4
World Precision Instruments
Calibrating the Sensor
Preparing Na
2
S Stock Solution
1. Use a 100mL volumetric flask. Dissolve 5mg EDTA in 100mL distilled water.
2 Purge the solution vigorously with argon gas for 15 minutes.
3. Weigh 24.0mg of sodium sulfide (Na
2
S .9 H
2
O) (reagent plus, 99.99+% from Sigma)
and dissolve it in the solution under argon atmospheres.
4. Seal the flask with a rubber stopper. The solution is 1.0mM Na
2
S. Store the solu-
tion in a dark bottle and refrigerate at 2–8°C.
Calibration Procedure
The sensor should be immersed in PBS and plugged into the free radical analyzer. Set
the range to 100nA and the poise to H
2
S (150mV). Once the sensor is polarized, It can
be calibrated. The following example describes the fundamental concepts behind a
standard calibration protocol. Known concentrations of H
2
S are generated in PBS by
adding a known volume of a the Na
2
S stock solution.
1. Place a 20mL vial of PBS on the magnetic stirring plate with the small stir bar still
inside. Turn on the stirrer so that the bar is stirring at a moderate rate.
NOTE
: This rate of the stirrer should NOT be modified during calibration.
NOTE
: The calibration should be carried out at the same temperature at which
the experimental measurements are to be made. This can be accomplished by
placing the vial and stand in a water bath at the appropriate temperature and
allowing the temperature of the solution in the vial to equilibrate with the water
bath.
2. Immerse the
ISO-H2S-2
sensor in the solution and secure it in an electrode
holder such as WPI’s
Pro-Guide
(WPI #
47510
,
47520
,
47530
,
47540
) or a micro-
manipulator. The sensor tip should be immersed about 2–3mm into the solution.
It should not contact the stir bar, which could damage the membrane.
3. Connect the H
2
S sensor into a channel of a free radical analyzer. Set the poise
voltage to +150mV. For the
Apollo 4000
, this is accomplished in the software.
Use the custom option to manually set the voltage to 150mV. For the
Apollo
1000
select poise from the main menu and set it to “internal”. Then, adjust the
poise voltage using a small screwdriver to turn the “poise adjust” potentiometer
on the front panel. For the
TBR4100
, simply set the “probe select” control on the
front panel to H
2
S. Allow the sensor to polarize for 12 hours.
The
quiescent baseline current is an indicator of the health of the sensor. A cor-
rectly operating sensor has a baseline current of < 75,000pA.
4. To the vial containing 20mL PBS, sequentially inject three aliquots of Na
2
S solution
(20µL, 40µL and 80µL). The current output increases after each addition and then
plateaus. As soon as it reaches a plateau, inject the next aliquot.
