The use of pH indicators to identify suitable environments for freezing samples in aqueous and mixed aqueous/nonaqueous solutions

The colours of frozen solutions containing pH indicators are shown to provide a test for changes in pH in the solvent environment which occur on freezing. Yeast alcohol dehydrogenase loses activity on freezing in phosphate buffer (a buffer in which pH indicator colour changes shows a marked decrease in pH on freezing) but when frozen in bis-tris, Hepes, or N-glycylglycine buffers (all of which show little change in the colour of universal pH indicator and hence of pH on freezing) is stable on freezing. The effects of freezing in different buffer systems on the rate of decomposition of NADPH, and on the rate hydrolysis of 4-nitrophenyl acetate, are rationalised in terms of the pH shifts in these buffers which were determined using universal pH indicator. It is proposed that a major reason for the instability of samples on freezing is the pH changes which occur when some systems are frozen. From the results a general scheme for selecting the best environment for safely freezing samples is proposed which is based on the use of pH indicators.