Stopped-Flow - A technique for studying rapid chemical kinetics

Stopped-flow is one of a number of techniques used to study the kinetics of reactions in solution. In the simplest form of the technique, two reactant solutions are rapidly mixed by being forced into a mixing chamber, and then through an observation cell. At some point in time, the flow is suddenly stopped, and the reaction monitored using a suitable spectroscopic probe, such as absorbance, fluorescence or circular dichroism. The change in the spectroscopic signal as a function of time is recorded.

Figure 1 shows the schematic of a typical stopped-flow apparatus. The stopping mechanism in this example is a stop-syringe. The flow fills the stop-syringe, until the plunger hits the trigger-switch. This simultaneously stops the flow and starts the data acquisition.

The rate constants which define the reaction kinetics, can be measured by fitting the data using a suitable model.

The performance of a stopped-flow instrument is determined to a large extent by the dead-time. This is defined as the minimum time after the reactant have mixed that the observation starts. The dead-time is essentially the age of the reaction as it enters the observation cell. The limiting factor in the dead-time of a particular stopped-flow apparatus is determined by the distance between the mixer and the cell, and the final velocity of the flow at the instant the flow is stopped. Another factor which can affect the dead-time is the efficiency of the mixer. Typically, dedicated stopped-flow instruments can achieve dead-times in the region of a millisecond. Using ultra-small observation cells, dead times of less than 0.5ms can be achieved. Typically however, the true experimental limitation is the rate of mixing of solutions of the two reactants.

Sequential mixing (or double mixing) is a variation of stopped-flow where two reactants (A and B) are mixed in a pre-mixer and then flow into an ageing loop. After a specified period of time, that mixture is forced to mix with a third reactant (C) and the reaction studied as previously. See figure 2 for a schematic of the stopped-flow apparatus used in sequential mixing mode. Syringes of different sizes can be used together to obtain mixing ratios other than simple 1:1 mixing. So-called asymmetric mixing or ratio mixing is a common requirement in stopped-flow work.

Applications

One area of particular interest at the moment is the study of protein folding. A circular-dichroism or fluorescence stopped-flow spectrophotometer such as the Applied Photophysics π*-180 or the Chirascan with SF.3 stopped-flow accessory can be used to study the rapid folding/refolding of proteins. An application note which describes such a study can be downloaded here.

Links to pages listing papers citing the use of Applied Photophysics’ stopped-flow instruments are listed below.

Stopped-Flow at Applied Photophysics

Applied Photophysics has a comprehensive range of stopped-flow instruments. The entry-level is the RX.2000 rapid mixing stopped-flow accessory which can be used with any existing spectrophotometer.  The highly successful SX-series stopped-flow is the world’s best selling stopped-flow. Recently, stopped-flow has been added to the Chirascan circular-dichroism (CD) spectrometer.

Stopped-flow is particularly flexible technique and Applied Photophysics can supply stopped-flow systems in many different configurations. Of primary importance is the ability to stabilize the temperature. All Applied Photophysics’ stopped-flow instruments have the ability to thermostatically control the reagents before they are mixed and the observation cell. Additionally, the need to work in an anaerobic environment is often required. Applied Photophysics can supply an anaerobic accessory for the p*-180 and SX-series stopped-flow instruments, or for a complete anaerobic environment, the stopped-flow head can be installed in a glove-box. A full list of accessories for the SX-series stopped-flow spectrometers is available here.

The Pro-Data software suite for SX-series, π*-180 and Chirascan provides simple 1-D fitting to many functions. Pro-K and Pro-K II offer full multivariate global analysis. Click here for more information on global-analysis and here for more information on Pro-K II.

Click here for a list of papers that reference the Applied Photophysics instruments.

If you would like to receive further information about any Applied Photophysics technology or instrument please fill out the information request form.

SX20 - the ultimate gold standard in stopped-flow