Oxygen Sensors

Fiber-optic and Contactless

USER MANUAL
OPERATING INSTRUCTIONS

O2

Version V1.07 www.pyroscience.com

Oxygen Sensors | Manual

Oxygen Sensors
Fiber-optic and Contactless
Document Version 1.07

The Oxygen Sensors are released by:

PyroScience GmbH
Kackertstrasse 11
52072 Aachen
Germany

Phone +49 (0)241 5183 2210

Fax +49 (0)241 5183 2299
Email [email protected]
Web www.pyroscience.com

Registered: Aachen HRB 17329, Germany

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Oxygen Sensors | Manual

TABLE OF CONTENT

1 Introduction ..................................................................................................................................... 5

2 Quick Start........................................................................................................................................ 6

3 Sensor Settings ............................................................................................................................... 7

3.1 Conditions in the sample ..............................................................................................................8

3.1.1 Temperature .........................................................................................................................8
3.1.2 Atmospheric Pressure ......................................................................................................9
3.1.3 Salinity....................................................................................................................................9

4 Sensor Calibration ....................................................................................................................... 10

4.1 Important parameters ................................................................................................................ 11

4.1.1 Temperature ...................................................................................................................... 12
4.1.2 Atmospheric Pressure ................................................................................................... 12
4.1.3 Relative Humidity ........................................................................................................... 13

4.2 Preparation of Calibration Standards .................................................................................. 13

4.2.1 Gas measurements: upper calibration .................................................................... 13
4.2.2 Gas measurements: 0% calibration .......................................................................... 14
4.2.3 Measurements in water: upper calibration .......................................................... 14
4.2.4 Measurements in water: 0% calibration ................................................................. 15
4.2.5 Custom Calibration: upper custom calibration .................................................... 16

4.3 Calibration Procedure ................................................................................................................. 16

4.4 Background Compensation ....................................................................................................... 18

5 SENSOR APPLICATION ................................................................................................................ 20

5.1 Oxygen Sensors ............................................................................................................................. 20

5.1.1 Fiber-based sensors ....................................................................................................... 20
5.1.2 Contactless sensors ........................................................................................................ 23
5.1.3 Combined sensors ........................................................................................................... 24

6 STERILIZATION, CLEANING AND STORAGE............................................................................ 25

6.1 Sterilization..................................................................................................................................... 25

6.2 Cleaning and Storage .................................................................................................................. 25

7 Related documents ..................................................................................................................... 27

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8 APPENDIX...................................................................................................................................... 28

8.1 Definition of Oxygen Units ....................................................................................................... 28

8.2 Oxygen Solubility ......................................................................................................................... 30
8.3 Oxygen Measuring Principle .................................................................................................... 31

8.4 Explanation of the Sensor Code ............................................................................................. 32

8.5 Available sensors and read-out devices ............................................................................. 35

8.5.1 FireSting devices ............................................................................................................. 35
8.5.2 PICO devices ..................................................................................................................... 36
8.5.3 SUB-connector Devices ................................................................................................ 36

8.6 Pt100 Temperature Sensor Calibration ............................................................................... 37

9 WARNINGS AND SAFETY GUIDELINES.................................................................................... 38

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1 INTRODUCTION

PyroScience offers a variety of fiber-based and contactless oxygen sensors. For an

overview see our homepage www.pyroscience.com .

These sensors can be read-out with different fiber-optic meters from PyroScience,
including

• the multi-channel PC-operated FireSting-O2 (FSO2-Cx (firmware 4 devices) with Pyro

Workbench and FSO2-x (firmware 3 devices) with Pyro Oxygen Logger software)

• the single channel PICO2 (with Pyro Oxygen Logger software)

• the multi-analyte & multi-channel PC-operated FireSting-PRO (with Pyro

Workbench)

• the (single channel) pocket meter FireSting-GO2 for stand-alone operation (with
FireSting-GO2 Manager software for data management or lab applications).

• the underwater AquapHOx Loggers and Transmitters (with Pyro Workbench) for
optical pH, O2 and temperature sensors with underwater connector (option -SUB).

All software versions are available as free downloads from the PyroScience website and
must be installed on the Windows PC/laptop before connecting the respective oxygen
meter for the first time. For details on the read-out devices, their software and user
interface, please see their respective manuals and handling guidelines.

This manual is intended to provide all necessary information on standard application of

optical oxygen sensors from PyroScience.

For more information concerning advanced applications, please contact us at

[email protected] .

Your PyroScience Team

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2 QUICK START

Step 1: For PC operation, download the respective software from our homepage. The
software can be found in the download tabs of the respective read-out device.
Download, unzip and start the installer, and follow the instructions.

Step 2: For PC operation, connect the PyroScience read-out device to the Windows
PC/laptop with the micro-USB cable.

Step 3: Carefully remove the protective caps from the sensor tip, fiber plug and from the
optical connector(s) at the read-out device.

Step 4: Connect the PyroScience oxygen sensor(s) to the optical connector(s) of the
device.

Step 5: For automatic temperature compensation, connect an appropriate Pt100

temperature sensor to the temperature port or, alternatively, an optical temperature
sensor to one of the remaining channel connectors (multi-channel devices only).

Step 6: Prepare appropriate oxygen calibration standards (see chapter 4.2).

Step 7: Start the respective PyroScience software by clicking on the short-cut on your
desktop or the LCD user interface of the FireSting-GO2 (stand-alone operation).

Step 8: Enter all required Sensor Settings, including the Sensor Code, the Fiber Length
(m) (sensor type: S, W, T, P, X, U, H), Medium and Oxygen Unit for each sensor, as well as
the compensation of environmental parameters (temperature, pressure, salinity, where
indicated/applicable).

Step 9: Perform a 1- or 2-point sensor Calibration.

Step 10: Start measurements and activate Data Logging.

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3 SENSOR SETTINGS

Each optical oxygen sensor comes with an individual Sensor Code, containing important
information for optimal sensor settings and for calibration. The first letter of the sensor
code defines the sensor type. Therefore, it is important to enter the Sensor Code of the
connected sensor into the Sensor Settings of the respective software. For multi-channel
devices, the number of the channel tab must correspond with the channel number at the
PyroScience read-out device.

Important: Enter the correct Sensor Code for sensors connected to a channel at
a PyroScience read-out device. The sensor code can be found on the label
attached to the cable (fiber-based sensors) or on the bag of contactless sensors
(see example below).

For contactless sensors (sensor spots, flow-through cells, respiration vials, nanoprobes;
sensor type: S, W, T, P) and for robust probes (sensor type: X, U, H), the Fiber Length (m)
of the connected optical fiber (e.g. SPFIB-BARE) or of the connected robust probe (e.g.
OXROB10) must be entered additionally (for automatic background compensation).

The Measuring Mode can be adjusted gradually between low drift and low noise of the
sensor signal by moving the arrow with the mouse along the scale. Typically, an
intermediate mode is default.

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3.1 Conditions in the sample

When entering the sensor settings, the Conditions in the Sample during measurements
have to be determined. There are three important parameters to be taken into account,
which can be automatically compensated:

• Temperature

• Atmospheric Pressure

• Salinity

3.1.1 Temperature

Several options for Temperature Compensation of optical oxygen sensors are available:

• External Temperature Sensor (Pt100, temperature port)

• Fixed Temperature (must be entered, kept constant and controlled!)

• Optical Temperature sensor connected to an optical channel (its channel number

must be selected) of a multi-channel read-out device (not for FSGO2, PICO2)

If External Temperature Sensor or Optical Temperature Channel is selected, automatic

compensation of temperature changes on the respective oxygen sensor readings is
activated. The Compensation Temperature will be displayed in the corresponding
channel panel of the main window.

Note: If an External or Optical Temperature Sensor was selected, the sensor has
to be fixed in the sample/calibration standard in which oxygen measurements/
calibration will be performed.

Important: For precise absolute oxygen measurements and optical temperature

sensor calibration using an External Temperature Sensor, please determine
manually if the external (Pt100) temperature has an offset. In case of an offset,
the Pt100 temperature sensors need to be calibrated first (see Appendix 8.6)
before calibrating the optical sensor.

If a Fixed Temperature was selected, the temperature in the sample/calibration

standard must be measured, adjusted and kept constant (needs to be
controlled)! Ensure constant and defined conditions!

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3.1.2 Atmospheric Pressure

Another parameter, which has to be defined in the settings is the atmospheric pressure
(for details please see chapter 8.1). Atmospheric pressure can be compensated by:

• the Internal Pressure Sensor for automatic compensation of pressure changes, e.g.
caused by weather changes. Possible with all FireSting-based devices if oxygen
sensor and device experience the same pressure conditions, or

• by entering a Fixed Pressure (mbar): for applications with PICO2 and for set-ups
with different pressure conditions experienced by the oxygen sensor and the
FireSting-based devices. The actual pressure at the sensor position needs to be
determined with e.g. a barometer and adjusted manually (default: 1013 mbar).

For older software versions it is also possible to enter the Elevation (m) above sea level.
Note that this option takes only the elevation-dependent pressure change into account,
but not the variations due to actual weather conditions. Thus, determining the actual
atmospheric pressure with a barometer gives more precise results (more information in
the respective read-out device manual).

3.1.3 Salinity

The Salinity (g/L) of the environmental sample (based on seawater salinity) is only
relevant if a concentration unit for dissolved oxygen DO measurements was selected
(e.g. mg/L or µmol/L). The sample salinity needs to be measured and entered, e.g. in case
of saline water/seawater. For measurements in gas samples this value has no relevance
(and is not active).

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4 SENSOR CALIBRATION

Ensure that the correct Sensor Code has been entered in the settings (refer to chapter 3)
and prepare appropriate calibration standards (see chapter 4.2). For calibration of
contactless sensors, refer also to chapter 4.4.

Oxygen sensor calibration can be performed in two different ways:

• 1-point calibration (required): upper calibration at ambient oxygen (standard) OR in

special applications 0% calibration (only for measurements exclusively at very low
O2, e.g. with trace range oxygen sensors; only possible with devices operated with
the Pyro Workbench)

• 2-point calibration (optional): upper AND 0% calibration; recommended for

measurements from air saturation/21% to low O2 and for accuracy measurements

Note: It is strongly recommended to perform a manual calibration at conditions

close to the environmental conditions during measurements. Ensure constant
conditions during calibration!

• Gas measurements: the sensor needs to be calibrated (temperature-controlled) in

ambient air (upper calibration) and in some cases also in nitrogen gas N2 (0%
calibration)

• Measurements in aqueous/water samples: the sensor needs to be calibrated

(temperature-controlled) in air-saturated water (upper calibration) and in some cases
also in de-oxygenated water (o% calibration)

Note: In most cases the upper calibration point is defined as the air calibration
point, which can be ambient air, air-saturated water or water-vapor saturated
air (with 100% RH).

Depending on the application (only for advanced users), the upper calibration point can
also be user-defined via a Custom Calibration.

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4.1 Important parameters

All air calibration standards described in the following rely on the virtually constant
oxygen content in the earth’s atmosphere of about 20.95% O2 in dry air. Slight
deviations might be given in closed rooms occupied by many people (or e.g. candles,
combustion engines) consuming the oxygen. So, if in doubt, ensure a good ventilation of
the room with fresh air, e.g. by opening a window for some minutes.

HUMIDITY

The relative humidity of the air causes deviations from the ideal value of 20.95% O2.
Simply speaking, the water vapor in humid air replaces a fraction of the oxygen,
resulting in a diminished oxygen level of e.g. 20.7% O2. For temperatures around and
below 20°C, this effect causes fortunately only a maximum deviation of about 0.5% O2.
However, for higher temperatures at 30°C or even 40-50°C, the humidity of the air gets
a significant influence on the actual oxygen level. For example, ambient air at body
temperature (37°C) with 100% relative humidity contains only 19.6% O2 compared to dry
air with 20.95% O2.

During the calibration of oxygen sensors, there are two possibilities to take the humidity
into account:

• The relative humidity and the temperature of the ambient air must be determined
during calibration. The respective software then calculates automatically the real
oxygen level under these conditions

• The calibration standard is prepared in a closed vessel either filled with water or
partly filled with e.g. wet cotton wool or a wet sponge. This ensures a constant
humidity of 100% RH and there is no need to measure the humidity

ATMOSPHERIC PRESSURE

Another parameter even more important for the air calibration standard is the
atmospheric pressure. The principle parameter measured by oxygen sensors is not the
partial volume (i.e. "% O2"), but the partial oxygen pressure (i.e. "mbar") (see also
appendix 8.1). So, an oxygen level of e.g. 20.7% O2 (determined as described above by a
given humidity and temperature) is converted internally by the respective software into
a partial pressure of oxygen essentially by multiplying the relative oxygen level with
the atmospheric pressure of e.g. 990 mbar:

0.207 x 990 mbar = 205 mbar

giving a partial oxygen pressure of e.g. 205 mbar. This is the essential calibration value
used internally by the software. The atmospheric pressure can be influenced

• by weather changes (e.g. varying between ca. 990 and 1030 at sea level) and

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• by the elevation above sea level (e.g. at 1000 m elevation the typical atmospheric
pressure is about 900 mbar compared to 1013 mbar at sea level)

TEMPERATURE

Precise temperature compensation of the oxygen sensor readings during calibration and
measurements is needed due to two reasons:

• the luminescence of the REDFLASH indicators is temperature dependent and

• the conversion of some oxygen units needs to be compensated for temperature

SUMMARY

There are three important parameters to be known for the air calibration standard:

• Temperature (°C)

• Relative Humidity (% RH)

• Atmospheric Pressure (mbar)

For the FireSting-based read-out devices, the built-in humidity and pressure sensors
together with the external temperature sensor will measure these parameters
automatically for most calibration types.

For the PICO2, these parameters need to be determined, entered and kept constant.

4.1.1 Temperature

It is crucial to determine exactly the temperature in the upper and 0% calibration

standards during the oxygen sensor calibration process via one of the following
possibilities:

• Manual adjustment of a Fixed Temperature (needs to be determined and kept

constant)

• Temperature Compensation with an External (Pt100) Temperature Sensor connected

to the temperature port of a FireSting-based read-out device or

• Temperature Compensation with an Optical Temperature Sensor connected to a

channel at a multi-channel FireSting device (its respective channel number needs to
be entered at Optical Temp. Channel)

4.1.2 Atmospheric Pressure

As for the oxygen measurements, the actual atmospheric pressure is an important

parameter for the calibration and needs to be compensated.

If the atmospheric pressure is read from the internal Pressure Sensor of a FireSting
device, it is important that the calibration standards are exposed to the same
atmospheric pressure as the FireSting device.

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For Pressure compensation with a PICO2 read-out device,

• the actual atmospheric pressure in the calibration standard must be measured and
entered manually. Normal conditions refer to 1013 mbar (default setting)

• Elevation (m) in meters above sea level can be entered (see above)

4.1.3 Relative Humidity

During the calibration of oxygen sensors, there are two possibilities to take the humidity
into account:

• The relative humidity of the ambient air must be determined during calibration. The
software then automatically calculates the actual oxygen level under these
conditions

• The calibration standard is prepared in a closed vessel either filled with water or
partly filled with e.g. wet cotton wool or a wet sponge. This ensures a constant
humidity of 100% RH and there is no need to measure the humidity

For precision calibrations, it is generally recommended to prepare calibration

standards with 100% Relative Humidity, which eliminates any possible error
source by the usage of the internal humidity sensor.

4.2 Preparation of Calibration Standards

4.2.1 Gas measurements: upper calibration

AMBIENT AIR

The dry oxygen sensor, optionally together with the dry external or optical temperature
sensor, is simply exposed to ambient air.

For precise calibrations in ambient air, it is important that the measuring tips of
the oxygen and temperature sensor are completely dry. Wet sensor tips will
cause undefined humidity levels around the sensor tips. And even worse, the
evaporation of water drops would cool down the sensor tips causing undefined
temperatures.

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WATER-VAPOR SATURATED AIR

Enclose wet cotton wool into a flask (e.g. DURAN flask) with a lid prepared with holes for
the oxygen sensor and a temperature sensor from PyroScience. Typically, about 1/3 to
1/2 of the flask volume is filled with wet cotton wool, while the other volume fraction is
left free for inserting the tip of the oxygen and temperature sensor.

After insertion of the sensors and equilibration, follow the calibration procedure given
by the software.

4.2.2 Gas measurements: 0% calibration

NITROGEN GAS

Flush 100% nitrogen gas through a glass flask (e.g. Duran flask) with a lid prepared with
holes for inserting the oxygen sensor and a temperature sensor. Ensure that all air has
been replaced by the nitrogen gas before performing the calibration. Insert the dry
oxygen and temperature sensor into the flask, let it equilibrate and perform the
calibration.

Important: Ensure that no ambient air enters the flask again during the
calibration process. Convectional gas transport is a very fast process! It is
therefore recommended to keep flushing the flask with nitrogen gas during the
complete calibration process!

Please consider that nitrogen gas from gas bottles might be significantly cooled
down by the decompression process. Ensure a correct temperature
determination of the calibration standard!

4.2.3 Measurements in water: upper calibration

AIR SATURATED WATER

For calibration in air saturated water, it is very important that the water is indeed 100%
saturated with air. Please follow one of the two options below to prepare an accurate
calibration standard:

• Fill an appropriate amount of water into a flask (e.g. Duran flask) with a lid prepared
with holes for inserting the oxygen sensor and a temperature sensor. Stream air
through the water with an air stone connected to an air pump (available as
commercial equipment for fish aquaria) for about 10 minutes

• Alternatively, if no air pump is available, fill water into the flask leaving >50% air in
the headspace, close it with a lid and shake the flask strongly for about 1 minute.

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Open the lid shortly for ventilating the headspace with fresh air. Close it again and
shake the flask for 1 more minute

In both cases insert the oxygen and temperature sensor into the flask and ensure that
the sensor tips are immersed in the water and are free of air bubbles. Afterwards follow
the calibration procedures given by the software.

Note: Streaming air through water may cause cooling of the water. Ensure a
correct temperature determination!

4.2.4 Measurements in water: 0% calibration

WATER MIXED WITH A STRONG REDUCTANT

Fill an appropriate amount of water into a glass flask (e.g. Duran flask) with a lid
prepared with holes for inserting the oxygen sensor and a temperature sensor.

Add a strong reductant, like sodium dithionite (Na2S2O4) or sodium sulfite (Na2SO3) at a
concentration of 30 g/L, creating oxygen-free water by chemical reaction. Please note
that 0% calibration capsules are available from PyroScience, giving 50mL 0% calibration
standard (item no.: OXCAL).

DO NOT use saline water (e.g. seawater) for this, but demineralized water. Saline
water prevents proper dissolution of the reductant and can lead to false 0%
sensor calibration.

Stir the solution until the salt is completely dissolved, then stop the stirring and leave
the solution for about 15 minutes. Ensure that there is no headspace and no air bubbles
in the closed flask.

Then insert the oxygen and temperature sensor into the flask, and ensure that the
sensor tips are completely immersed into the water and free of air bubbles. Let
equilibrate and perform the calibration.

Important: Do not store the sensors in this solution and rinse them carefully
after the calibration with demineralized water. Especially the retractable
needle-type sensors (item no. OXRxx and TROXRxx) need to be rinsed very
thoroughly, because salt crystallization within the needle might damage them
irreversibly.

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4.2.5 Custom Calibration: upper custom calibration

Instead of air (ambient air, air saturated water, water-vapor saturated air) for the upper
calibration point, a custom calibration can be performed if custom calibration gases are
used. There are two applications, where custom calibration mode is recommended:

• using trace range sensors in the range of 0-10% O2

• measurements at high oxygen levels (>21% O2)

For a custom calibration, the oxygen level in the calibration standard can be freely
chosen in Oxygen (%O2). Here, the correct value has to be adjusted if custom calibration
gases are used, of e.g. 5% O2, which is useful when using trace range oxygen sensors.

Important: Custom calibration is only recommended for advanced

applications/users! The relevant parameters (%O2, humidity, pressure,
temperature) must be entered correctly (and need to be controlled)!

4.3 Calibration Procedure

Calibration should be performed following the instructions of the software (Pyro

Workbench) or read-out device manual. We generally recommend to perform a two-
point calibration in gas (water) for gas (water) measurements. A one-point calibration
close to environmental conditions is obligatory.

Important: The device and sensors must be placed for >30 min. under constant
environmental conditions before the calibration is performed.

Each time the sensor is placed into a new calibration standard, wait until the sensor
reading is stable by observing the graph and the numerical display of the oxygen sensor
reading. Ensure also stable temperature readings of the External or Optical Temperature
Sensor indicated at Compensation Temperature (°C).

For calibration of optical oxygen sensors from PyroScience, it is important to follow

these steps:

Step 1: Connect the sensor to the respective read-out device and remove the protective
caps from the sensor tip, from the fiber plug and from the optical connector(s) at the
read-out device.

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Step 2: Connect an appropriate Pt100 temperature sensor to the temperature port or,
alternatively, an optical temperature sensor to one of the remaining channel connectors
(multi-channel devices only) for automatic temperature compensation of the oxygen
measurements.

Step 3: Enter the correct Sensor Code for sensors connected to a channel at a
PyroScience read-out device and their Fiber Length (m) (only for sensor type: S, W, T, P,
X, U, H).

Step 4: Prepare appropriate oxygen calibration standards:

• For measurements in GAS: ambient air (upper calibration); nitrogen gas N2 (0%
calibration)

• For measurements in WATER/AQUEOUS samples: air-saturated de-mineralized water

(upper calibration); de-oxygenated water (0% calibration) using sodium dithionite
(Na2S2O4) or sodium sulfite (Na2SO3)

• For measurements in SEAWATER/SALINE WATER: DO NOT use saline water for

preparation of 0% calibration standards, but de-mineralized water

Step 5: Insert the oxygen and temperature sensor into the flask, and ensure that the
sensor tips are completely immersed into the water and free of air bubbles. Let
equilibrate and perform a 1- or 2-point oxygen sensor calibration.

Note: It is strongly recommended to perform a manual calibration at conditions

close to the environmental conditions during measurements. Ensure constant
conditions during calibration! Rinse the sensors carefully after calibration with
demineralized water.

Step 6: After successful 1- or 2-point calibration at constant and comparable

temperature conditions of the successive measurements, perform the measurements in
your samples. Ensure a sufficiently high signal intensity of the sensor (>50), regular
cleaning, re-calibration and careful handling of the sensors.

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4.4 Background Compensation

A background compensation is recommended for optical fibers used for read-out of

contactless sensors and for robust probes.

• For robust probes, respiration vials, flow-through cells and sensor spots with a
black optical fiber (sensor type: S, W, T, P, X, U, H), the FIBER LENGTH needs to be
entered into the software for an automatic background compensation
(recommended for most applications).

• For precision applications, for applications with low signal intensities and for
application of nanoprobes, the option MANUAL background compensation must be
used

FIBER LENGTH

Based on the Fiber Length (m) entered into the Settings, a background signal for
compensation is estimated automatically by the software. For standard applications, this
is the preferred procedure.

MANUAL

For precision applications, for measurements at low signal intensities and for application
of oxygen nanoprobes in microfluidic applications, a Manual background compensation
must be performed to determine the individual luminescence background of the applied
optical fiber. Especially in the case of oxygen nanoparticles (item no. OXNANO) the
luminescence background compensation is important.

Ensure that during manual background compensation the Optical Fiber is

connected to the medium WITHOUT oxygen nanoprobes.

For other contactless sensors, it is important that the fiber is NOT attached to
the sensor spot (i.e. disconnect this end from the adapter, adapter ring or from
the flow-through cell).

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Please ensure that during the subsequent calibration process the Optical Fiber
is again attached to the medium WITH oxygen nanoprobes or to the position
with sensor spots.

Remind that the position of the spot adapter or adapter ring should not be
changed after calibration of the sensor spot; otherwise it has to be calibrated
again.

DISABLE

This option disables the background compensation and is only recommended for expert
users.

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5 SENSOR APPLICATION

PyroScience oxygen sensors can be applied in gas phases, water and aqueous solutions.
Organic solvents (like e.g. acetone), bleach and gaseous chlorine (Cl2) induce
interferences with the sensor reading and potentially destruction of the sensor. No
cross-sensitivity is found for pH 1-14, CO2, CH4, H2S and any ionic species.

For application in organic solvents, a special solvent-resistant oxygen probe (item no.
OXSOLV or OXSOLV-PTS) is available.

Specific application instructions are listed for different sensors in the table below.

5.1 Oxygen Sensors

5.1.1 Fiber-based sensors

Sensor item Sensor-specific application instructions

OXROB… Application: water & gas

Calibration: 1- or 2-point calibration*
Features: optical isolation
Sterilization: short term treatment with 70% ethanol (EtOH)
or 70% isopropanol (IPP); ethylene oxide (EtO, EO)
sterilization (details on request)
Note: Remove air bubbles from sensor surface, stirring is
obligatory for application in water/aqueous samples.

OXR… Application: water & gas and semi-solid samples

OXF… Calibration: 1- or 2-point calibration* in same application
medium obligatory: gas (water) calibration for gas (water)
measurements
Sterilization: short term treatment with 70% ethanol (EtOH)
or 70% isopropanol (IPP); ethylene oxide (EtO, EO)
sterilization (details on request)
Note: Handle with care! Unprotected fragile sensor tip.
Extend sensor tip for calibration and measurements.

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OXF…-PT Application: gas

Calibration: 1- or 2-point calibration* in gas
Sterilization: short term treatment with 70% ethanol (EtOH)
or 70% isopropanol (IPP); ethylene oxide (EtO, EO)
sterilization (details on request)
Note: Handle with care! Piercing of packaging
materials/septa.

OXB… Application: water & gas, semi-solid & custom samples

Calibration: 1- or 2-point calibration* in same application
medium obligatory: gas (water) calibration for gas (water)
measurements
Sterilization: short term treatment with 70% ethanol (EtOH)
or 70% isopropanol (IPP); ethylene oxide (EtO, EO)
sterilization (details on request)
Note: Handle with care, especially during custom
integration! Unprotected fragile sensor tip. Avoid breakage!

TROX…. Application: water & gas at low oxygen concentration

around 0% O2 (max. 10% O2)
Calibration: 1- or 2-point calibration** in application
medium, manual 0% calibration obligatory
Sterilization: short term treatment with 70% ethanol (EtOH)
or 70% isopropanol (IPP); ethylene oxide (EtO, EO)
sterilization (details on request)
Note: Low signal intensity/signal-to-noise at air-saturated
conditions during upper calibration!

OXIMP… Application: water & semi solid samples with oxygen micro
heterogeneity
Calibration: 1- or 2-point calibration* in water
Sterilization: 3% H2O2, ethylene oxide (EtO, EO) sterilization
(details on request)
Note: Low signal intensity/signal-to-noise at air-saturated
conditions during upper calibration!

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OXSOLV… Application: approved polar and non-polar solvents

Calibration: 2-point calibration in air-saturated water (air)
and de-oxygenated water for measurements in approved
solvents (solvent vapor)
Sterilization: 70% EtOH, 70% ISPP
Note: Only measurement in hPa or mmHg for max. 1 h.
Handle with care and mind air bubbles!

* depending on application: 1-point for measurements around 21%/air saturation, 2-point for
complete range between 0% and 21%/air saturation
** 0% calibration obligatory. For measurements around 0%, 1-point calibration at 0% O2 or custom
calibration at custom <21% O2 upper and at 0% O2 recommended.

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5.1.2 Contactless sensors

Sensor item Sensor-specific application instructions

OXSP5 Application: water & gas

Calibration: 1- or 2-point calibration*
Features: optical isolation
Sterilization: ethylene oxide (EtO), 70% ethanol (EtOH), 70%
isopropanol (ISPP), can be autoclaved few cycles at 121°C
for 15 min with special precautions (details on request)
Note: Mind air bubbles! Glue carefully with silicone glue and
let dry for 24h.

OXVIAL… Application: water & gas

Calibration: 1- or 2-point calibration*
Features: optical isolation
Sterilization: EtO, 70% EtOH, 70% ISPP
Note: Remove air bubbles! Determine specific volume
before measurements. Ensure stable temperature
conditions.

OXFLOW… Application: water & gas

Calibration: 1- or 2-point calibration*
Features: optical isolation
Sterilization: EtO, 70% EtOH, 70% ISPP
Note: Flow rate 1-500 mL/min. Remove air bubbles! Clean
regularly.

OXFTC… Application: water & gas

Calibration: 1- or 2-point calibration*
Sterilization: EtO, 70% EtOH, 70% ISPP
Note: Flow rate 10-100/20-500 mL/min. Remove air
bubbles! Clean regularly.

OXNANO Application: water/aqueous samples

Calibration: 2-point calibration in application medium
Sterilization: can be autoclaved few cycles at 121°C for 15
min with special precautions (details on request)
Note: Manual background compensation necessary in
microfluidic applications. Not in colored, illuminated or
fluorescing samples.

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TROX… Application: water & gas at low oxygen concentration

around 0% O2 (max. 10% O2)
Calibration: 1- or 2-point calibration** in application
medium, manual 0% calibration obligatory
Note: Low signal intensity/signal-to-noise at air-saturated
conditions during upper calibration!

* depending on application: 1-point for measurements around 21%/air saturation, 2-point for
complete range between 0% and 21%/air saturation
** For measurements around 0%, 1-point calibration at 0%O2 or custom calibration at custom
<21% O2 upper and at 0% O2 recommended.

5.1.3 Combined sensors

Sensor item Sensor-specific application instructions

TOVIAL… Application: water & gas

Calibration: 1- or 2-point calibration for each sensor
necessary*
O2 T Features: optical isolation
Note: Remove air bubbles! Determine specific volume
before measurements. Ensure stable temperature
conditions.

PHTOVIAL … Application: water

Calibration: 1- or 2-point calibration in specified
buffers/calibration standards for each sensor necessary**
O2 pH T Features: optical isolation
Note: Remove air bubbles! Ensure stable conditions.

TOFTC2 Application: water & gas

Calibration: 1- or 2-point calibration in application medium
for each sensor necessary*
O2 T Note: Flow rate 20-500 mL/min
Remove air bubbles! Clean regularly.

* depending on application: 1-point for temperature sensors, 1-point for oxygen measurements
around 21%/air saturation, 2-point for complete range between 0% and 21%/air saturation
** 1-point for temperature sensors, 1-point for oxygen measurements around 21%/air saturation,
2-point for complete range between 0% and 21%/air saturation, 2-point calibration for pH sensors
at pH 2 and pH 11, using PyroScience buffer capsules

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6 STERILIZATION, CLEANING AND STORAGE

6.1 Sterilization

Most oxygen sensors can be sterilized with ethylene oxide (EtO) and cleaned with
peroxide (3% H2O2), soap solution or ethanol.

Please refer to the specifications on the respective PyroScience website.

The oxygen sensor spots (item no. OXSP5) and nanoprobes (item no. OXNANO) can be
autoclaved (few cycles at 121°C for 15 min) with special precautions. More details on
request.

Important: do not use bleach, acetone or any solvent/agent not approved by

PyroScience!

6.2 Cleaning and Storage

After finalization of the measurements, the sensor tip of the needle-type and bare fiber
sensors, as well as the robust probes should be rinsed carefully with demineralized
water. After cleaning, let dry and put on the protective cap / tubing for storage in a dry,
dark and secure place at room temperature. For all sensors and fibers, put the black caps
on the fiber plug to prevent that light is entering the fiber possibly causing photo-
bleaching of the indicator.

In case of retractable sensors and application in seawater / aqueous samples with

dissolved salts, the sensor has to be cleaned thoroughly with demineralized water to
prevent salt crystallization in the needle which can cause breaking of the sensor tip.
After drying, retract the sensor tip into the needle and put on the protective cap onto
the needle to protect the sensor tip and to avoid injuries.

Store the sensor in a dry, dark and secure place at room temperature.

A signal drift of the sensor can indicate photo-bleaching of the oxygen-sensitive

REDFLASH indicator depending on the ambient light intensity, as well as the intensity of
the excitation light and the sample frequency. This can necessitate new calibration of

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the sensor and possibly also a re-adjustment of the Sensor Settings. In case of sensor
spots, this could require a re-positioning of the optical fiber on the sensor spot and a
subsequent new calibration.

If the signal intensity is getting below 50 mV, the sensor needs to be replaced, as
indicated by the respective warning.

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7 RELATED DOCUMENTS

Related documents for more detailed instructions on fiber-optic read-out devices,

software and optical sensors are available:

• manual for logger software "Pyro Workbench" (Windows)

• manual for multi-analyte meter FireSting-PRO

• manual for oxygen meter FireSting-O2 (with Oxygen Logger software)

• manual for portable oxygen meter FireSting-GO2 (with FireSting-GO2 Manager

software)

• manual for oxygen meter PICO2 (with Oxygen Logger software)

• manual for the AquapHOx Loggers or Transmitters

• manual for optical pH sensors

• manual for optical temperature sensors

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8 APPENDIX

8.1 Definition of Oxygen Units

• phase shift dphi

The phase shift dphi is the fundamental unit measured by the optoelectronics in the
PyroScience read-out device (see chapter 8.3). Please note, that dphi is not at all linearly
dependent on the oxygen units, and increasing oxygen levels correspond to decreasing
dphi values, and vice versa! As a thumb of rule, anoxic conditions will give about dphi =
53, whereby ambient air will give about dphi = 20.

• raw value raw value

Definition: raw value = %O2 (uncalibrated)

The unit raw value is the default unit for uncalibrated sensors and shows only
qualitative oxygen sensor readings.

• partial pressure pO2 hPa = mbar

Used in: gas and water phase

For a calibrated sensor, the partial oxygen pressure pO2 in units of hPa (equivalent to
mbar) is the fundamental oxygen unit measured by the PyroScience read-out device.

• partial pressure pO2 Torr

Definition: pO2 [Torr] = pO2 [hPa] x 759.96 / 1013.25

Used in: gas or water phase

• volume percent pV %O2

Definition: pV = pO2 [hPa] / patm x 100%

Used in: gas

with patm: actual barometric pressure

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• % air saturation A % a.s.

Definition: A[%a.s.] = 100% x pO2 / p100O2

Used in: water phase

with p100O2 = 0.2095 (patm – pH2O(T))

pH2O(T) = 6.112mbar x exp (17.62 T[°C] / (243.12 + T[°C]))

pO2: actual partial pressure

patm: actual barometric pressure

T: actual temperature

pH2O(T): saturated water vapor pressure at temperature T

• Dissolved O2 concentration C µmol/L

Definition: C [µmol/L] = A[%a.s.] / 100% x C100(T, P, S)

Used in: water phase

with C100(T, P, S): interpolation formula for dissolved oxygen concentration in

units of µmol/L at temperature T, atmospheric pressure P and Salinity S (see chapter
8.2).

• Dissolved O2 concentration C mg/L = ppm

Definition: C [mg/L] = C [µmol/L] x 32 / 1000

Used in: water phase

• Dissolved O2 concentration C mL/L

Definition: C [mL/L] = C [µmol/L] x 0.02241

Used in: water phase

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8.2 Oxygen Solubility

The calculation of the equilibrium oxygen concentration C100(T, P=1013mbar, S) in units

of µmol/L is done at standard atmospheric pressure of 1013 mbar as a function of water
temperature in units of °C and salinity in units of PSU ("practical salinity unit" ≈ g/L). In
order to correct these for the actual atmospheric pressure patm, the following formula
has to be applied:

C100(T, P, S) = C100(T, P=1013mbar, S) x patm / 1013mbar

References: Garcia, HE and Gordon, LI (1992)

Oxygen solubility in seawater: Better fitting equations.

Limnol. Oceanogr. 37: 1307-1312

Millero, FJ and Poisson, A (1981)

International one-atmosphere equation of state of seawater.

Deep Sea Res. 28A: 625-629

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8.3 Oxygen Measuring Principle

The new REDFLASH technology is based on the unique oxygen-sensitive REDFLASH

indicator showing excellent brightness. The measuring principle is based on the
quenching of the REDFLASH indicator luminescence caused by collision between oxygen
molecules and the REDFLASH indicator immobilized on the sensor tip or surface. The
REDFLASH indicators are excitable with red light (more precisely: orange-red at a
wavelength of 610-630 nm) and show an oxygen-dependent luminescence in the near
infrared (NIR, 760-790 nm).

The REDFLASH technology impresses by its high precision, high reliability, low power
consumption, low cross-sensitivity, and fast response times. The red-light excitation
significantly reduces interferences caused by autofluorescence and reduces stress in
biological systems. The REDFLASH indicators show much higher luminescence brightness
than competing products working with blue light excitation. Therefore, the duration of
the red flash for a single oxygen measurement could be decreased from typically 100
ms to now typically 10 ms, significantly decreasing the light dose exposed to the
measuring setup. Further, due to the excellent luminescence brightness of the REDFLASH
indicator, the actual sensor matrix can be now prepared much thinner, leading to fast
response times of the PyroScience oxygen sensors.

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The measuring principle is based on a sinusoidally modulated red excitation light. This
results in a phase-shifted sinusoidally modulated emission in the NIR. The PyroScience
read-out device measures this phase shift (termed "dphi" in the software). The phase
shift is then converted into oxygen units based on the Stern-Vollmer-Theory.

8.4 Explanation of the Sensor Code

The oxygen sensors are delivered with an attached sensor code which must be entered
in the Settings (refer to chapter 3). The following figure gives a short explanation about
the information given in the sensor code.

Example Code: XB7-532-205

Sensor Type

LED Intensity

Amplification

Pre-Calibration 0%

Pre-Calibration 21%

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Sensor Type

Z Oxygen Micro / Minisensor

Y Oxygen Minisensor

X Robust Oxygen Probe

V Oxygen Minisensor (TRACE range)

U Robust Oxygen Probe (TRACE range)

T Oxygen Sensor Spot / FTC (TRACE range)

S Oxygen Sensor Spot / FTC

Q Solvent-Resistant Oxygen Probe

P Oxygen Nanoprobes

H Insertable Oxygen Miniprobe

LED Intensity

A 10% E 40%

B 15% F 60%

C 20% G 80%

D 30% H 100%

Amplification

4 40x

5 80x

6 200x

7 400x

OXYGEN SENSORS

C0 (Pre-Calibration at 0% O2)

dphi0 = C0 / 10

C100 (Pre-Calibration at 100% O2)

dphi100 = C100 / 10

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The values of the pre-calibration are valid for the following calibration conditions:

Partial Volume of Oxygen (% O2) 20.95

Temperature at both calibration points (°C) 20.0

Air Pressure (mbar) 1013

Humidity (% RH) 0

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8.5 Available sensors and read-out devices

8.5.1 FireSting devices

Sensor Type Compatible read-out devices

FSO2-Cx/FSO2-x FSGO2 FSPRO

OXR…

OXF…

OXF…PT

OXB…

OXROB(SC)…

OXIMP…

OXSOLV…

OXSP5
with SPFIB-BARE / SPFIB-LNS

OXFLOW…
with SPFIB-BARE

OXFTC…
with SPFIB-BARE

TOFTC2
with SPFIB-BARE

OXVIAL..
with SPFIB-BARE

TOVIAL20
with SPFIB-BARE

OXNANO
with SPFIB-BARE / SPFIB-LNS

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8.5.2 PICO devices

Sensor Type Compatible read-out devices

PICO-O2

OPROB3

OPDIP20

OXSP5
with PICFIB2 / PICROD2

OXFLOW…
with PICFIB2

OXFTC…
with PICFIB2

OXVIAL..
with PICFIB2

OXNANO
with PICFIB2 / PICROD2 (cultures) or
PICFIB2-LNS/ PICROD2-LNS (Microfluidics)

8.5.3 SUB-connector Devices

Sensor Type Compatible read-out devices

PICO-O2-SUB APHOX-x

OXR…-SUB

OXF…-SUB

OXB…-SUB

OXROB(SC)-SUB

OXSP5-SUB

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with SPFIB-LNS-SUB

OXFLOW-HS
with SPFIB-BARE-SUB

8.6 Pt100 Temperature Sensor Calibration

For precise absolute temperature readings, an optional 1-point calibration of the

external temperature sensor is recommended (except for AquapHOx devices).

For this, check the reading of the external temperature Pt100 probe periodically in
stirred water / water bath / incubator of known temperature at steady state. It is also
possible to prepare a water-ice-mixture giving 0°C, where at least 50 mm of the Pt100
temperature probe tip is submerged. After calibration of the Pt100, a new optical sensor
calibration must be performed.

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9 WARNINGS AND SAFETY GUIDELINES

Before using PyroScience oxygen sensors, carefully read the instructions and
user manuals for the respective PyroScience read-out device. The manuals are
available for download on www.pyroscience.com

Prevent mechanical stress (e.g. scratching) to the sensing surface at the tip of
the oxygen sensor! Avoid strong bending of the fiber-optic cables. They might
break!

Ensure that the complete sensing surface at the tip is always covered by the
sample and is free of air bubbles, and that liquid samples are stirred.

Calibration and application of the oxygen sensors is on the user’s authority, as

well as data acquisition, treatment and publication!

PyroScience oxygen sensors and read-out devices are not intended for medical
or military purposes or any other safety-critical applications. They must not be
used for applications in humans; not for in vivo examination on humans, not for
human-diagnostic or therapeutic purposes. The sensors must not be brought in
direct contact with foods intended for consumption by humans.

The sensors must be used in the laboratory by qualified personnel only,

following the user instructions and the safety guidelines of the manual, as well
as the appropriate laws and guidelines for safety in the laboratory!

Keep the PyroScience oxygen sensors and read-out devices out of reach of
children! Store the oxygen sensors in a secure, dry and dark place at room
temperature.

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CONTACT

PyroScience GmbH Tel.: +49 (0)241 5183 2210

Kackertstraße 11 Fax: +49 (0)241 5183 2299
52072 Aachen [email protected]
Deutschland www.pyroscience.com

www.pyroscience.com