Oxygraph-2k
1.4.A. Mitochondrial Physiology Network 6.3: 1-6 (2001-2008)
2008 OROBOROS
Last update: 2008-07-08
Oxygen Solubility in
Experimental Media
Erich Gnaiger
Medical University of Innsbruck, Department of Transplant Surgery,
D. Swarovski Research Laboratory, Innrain 66/6, 6020 Innsbruck, Austria
OROBOROS INSTRUMENTS, high-resolution respirometry, Schpfstr. 18,
A-6020 Innsbruck, Austria.
Email: [email protected]; http://www.oroboros.at
Summary: High-resolution respirometry critically depends on accurate
calibration of the polarographic oxygen sensors, which respond to partial
oxygen pressure. Expressing the oxygen signal in terms of partial pressure
has several advantages, but respiration must be expressed in molar units
related to biochemical stoichiometries. Conversion of oxygen partial
pressure to oxygen concentration requires accurate information on oxygen
solubilities in experimental incubation media. Absolute errors up to 10 %
are commonly found in the established literature. The oxygen solubility of
mitochondrial respiration medium MiR05 relative to pure water (oxygen
solubility factor, FM) is 0.92, accurately determined at 37 C and 30 C.
Section
1.
2.
3.
O2 Solubility and Concentration at Air Saturation.........1
The O2 Solubility Factor in Salt Solutions....................3
References ............................................................5
Page
Oxygen calibration in high-resolution respirometry requires a fundamental
analysis of oxygen solubilities based on a compilation of the pertinent
physicochemical background. Consideration of this thermodynamic review helps
to express instrumental specifications of the Oxygraph in generally valid units,
e.g. using oxygen concentrations at standard rather than local barometric
pressure as a reference.
1. O2 Solubility and Concentration at Air Saturation
1.1. Oxygen Pressure and Concentration
It is practical to calculate the saturation concentration for pure water, which
then is corrected by the solubility factor of the medium, FM, to account for the
reduced O2 solubility in salt media. Owing to the salting-out effect, FM must be
<1.0 in salt media used for respiratory studies of mitochondria, cells and tissues.
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1.4.A. Oxygen Solubility
FM is typically near 0.9 for Oxygraph media (0.92 for MiR05). Several
oxygen solubilities reported in the literature must be critized on the basis of
physicochemical considerations.
Water in equilibrium with air contains an oxygen concentration proportional
to the oxygen solubility and the partial oxygen pressure of air. In the gas-liquid
boundary, air is saturated with water vapor at the partial pressure of pH2O*. The
water vapor pressure is subtracted from the total barometric pressure, pb, to
obtain the partial pressure of dry air, pb-pH2O*. The volume fraction of dry air is
constant at O2 = 0.20946. Therefore, the partial oxygen pressure at air
saturation is, for any temperature and barometric pressure,
Eq. 1
pO2* = (pb - pH2O*) 0.20946
The saturation O2 concentration depends on the O2 solubility, SO2
[moldm-3kPa-1],
Eq. 2
cO2* = pO2* SO2
Oxygen solubility is a function of temperature and composition of the medium. In
other words, oxygen solubility, SO2, is defined as the ratio of partial oxygen
pressure and concentration,
Eq. 3
SO2 = cO2*/pO2*
1.2. Temperature Effect on Saturation O2 Concentration
pH2O* (Eq. 1) is the saturation water vapor pressure at experimental
temperature. pH2O* is a function of absolute temperature, T [K], obtained from
the experimental temperature, , recorded in units oC,
Eq. 4
T = + 273.15
The saturation water vapor pressure [kPa] is (Table 1),
Eq. 5
pH2O* = exp[(-216961 T-1 - 3840.7) T-1 + 16.4754]
Until recently, the atm-standard pressure has been used: 1 atm = 760
mmHg = 101.325 kPa. For pure water in equilibrium with air at this atmstandard pressure, the 'unit standard concentration' of oxygen, C*, is calculated
by the polynomial expression,
Eq. 6
C* = exp<{[(-8.6219491011T -1 + 1.2438001010)T -1
-6.642308107]T -1 + 1.575701105}T -1 - 135.90202>
1.3. Barometric Pressure Effect on Saturation O2 Concentration
The unit standard concentration and the oxygen concentration at air
saturation and actual barometric pressure are related by (compare f o in Table 1),
Table 1. Saturation water vapor pressure, pH2O*, oxygen pressure, pO2*, and
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1.4.A. Oxygen Solubility
oxygen concentration, cO2*, at air saturation and standard barometric pressure,
pbo = 100 kPa, in pure water as a function of temperature. SO2 is the oxygen
solubility, independent of choice of standard pressure. f o is the multiplication
factor to convert partial O2 pressures and concentrations given at atm-standard
pressure (1 atm = 101.325 kPa) to the IUPAC standard pressure of 100 kPa
(compare Eq. 6),
f o = (100-pH2O*) / (101.325-pH2O*)
Eq. 7
pH2O*
pO2*
cO2*
kPa
kPa
moldm-3
fo
SO2
moldm-3kPa-1
40
313.15
7.38
19.40
197.6
0.9859
10.18
37
310.15
6.27
19.63
207.3
0.9861
10.56
35
308.15
5.62
19.77
214.2
0.9862
10.83
30
303.15
4.24
20.06
233.0
0.9864
11.62
25
298.15
3.17
20.28
254.8
0.9865
12.56
20
293.15
2.34
20.46
280.4
0.9866
13.70
15
288.15
1.70
20.59
310.9
0.9867
15.10
10
283.15
1.23
20.69
348.1
0.9868
16.83
278.15
0.87
20.76
393.9
0.9868
18.97
277.15
0.81
20.78
404.3
0.9868
19.46
cO2* = C* pO2*/[(101.325-pH2O*) 0.20946] FM
= C* (pb-pH2O*)/(101.325-pH2O*) FM
Values for characteristic temperatures are given in Table 1.
2. The O2 Solubility Factor in Salt Solutions
The salting out effect is responsible for the reduced oxygen solubility in
aqueous solutions compared to pure water (Fig. 1). Detailed equations are
available for calculating the oxygen solubility of sea water at different salinities
(Forstner and Gnaiger 1983).
Physiological solutions commonly used in Oxygraph studies (Rasmussen,
Rasmussen 2003, Reynafarje, Costa, Lehninger 1985) are compared with pure
water and 20 sea water in Fig. 1. The corresponding polynomial equations are
summarized in Table 2 for calculating the oxygen saturation concentration in
equilibrium with air at various temperatures and standard pressure (Table 3).
Characteristic temperatures are commonly used in experimental studies. Under
these conditions it is convenient to use oxygen solubility factors for the medium,
FM (Fig. 1). This factor is independent of barometric pressure, but FM changes
with temperature (compare Fig. 1). The solubility factors are compiled in Table 4
for different salinities of sea water (Forstner and Gnaiger 1983) and two typical
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1.4.A. Oxygen Solubility
O2 concentration [M]
450
400
350
300
250
200
150
0
10
20
30
40
Temperature [C]
O2 solubility factor, Fm
1.05
1.00
0.95
0.90
0.85
0.80
0
10
20
30
40
Temperature [C]
Figure 1. Oxygen concentration at air saturation and standard barometric pressure
(100 kPa; top) and oxygen solubility factor (bottom) in MiR05 (diamonds), KCl
medium (open trianlges, full line; 150 mmoldm-3 KCl) and sucrose medium (open
circles, dashed line; 250 mmoldm-3 sucrose; data for both media from Reynafarje
et al., 1985), compared to pure water (upper full line) and 20 sea water (lower
dotted line). For the parameters of the polynomials see Table 2. The solubility factor
for serum is shown by the full square (bottom). Literature data (bottom) on KCl
media (closed triangles) and sucrose media (closed circles) show (i) the wide
scatter of solubility data, (ii) the erroneous use of values even higher than solubility
established for pure water, and (iii) a trend to higher values, particularly in sucrose
medium, compared to Reynafarje et al. (1985).
media used with isolated mitochondria (Reynafarje, Costa, Lehninger 1985). The
latter values have been criticized on methodological grounds by Rasmussen and
Rasmussen (2003), and the complex temperature dependence of FM compared to
sea water is doubtful from a thermodynamic perspective Fig. 1).
Table 2. Parameters of the polynomial fits of oxygen saturation concentration in
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1.4.A. Oxygen Solubility
equilibrium with air at pbo = 100 kPa, for sea water (0 and 20) and typical
Oxygraph incubation media, in the range of from 5 to 40 oC. Instead of the
theoretically based plot of ln(SO2) versus T -1, the fits were performed on the
untransformed data, with temperature, , in units of oC (r 2 0.999 in all cases).
The equation in nested form is,
cO2* = {[(b4 + b3) + b2] + b1} + a
Medium
B1
b2
b3
b4
450.5946
-12.60381
0.2712233
-0.003808
2.37910-5
20
390.8769
-10.2165
0.2051415
-0.002746
1.62110-5
KCl
401.9152
-10.70002
0.2291496
-0.003283
2.49210-5
Sucrose
427.411
-14.4983
0.2762108
-0.0003628
-3.60610-5
The oxygen solubility factor of MiR05 is 0.92, at 30 C and 37 C (Rasmussen,
Rasmussen 2003), corresponding to an oxygen concentration in equilibrium with
air under standard conditions (cO2*) of 214.4 and 190.7 M, respectively. The
oxygen solubility of serum is 9.4 nmol O2.cm-3.kPa-1 at 37 oC (Baumgrtl and
Lbbers 1983). In comparison to the oxygen solubility in pure water (10.56 nmol
O2.cm-3.kPa-1 at 37 oC; Table 1), this corresponds to a solubility factor for serum
of FM = 0.89 (Fig. 1) and cO2* of 184.5 M.
3. References
Baumgrtl H, Lbbers DW (1983) Microaxial needle sensor for polarographic
measurement of local O2 pressure in the cellular range of living tissue. Its
construction and properties. In: Polarographic Oxygen Sensors, Gnaiger E, Forstner
H (eds) Springer, Berlin, Heidelberg, New York: 37-65.
Beavis AD, Lehninger AL (1986) Eur. J. Biochem. 158: 315-322.
Brand MD; Harper ME, Taylor HC (1993) Control of the effective P/O ratio of oxidative
phosphorylation in liver mitochondria and hepatocytes. Biochem J. 291: 739-748.
Chapell JB (1964) Biochem. J. 90: 225-237.
Forstner H, Gnaiger E (1983) Calculation of equilibrium oxygen concentration. In:
Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Gnaiger E,
Forstner H (eds) Springer, Berlin, Heidelberg, New York: 321-333.
Gnaiger E, Steinlechner-Maran R, Mndez G, Eberl T, Margreiter R (1995) Control of
mitochondrial and cellular respiration by oxygen. J. Bioenerg. Biomembr. 27: 583596.
Hinkle PC, Yu ML (1979) J. Biol. Chem. 254: 2450-2455.
Lemasters JJ (1984) J. Biol. Chem. 259: 13123-13130.
Rasmussen HN, Rasmussen UF (2003) Oxygen solubilities of media used in
electrochemical respiration measurements. Analyt. Biochem. 319: 105-113.
Rettich TR, Battiono R, Wilhelm E (2000) Solubility of gases in liquids. 22. High-precision
determination of Henrys law constants of oxygen in liquid water from T=274 K to
T=328 K. J. Chem. Thermodynam. 32: 1145-1156.
Reynafarje B, Costa LE, Lehninger AL (1985) O2 solubility in aqueous media determined
by a kinetic method. Anal.Biochem. 145: 406-418
Stoner CD (1987) J. Biol. Chem. 262: 11445-11453.
Wilhelm E, Battino R, Wilcock RJ (1977) Low-pressure solubility of gases in liquid water.
Chem. Rev. 77: 219-262.
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1.4.A. Oxygen Solubility
Appendix
Table 3. Oxygen solubility, SO2 [M.kPa-1], for seawater at various
salinities (10, 20, 30 and 36), and for two typical Oxygraph
media (concentrations given in mmoldm-3); Sucrose: 250 sucrose, 5
KCl, 3 K-Hepes, pH 7.05; KCl: 150 KCl, 3 K-Hepes, pH 7.05.
SO2 for sea water
10
20
30
SO2 for exp. Medium
36
Sucrose
KCl
40
9.62
9.08
8.58
8.29
8.96
10.01
37
9.98
9.43
8.90
8.61
9.33
10.19
35
10.24
9.67
9.14
8.83
9.54
10.36
30
10.98
10.37
9.80
9.47
10.07
10.90
25
11.86
11.20
10.57
10.21
10.74
11.64
20
12.92
12.19
11.49
11.09
11.70
12.58
15
14.21
13.38
12.59
12.14
13.07
13.75
10
15.79
14.82
13.91
13.39
14.95
15.22
17.75
16.60
15.53
14.92
17.42
17.04
18.19
17.00
15.89
15.26
17.99
17.45
Table 4. Oxygen solubility factor of the medium, FM, for seawater at
various salinities (10, 20, 30 and 36), and for two typical
Oxygraph media (concentrations given in mmoldm-3); Sucrose: 250
sucrose, 5 KCl, 3 K-Hepes, pH 7.05; KCl: 150 KCl, 3 K-Hepes, pH
7.05.
FM for sea water
10
20
30
FM for exp. Medium
36
Sucrose
KCl
40
0.945
0.892
0.842
0.814
0.880
0.983
37
0.945
0.893
0.843
0.815
0.884
0.966
35
0.945
0.893
0.844
0.815
0.881
0.956
30
0.945
0.893
0.843
0.815
0.867
0.938
25
0.944
0.892
0.842
0.813
0.855
0.926
20
0.943
0.889
0.838
0.809
0.853
0.918
15
0.941
0.886
0.833
0.804
0.865
0.911
10
0.939
0.881
0.827
0.796
0.889
0.904
0.936
0.875
0.819
0.786
0.918
0.898
0.935
0.881
0.817
0.784
0.925
0.897
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