Stopped-flow
Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance [1][2] and extended by Quentin Gibson [3] (Other techniques, such as the temperature-jump method,[4] are available for much faster processes.)
Description of the method
Summary
Stopped-flow spectrometry allows chemical kinetics of fast reactions (with half times of the order of milliseconds) to be used to study the fast reactions in solution. It is applied especially to enzyme-catalyzed reactions. The solutions are first forced from syringes into a mixing chamber that results in complete mixing. After perhaps a dead time less than 1 ms the observation cell is filled by a piston linked to a sensing switch that triggers the measuring device and the flow is stopped suddenly.
Reactant syringes
Two syringes are filled with solutions that do not undergo a chemical reaction until mixed together. These have pistons that are driven by a single drive piston, so that they are coupled together and their contents are forced out simultaneously into a mixing device.
Mixing chamber
Once the two solutions are forced out of their syringes they enter a mixing system that has baffles to ensure complete mixing, with turbulent flow rather than laminar flow, which would allow the two solutions to flow side by side with incomplete mixing.
Dead time
It is impossible for the mixing of reactants to be achieved instantaneously. As a result there is a dead time before which measurements can be made. In older instruments this could be of the order of 1 ms, but improvements now allow a dead time of about 0.3 ms.[5]
Observation cell
The mixed reactants pass an observation cell that allows the reaction to be followed spectrophotometrically, typically by ultraviolet spectroscopy, fluorescence spectroscopy, circular dichroism or light scattering.[6]
Stopping
Once through the observation cell the mixture enters a third syringe that contains a piston that is driven by the flow to activate a switch to stop the flow and activate the observation.
Quenched-flow
In a quenched-flow instrument, the reaction is stopped after a certain amount of time has passed after mixing. The stopping of the reaction is called quenching and it can be achieved by various means, for example by mixing with another solution, which stops the reaction (chemical quenching), quickly lowering the temperature (freeze quenching) or even by exposing the sample to light of a certain wavelength (optical quenching).
See also
- Syringe driver - sometimes used as part of a stopped-flow device.
- Continuous-flow - alternative to stopped flow.
References
- ^ Chance, Britton (1951). "Rapid and Sensitive Spectrophotometry. I. The Accelerated and Stopped‐Flow Methods for the Measurement of the Reaction Kinetics and Spectra of Unstable Compounds in the Visible Region of the Spectrum". Review of Scientific Instruments. 22 (8): 619–627. doi:10.1063/1.1746019.
- ^ Chance, Britton; Legallais, Victor (1951). "Rapid and Sensitive Spectrophotometry. II. A Stopped‐Flow Attachment for a Stabilized Quartz Spectrophotometer". Review of Scientific Instruments. 22 (8): 627–634. doi:10.1063/1.1746020.
- ^ Gibson, Q. H. (1954). "Stopped-flow apparatus for the study of rapid reactions". Discussions of the Faraday Society. 17: 137. doi:10.1039/df9541700137.
- ^ Eigen, M. (1954). "Methods for investigation of ionic reactions in aqueous solutions with half-times as short as 10–9sec. Application to neutralization and hydrolysis reactions". Discuss. Faraday Soc. 17: 194–205. doi:10.1039/df9541700194.
- ^ Clark, Charles R. (1997). "A Stopped-Flow Kinetics Experiment for Advanced Undergraduate Laboratories: Formation of Iron(III) Thiocyanate". Journal of Chemical Education. 74 (10): 1214. Bibcode:1997JChEd..74.1214C. doi:10.1021/ed074p1214.
- ^ West, J.M.; Xia, J.; Tsuruta, H.; Wenyue, G.; O'Day, E.M.; Kantrowitz, E.R. (2008). "Time Evolution of the Quaternary Structure of Escherichia coli Aspartate Transcarbamoylase upon Reaction with the Natural Substrates and a Slow, Tight-Binding Inhibitor". Journal of Molecular Biology. 384 (1): 206–218. doi:10.1016/j.jmb.2008.09.022. PMC 2656920. PMID 18823998.
Further reading
- IUPAC, Compendium of Chemical Terminology, 2nd ed. (the "Gold Book") (1997). Online corrected version: (2006–) "stopped flow". doi:10.1351/goldbook.S06033
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