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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.

				
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