Unisense A S 2024.01-N2O Nitrous Oxide Sensor User Manual

NITROUS OXIDE SENSOR
USER MANUAL
NITROUS OXIDE SENSOR MANUAL
Copyright © 2024 · Unisense A/S
Version January 2024
UNISENSE A/S

WARRANTY AND LIABILITY

NOTICE TO PURCHASER
This product is for research use only. Not for use in human diagnostic or therapeutic procedures.

WARNING
Microsensors have very pointed tips and must be handled with care to avoid personal injury and only by trained personnel.
Unisense A/S recommends users to attend instruction courses to ensure proper use of the products.

WARRANTY AND LIABILITY
The Nitrous Oxide sensor is covered by a 60 days limited warranty.
Microsensors are a consumable. Unisense will only replace dysfunctional sensors if they have been tested according with the instructions in the manual within 14 days of receipt of the sensor(s).
The warranty does not include repair or replacement necessitated by accident, neglect, misuse, unauthorized repair, or modification of the product. In no event will Unisense A/S be liable for any direct, indirect, consequential or incidental damages, including lost profits, or for any claim by any third party, arising out of the use, the results of use, or the inability to use this product.
Unisense mechanical and electronic laboratory instruments must only be used under normal laboratory conditions in a dry and clean environment. Unisense assumes no liability for damages on laboratory instruments due to unintended field use or exposure to dust, humidity or corrosive environments.

REPAIR OR ADJUSTMENT
Sensors and electrodes cannot be repaired. Equipment that is not covered by the warranty will, if possible, be repaired by Unisense A/S with appropriate charges paid by the customer. In case of return of equipment please contact us for return authorization.
For further information please see the documents General Terms of Sale and Delivery of Unisense A/S as well as the manuals for the respective products.

CONGRATULATIONS WITH YOUR NEW PRODUCT!

SUPPORT, ORDERING, AND CONTACT INFORMATION
The Unisense nitrous oxide microsensor is a miniaturized Clark-type nitrous oxide sensor with a guard cathode designed for research applications within environmental sciences.
If you wish to order additional products or if you encounter any problems and need scientific/technical assistance, please do not hesitate to contact our sales and support team. We will respond to your inquiry within one working day.
E-mail: [email protected]
Unisense A/S
Langdyssen 5
DK-8200 Aarhus N, Denmark
Tel: +45 8944 9500
Fax: +45 8944 9549
Further documentation and support is available at our website www.unisense.com.

REPLACEMENT OF SENSORS
Unisense will replace sensors that have been damaged during shipment provided that:

• The sensors were tested immediately upon receipt in accordance with the delivery note and the manual
• The seal is still intact.
• The sensors are returned to Unisense for inspection within two weeks.
• The sensors are correctly packed for return to Unisense, in accordance with the note included in the sensor box.

OVERVIEW

This manual covers all the Unisense N2O sensors.
With its minute tip size, excellent response time, and insignificant stirring sensitivity the Unisense nitrous oxide sensor makes it possible to make reliable and fast measurements with a high spatial resolution.

WARNING
Unisense sensors are neither intended nor approved for use on humans

TABLE 1:
N2O SENSOR CHARACTERISTICS

Type| Tip diameter| Measuring range| Detection
limit| Response
time (90%)
---|---|---|---|---
N2O-50| 40-60 µm| 0-200 µm| 0.3 µM| < 30 sec
N2O-100| 90-110 µm| 0-200 µm| 0.1 µM| < 30 sec
N2O-500| 400-600 µm| 0-200 µm| 0.1 µM| < 35 sec
N2O-R-LR| 400-600 µm with cap| 0-10 µm| 25 µM| < 65 sec
N2O-R-SR| 400-600 µm with cap| 0-50 µm| 0.1 µM| < 65 sec
N2O-R-IR| 400-600 µm with cap| 0-300 µm| 0.1 µM| < 65 sec
N2O-R-HR| 400-600 µm with cap| 0-4 µm| 1 µM| < 65 sec
N2O-MR| 500 µm for Microrespiration system| 0-50 µm| 0.1 µM| < 65 sec
N2O-NP| 1.6 x 40 mm needle for piercing| 0-50 µm| 0.1 µM| < 65 sec
N2O-FT| With glass flow cell, 6 or 8 mm OD| 0-50 µm| 0.1 µM| < 65 sec
N2O-SL| With Swagelok flow cell, 1/8” or 1/4”| 0-50 µm| 0.1 µM| < 65 sec
N2O-ST| With 1/4” x 4 cm steel tube| 0-50 µm| 0.1 µM| < 65 sec
N2O-PEEK| For 1/8” or 1/6” PEEK tube| 0-50 µm| 0.1 µM| < 65 sec

*Stainless steel cap, 10 mm outer diameter
Stirring sensitivity for all types < 2%
Sensors must never be exposed to N2O concentrations above their working range.
LR: Low Range; SR: Standard Range; IR:Intermediate Range; HR: High Range.
The Unisense nitrous oxide microsensor is a miniaturized Clark-type sensor with an internal reference and a guard cathode. In addition, the sensor is equipped with an oxygen front guard, which prevents oxygen from interfering with the nitrous oxide measurements. The sensor is connected to a high- sensitivity picoammeter and the cathode is polarized against the internal reference. Driven by the external partial pressure, nitrous oxide from the environment will penetrate through the sensor tip membrane and be reduced at the metal cathode surface. The picoammeter converts the resulting reduction current to a signal. The internal guard cathode is also polarized and scavenges oxygen in the electrolyte, thus minimizing zero-current and stabilization time.

GETTING STARTED

UNPACKING AND CONNECTING A NEW SENSOR
When receiving a new microsensor, first remove the shock-absorbing grey plastic net. Then secure the sensor in a safe position (e.g. micromanipulator or calibration chamber) before connecting it to the measuring meter.
The signal from the nitrous oxide sensor is generated in picoamperes. Therefore the nitrous oxide sensor must be connected to a picoammeter amplifier unit during measurements.

WARNING
Do not remove the seal and protective plastic tube before these steps and calibration are successfully completed.

PRE-ACTIVATION AND POLARIZATION
Please see procedure below.
Pre-activation period: Nitrous oxide sensors have a metal cathode which during normal exposure to oxygen over time will acquire an oxidized surface layer. If the sensor is new or has not been used recently, this layer must be “pre-activated” in order for the sensor to work. This is done by applying a voltage of -1.3 V for 30 minutes prior to the period of  polarization which is required for most electrochemical sensors.

WARNING
Incorrect polarization may destroy the sensor.

Stabilization period: The electrolyte inside the sensor can contain large amounts of oxygen which must always be removed before the sensor is stable. This is done by the guard  cathode inside the sensor whenever the sensor is polarized, i.e., connected to an amplifier. This process requires some time, depending on the dimensions and exact proportions of the individual sensor.
IMPORTANT:
If the sensor is new or has been disconnected for > 4 hours, follow the PRE- ACTIVATION PROCEDURE below. For recently used N2O sensors, follow the POLARIZATION  PROCEDURE below.
Please read both sections before starting the relevant procedure.

For directions on adjusting the polarization, please consult the relevant instrument manual.

PRE-ACTIVATION PROCEDURE

1. Secure the nitrous oxide sensor with its tip immersed in nitrous oxide free water. (It does not harm the sensor to be in air instead, but pre-activation, polarization, and calibration might as well be made with the same set-up).
2. Using an fx-6, fx-3 pA, x-5 or N2O UniAmp:
   Change the polarization to -1.3 V in the Calibration tab of SensorTrace Suite.
   Using a Microsensor Multimeter or Monometer:
   Connect the sensor to the amplifier.
   Turn the polarization to -1.3V.
   Using a PA2000 amplifier:
   Connect ONLY the BNC connector of the adaptor cable, not the yellow banana plug and turn the polarization voltage to -1.3 V.

NOTE
The software converts the pA signal into mV. Standard setting is 1 mV = 1 pA. This may be modified by changing the Pre-Amp setting (mV/pA) in the software (UniAmp) or on the  instrument (Multimeter, Monometer, and Field Multimeter).

Leave the polarization at -1.3 V for approx 30 min. You will see a very high signal in the beginning but this will decrease after 5 – 10 minutes.

POLARIZATION PROCEDURE

1. Using a fx-6, fx-3 pA, x-5, or N2O UniAmp
   Change the polarization back to -0.8 V.
   Using a Microsensor Multimeter or Monometer:
   Turn the polarization voltage to -0.8 V.
   Using a PA2000 amplifier:
   Turn the polarization voltage to -0.8 V.
   Connect the yellow banana plug to the yellow connection on the meter.

2. You will see a sharp decrease followed by a rapid increase in the signal. After this, the signal will stabilize with an initial rapid and then a more slow decrease. Wait until the  signal is below 20 mV and stable. This may take many hours, e.g. 12 hours (see below). If the sensor signal does not reach this value, please go to the Troubleshooting section.

In general the sensor should be polarized and allowed to stabilize for as long as possible before calibration and measurements to get maximum stability. Unisense recommends that you perform the pre-activation the day before measurements and then leave the sensor polarized overnight. If the signal does not stabilize or is too high or too low, see ‘Trouble-shooting’.

IMPORTANT
Calibration must first be performed after the sensor signal has stabilized.

CALIBRATION PRINCIPLES
Calibration must be performed after the sensor signal has stabilized. The N2O sensors respond linearly to the N2O concentration within their measuring range (see Table 1) and signals can thus be linearly converted to partial pressure. Check and repeat calibration at appropriate intervals. Shortly after taking a sensor into use, the appropriate interval may be 2 hours; when the sensor has been used for some time, it may be 24 hours. To minimize the need for calibrations, keep the sensor polarized between measurements, unless the time between measurements exceeds several days.

NOTE
The lifetime of the N2O sensor is the same whether the sensor is continuosly polarized or store un-polarized.

The membrane permeability of nitrous oxide microsensors changes with time, so a change in signal of up to 50% may occur over months.

CALIBRATION
It is recommended to use the Unisense N2O Sensor Calibration Kit for calibrating the N2O sensors (https://www.unisense.com/calibration_kits/). This kit ensures accurate and  simple calibration both in the lab and in the field. The calibration kit can be shipped as normal cargo and does not require dangerous goods shipping.

Therefore, it is ideal also for shipping to field work, research cruises etc. The detailed calibration procedure can be found in the Calkit-subscript Manual (https://www.unisense.com/manuals/)

ALTERNATIVE CALIBRATION
ZERO CALIBRATION POINT
Place/keep the sensor tip in nitrous oxide free water and record the signal. This signal is your calibration value for zero nitrous oxide. The temperature in the solutions for zero calibration and for known N2O concentration must be the same.

WARNING
Never expose the N2O sensors to concentrations above their measuring range. This will destroy the senor! See Table 1 for the measuring range for each type of N2O sensor.

KNOWN CONCENTRATION CALIBRATION POINT
The nitrous oxide sensor responds linearly to N2O within its measuring range (see Table 1 for the measuring range for your type of sensor). Therefore, a two-point calibration is sufficient. Prepare water with a defined nitrous oxide concentration (partial pressure), which is slightly above the maximum expected nitrous oxide concentration (partial pressure) in  the samples. Never expose the sensor to concentrations more than the specified measuring range – it may cause irreversible damage to the sensor (see Table 1 for the measuring range for your sensor).
A defined nitrous oxide concentration can be obtained by two different procedures:

WARNING
Bubbling of water with any gas may cause the water to cool considerably. Monitor the temperature to find a suitable bubbling rate, which does not cool the water significantly.

a) Use a gas mixture controller to obtain a defined mixture of nitrous oxide and nitrous oxide-free inert bulk carrier gas from a gas tank (e.g. N2) . For instance, to obtain a nitrous oxide concentration of 100 μM in the calibration chamber at 22°C, bubble the water in the calibration chamber with a gas mixture containing 1 atm N2 and 0,003679 atm N2O (nitrous oxide partial pressure 0.003679 atm × solubility 27,05 × 10-3 mol/liter/atm. = 100 μmol/liter, see Table 2).
For a Unisense 300 ml calibration chamber CAL300, 5 minutes of bubbling at a rate of 5 L per minute is sufficient time to achieve 99% of the concentration. If the equipment (gas mixture controller) is available, this method can be convenient, because it is possible to switch between different nitrous oxide levels without changing the water.
Calculate the appropriate mixture from the solubilities given in Table 2. Alternatively, use the N2O calculator in the Unisense software (Tools – N2O Calculator) or the Unit Converter on the Unisense website: Click “Knowledge” -“Unit Converter”.
For obtaining correct concentrations it is important that the headspace above the water in the calibration chamber is closed except for one hole, which should be only slightly larger than the microsensor shaft. This effectively prevents ambient air from entering the vessel. We recommend the Unisense Calibration chamber, CAL300, and rubber stoppers for calibrations.

b) Add a defined volume of nitrous oxide saturated water to a defined volume of water in a calibration chamber. For instance adding 3,679 ml of nitrous oxide saturated to 996,32 ml water gives a concentration of 100 μM at 22°C (calculated from Table 2), as 3,679 ml of nitrous oxide saturated water contains 100 μmol nitrous oxide. Using a calibration chamber containing 200 ml, reduce the volume of added nitrous oxide saturated water by a factor of 5 for obtaining 100 μM, and so on. After adding nitrous oxide-saturated water to the calibration chamber, mix it thoroughly for a few seconds and read the signal when it is stable.

Do not stir bubbles into the water and do not mix by bubbling, as this will flush nitrous oxide from the water. A magnetic stirrer is also not recommended as a mixing tool because it can introduce electrical noise to the signal. The nitrous oxide in the water will slowly escape to the atmosphere and the concentration can only be considered constant for a few  minutes. concentration can only be considered constant for a few minutes.
APPROVAL OF NEW SENSOR
If the sensor functions according to the criteria given in the delivery note, carefully remove the seal and the protective tube before making measurements.

MEASUREMENTS

Nitrous oxide sensors can be used for a wide variety of measurements (please see our web page www.unisense.com for further information). They are for example used for making profiles in sediments or tissues where a high spatial resolution is required or for nitrous oxide measurements in water samples.

TEMPERATURE
Closely monitor the temperature. The temperature coefficient varies from sensor to sensor but is approximately 2-3% per ºC.
UniAmp instruments has automatic temperature compensation within ±3°C from the calibration temperature, if a temperature sensor is connected.

MOUNTING THE SENSORS
Although the Unisense microsensors are made of glass, the tip is flexible and can adjust slightly around physical obstacles. The sensor is thus rather sturdy in the longitudinal direction. However, large obstacles like stones or lateral movements of the sensor when the tip is in contact with a solid substrate may cause the tip to break.
Furthermore, due to the small size of the microsensor tip and to the steepness of gradients in many environments, a displacement of the sensor tip of only a few microns may change its environment.
Therefore, when working with glass-tip microsensors, we recommend that measurements are performed only in a stable set-up free of moving or vibrating devices. We recommend the Unisense laboratory stand LS and the Unisense micro- manipulator MM33 (double MM33-2) for laboratory use. For in situ use we recommend our in situ stand (IS19) and a micromanipulator.

TEMPERATURE
Closely monitor the temperature. The temperature coefficient varies from sensor to sensor but is approximately 2-3% per °C. UniAmp instruments has automatic temperature compensation within ±3°C from the calibration temperature, if a temperature sensor is connected.

ELECTRICAL NOISE
The signal of the microsensor is in the 10-13 to 10-10 ampere range.
Although both the Unisense picoammeter and the Unisense nitrous oxide microsensors are quite resistant to electrical noise from the environment, electrical fields may interfere with the sensor signal. Therefore, we recommend that unnecessary electrical/mechanical equipment is switched off and the sensor or wires are not touched during measurements.

INTERFERENCE
Exposure to high concentrations of sulfide should be avoided as it can affect the sensitivity of the nitrous oxide microsensor.
Nitric Oxide (NO) is interfering the N2O sensor signal.
CO2 in mM concentrations may affect the zero signal of the sensor.
On suspicion of sensor damage, repeat calibration and consult ‘Trouble- shooting’.

ADVANCED USE OF THE N2O SENSOR

Unisense can construct nitrous oxide sensors for customer requested applications at an additional cost. Unisense provides several options for customizations and adaptions (e.g. tip  size, response time, pressure tolerance, and stirring sensitivity) making accurate measurements possible for even more applications.
Please visit our website for more information (unisense.com/products/customizations)

STORAGE AND MAINTENANCE

Always store the sensor in the protective plastic tube used for shipping. The nitrous oxide microsensor can be stored with the tip exposed to water or air. The sensor can be stored dry at room temperature (15-30°C) or in the fridge (approx. 4°C) which may give a longer lifetime. If the sensor is used regularly it can be stored polarized.

CLEANING THE SENSOR
Depending on which substance is present on the sensor tip or membrane, the sensor can be cleaned with different solutes.
The standard method is to rinse with 96% ethanol, then rinse with 0.01 M HCl and rinse with water. This will remove most substances.
Alternatively it is possible to rinse with 0.1M NaOH, isopropanol, or other detergent

REFERENCES

• Andersen, K., T. Kjaer, and N. P. Revsbech. 2001. An oxygen insensitive microsensor for nitrous oxide. Sensors and Actuators B-Chemical. 81(1):42-48.

• Elberling, B., Christiansen, H.H. and Hansen, B.U. 2010. High nitrous oxide production from thawing permafrost. Nature geoscience 3:332-335. Abstract

• Horn, M., A. Schramm, and H.L. Drake. 2003. The earthworm gut: an ideal habitat for ingested N2O-producing microorganisms.
  Appl. Environ. Microbiol. 69, 1662-1669.

• Revsbech, N. P., and B. B. Jørgensen. 1986. Microelectrodes:
  Their Use in Microbial Ecology, p. 293-352. In K. C. Marshall (ed.), Advances in Microbial Ecology, vol. 9. Plenum, New York.

TROUBLESHOOTING

thermometer. See video guide here: unisense.com/video- guides/
Problem| A slow response
Possible cause| Insoluble compounds deposited at the sensor tip
Solution| Rinse with 96% ethanol, rinse with 0.01M HCl and rinse with water
Problem| An unstable signal or the signal fluctates if the setup is touched or equipmen is introduced in the medium you are measuring in
Possible cause| Electrical disturbance of the sensor through the  tip membrane
Solution| Ground the set-up using the blue grounding cable supplied with the picoammeter. Connect the reference plug on the picoammeter (blue plug) with the medium you are measuring in.
Problem| High and drifting signal
Possible cause| The sensor tip is broken
Solution| Replace the microsensor
Problem| A high signal
Possible cause| Possible oxygen interference if the oxygen front guard is damaged
Solution| Replace the microsensor
Problem| A high signal (2)
Possible cause| Air is trapped in the tip
Solution| Degas water by boiling and subsequent cooling or by 10 min of vacuum treatment. Immerse the sensor tip for 20 min in the degassed water. Repeated or prolonged treatment may be necessary.
Problem| The signal is very low
Possible cause| Contamination of the cathode surface (e.g. by sulfide) or loss of the cathode material due to excessive vibration.
Solution| Replace the microsensor.

If you encounter other problems and need scientific/technical assistance, please contact [email protected] for online support (we will answer you within one workday)

APPENDIX: EQUILIBRIUM N2O CONCENTRATIONS

TABLE 2:
Nitrous oxide (mmol N2O/liter) at ambient partial pressure of 1 atm. in water as a function of temperature and salinity.

(‰) / ºC| 0| 5| 10| 16| 22| 26| 30| 36| 40
---|---|---|---|---|---|---|---|---|---
0| 59.35| 48.46| 40.16| 32.66| 27.05| 24.09| 21.61| 18.61| 16.98
10| 55.85| 45.73| 37.99| 30.96| 25.69| 22.91| 20.57| 17.73| 16.18
20| 52.58| 43.15| 35.93| 29.35| 24.40| 21.78| 19.58| 16.89| 15.42
30| 49.50| 40.73| 33.98| 27.82| 23.18| 20.71| 18.63| 16.09| 14.70
35| 48.03| 39.56| 33.05| 27.09| 22.59| 20.19| 18.18| 15.70| 14.35
38| 47.17| 38.88| 32.50| 26.66| 22.24| 19.89| 17.91| 15.48| 14.15
40| 46.60| 38.43| 32.14| 26.37| 22.01| 19.69| 17.73| 15.33| 14.01

Source: Weiss, R.F; Price, B. A.: Marine, Chemistry, 1980, 8, 347-359
For conversion between units go to the Unit Converter on Unisense website: unisense.com/unit-converter/#
The Unisense software has a built-in calculator, giving the solubility of N2O at a given combination of temperature and salinity (“Tools -N2O Calculator” or the “N2O table” button in the calibration dialog).

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