is
Ag to form AgzO. turned on a current passes which is the
same as if a bare clcctrode were used, since
2Ag + 20H- -+ AgaO + Hz0 + 2e- (2) the KOH solution has previously established
oxygen tension equilibrium across the film.
The end result is the formation of AgzO The current decreases in a few seconds as
which appears as a dark brown or black the small amount of oxygen in solution bc-
coating on the surface of the silver. The hind the film is consumed by the above
Ag,O then forms the half cell which makes reaction. Oxygen from the medium begins
contact with the solution to complete the to diffuse across the film bccausc of the
circuit with the pla,tinum clectrodc. Since gradient produced. A steady state is soon
the Ag can be made thick such an electrode reached where the oxygen consumed at the
212 JOHN KANWISHER
platinum equals that diffusing across the the current and thus the size of the electrode
film. With a 0.0015” thick polyethylene to bc used in a small closed system, where
lilm the current will now bc about x 000 the total quantity of dissolved oxygen is
as large as if the platinum were uncovered. small.
The 02 tension behind the film must be Most of the electrode systems have been
reduced from that outside by this factor. made from a length of silver tubing and a
Thus in air-saturated water this tension is disc of platinum. These are soldered onto a
only a fraction of a mm of Hg when a cable and then cast in an epoxy plastic so
steady state is reached. that only the two desired metals are exposed.
If the electrode is turned off and then Lucite has also been used successfully as
turned on again, the current is initially an insulating support for the electrodes.
very large. Oxygen tension equilibrium The silver is then electrolytically coated
has been partly reestablished across the with oxide until it is black. This elimi-
film in the interim. If a sensitive meter is nates a period of several hours initial
used as a current indicator, it is necessary equilibration. The area of the silver should
to protect it from this initial surge of current. be at least ten times that of the platinum
The tension increment across the film is for the quickest response.
nearly equal to the value of the tension in The polarographic cell is an amperometric
the medium, since it has been shown that device in which the oxygen concentration
that in the KOH is very low. So little rather than the: voltage applied determines
oxygen diffuses from the medium into the the current that flows. With a given oxygen
film the system is much less dependent concentration this current increases about
upon the flow of water past the electrode. 5 per cent for every degree centigrade.
Going from an absolutely quiescent con- Unless measurements are made at the
dition to vigorous stirring increases the calibration tempe.rature, they must be
current only 25 to 50 per cent. A very corrected. Automatic compensation can be
slight motion of about 1 cm/set. brings the effected by taking the voltage drop of the
current nearly up to saturation. This current through a thermistor with a tem-
much movement is supplied easily by perature coefficient equal in magnitude and
random motion when an electrode is dangled opposite in sign to the cell. The thermistor
on the end of a cable over the side of a boat. is exposed to the same temperature as the
The platinum electrode “sees” more than solution being measured and can con-
its own area of polyethylene, and diffusion veniently be cast in the plastic electrode
takes place through this increased arca. block. The relatively high temperature
Due to this edge effect the polarographic coefficient represents a combination of the
current does not increase linearly with the effect of temperature on the diffusion and
area of platinum. For electrodes of less solubility of oxygen, and also the tempera-
than 2 mm diameter, it is more nearly a ture kinetics of the chemical reactions
function of the diameter. This gradually involved.
changes to an area dcpendencc in larger A l-cm platinum electrode covered with a
electrodes as the edge area becomes a 0.0015” polyethylene film will give a current
smaller fraction of the whole. Platinum of about 20 microamperes at 25”. This can
electrodes from 0.2 to 2 cm in diameter have be measured directly on a meter. For
been used. There seems to be no reason smaller electrodes it is necessary to use
why larger electrodes with correspondingly either a galvanomcter or recording po-
greater currents are not feasible. tentiometer. The latter instrument can be
Equation 1 states that the electrode driven from the voltage drop across a
reaction consumes a molecule of oxygen for ten-turn potcntiomleter. With provision for
every 4 electrons of current flow. If one offsetting the zero, the recorder can be
microamp of current flows for one hour made to cover only the range of interest.
the amount of oxygen consumed is about If small changes in oxygen are to be
2 x 10-4 ml. This consideration may limit recorded, the flow past the electrode must
POLAl~OGRAPIIIC OXYGEN ELECTRODE 213
be constant. To accomplish this in closed driving force of diffusion, it is sometimes a
bottles, magnetic stirring has been used. more pertinent variable than oxygen con-
A large magnetron magnet turned by a tent.
synchronous motor will rotate a small The overall analytical performance of
magnet inside a bottle in a water bath from these clcctrodes has been investigated with a
as far as a foot away. recording potentiometer and independent
Small fluctuations in cell current have physical determinations of the oxygen ten-
been traced to small bubbles on the poly- sion (Scholandcr et al. 1955). Since these
ethylene. Unfortunately bubbles seem to latter measurements were accurate only to
form preferentially on plastic rather than 1 or 2 per cent, knowledge of the electrode is
glass. The trouble usually develops when correspondingly limited. Operation of the
the water being measured has been warmed device is as follows :
up from a lower temperature, and is thereby 1. Current is a linear function of oxygen
super-saturated. For coarse work a few tension over at least the range of 0 to 1
small bubbles do not seem to matter. atmosphere oxygen.
A cell for continuous flow work is shown 2. It is stable to a few per cent over a
in Figure 1B. The entering flow is directed period of days, and changes less than 25
against the area of plastic directly over the per cent in a period of several WC&S. Dur-
platinum. By bringing the entering tube ing the time there are no abrupt changes.
to within 0.5 mm of the electrode surface, Thus, standardization every few hours is
a flow of 5 to 10 ml/min, can produce a sufficient.
saturation current. 3. Short period changes of 0.01 ml per
It must be kept in mind that a polar- liter in sea water can bc detected. This is
ographic electrode is a diffusion-dependent about 0.2 per cent of the amount in solution
device and therefore measures oxygen when equilibrated with air.
tension rather than content. Fresh and 4. The electrode as described gives 90
salt water equilibrated with the same gas per cent of full response in about 20 seconds,
mixture will give the same reading despite and has reached 99 per cent in 2 minutes.
the salt water having less oxygen. If 5. The electrode has operated success-
an instrument is to read in amount rather fully in dilute acids and alkalies, sea water,
than in tension, it must be calibrated with a and a variety of polluted waters. No
solution of the same gas solubility. Fortu- chemical interference has been noted. Only
nately the salting out effect on gas solubility gross fouling on the plastic surface ap-
is not large. A change of 0 to 3.5 per cent preciably reduces the current. It is in-
salt decreases the oxygen solubility by only sensitive to the osmotic concentration of the
20 per cent. When solutions of blood pig- medium.
ments are involved the oxygen content is
II. EXPERIMENTAL USES
not linearly related to tension. Reference
must then be made to the loading curve of A. Algal respiration and photosynthesis
the pigment for the pH and tempcraturc of The algal sample is placed in a bottle of
the measurement, Since tension is the sea water with a stirring magnet. An
I
I
$3~. 2. Continuous time curve of oxygen in a closed bottle containing algae. The change in slope
represents different rates of respiration and photosynthesis.
214 JOHN KANWISHER
oxygen electrode is used as the stopper. diffusion limiting tlhe supply of oxygen to
Changes in dissolved oxygen arc recorded the cells at the center of the relatively thick
while the alga is exposed to different light thallus. Thin species such as Ulva main-
intensities in a temperature bath. The tain a nearly constant respiratory rate to
resulting curve for Fucus vesiculosus in lower values of oxygen.
Figure 2 shows an initial decrease of oxygen The rates of oxygen production at differ-
in the dark due to respiration. The oxygen ent light levels arc indicated by changes in
tension dependence of respiration is shown slope from that in the dark. The slope was
by the decrease in slope as the oxygen con- zero at 200 foot-c.andles, indicating that
tent becomes smaller. This is plotted in photosynthesis just balanced respiration.
Figure 3. The drop is probably due to In Figure 4 it is clear that at 1200 foot-
. candles there was no sign of saturation.
Quantum efficiency studies in the ultra
. violet arc being done in an analogous manner
with a quartz chamber.
Cl4 labcllcd carbon dioxide is being used
to follow carbon fixation simultaneously
with oxygen production during photo-
synthesis in unicellular algal cultures. The
culture is confined in a hypodermic syringe
w with an electrode in the end of the plunger.
>
With such a container serial samples can be
5
-I withdrawn for counting while the oxygen
k! ml/LITER O2 in solution is continuously recorded.
0 2 3 4
FIG. 3. Curve showing variation in respiration 13. A nimal respirometry
with oxygen tension in Figure 2. The oxygen consumption of many in-
vertebrates and fislhes has also been meas-
FUCUS VESICULOSUS ured in closed containers. If the animal is
SEPT. 4, 1957 allowed to remove all the oxygen, an anal-
+30 -
ogous curve relating respiration to oxygen
ILLUMINATION tension can be calculated. The electrode
W VERSUS has also been used in a continuous flow
- > O2 PRODUCTION respirometcr in which the water in the
K
3
0 animal chamber is continously replaced.
The animal is thu;+ not exposed to an in-
creasing accumulation of its own waste
products. Flow rate times the increment
of oxygen between ingoing and outgoing
water indicates the instantaneous rcspira-
tion rate. The time resolution of such a
system depends on how fast the water in
the animal chamber is replaced, and thus is
a function of flow rate and chamber volume.
FOOT CANDLES
If the flow rate and oxygen content of the
entering water are constant, a single clec-
trade in the water leaving the chamber will
record the time course of metabolism. The
method has been useful with a variety of
FIG. 4. Change of slope with illumination in
Figure 2. This is a measure of the rate of oxygen animals ranging from a 0.5-g snail to a
production and therefore of photosynthesis. SO-kg shark.
POLAROGRAPE-IIC OXYGEN ELECTRODE 215
An arrangement for measuring the me- placed in the syringe. Stirring for two
tabolism of a fish weighing a few grams or minutes produces a maximum reading and
more is shown in Figure 1C. The fish is then a gradual decline as the electrode
placed in a tube small enough so that it reaction slowly consumes the dissolved
cannot turn around. Its head faces the oxygen. It is necessary to handle the
incoming water. Some of the water goes sample quickly and with a minimum cx-
into the fishes mouth and through its gills. posure to the air. A series of standards
The rest passes around the fish. These arc can bc kept in large syringes for running
mixed in the glass beaded chamber at the before and after an unknown. Since the
end of the tube, and pass to the clcctrode. tension measurement does not destroy or
One can see wide excursions in the oxygen consume the sample any additional opera-
uptake when the fish is frightened by a tion can be performed on it afterwards.
light or a noise. A motor driven syringe This technique has been convenient with
has been used to deliver a toxic substance human blood. After a reading part of the
to the inflowing water. The time course sample can bc injected directly into a Van
and severity of its effect on oxygen con- Slykc analyzer to measure oxygen content
sumption could be recorded. as opposed to oxygen tension.
A Venus clam was kept continuously in a
flow respirometcr for several days. As Il. Field experiments
would be expected there was negligible drop The electrode has been used as a field
in the oxygen of the water leaving the instrument to obtain vertical profiles of
chamber when the shell was closed. Upon oxygen in natural waters. Accessory tcm-
opening there was an abrupt decrease as the pcraturc information is necessary to cor-
sea water enclosed in the shell mixed with rect the readings. The electrode and a
that in the rest of the chamber. The drop thermistor have both been operated on the
was always the same amount and indicated same cable. Calibration just bcforc lower-
that the oxygen tension must be close to ing is done by immersing the electrode in a
zero inside the shell when it had been closed container of water that has been air-satu-
for more than 30 minutes. Even when the rated by swirling for several minutes.
shell was open with the animal pumping, The hypolimnion and epilimnion clearly
the instantaneous oxygen consumption showed in a pond used for the local water
varied considerably. An average would supply. Water was being drawn from
have to be taken over a considerable length hclow t,he thermocline so that tap water
of time to be an accurate measure of mc-
was only about one-third saturated in late
tabolism in such an animal. summer. An electrode was being used to
C. Small sample techniques monitor this when a fall storm produced
considerable vertical mixing. The tap
A liquid sample in a syringe can be water oxygen content doubled in two days.
ejected through the cell in Figure 1B to The diurnal oxygen variation in a shallow
measure its oxygen tension. A small pond in mid summer was followed by an
electric motor has been conveniently used instrument supported on a pole pushed into
to advance the plunger at a constant rate. the bottom and read by a telescope from
A single determination takes two minutes, short. A thick plant growth covering the
and needs 5 to 10 ml. Frequent use of bottom was the main source of photo-
calibrating samples make it unnecessary to synthetic oxygen. Bubbles formed on the
reach saturation velocity at the clcctrodc plants in the sunshine and were analyzed
surface. and found to bc largely oxygen. The
It has also been possible to measure the electrode read off scale when pushed in
oxygen tension in a 2-ml sample drawn into a among the plants. The electrode was than
syringe with an electrode in the plunger. hung at mid-depth and moved horizontally
A small magnetic rod has been previously lrom shore by a string to produce a satum-
216 JOHN I~ANWISIIER
S hallow Pond
Woods Hole
x X
x= MEASURED OXYGEN
OC SATURATION VALUE
8 I I
0600 1200 18 00 2400- 0600
TIME
FIG. 5. Diurnal change in dissolved oxygen in a shallow pond is shown by the X’s. The satura-
tion value (small circles) changes due to temperature change.
tion current. This movement is only ncccs- An arrangement for recording in situ with a
sary just before taking a reading. lowered instrument is being planned.
The record for one day in Figure 5 shows
III. CONCLUSION
oxygen supersaturation produced photo-
synthetically during the day and a cor- Some possible uses of the polarographic
responding undersatura tion from night oxygen electrode have been explored in the
time respiration. A strong wind in the experiments described. Others will be re-
afternoon mixed the water and unloaded ported in separa<te publications, As a
some of the cxccss gas. The increasing laboratory instrument the electrode will
concentration during the morning can be indicate oxygen tension to 1 or 2 per cent.
used to estimate the productivity per Because of its marked temperature sensi-
unit area when the entire water column is tivity no effort has been made to equal this
taken into consideration. The uncertain in field experiments. There seems to be no
amount of atmospheric exchange makes reason why such accuracy cannot be realized
this a poor method with strong winds. if attention is paid to such factors as flow
On one instance a submersible pump was past the electrode and temperature of
used to deliver a constant stream of water the water. The rneasurement is no more
from depths up to 100 feet in the ocean. difficult or involvod to perform than that
An electrode was inserted in the lint on of pH.
deck and the pump was gradually lowered. The development of the clcctrodc has
Winkler samples taken at the same time been done in cooperation with Dr. Dayton
showed that the clcctrode agreed within 3 Carritt of the Chesapeake Bay Institute.
per cent. Such an arrangement is only He will report more fully on its chemical
useful to moderate depths in still water. functioning and on additional uses.
POLhROGRhPIIIC OXYGEN ELECTRODE 217
The electrode will be made commercially CI,ARK, L. C., It. WOLF, D. GRANGER, ANI) A.
TAYLOR. 1953. Continuous recording of
by the Jarrell-Ash Company, Newtonvillc, blood oxygen tensions by polarography. J.
Massachusetts. Rppl. Physiol., 6: 189-193.
SYMPOSIUM ON TISSUE OXYGEN TENSION. 1957.
REFERENCES Fed. I’roc., Fed. Am. Sot. Exp. Biol., 16:
665-703.
DAVIES, P. W., AND F. BRINK. 1942. Micro- SCHOLANDER, P. F., L. VANDAM,C.LLOYDCLAFF,
electrodes for measuring local oxygen tension AND J. W. KANWISHER. 1955. Micro gaso-
in animal tissues. Rev. Sci. Instr., 13: 524- metric determination of dissolved oxygen and
533. nitrogen. Biol. Bull., 109: 328-324.
