Difference between revisions of "Air calibration"
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|description='''Air calibration''' of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, ''c''<sub>O2</sub> [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, ''S''<sub>O2</sub> [µM/kPa]. ''S''<sub>O2</sub> is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, ''F''<sub>M</sub>, expresses the oxygen solubility relative to pure water at any experimental temperature. ''F''<sub>M</sub> is 0.89 in serum (37 °C) and 0.92 in [[MiR06]] or [[MiR05]] (30 °C and 37 °C). | |description='''Air calibration''' of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, ''c''<sub>O2</sub> [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, ''S''<sub>O2</sub> [µM/kPa]. ''S''<sub>O2</sub> is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, ''F''<sub>M</sub>, expresses the oxygen solubility relative to pure water at any experimental temperature. ''F''<sub>M</sub> is 0.89 in serum (37 °C) and 0.92 in [[MiR06]] or [[MiR05]] (30 °C and 37 °C). | ||
|info=[[MiPNet06. | |info=[[MiPNet06.03 POS-Calibration-SOP]] | ||
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{{MitoPedia methods | {{MitoPedia methods | ||
Revision as of 13:24, 10 July 2014
- high-resolution terminology - matching measurements at high-resolution
Air calibration
Description
Air calibration of an oxygen sensor (polarographic oxygen sensor) is performed routinely on any day before starting a respirometric experiment. The volume fraction of oxygen in dry air is constant. An aqueous solution in equilibrium with air has the same partial pressure as that in water vapour saturated air. The water vapour is a function of temperature only. The partial oxygen pressure in aqueous solution in equilibrium with air is, therefore, a function of total barometric pressure and temperature. Bubbling an aqueous solution with air generates deviations from barometric pressure within small gas bubbles and is, therefore, not recommended. To equilibrate an aqueous solution ata known partial pressure of oxygen [kPa], the aqueous solution is stirred rigorously in a chamber enclosing air at constant temperature. The concentration of oxygen, cO2 [µM], is obtained at any partial pressure by multiplying the partial pressure by the oxygen solubility, SO2 [µM/kPa]. SO2 is a function of temperature and composition of the salt solution, and is thus a function of the experimental medium. The solubility factor of the medium, FM, expresses the oxygen solubility relative to pure water at any experimental temperature. FM is 0.89 in serum (37 °C) and 0.92 in MiR06 or MiR05 (30 °C and 37 °C).
Abbreviation: R1
Reference: MiPNet06.03 POS-Calibration-SOP
MitoPedia methods:
Respirometry
MitoPedia topics:
Media for respirometry
Air calibration in the O2k
Air calibration of the polarographic oxygen sensor is performed routinely on any day before an experiment. Switch on the O2k, select the experimental temperature and gain setting (Oxygraph control window [F7]). Clean the chamber and add experimental medium (at least 2.1 ml for a 2 ml chamber). Then the stopper is fully inserted while the stirrer is on, and excess medium is siphoned off the receptacle of the stopper. Now the stopper is partially opened and positioned with the stopper spacer.
Then the stirred aqeous phase is equilibrated with the oxygen contained in the small air space included in the chamber. Use Graph layout "01 Calibration Exp. Gr3-Temp" or "02 Background Experiment".
After stability is obtained within 20-30 min (the uncorrected slope is close to zero (less than +/- 1 pmol.s-1.ml-1), a mark is set on the blue trace of the oxygen signal (R1), and inserted in the calibration window [F5].
Perform the oxygen calibration [F5] on-line, such that the calibration parameters are carried over automatically as a default calibration in subsequently opened DatLab files.
Calibration parameters are copied to clipboard, for insertion into the Excel Template "O2k-Calibration-List.xls". This template can be copied from www.oroboros.at.
Air calibration for high O2 experiments
Question: For an experiment with with high (above atmospheric levels) O2 concentrations I have to change the gain and therefore to perform a new "air calibration". How to do this?
Answer: The air calibration has always to be done at atmospheric saturation (a point of known O2 conc.) at the final Gain used in the experiment.
- Set the gain to 1.
- Perform air calibration at atmospheric saturation, open chamber, near zero flux at open chamber, as usually.
- Close the chamber.
- Wait until you observe a stable flux at closed chamber near to air saturation of 2 to 4 pmol/s ml (quality control for biological contamination).
- Increase the O2 concentration either with a gas phase method or (in MiR06) with H2O2 injections.
