Fixing Initial Water Activity of Reaction Components

Fixing Initial Water Activity of Reaction Components 

The reaction mixture for biocatalysis will be prepared by combining several components. To ensure defined water conditions in the final mixture, all these components should preferably be brought to known water activity beforehand. (It may be safe to disregard this for a component that has a limited affinity for water and makes up only a small proportion of the final mixture.) 

Often the easiest way to set the initial water activity of components of the reaction mixture is by pre-equilibration with a saturated salt solution. The relative humidity or water activity is fixed above a saturated solution of a given salt at a known temperature. As water equilibrates in or out of the solution, solid salt will tend to dissolve or crystallize to maintain saturation and hence the fixed water activity in the headspace. Any other material placed in contact with the headspace will eventually equilibrate to the same water activity. The reaction mixture component can simply be placed inside a closed vessel together with the salt solution, such that water can transfer between the two via the vapour phase. Wide-mouth screw cap jars are convenient, with salt solution over the base and an open vial containing the sample (Fig. 8-3). The rate of equilibration depends on the surface areas exposed and the amount of water that must be transferred. Typically 1-2 days is sufficient for either solid biocatalyst preparations or liquid phases based on relatively non-polar organic solvents. The rate of equilibration may be checked by weighing or Karl Fischer analysis respectively. 

Jar with reasonable seal Sample being equilibrated Saturated salt slush 

For water activities below 0.05, drying agents are required rather than salt solutions. Agents suitable for exhaustive drying are described in many conventional reference sources. Molecular sieves are popular in applied biocatalysis, but I would note two cautions. Firstly, if they are reactivated by heating, about 350 °C is required to obtain maximal drying efficiency. Secondly, if placed in direct contact with a liquid phase, they can have significant acid-base effects. 

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