UNISENSE N2O Sensor Calibration Kit Instruction Manual

N2O sensor calibration kit
Standard concentration version
Manual

N2O SENSOR CALIBRATION KIT – STANDARD CONCENTRATION VERSION
Copyright © Unisense A/S
Version: September 2020

Warranty and liability

1.1 Notice to Purchaser
This product is for research use only. Not for use in human diagnostic or therapeutic procedures.
1.2 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.
1.3 Warranty and Liability
The Nitrous Oxide Calibration Kit is guaranteed to give the concentration indicated on the package label until expiry as indicated on the package label. The warranty does not include 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.

Support, ordering, and contact information

If you wish to order additional products or if you encounter any problems and need scientific or 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: sales@unisense.com
Unisense A/S
Tueager 1
DK-8200 Aarhus N, Denmark
Tel: +45 8944 9500
Fax: +45 8944 9549
Further documentation and support are available at our website: www.unisense.com.

Content of the calibration kit

figure 1: Calibration kit contents: A: Calibration kit box with Exetainers and ampoules, B: 10 ml syringe, C: 1 ml syringe, D: 80 x 2.1 mm needle (green), E: 50 x 1.2 mm needle (red), F: 30 x 0.6 mm needle (blue), G: O-ring, H: Calibration Cap with tubing.

Principle of calibration

Unisense N2O sensors respond linearly to N2O concentrations within their linear range (see specifications for your sensor at https://www.unisense.com/N2O). Therefore, a two-point calibration is sufficient. One calibration point is the signal for zero N2O, which is water equilibrated with atmospheric air, and the other calibration point is the signal for one known N2O concentration.
Note that the atmospheric concentration of N2O is approximately 330 ppb which corresponds to a concentration of around 9 nM in water. When working with the low-range sensor, with a detection limit of approximately 25 nM N2O, this should be considered. This can be done by entering 9 nM as the concentration for air-saturated water. For normal range sensors, it can be assumed that such water is free of N2O.
This calibration kit contains an ampoule with water equilibrated with a gas with a certified N2O content around 45000 ppm (vol) N2O in N2, giving a concentration in the water in the ampoule of approximately 1200 µM. One ml of this water is diluted in approximately 12 ml of the water or medium in which the measurements will be performed. This gives a final concentration of approximately 100 µM.
4.1 The actual concentration
The actual concentration of N2O in the ampoule is calculated from the certified N2O content in the N2O gas mixture used and the temperature at equilibration (see 7 Specifications). This, and the actual volume of the Exetainers, is used for calculation of the final concentration after dilution and this is shown on the sticker on the calibration kit box.
The concentration is given both in µmol N2O/l and mg N2O/l. The concentration in mg N2O-N/l can be
obtained by multiplying the N2O concentration in mg N2O/l by the ratio of the N content in N2O to the
molar mass of N2O:
Concentration in mg N2O-N/l = Concentration in mg N2O/l x (28.0136 (g N/mol N2O) / 44.013 (g/mol))
Concentration in mg N2O-N/l = Concentration in mg N2O/l x 0.6365
The empty vials (Exetainers) in the calibration kit should be filled with the water or medium in which the measurements will be done. This will ensure that the calibration and measurements are performed in the same environment, i.e. same salinity, composition of salts, proteins etc. This is important because the sensor reacts to the partial pressure of the gas, not the concentration as such, and in the software, this is recalculated into a concentration. The relationship between concentration and partial pressure depends on the salinity and temperature.

Standard calibration procedure

For calibration of N2O sensors a high and a low calibration point are needed. Note that sensors with piercing needle will not fit the calibration cap and must be calibrated as described in section 5.4. For calibrating High Range N2O sensors that work in the mM range, use the N2O Calibration kit – High Range.

5.1 Preparation of the calibration solution

1. Mount the 50 x 1.2 mm needle (red) on the 1 ml syringe and mount the 80 x 2.1 mm needle (green) on the 10 ml syringe.
2. Open the Exetainer and put two glass beads into the vial.
3. Fill the Exetainer with water of the same composition as the water where the measurements will be done.
4. Close the Exetainer with the lid, avoiding any bubbles or headspace.
5. Adjust the Exetainer temperature to the sample/measuring temperature.
6. Insert the 30 x 0.6 mm needle (blue) needle through the septum so the tip is just below the septum.
7.  Open the ampoule with the N2O containing water by breaking the top off (see note A below). Make sure that you don’t shake or mix the content of the ampoule.
8. Fill the 1 ml syringe with 1.0 ml of the water from the ampoule (see note B below).
9. Inject the 1.0 ml N2O containing water into the Exetainer by inserting the needle fully. The excess water from the Exetainer will be expelled through the open needle (see note C below).
10. Remove first the open needle and then the 1 ml syringe from the Exetainer.
11. Shake the Exetainer vigorously to ensure an even distribution of the N2O.
12. The calibration solution is now ready and should be used within a couple of hours.

Figure 2: Open the ampoule. Leave the tubing on for protection.

5.2 Preparation of the N2O sensor

IMPORTANT:

• The pre-activation and polarization of the N2O sensor must have been completed before doing the calibration. See the N2O sensor manual for details: https://www.unisense.com/manuals/
• The temperature of the two calibration solutions must be the same.
• Perform the calibration at the same temperature as the measurements if possible. The UniAmp series of amplifiers has a built-in temperature compensation within ±3°C of the calibration temperature.
• It is recommended to obtain the low calibration point first to avoid carry over from the N2O standard.

5.3 Calibrating most N2O sensors
For calibrating the N2O sensor a low and a high calibration point are needed.

5.3.1 Obtaining the low calibration point

1. Mount the calibration cap on the protection tube with the N2O sensor. Make sure that the Oring is in place at the bottom of the calibration cap creating a seal between this
   and the protection tube.

2. Use the 10 ml syringe to inject N2O free water until the sensor tip is immersed at least 2-3 cm.

3. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/)

4. Remove the N2O free water with the syringe.

5.3.2 Obtaining the high calibration point

1. Open the Exetainer with the N2O calibration solution, prepared as described in 5.1 above.
2. With the 10 ml syringe, aspirate slowly, avoiding bubble formation, 10 ml of the N2O containing water.
3. Keep the syringe vertical and avoid mixing of the water with the air bubble inside.
4. Remove the needle and attach the 10 ml syringe to the calibration cap tubing.
5. Inject the calibration solution slowly until the sensor tip is immersed at least 2-3 cm.
6. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/)

NOTES:
A. Opening the ampoule with N2O containing water: Hold the bottom of the ampoule firmly while grabbing the tubing on the top. Break the top off the ampoule (Figure 2).
IMPORTANT: Once an ampoule is opened it must be used immediately. It is only possible to prepare one calibration vial from one ampoule.
B. Pull up around 0.3 ml of N2O containing water from the ampoule. Point the needle upwards and knock the syringe gently to get all air bubbles to the top. Press the piston to eject these bubbles. Once the syringe is free of bubbles, empty the syringe. Pull up 1.1 ml of water from the ampoule.
IMPORTANT: Insert the needle fully in the ampoule and pull up water slowly to avoid bubble formation. Adjust the volume to exactly 1.0 ml.
C. Injection of the N2O containing water is done with the needle inserted fully while the open needle in inserted just below the septum. Thereby the injected N2O containing water will not be lost.
5.4 Calibrating N2O sensors with piercing needle
Sensor of the piercing needle type (N2O-NP) cannot be calibrated using the calibration cap. Therefore, the calibration points are obtained by inserting the needle directly into N2O free water and the calibration solution.
Pay attention to the general information in section 5.2 and follow the procedure outlined below.
5.4.1 Obtaining the low calibration point

1. Place the sensor in N2O free water.
2. Allow the sensor to respond and stabilize. Then record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/)

5.4.2 Obtaining the high calibration point

1. Open the Exetainer with the N2O calibration solution prepared as described in section 5.1.

2. Insert the needle of the sensor fully into the N2O calibration solution.
   a. Use a clamp to hold the sensor stable to avoid handling noise

3. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/)

4. Remove the sensor and rinse the needle.

5.5 Calibrating N2O sensors for the Microrespiration system
Sensors of the Microrespiration type (OX-MR) cannot be calibrated using the calibration cap. Instead it is recommended to follow the procedure outlined below.
5.5.1 Obtaining the low calibration point
5.5.1.1 Using the Unisense Cal300 Calibration Chamber

1. Place the sensor in the Cal300 Calibration Chamber containing N2O free water (see the N2O microsensor manual (https://www.unisense.com/manuals/).
   • The N2O sensor must be mounted in the blue Microrespiration guide and the tip must be retracted (Figure 3).
   • Temperature of the water must be the same as where the measurements are done.

2. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (seethe SensorTrace manual for details: https://www.unisense.com/manuals/)

5.5.1.2 Using a Microrespiration Chamber

1. Prepare a volume of N2O free water at the same temperature as the N2O calibration solution used in 5.5.2.
2. Transfer this water to a microrespiration chamber and mount the lid.
3. Place the microrespiration chamber in the stirrer rack
4. Place the N2O sensor in the stirrer rack with its plastic tip in the opening of the lid.
5. Insert the sensor into the chamber.
6. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/).
7. Retract the sensor tip and remove the sensor from the stirrer rack.

Figure 3: Microsensor mounted in the Microrespiration guide.

5.5.2 Obtaining the high calibration point

1. Prepare the N2O calibration solution as described in section 5.1.
2. Aspirate a suitable volume of the N2O calibration solution with the syringe and needle. Do this slowly to avoid bubble formation.
3. Dispense the N2O calibration solution into a microrespiration chamber. Place the needle at the bottom of the chamber, filling slowly from below, to avoid bubbles and splashing.
4. Mount the lid in the microrespiration chamber making sure that no air bubbles are trapped.
5. Place the microrespiration chamber in the stirrer rack.
6. Place the N2O sensor in the stirrer rack with its plastic tip in the opening of the lid.
7. Insert the sensor into the chamber.
8. Allow the sensor to respond and stabilize and record the calibration value in SensorTrace (see the SensorTrace manual for details: https://www.unisense.com/manuals/).
9. Retract the sensor tip and remove the sensor from the stirrer rack.

6 Calibrating at other concentrations
The calibration kit may be used to calibrate at other concentrations than described in section 5.
NOTE: N2O sensors must not be exposed to concentrations higher than they are designed for as this may destroy the sensor.
6.1 Higher concentrations
It is possible to inject more than 1 ml of the ampule-solution into the Exetainer which will create a higher concentration than that obtained using the standard procedure in section 5. The concentration is directly proportional to the amount injected: Final conc. (µM) = Inj. vol (ml) x Certified conc. (µM) where Final conc. (µM) is the concentration obtained in the Exetainer, Inj. vol (ml) is the volume injected into the Exetainer and Certified conc. (µM) is the N2O concentration obtained when following the standard procedure in section 5, which is the concentration shown on the label on the calibration kit box.
The dilution of the stock solution into the Exetainer requires that the injected N2O containing water is not spilled out of through the open needle. Therefore, it is important that the tip of the open needle is right below the septum and the injection needle is as deep as possible. Furthermore, if injecting more than approximately 3 ml, there is a risk that some of the N2O is mixed with the liquid in the exetainer and lost through the open needle. Injection should only be done once. Therefore, a larger syringe is needed for injecting more than 1 ml.
Please note that when injecting less than the full volume of a syringe, the accuracy of the injection will become lower. Therefore, always use a syringe with a full volume that is close the amount to be injected. E.g. if 3 ml is injected with a 10 ml syringe the accuracy is low.
6.2 Lower concentrations
It is possible to obtain a lower concentration than that obtained using the standard procedure in section 5. This may be done either injecting less than 1.0 ml or by diluting the calibration solution obtained using the standard procedure in section 5.
When injecting less than 1.0 ml it is important to use a precision syringe. The resulting concentration can be calculated using the formula in section 6.1.
Lower concentration may also be obtained by dilution of the solution made in section 5.1.

1. Fill two Exetainers with water of the same composition as the water where the
   measurements will be done.

2. Inject the 1.0 ml N2O containing water into first Exetainer as described above in section 5.1.

3. After shaking the Exetainer vigorously to obtain a homogenous distribution of N2O, open the Exetainer and aspirate a volume with a syringe.

4.  Inject a known volume into the second Exetainer with the blue needle inserted just below the septum to allow excess liquid to run out.

5. Shake the Exetainer vigorously.

Now the sensor may be calibrated as described in section 5.2 using the calibration solution in the second Exetainer.
The concentration of N2O in the second Exetainer may be calculated as: Final conc. (µM) = Inj. vol. (ml)/Exetainer vol. (ml) x Certified conc. (µM) where Final conc. (µM) is the concentration obtained in the second Exetainer, Inj. vol (ml) is the volume injected transferred from the first to the second the Exetainer, Exetainer vol. (µM) is the
volume of the Exetainer and Certified conc. (µM) is the N2O concentration obtained when following the standard procedure in section 5. The Certified conc. and the Exetainer volume are shown on the label on the calibration kit box.
Please note that when injecting less than the full volume of a syringe, the accuracy of the injection willbecome lower. Therefore, always use a syringe with a full volume that is close the amount to be injected. E.g. if 3 ml is injected with a 10 ml syringe the accuracy is low. The Exetainers may be cleaned and re-used for serial dilutions.

Specifications

Volume of calibration solution in one ampule:
Content of the ampoule:
Lifetime of the calibration kit:
N2O concentration in final calibration solution
Certificate of N2O gas concentration:| 5 ml
Slightly acidic water with N2O
See label on the calibration kit box
See label on the calibration kit box
See label on the calibration kit box
---|---

• The ampoule contains water equilibrated with a gas with a certified N2O content. The concentration is calculated according to Weiss R.F. & Price B.A. 1980. Nitrous oxide solubility in water and seawater. Marine Chemistry 8:347-359. The concentration of N2O in the calibration solution is specified on the label on the calibration kit box. The certificate of the N2O gas mixture used to produce the ampoule water can be requested from Unisense. See the ID of this certificate on the label on the calibration box.

References

• Microsensors, instruments, and meters for microscale measurements
• Microsensors, instruments, and meters for microscale measurements
• Manuals - Unisense
• N2O microsensor to measure nitrous oxide - Unisense

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