pyroscience Oxygen Sensors Fiber Optic and Contactless User Manual

pyroscience Oxygen Sensors Fiber Optic and Contactless

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 fibre-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 the standard application of optical oxygen sensors from PyroScience. For more information concerning advanced applications, please contact us at info@pyroscience.com. Your PyroScience Team

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, fibre 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), 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.

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 (fibre-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), the Fiber Length (m) of the connected optical fibre (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 the default.

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

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!)
• An 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 an 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!

Atmospheric Pressure

• Another parameter, that 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 the 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).

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 the case of saline water/seawater. For measurements in gas samples, this value has no relevance (and is not active).

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.

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 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 vapour 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 has 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
• 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 RED FLASH 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.

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)

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. 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)

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.

Preparation of Calibration Standards

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. Even worse, the evaporation of water drops would cool down the sensor tips causing undefined temperatures.

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.

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. Conventional 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!

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. Open the lid shortly to ventilate 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!

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 sulphite (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. 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

Custom Calibration: upper custom calibration Instead of air (ambient air, air-saturated water, water-vapour 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)!

Calibration Procedure
Calibration should be performed following the instructions of the software (Pyro Workbench) or read-out device manual. We generally recommend performing 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.
• 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).
• 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.

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), 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).

Oxygen Sensors | Manual

• 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.

SENSOR APPLICATION

PyroScience oxygen sensors can be applied in gas phases, water, aqueous solutions and in ethanol, methanol and isopropanol (robust probes: only short- term application in diluted ethanol, methanol or isopropanol). Other 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.

Oxygen Sensors
Fiber-based sensors

Calibration: 1- or 2-point calibration*

Features: optical isolation

Sterilization: short term treatment with ethylene oxide (EtO), 70% ethanol (EtOH), 70% isopropanol (IPP)

Note: Remove air bubbles from sensor surface, stirring is obligatory for application in water/aqueous samples.

OXR… OXF…| Application: water & gas and semi-solid samples Calibration: 1- or 2-point calibration* in same application medium obligatory: gas (water) calibration for gas (water) measurements

Sterilization: EtO, 70% EtOH (not for option -OI), 70% ISPP (not for option -OI)

Note: Handle with care! Unprotected fragile sensor tip. Extend sensor tip for calibration and measurements.

OXF…-PT| Application: gas

Calibration: 1- or 2-point calibration* in gas

Sterilization: EtO, 70% EtOH (not for option -OI), 70% ISPP (not for option -OI)

Note: Handle with care! Piercing of packaging materials/septa.

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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: EtO, 70% EtOH (not for option -OI), 70% ISPP (not for option -OI)

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

Note: Low signal intensity/signal-to-noise at air-saturated conditions during upper calibration!

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)

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.

Contactless sensors

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.

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!

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Combined sensors

TOVIAL…

| Application: water & gas

Calibration: 1- or 2-point calibration for each sensor necessary*

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** 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*

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.

STERILIZATION, CLEANING AND STORAGE

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!

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 photobleaching 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 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.

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

APPENDIX

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

• 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

% 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

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

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.

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.

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%

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

LED Intensity

• A: 10%
• B: 15%
• C: 20%
• D: 30%
• E: 40%
• F: 60%
• G: 80%
• 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
• 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

Available sensors and read-out devices

FireSting devices

PICO devices

SUB-connector Devices

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.

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.

CONTACT

• PyroScience GmbH
• Kackertstraße 11
• 52072 Aachen
• Deutschland
• Tel.: +49 (0)241 5183 2210
• Fax: +49 (0)241 5183 2299
• info@pyroscience.com
• www.pyroscience.com

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 info@pyroscience.com
• Web www.pyroscience.com
• Registered: Aachen HRB 17329, Germany

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