The oxygen signal of the Oroboros O2k is transmitted from the electrochemical polarographic oxygen sensor (OroboPOS) for each of the two O2k-chambers to DatLab. The primary signal is a current [µA] which is converted into a voltage [V] (raw signal), and calibrated in SI units for amount of substrance concentration [µmol·L-1 or µM].
Reference: MiPNet06.03 POS-calibration-SOP
MitoPedia O2k and high-resolution respirometry: O2k-Open Support
Oxygen signal without calibration
- Without oxygen calibration in DatLab, the default oxygen signal is shown in units of O2 concentration [µM], but the numbers shown as a default are different from the real O2 concentration. The O2 calib. buttons on the lower left and right corners of the DatLab window are yellow, indicating that a proper calibration should be performed. The non-calibrated default values are calculated on the basis of the following default settings, which apply initially when no calibration was performed previously in DatLab, or when the Reset to system default button is clicked in the O2 calibration window:
- These default settings result in a calculated O2 concentration at air saturation of 207.32 µM.
Oxygen signal after calibration
- After performing an oxygen calibration in DatLab and pressing Calibrate and copy to clipboard in the O2 calibration window, the default settings are replaced by the actually measured and recorded signals of oxygen (you have to select the marks for air calibration and zero oxygen calibration), temperature, and barometric pressure. The oxygen solubility factor has to be entered digitally. Subsequently, the new default settings correspond always to the latest calibration (clicking Calibrate and copy to clipboard) performed while DatLab is connected to the O2k which must be switched on. When clicking Calibrate and copy to clipboard, the oxygen signal of the entire plot in the DatLab file is calibrated, and the O2 calib. buttons on the lower left and right corners of the DatLab window are switched to green. When performing a series of experimental runs recorded in separate DatLab files, the correct default calibration parameters are carried over, and the yellow O2 calib. buttons on the lower left and right corners of the DatLab window can be ignored.
- The raw signal of the polarographic oxygen sensor (POS) depends on the Gain setting. The default is Gain 1 in O2k-Series G and higher (Gain 2 in previous O2k-Series). The maximum signal is 10 volt (10 V). Above 9.99 V, the amplifier is in the saturation range, the apparent signal will remain constant at this value, and the slope will appear to be zero. The raw signal can be modified by choosing an appropriate Gain setting.
- The raw signal observed during calibration at air saturation depends on experimental temperature, barometric pressure (lower values are obtained above sea level), and the sensitivity of the individual POS. The raw signal is converted to oxygen concentration [µM or nmol/mL] or partial oxygen pressure [kPa] by calibration of the POS. Each gain requires a separate calibration of the oxygen signal. Therefore, after changing the gain you should not only perform a calibration at air saturation (as always before an experiment), but also a new zero oxygen calibration.
- Raw signal at air calibration, R1
- The voltage recorded at air saturation and with an open chamber is an important instrumental quality parameter. It is one result of a full oxygen sensor test.
- » Oxygen calibration - DatLab
- If the recorded value is too high, first of all the zero current should be checked. A high zero current will also give a correspondingly higher voltage at air saturation; e.g., if the zero current corresponds to a voltage of 1 V at Gain 1, the voltage at air saturation will typically be 3 V instead of 2 V.
- Depending on whether or not a high zero current is found the problem has to be further located by switching components. Typically the problem will be located either on the POS or on the POS connector (check here instructions to clean and dry the OroboPOS-Connector).
- If the zero current if ok, consider the following potential reasons for changes in R1:
- The stirrer speed (rpm) may have been changed, or the stirrer may have been left in the off mode. - Re-set the stirrer speed and re-calibrate.
- The gain setting may have been changed. - Compare calibration values at identical gain settings.
- The block temperature may have been changed. - Compare calibration values at identical temperatures.
- R1 is similar, but the oxygen calibration factor of the medium, FM.
- The barometric pressure has changed (e.g., experiments carried out at high altitude).
- The problem occurred after application of a new membrane:
- Lower R1: The membrane thickness may have changed, possibly by inadvertently using two superimposed membranes.
- Lower R1: The membrane may have wrinkles in the area of the cathode.
- Lower R1: The sensor cap may have not been applied properly in the centre, now partially blocking the cathode.
- Higher R1: The membrane may have been overstretched, or a thinner membrane is used.
- The problem occurred after re-assembly of the O2k-chamber:
- Lower R1: The chamber may have been improperly positioned, with the blue POS holder (on the left or right side of the copper block) screwed too far into the chamber housing. This would allow only partial contact between the stirred medium and the cathode. - Follow the O2k-Start instructions (MiPNet22.11 O2k-FluoRespirometer manual) to insert the chamber properly.
- The electrolyte in the POS may have partially dried out. - Apply a new membrane, following POS-Service instructions: MiPNet19.18B POS-service.
Keywords: Oxygen signal
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Problems - solutions
Instability of the oxygen signal
- The stability of the oxygen signal of the O2k is evaluated by the (negative) uncorrected slope over time. After smoothing, the slope of a stable sensor should be less than ± 1 pmol·s-1·mL-1 in a 2-mL O2k chamber.
- Instability of the oxygen signal
- There is drift of the oxygen signal over short or long periods of time.
- The signal at air saturation is abnormally high for a given gain setting.
- The zero current is high (>2.5%) and may show drift.
- Follow the SOP instructions: MiPNet06.03 POS-calibration-SOP.
Oxygen signal does not respond
Q1: The Oxygen Concentration couldn't go down while I tried zero oxygen calibration. No matter how much solution I added, the oxygen concentration still remain the same. I reassembled, but it still keeps the same. The data is attached.
Q2: The chamber B's oxygen slope is not stable. This is a new problem I have never seen. Please see the attachment.(2019-04-26)
Q3. The maximum Oxygen concentration of another Oroboros O2k is only about 140 μM. I don't how to increase the starting point of oxygen concentration.
A1: I suspect that the dithionite solution was not correctly prepared or not fresh. Dithionite loses activity quite fast, so you need to make it freshly. Please check this site: http://bioblast.at/index.php/Dithionite
A2: While there is a difference between chambers A and B, the stability of the signal is still within the acceptable range of noise given by the specifications of the instrument, see: Oxygen_sensor_test.
A3: You may simply need to calibrate, please see here: http://wiki.oroboros.at/index.php/O2_calibration_-_DatLab
MitoPedia methods: Respirometry