Gravimetric measurement of hydration

All four measurement techniques require careful, accurate measurement of the hydration fraction h at the time of biophysical measurement. Wet sample mass is calculated by subtracting the weighing boat mass from the boat + sample mass. Samples are then dried to equilibrium in a vacuum oven at room temperature to avoid denaturing damage and possible loss or gain of mass from chemical reactions with the atmosphere. The temperature is then raised to 50° in a vacuum for 1 day and then to 90° for 4 additional days to achieve removal of the most tightly bound water fraction. The vacuum oven is then allowed to cool to room temperature and the chamber is back filled with dry nitrogen. The mass of the dry sample + boat is measured rapidly to minimize exposure to reabsorption of atmospheric water on the sample. The mass of the water is calculated from the mass (boat + wet sample) — mass(boat + dry sample), whereas the mass of dry protein is calculated from the mass(boat + dry sample) — mass(boat). The hydration is h = (mass water)/(mass protein). Careful adherence to a systematic gravimetric drying protocol is crucial to avoid loss or gain of sample mass as a consequence of chemical interaction with the atmosphere and/or unexpected regain of water from the laboratory atmosphere.

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