Performance Comparison of different Rapid Freeze-Quench Strategies for Electron Paramagnetic Resonance

This work addresses the development of a custom-made home-built rapid freeze-quench (RFQ) device and the comparison of its performance to the one of a commercial RFQ setup that was in-house custom adapted. Both systems consist of two syringes that push the reactants into a mixing chamber and the products to a subsequent freezing setup. Using the binding of azide to myoglobin as a calibration reaction, the quenching times of the different setups were compared, evaluating different instrumental parameters, such as software-controlled variation of the ageing time, variations of the flow rate and variations of the distance travelled by the mixed sample before freezing. In addition to minimal sample consumption, the home-built RFQ device was found to lead to the shorter reaction times which could be controlled in a time range from 10 to 25 ms. The commercial RFQ system yielded optimal reaction control in a time range from 50 to 200 ms, although a larger volume of reactants needed to be used due to the significant dead volume of the system. Three different freezing methods were also evaluated, among which, in our hands, freezing the jet directly in a deep bath of cold isopentane yielded shorter and reproducible freezing times.