Oroboros Instruments Oxygraph-2k User manual

[e-mail] www.oroboros.at
Service of the
polarographic
oxygen sensor
OroboPOS
Gnaiger E
OROBOROS INSTRUMENTS Corp
high-resolution respirometry
Schöpfstr 18, A-6020 Innsbruck, Austria
Email: [e-mail]
www.oroboros.at
Section 1. Accessories ............................................. 20 Page
2. Cleaning ................................................. 20
3. Membrane mounting ................................ 22
4. Electrical cable connection ........................ 23
5. Storage of the POS .................................. 23
6. Replacement of the POS head ................... 24
7. References.............................................. 24
Summary: Service of the polarographic oxygen sensors (OroboPOS,
POS) is the basis for signal stability, low noise and high time resolution.
Sensor service may not be required for several months of operation.
Performance of the OROBOROS Oxygraph-2k according to instrumental
specifications is obtained only with oxygen sensors which are maintained
in a good functional state.
POS service is required if (1) a new
sensor is prepared, (2) the raw
signal at air calibration declines, (3)
signal noise is high, (4) the time
response is prolonged (time
constant >10 s) and biphasic, (5)
the oxygen signal at zero calibration
does not decline rapidly to a low
level of zero current (0% to max. 5%). For each sensor, the frequency of
POS service can be optimized on the basis of a long-term record of
calibration values as a quality control (O2k-Calibration-List.xls).
Mitochondrial Physiology Network 19.01(B01): 19-24 (2014) 2014 OROBOROS
B: www.bioblast.at/index.php/MiPNet19.01B_POS-Service Version B01: 2014-03-06
Oxygraph-2k Manual
B

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OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
Figure 2. Removal of a used
membrane from the POS.
2
2. Grab the PEEK
membrane ring
on the sensor
head
3
4
3. Pull the PEEK
membrane ring from
the sensor head
4. Remove and
discharge the old
membrane
Remove an old
membrane from
the POS
Membrane
mounting tool
POS on POS
connector
1. Remove
POS seal tip
1
1. Accessories
Accessories for sensor service are provided in the OroboPOS-Service Kit
(Fig. 1). In addition, you need distilled water and 25% ammonia solution
(fresh). Store the OroboPOS in the dark.
2. Cleaning
Prevent damage by electrostatic
discharge (ESD) when handling the POS
connectors or cable connections to the
O2k (MiPNet14.01).
For sensor service, remove the black
POS seal tip (1). It is normal to see many
small bubbles in the electrolyte reservoir.
This does not indicate that this caused a
problem while the sensor was actually in
use. Remove the PEEK membrane ring
(2,3) and membrane (4; Fig. 2). Wash
off the electrolyte with distilled water.
For cleaning of the anode and
cathode, the sensor head is best
removed from the OroboPOS-Connector
Fig. 1. OroboPOS-Service Kit - Oxygraph-2k
26300-01 OroboPOS-Electrolyte powder, KCl
26200-01 OroboPOS-Membranes, FEP 25 µm; 40/Pck.
26520-01 OroboPOS-Polishing Powder for cathode cleaning
26510-01 OroboPOS-Polishing Cloth for cathode cleaning
26400-01 OroboPOS-Mounting Tool for membrane application
26800-01 Pipette\Plastic\1 ml ungraded for electrolyte
26600-01 O2-Zero Powder, dithionite (Na2S2O4)
26550-01 Pen-Contact Oil for stable low contact resistance
1
2
3
4
5
6
7
1
2
3
4
5
6
7

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B
Figure 3. Cathode cleaning.
and mounted to the blue base of the Perspex housing of the POS
(OroboPOS-Service kit; page 1).
The cathode should be cleaned when its gold surface appears to be
coated by a colored layer. The silver/silver chloride anode darkens after
long-term operation, inadequate storage of the sensor or contact with
hydrogen sulfide. This may cause high signal noise or reduces the signal
output by >30%, reflected by the requirement to increase the gain. Such
sensors can be improved by cleaning the cathode, anode and gold
connections.
2.1. Cathode cleaning
The cathode must be treated with extreme care. It must not be
touched with the fingers, nor exposed to detergents or greasy liquids.
Wash off the electrolyte from the POS
with distilled water. Place the Petri dish
with the polishing cloth supplied in the POS
recharge kit (Fig. 1) on a flat surface, add
some fine aluminum oxide powder (0.3
m) with the tip of a spatula, and moisten
it with a few drops of distilled water to
obtain a thin paste (Fig. 3). Hold the
sensor in a vertical position and polish the
cathode for one minute in a figure-eight
motion (Fig. 3). Wash the aluminum oxide
powder carefully off the sensor with
distilled water, and wash the cleaning
tools.
You may further clean the gold cathode with ultrasonic treatment at
low power for up to 30 seconds while immersing the tip of the sensor head
in distilled water. In extreme and rare cases, the cathode may be cleaned
by adding a drop of 50-75% nitric acid onto the surface of the cathode for
only 15 seconds (no longer) with care. Remove carefully any traces of nitric
acid by washing with distilled water, and proceed as described above. After
cleaning the cathode, the anode must be cleaned as well.
2.2. Anode cleaning
Fill the electrolyte reservoir of the sensor with concentrated (25%)
ammonia solution. After up to 10 minutes the silver/silver chloride should
appear bright gray. Wash the sensor carefully with distilled water. Repeat
the application of ammonia solution twice. With severely aged sensors it
might be necessary to prolong the exposure to ammonia up to several
hours (overnight), sealing the ammonia under a membrane and under the
POS cover slip in long-term applications. Protect the POS from light, since
the silver/silver chloride anode is light sensitive.

MiPNet19.01 B: OroboPOS service 22
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
The response time of the sensor signal may remain high with a
biphasic pattern (exponential phase followed by a slow drift) even after
polishing and cleaning with ammonia. Repeat the cathode/anode cleaning
cycle several times.
2.3. Cleaning of the electrical connection
Unscrew the POS head and inspect both sides of the electrical connection
(gold pin and threads). Remove any contamination such as grease and
moisture with a fine paper cloth. If necessary, wash with distilled water and
then with pure alcohol, and apply contact oil from the OroboPOS-Service kit
to the connections. Before screwing the POS head onto the POS connector
used for membrane application, clean the POS connector of moisture and
any other contamination (particularly any salt crystals from the electrolyte).
Similarly, clean the electrical cable connection to the O2k-Main Unit.
3. Membrane mounting
For mounting a new membrane, the POS head is screwed on the sensor
connector. Check the O-ring on the POS head to ensure that it fits properly
and its surface is smooth and intact. You may apply a tiny amount of
grease to the O-ring of the sensor head (Fig. 2). Rinse the mounting tool
(OroboPOS service kit) with distilled water to wash off any electrolyte
crystals (Fig. 4).
As electrolyte, use KCl solution (1 mol∙dm-3; 74.56 g potassium
chloride per litre, in distilled water). Add distilled water to the electrolyte
powder (Fig. 1) up to the 10 ml mark. Alternatively, dissolve 1.49 g KCl in
distilled water with a total volume of 20 ml. Store at room temperature or
4 °C in a closed bottle. To prevent the formation of gas bubbles in the
electrolyte, heat the electrolyte solution by shaking the sealed electrolyte
container in hot water (40-70 °C) before filling the electrolyte reservoir of
the POS.
POS membranes are contained in a small box (Fig. 1, item 6). They
are fully transparent. Each membrane is separated by a non-transparent
white paper sheet. Do not add the paper to the oxygen sensor. Separate
the membrane from the paper sheets, carefully avoiding any mechanical
damage of the transparent membrane. Do not touch the central area of the
membrane with your fingers. The pair of forceps is used for separating the
membrane and positioning a new membrane into the membrane guide of
the POS mounting tool (Fig. 4 [1]).
Fix the PEEK membrane ring (which seales the membrane against the
sensor body) to the membrane ring holder (Fig. 4 [2]). With the POS head
filled with electrolyte, slide the membrane guide down the POS connector,
and move the lower ring on the POS connector down to bring the
membrane guide into a fixed position (Fig. 4 [3]). Then slide the membrane
ring holder into the membrane guide and press firmly down to slide the
PEEK membrane ring over the POS head (Fig. 4 [4]).

MiPNet19.01 B: OroboPOS service 23
OROBOROS INSTRUMENTS O2k-Core Manual
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B
•
Figure 4. Mounting of a membrane onto the POS,
using the membrane mounting tool which consists of
two parts, the membrane guide and the membrane
ring holder.
No gas bubbles
should be trapped
in the electrolyte
reservoir after
membrane
application. As a
check, you may
inspect the
electrolyte
reservoir under a
binocular. No
folds should be
visible in the
membrane in the
central area.
Small folds in the
membrane near
the outer
circumference
have no influence, but large folds should be avoided. Wash excess
electrolyte off the outside of the sensor and POS connector and wipe dry,
before attaching the POS connector to the POS holder at the O2k-Main Unit.
After sensor service and membrane mounting, the POS needs some
time in operation to stabilize while the O2k is switched on. Such
stabilization may require several hours. For this purpose the O2k may be
left on overnight.
4. Electrical cable connection
Connect the POS cable to the O2k-Main Unit,
avoiding bending and torsions of the cable.
Insert the male plug of the POS cable into the
female connector next to the control light of the
stirrer. The red dot on the male plug has to face
accurately upwards when inserting the plug.
See Supplement for O2k Series B to C.
5. Storage of the OroboPOS
5.1. Short-term storage in the Oxygraph-2k
For short periods of days or several weeks, the POS is stored in the
Oxygraph-2k chamber. The chamber is washed with distilled water and
completely filled with 70% ethanol for chemical sterilization. The stopper
is inserted loosely without pushing it down beyond the point where the
sealing ring is inserted into the glass chamber. This ensures a longer life
time of the sealing rings. The receptacle of the stopper is completely filled
with ethanol from the top, and is sealed with a black cover slip to avoid

MiPNet19.01 B: OroboPOS service 24
OROBOROS INSTRUMENTS OROBOROS Oxygraph-2k
evaporation of ethanol. Before an experiment, the ethanol is siphoned off
and the chamber is washed with distilled water (MiPNet06.03).
5.2. Short-term shelf storage
For shelf storage, unplug the POS-Connector from the O2k-Main Unit.
Clean the sealing tip and membrane with distilled water. The POS head is
maintained moistened by applying a cup on the sensor head to prevent
the drying out of the electrolyte. Store in the dark.
5.3. Long-term storage
For storage of the POS for several months, the sealing tip and membrane
are removed. Wash the electrolyte off the POS with distilled water. Even if
the membrane is not damaged, remove it by gripping the membrane
holding ring with the groove in the lower end of the membrane ring holder
of the mounting tool (Fig. 2). The POS head is stored dry and in the
dark. Check for any moisture and salt contamination in the electrical
connector of the POS head. In this case, wash with distilled water and
subsequently with pure methanol, dry at 60 oC for 24 h.
6. Replacement of the OroboPOS head
After cleaning and drying the gold cathode contact or drying the sensor
body at 60 °C for a few days, the zero current should be reduced. In
addition, the zero current of the bare cable, without sensor head
connected, is tested for any leak currents. If the latter test excludes any
sources of leak currents other than the POS, and POS service is not
successful, the sensor head must be replaced.
A new sensor head can be screwed onto the sensor connector, if the
old sensor head has been irreversibly damaged or should be replaced.
7. References
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and
high-resolution respirometry to assess mitochondrial function. In:
Mitochondrial dysfunction in drug-induced toxicity (Dykens JA, Will Y
eds) John Wiley: 327-352.»
Gnaiger E, Forstner H, eds (1983) Polarographic Oxygen Sensors.
Aquatic and Physiological Applications. Springer, Berlin, Heidelberg,
New York: 370 pp. »
Hitchman ML (1983) Calibration and accuracy of polarographic oxygen
sensors. In: Polarographic oxygen sensors, Gnaiger E and Forstner H,
eds., Springer, Berlin, Heidelberg, New York: 18-30. »
Hitchman ML, Gnaiger E (1983) A thermodynamic consideration of
permeability coefficients of membranes. In: Polarographic oxygen
sensors, Gnaiger E and Forstner H, eds., Springer, Berlin, Heidelberg,
New York: 31-36. »
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MiPNet19.01 B: OroboPOS service: Supplement 1
[e-mail] www.oroboros.at
Supplement: O2k-Series B to C
Electrical Cable Connection: O2k-Series B to C
Insert the male plug of
the POS cable into the
female plug positioned
between the two control
lights of the Oxygraph-2k (left chamber: between
the MAINS and STIRRER lights; right chamber:
between the STIRRER and COMM lights). The
ridge on the male plug has to face accurately
downwards when inserting the plug, before
rotating the connector screw and fixing it finger
tight.
O2k-Manual
» MiPNet19.01A Oxygraph-2k: start high-resolution respirometry.
» MiPNet14.01 Electrostatic discharge (ESD): damage and protection.
O2k-Protocols
» MiPNet06.03 POS-Calibration-SOP.
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