Regarding the modification of hyaluronan with EDC/NHS, in order to wash off the unbound EDC/NHS, common method is dialysis against water, but I heard of an alternative method is using ethanol precipitation:

After the mixture of HA and EDC/NHS, the mixture was put frozen (-80), then washed with -20 ethanol to wash off the unbound EDC, NHS and other byproducts.

Just curious about the frozen part, why can't it be done in room temperature