quenchedflow

Quenched-flow is a laboratory technique used to study fast chemical and enzymatic reactions by rapidly mixing reactants and then stopping the reaction at precisely defined time points. This approach allows researchers to capture transient intermediates and measure short-lived kinetic phases that are difficult to resolve by conventional methods.

The method relies on a rapid-mixing device that combines reagents, followed by a reaction channel or delay line that lets the reaction proceed for a controlled interval. A quench system introduces a reagent that effectively halts the ongoing reaction, after which the quenched samples are collected and analyzed. Common downstream analyses include high-performance liquid chromatography, mass spectrometry, radiolabeling assays, electrophoresis, or spectroscopic measurements.

Timing in quenched-flow is controlled by the lengths of tubing, the arrangement of flow paths, and the flow rates, enabling experiments with millisecond to second time resolution. Quench reagents are chosen to irreversibly stop the reaction, for example by denaturing the enzyme, inactivating catalytic activity, or altering conditions to prevent further progress.

Applications of quenched-flow span pre-steady-state kinetic studies in enzymology, nucleic acid chemistry, and protein folding, as well as investigations into fast-capturing reaction intermediates. It complements stopped-flow techniques by providing discrete time-point data after quenching rather than continuous real-time observation.

Limitations include the potential for incomplete quenching, dilution effects, or perturbation of the system by the quench reagent. The method also requires specialized instrumentation and careful calibration to ensure accurate time assignments and reliable downstream analysis.