The traditional experimental methods described above all assume the possibility of following the reaction after its components have combined into a homogeneous mixture of known concentrations. But what can be done if the time required to complete the mixing process is comparable to or greater than the time needed for the reaction to run to completion?
For reactions that take place in milliseconds, the standard approach since the 1950s has been to employ a flow technique of some kind. An early example was used to study fast gas-phase reactions in which one of the reactants is a free radical such as OH that can be produced by an intense microwave discharge acting on a suitable source gas mixture. This gas, along with the other reactant being investigated, is made to flow through a narrow tube at a known velocity.
If the distance between the point at which the reaction is initiated and the product detector is known, then the time interval can be found from the flow rate. By varying this distance, the time required to obtain the maximum yield can then be determined.
Although this method is very simple in principle, it can be complicated in practice, as the illustration shows. Owing to the rather large volumes required, his method is more practical for the study of gas-phase reactions than for solutions, for which the stopped-flow method described below is generally preferred.
Stopped-flow and Quenched-flow methods
These are by far the most common means of studying fast solution-phase reactions over time intervals of down to a fraction of a millisecond. The use of reasonably simple devices is now practical even in student laboratory experiments. These techniques make it possible to follow not only changes in the concentrations of reactants and products, but also the buildup and decay of reaction intermediates.
The basic stopped-flow apparatus consists of two or more coupled syringes that rapidly inject the reactants into a small mixing chamber and then through an observation cell that can be coupled to instruments that measure absorption, fluorescence, light scattering, or other optical or electrical properties of the solution. As the solution flows through the cell, it empties into a stopping syringe that, when filled, strikes a backstop that abruptly stops the flow. The volume that the stopping syringe can accept is adjusted so that the mixture in the cell has just become uniform and has reached a steady state; at this point, recording of the cell measurement begins and its change is followed.
Of course, there are many reactions that cannot be followed by changes in light absorption or other physical properties that are conveniently monitored. In such cases, it is often practical to quench (stop) the reaction after a desired interval by adding an appropriate quenching agent. For example, an enzyme-catalyzed reaction can be stopped by adding an acid, base, or salt solution that denatures (destroys the activity of) the protein enzyme. Once the reaction has been stopped, the mixture is withdrawn and analyzed in an appropriate manner.
The quenched-flow technique works something like the stopped-flow method described above, with a slightly altered plumbing arrangement.
The reactants A and B are mixed and fed directly through the diverter valve to the measuring cell, which is not shown in this diagram. After a set interval that can vary from a few milliseconds to 200 sec or more, the controller activates the quenching syringe and diverter valve, flooding the cell with the quenching solution.