Inlab cool

To monitor pH shifts in the intermediate bulk during freezing, a preliminary examination was done using a simplified medium. All substances without significant contribution to the pH were omitted leaving only phosphate and Tris/HCl. To show the dependency between pH-stability and buffer concentration, 3 different concentrations of Tris/HCl, namely 0.1 M, 0.5 M and 1.0 M, were analysed. We prepared the respective Tris solutions, before phosphoric acid was added to obtain a phosphate concentration of 1.57 × 10^–2 mol/l (see also Table 1) and then pH was adjusted to 7.0 by addition of HCl. These solutions were frozen as described above with the exception of a lower freezing temperature of −25 °C which was chosen for a more gentle treatment of the measuring system. The pH was monitored during the whole freezing process using a low temperature pH-electrode (InLab Cool, Mettler Toledo) with an added thermocouple for correction of temperature dependent characteristics of the measuring system. The pH electrode was placed in the centre of the freezing container. All measurements were done in duplicates.

The pH value of filtrates was measured by mixing 10 g of a minced sample with 50 mL of deionised water for 1 min using a homogeniser (Bamix 200, Greifensee, Switzerland). The pH was measured using a digital pH meter (Seven Compact S220, Mettler Toledo, Greifensee, Switzerland) equipped with a pH electrode (Seven Compact S220, InLab Cool, Mettler Toledo, Greifensee, Switzerland). The pH readings were recorded exactly 4 min after the insertion of the electrode into the sample. The pH meter was standardised with buffer solutions at pH 2.0, 4.0, 7.0, and 10.0.