In this context, it is vital to clearly understand what definitions of gaseous and liquid oxygen concentrations refer to. For example, the use of percentage will not always correlate to concentration. While the percentage of atmospheric oxygen is the same (20.9%) at both high and low altitudes (and corresponding pressure), the concentration (ppm or mg/L) decreases at extremely high altitudes as the decrease in pressure reduces the number of gas molecules present. Additionally, the concentration of oxygen within the gaseous atmosphere of the workstation or incubator is not the same as that within the liquid phase of the cell culture media. In fact, defining what is meant by an amount of oxygen is even more critical when discussing a solution of oxygen in a liquid.
The degree to which oxygen dissolves within a liquid is determined by a number of laws and factors, all of which must be considered when making measurements of dissolved oxygen in liquid and working in reduced oxygen environments. Commonly, when programming workstation or incubator set points, reports of a chosen oxygen percentage refer to the percentage chosen solely for the atmosphere inside the chamber, regardless of the temperature, altitude and salinity of the chosen cell culture media, all of which affect the solubility of oxygen in a liquid. The relevant parameter, which is the amount of oxygen within the cell culture itself, is often ignored. This can be reported in either relative or absolute terms.
Three laws govern the extent to which a gas diffuses into a liquid:
Dalton’s Law rules that the pressure of an individual gas within a mixture of gases is the same as if it was the only gas present within the same system. Hence, the total pressure of a mixture of non-reactive gases equals the sum of the partial pressure of each individual gas.
Henry’s Law states that at constant temperature, the concentration of a given gas in a liquid is directly proportional to the partial pressure at which the gas is applied. As pressure above the liquid increases, the amount of dissolved oxygen within the liquid increases proportionally.
The ideal gas law states that the partial pressure of each gas is dependent and proportional to the number of gas molecules present. Henry’s Law must be considered in this aspect; any alteration to the number of gas molecules (for example, by a change in altitude) will alter its partial pressure and hence affect the ability of a gas to dissolve within a liquid.
In summary, the extent to which a gas diffuses into a liquid is directly proportional and dependent on, the partial pressure at which the gas is applied.
Three additional factors affect the concentration of dissolved oxygen in liquid specifically.
The solubility of oxygen within a liquid is inversely proportional to the temperature of the liquid, which must be taken into account when considering percentage saturation. For example, at 15°C, water will hold a maximum of 11.24mg/L, while at 30°C it will hold 7.54mg/L. Water holding half the possible amount of oxygen at both temperatures will continually show as 50% saturation although the concentration would be vastly different. Additionally, a common tool for measuring dissolved oxygen is an electrochemical probe whereby oxygen must diffuse across a semi-permeable membrane, a process affected by temperature (Lighton 2008).
The solubility of oxygen is also inversely correlated with the salinity of the given liquid. As salinity increases, oxygen must compete for space between water molecules to dissolve. Again, as this affects the maximum possible oxygen saturation, salinity must be considered when assessing percentage saturation.
As altitude increases, pressure decreases. This decrease in pressure causes a reduced number of gas molecules present in air, including oxygen, impinging on both Henry’s Law and The Ideal Gas Law (detailed above). Large increases in altitude above sea level must be taken into account when measuring percentage saturation of oxygen within a liquid.
The concentration of a gas, either in a mixed population of gases or when dissolved into a liquid, can be defined in multiple ways. A common method is as percentage of air saturation (i.e. 20.9% O2) or the use of partial pressure (pO2). In dry atmospheric air, this relates to the total barometric pressure (760 mmHg) and the proportion of that which is contributed to by oxygen (20.9%); 760 x 0.209 = 160 mmHg.
When gases become dissolved in a liquid, both of the above definitions and their meanings shift. In this sense, the use of percentage relates the amount of dissolved oxygen present to the maximum amount possible in water; when a volume of water at fixed temperature, salinity and pressure reaches equilibrium and is completely saturated with air, it can be defined as containing 100% oxygen. These are commonly recognised as relative dissolved oxygen measurements.
As more precise alternatives, absolute measurements of concentration are calculated relative to the total number of molecules present. These commonly include parts per million (ppm, the number of O2molecules per million of all samples present in a sample), mg/L (mg of O2 per litre of liquid volume) and Molar (the concentration relative to fixed volume).
Liquid tissue cultures exposed solely to a gaseous phase with reduced oxygen concentration (for example, due to a change in content in an incubator or workstation) will take ~5-10 hours of incubation to reach equilibrium without shaking/stirring the media (Fernandes et al. 2010), reportedly forming O2gradients within tissue culture dishes. A further decrease in oxygen concentration may be observed in pericellular spaces within cultures themselves (Pettersen et al. 2005).
The effects on cultures of exposure to higher-than-desired and inconsistent oxygen levels are significantly minimized when oxygen levels in the media are reduced prior to use. HypoxyCOOL™ is a tested, repeatable protocol that quickly and precisely reduces dissolved oxygen in culture media in as little as three hours.