WPI ISO-HPO-2 User manual

WORLD
PRECISION
INSTRUMENTS
ISO-HPO-2
Hydrogen Peroxide Sensor for use with WPI Analyzers
Serial No._____________________
www.wpiinc.com
INSTRUCTION MANUAL
031919

ISO-HPO-2
World Precision Instruments i
CONTENTS
ABOUT THIS MANUAL ................................................................................................................... 1
INTRODUCTION .............................................................................................................................. 1
Notes and Warnings................................................................................................................. 1
Parts List...................................................................................................................................... 2
Unpacking................................................................................................................................... 2
INSTRUMENT DESCRIPTION ........................................................................................................ 2
OPERATING INSTRUCTIONS......................................................................................................... 3
Polarizing the Sensor ............................................................................................................... 3
Calibrating the Sensor ............................................................................................................. 4
Items Required..................................................................................................................... 4
Making Standard 1mM HPO Solution ............................................................................ 4
Making of 0.1M PBS Buer Solution............................................................................... 5
SENSOR CARE AND MAINTENANCE........................................................................................... 6
Durability and Handling........................................................................................................... 6
Storing the Sensor.................................................................................................................... 7
Cleaning the Membrane.......................................................................................................... 7
Replacing the Membrane Sleeve .......................................................................................... 7
ACCESSORIES................................................................................................................................... 9
TROUBLESHOOTING ...................................................................................................................10
SPECIFICATIONS............................................................................................................................10
WARRANTY .....................................................................................................................................11
Claims and Returns ................................................................................................................11
Repairs.......................................................................................................................................11
Copyright © 2019 by World Precision Instruments. All rights reserved. No part of this publication may
be reproduced or translated into any language, in any form, without prior written permission of World
Precision Instruments.

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ISO-HPO-2
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ABOUT THIS MANUAL
The following symbols are used in this guide:
This symbol indicates a CAUTION. Cautions warn against actions that can cause
damage to equipment. Please read these carefully.
This symbol indicates a WARNING. Warnings alert you to actions that can cause
personal injury or pose a physical threat. Please read these carefully.
NOTES and TIPS contain helpful information.
Fig. 1—The ISO-HPO-2 sensor works with WPI free radical analyzers to monitor hydrogen
peroxide.
INTRODUCTION
The ISO-HPO-2 is a 2.0 mm stainless steel sensor, with replaceable membrane
sleeves (#600012) and an internal rellable electrolyte (#100042). The sensor is
designed for use in cell cultures and similar applications.
The basic structure of the ISO-HPO-2 sensor is shown in Fig. 2. It consists of an
internal H2O2-sensing working/reference electrode combination. This electrode ts
inside a disposable protective stainless steel sleeve (WPI #600012) which must contain
fresh electrolyte (WPI #100042) and is separated from the external environment by a
gas-permeable polymeric membrane covering the end of the sleeve. The other end of
the sleeve is anged. The locking cap is used to attach the sleeve to the probe handle.
Notes and Warnings
NOTE: The sensor must be polarized for at least 2 hours in 0.1M PBS solution prior to use.
NOTE: The sensor membrane is extremely delicate. Improper handling will lead to
damage of the sensor.
NOTE: For optimal use of each sensor and sensor membrane sleeve, the sensor must
be stored properly. See “Sensor Care and Maintenance” on page 6. If your sensor
was shipped wet, it includes a uorescent sticker on the box that says, “THE SENSOR
SLEEVE IS FILLED WITH ELECTROLYTE. IF THE SENSOR IS NOT TO BE USED WITHIN
ONE WEEK, REMOVE THE SLEEVE, CLEAN THE SENSOR WITH DEIONIZED WATER, AND
STORE THE SENSOR DRY.” See “Cleaning the Membrane” on page 7.

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Parts List
After unpacking, verify that there is no visible damage to the sensor. Verify that all
items are included:
(1) ISO-HPO-2 H2O2sensor
(1) Probe Unpacking Instructions (Read this before handling the probe.)
(1) Spare membrane sleeve
(1) 1 cc Syringe
(1) MF28G67 MicroFil
(1) 100042 10mL vial of lling solution
(1) Instruction Manual
(1) Sensor Performance Evaluation
Unpacking
Upon receipt of this sensor, make a thorough inspection of the contents and check
for possible damage. Missing cartons or obvious damage to cartons should be noted
on the delivery receipt before signing. Concealed damage should be reported at once
to the carrier and an inspection requested. Please read the section entitled “Claims
and Returns” on page 11 of this manual. Please contact WPI Customer Service if any
parts are missing at 941.371.1003 or [email protected].
Returns: Do not return any goods to WPI without obtaining prior approval (RMA
# required) and instructions from WPI’s Returns Department. Goods returned
(unauthorized) by collect freight may be refused. If a return shipment is necessary,
use the original container, if possible. If the original container is not available, use a
suitable substitute that is rigid and of adequate size. Wrap the instrument in paper or
plastic surrounded with at least 100mm (four inches) of shock absorbing material. For
further details, please read the section entitled “Claims and Returns” on page 11 of
this manual.
INSTRUMENT DESCRIPTION
combination
working / counter
electrode
probe
handle
stainless steel
sleeve
gas permeable
membrane
connection
to meter
locking cap
Fig. 2—The locking cap slides over the sensor sleeve. The sleeve is lled with electrolyte and
slides over the ISO-HPO-2 electrode. The locking cap securely attaches the sleeve to the
sensor handle.

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Gas permeable membrane–The membrane covers the end of the stainless sleeve
to separate it from the external environment.
Stainless steel sleeve (WPI#600012, pkg. of 4)–The sleeve houses the sensitive
electrode pair. The sleeve is anged to properly connect with the locking cap. It must
contain fresh lling solution (WPI#100042).
Working/Counter electrode–This is an internal H2O2-sensing pair of working and
counter (reference) electrodes.
Locking cap–This cap attaches the sleeve to the probe handle.
When the sensor is assembled (with locking cap and sleeve in place) the internal
electrode should press gently against the polymeric membrane, which will be slightly
stretched. This ensures that the electrolyte diusion is as thin as possible, minimizing
sensor response time.
NOTE: Once a membrane is stretched it is permanently deformed and cannot be
reused if the sleeve is removed from the electrode.
Additional membrane sleeves are available in packages of 4 (WPI #600012). The
ISO-HPO-2 start-up kit (WPI #600011) also includes replacement membrane sleeves,
along with all the accessories to ll them properly with lling solution (electrolyte).
OPERATING INSTRUCTIONS
Polarizing the Sensor
1. Measure an appropriate volume (for example, 10mL) of 0.1M PBS buer solution
into a glass vial. Place the vial on a magnetic stirring plate and put a small stir bar
into the vial. Set the stir rate at a medium speed.
2. Remove the sensor from the lling solution in which the tip has been immersed
during storage. See the Probe Unpacking Instructions included with your sensor.
NOTE: Some sensors are shipped or stored dry. For these sensors, you will need
to install the membrane sleeve (included). See “Replacing the Membrane Sleeve”
on page 7.
3 Immerse the ISO-HPO-2 sensor tip in PBS buer solution. The sensor tip should
be immersed about 0.3-0.5mm into the solution, and should not be touched by
the stir bar.
4. Connect the ISO-HPO-2 sensor to the input on the free radical analyzer.
5. Turn on the free radical analyzer (TBR4100/1025).
6. Select the poise voltage for hydrogen peroxide on the free radical analyzer. On
the TBR4100/1025, set the Probe Select switch to HPO (+450mV).
NOTE: It is important that the poise be set correctly to avoid erroneous results.
7. Set the current range to 10nA

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8. Allow the sensor to polarize for at least 2 hours to reach a stable baseline current
of about 800–900pA before it is used for measurement.
9. During initial polarization the current is typically high, but within an hour it will be-
gin to decrease rapidly. After several hours the current will fall. The range should
always be set to 10nA, regardless of how low the baseline falls.
10. In two hours or less, the sensor should achieve a stable baseline current value.
A typical baseline value is about 800–900pA. At this time, the sensor is ready for
calibration. If the baseline value does not drop within 2 hours, see “Replacing the
Membrane Sleeve” on page 7.
NOTE: This polarization procedure assumes the temperature is 25ºC. At 37ºC the
baseline current is higher.
Calibrating the Sensor
Hydrogen peroxide (H2O2) is a very important product in the biological system. The
determination of H2O2requires an accurate method of calibration. Amperometeric
(electrochemical) determination using the TBR4100/1025 is a very reliable method to
measure H2O2. The instrument measures the amount of H2O2oxidized on the surface
of the sensor using a poise voltage of +450mV. The oxidation of H2O2at the sensor
surface produces a small current (pA), which is detected by the TBR4100/1025.
The amount of current produced is linearly proportional to amount of H2O2in the
experiment.
Items Required
• One glass vial
• 1.0mM H2O2. standard solution (To make standard solution, see “Making Standard
1mM HPO Solution” on page 4.)
• 0.1M PBS buer solution (PBS buer tablets) (To make standard solution, see
“Making of 0.1M PBS Buer Solution” on page 5.)
NOTE:The multi-port measurement chamber (WPI #NOCHM-4) can be used as
an alternative calibration kit, specically for use at dierent temperature condition.
Calibration temperatures from 4 - 40°C can be controlled using an external circulating
bath (contact WPI for information).
Making Standard 1mM HPO Solution
Hydrogen peroxide solution is commercially available and is commonly sold with
a stabilizer compound in solution. Usually these solutions are within a known
concentration range (for example, 2-4% H202). There are many published procedures
for standardizing H2O2 solutions, such as the titration method using potassium
permanganate. The H2O2 sensor can detect low nM concentrations, so, WPI
recommends the following procedure.
1. Weigh 67.5mg Acetanilide (Sigma-Aldrich 397237) and dissolve it into 250mL of
deionized water in a volumetric ask.
2. Remove 1mL of this solution and put it in a clean container.

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3. Add 231mL of 2-4% H2O2 (Sigma-Aldrich 323381) to the ask.
4. Then, use the removed 1mL of solution from step 2 to dilute the solution in the
ask to exactly 250mL again.
5. Store this standard solution in an amber bottle, if available, or alternatively, wrap
aluminum foil around the bottle to limit light intrusion. This solution should be
refrigerated.
Making of 0.1M PBS Buer Solution
Dissolve 1 Sigma tablet (Sigma-Aldrich P4417-100TAB) into 200mL deionized water
(DIW). This solution yields 0.01M phosphate buer, 0.0027M potassium chloride and
0.137M sodium chloride, pH 7.4, at 25 °C.
Calibration Procedure
1. Polarize the sensor. See “Polarizing the Sensor” on page 3.
2. Record the current value after a 10–15 minute settling period. If the current is
oscale or unstable after a half hour in solution, it is likely that the membrane has
been damaged and the sleeve needs to be changed. (See “Replacing the Mem-
brane Sleeve” on page 7.)
NOTE: The calibration should be carried out at the temperature at which the
samples of H2O2are to be measured. 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 bottle to equilibrate with the water bath.
Creating a Calibration Curve
To create a calibration curve, measure the current (pA) generated by the addition of
increasing amounts of H2O2to the calibration solution.
1. Wait for current output to stabilize.
2. Consecutively add known volumes of the H2O2standard solution to the PBS buf-
fer solution. For example, add 0, 5, 10, 20, 40 and 80mL H2O2standard solution
(1.0mM) into the 10mL PBS buer solution. The additions can be added once
every 20-30 seconds. The resulting output should look similar to the example in
Fig. 3.
Fig. 3—Calibration Curve

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From this output, a calibration curve Fig. 4 can be created by plotting the changes
in current (pA) against the changes in concentration (mM). The slope of this curve
indicates sensitivity of the probe.
Fig. 4—Calibration Data
Interference Temperature
The background current of the sensor will usually increase with increasing
temperature of the experiment. Although, the sensitivity of the sensor does not
change signicantly within the range 20-37°C, it is recommended that any calibration
should be performed at the same temperature as the experiment.
The sensor works best between pH 3–10. Changing the pH of the solution does not
aect the sensitivity. However, if the pH is below 3.0, the noise of the sensor will
increase. At pH 10.0 and higher, the response of the sensor will diminish signicantly.
SENSOR CARE AND MAINTENANCE
Durability and Handling
The sensor is relatively durable, except for the membrane sleeve. Exercise caution
when handling any sensor to avoid actions that could damage the sensor tip. Pay
particular attention to the sensor membrane, because the membrane is extremely
delicate and improper handling will lead to damage. With proper care and by following
the instructions, a membrane sleeve should last more than one month.
Refer to the Probe Unpacking Instructions that came with your sensor for handling
instructions.
CAUTION: Do NOT scratch the sensor membrane sleeve. Do NOT wipe the
sensor membrane with anything, even Kimwipes. If necessary, squirt it with
distilled water or compressed air.