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Zero calibration

From Bioblast

high-resolution terminology - matching measurements at high-resolution

Zero calibration


Zero calibration is together with air calibration one of the two steps of the OroboPOS calibration. It is performed in the closed chamber after all the oxygen has been removed by the addition of dithionite or by respiration of imt or cells. Any incubation medium can be used for zero calibration with dithionite or sample. Unlike air calibration it is not necessary to perform a zero calibration each day.

Abbreviation: R0

Reference: MiPNet06.03 POS-calibration-SOP, O2 calibration - DatLab

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MitoPedia O2k and high-resolution respirometry: O2k-Open Support 


A zero calibration should be performed
A zero calibration has to be performed
  • When measurements are performed at low oxygen (<10 µM).
  • Bracketing zero calibrations are required when measurements are performed near the limit of detection at oxygen concentrations close to 5 nM (0.005 µM).
  • When a new or serviced OroboPOS is used for the first time.
  • When a new membrane was applied to the sensor.
  • When the gain is changed.
  • When a new OroboPOS-Connector is used.

Incubation medium for zero O2 calibration with dithionite

Any incubation medium can be used for zero calibration with dithionite. MiR05 or a highly concentrated phosphate buffer are needed for measurement of instrumental background oxygen flux when using dithionite, to avoid secondary oxygen-consuming reactions after a step titration of dithionite. For zero calibration, however, an excess dithionite concentration is applied which consumes all dissolved oxygen in the medium, and secondary reactions are not an issue.

Zero current

Each polarographic oxygen sensor has a zero current, i.e., a current [µA] that flows even at an oxygen concentration of zero, which is converted into a voltage [V] as the raw signal. This zero oxygen signal is taken into account by the zero calibration and can be expressed as % of the raw signal at air saturation. New OroboPOS have a zero signal below 2 % of the signal at air saturation. For accurate calibration, the zero signal must be stable, which may be the case for zero currents up to 5 % of the signal at air saturation. The stability of the zero current can be assessed by keeping track of calibration values using the "O2k-Calibration-list.xls" template.
A high zero current is caused either by the O2k-Main Unit, the OroboPOS-Connector, the OroboPOS, or by a bad electrical connection between those parts.
» Cleaning the electrical connections of the OroboPOS-Connector frequently solves the problem.
» Further details: High signal at zero oxygen


» MiPNet06.03 POS-calibration-SOP

Keywords: Oxygen signal


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MitoPedia methods: Respirometry 

MitoPedia O2k and high-resolution respirometry: DatLab