A general method for performing biotransformations at known water activity consists of exposing each component of the reaction mixture to a vapor phase characterized by a constant known relative humidity (33,34). In principle, the phases, once mixed, reach the equilibrium water activity.
Figures 5 and 6 show the rate of equilibration of dry Celite R-640 (batch 3, Fig. 1) exposed to atmospheres generated by saturated salt solutions or pure water
inside carefully capped vessels. The adsorption isotherms are obtained by keeping three rods (95 mg) in contact with vapor phases characterized by different known relative humidities, and then weighing the Celite every 24 h. Figure 5 indicates that most of the moisture is adsorbed within the first 24 h of exposure of the rods to the vapor phase; there are negligible increments of weight over subsequent days. In all cases, Celite R-640 has a limited capacity of adsorbing water from vapor phases, ranging from 1% w/w (aw = 0.32) to 4% w/w (aw = 0.86).
The rods equilibrated at aw = 1 behave differently, as they progressively adsorb water up to 60% of their weight (Fig. 6), a result that is quite far, however, from the adsorption capacity exhibited by Celite R-640 in toluene (up to 120% w/w).
The possibility that the plateau reported in Figs. 5 and 6 represents merely an apparent equilibrium and that the actual equilibrium requires extremely prolonged equilibration times cannot be excluded. Even so, the physical and chemical processes governing the adsorption of vapor water and liquid water in toluene on Celite R-640 appear very different. The difference is even clearer when Celite rods and toluene, previously equilibrated at the same value of water activity, are mixed. Figure 7 shows how the relative humidity (RH) of the gas phase in the headspace above these mixtures decreases with time, and no equilibrium is reached even after 300 h of incubation (Fig. 7). For instance, in the case of the Celite equilibrated at aw = 0.86, the relative humidity after 24 h of incubation is 0.75 and decreases to 0.51 after 300 h of incubation.
Most probably, upon moving from the gas phase to the apolar solvent, the variation of surface forces promotes a redistribution of the water inside the rods, so that the system reaches the equilibrium in the new environment very slowly. Therefore, the equilibration of this porous matrix in vapor phase is not advisable when Celite rods have to be used in apolar medium.
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