# Quantifying the liquid–liquid transition in cold water/glycerol mixtures by intermolecular hyperfine relaxation-induced dipolar modulation enhancement (ih-RIDME)

## TL;DR

A new EPR technique called ih-RIDME is used to study phase transitions in frozen water/glycerol mixtures, revealing consistent phase compositions and potential for studying macromolecules.

## Contribution

The novel ih-RIDME EPR technique enables quantification of liquid–liquid transitions in cryogenic water/glycerol mixtures.

## Key findings

- Frozen water/glycerol mixtures show nearly equal phase composition regardless of isotope balance.
- ih-RIDME provides a consistent picture of phase transitions and glass transition data in these mixtures.
- The technique has potential for investigating solvation shells of spin-labelled macromolecules.

## Abstract

Water/glycerol mixtures are commonly used for experiments with biomacromolecules at cryogenic temperatures due to their vitrification properties. Above the glass transition temperature, they undergo liquid–liquid phase separation. Using a novel EPR technique called intermolecular hyperfine relaxation-induced dipolar modulation enhancement (ih-RIDME), we quantified the molar composition in frozen water/glycerol mixtures with one or the other component deuterated after the phase transition. Our experiments reveal a nearly equal phase composition regardless of the proton/deuterium isotope balance. With the new ih-RIDME data, we can also revisit the previously reported body of glass transition data for these mixtures and build a consistent picture of water/glycerol freezing and phase transitions. Our results also indicate that ih-RIDME has the potential to be used for investigating the solvation shells of spin-labelled macromolecules.

Pulse EPR allows to quantify the liquid–liquid transition in water/glycerol mixtures above glass transition temperature. This is achieved using Hahn echo decay and ih-RIDME experiments.