Stopped-flow
The change in spectroscopic signal as a function of time is recorded, and the rate constants that define the reaction kinetics can then be obtained by fitting the data using a suitable model.
Initially, it was primarily used for investigating enzyme-catalyzed reactions but quickly became a staple in biochemistry, biophysics, and chemistry laboratories for tracking rapid chemical processes.
The timing of the trigger is software-controlled, allowing users to synchronize it with the flow stop or slightly earlier to confirm that the stationary state has been reached.
Broad-spectrum xenon lamps are highly versatile, allowing users to select virtually any wavelength for absorbance or fluorescence studies, making them ideal for applications such as monitoring structural changes in proteins over time.
For far-UV applications, ozone-producing xenon arc lamps are available, but they require purging with pure nitrogen gas to prevent ozone buildup and optical degradation.
Alternatively, mercury-xenon (Hg-Xe) lamps are well-suited for fluorescence experiments where the desired excitation wavelength corresponds to one of the intense mercury emission lines.
[4] Dead time can be minimized by reducing the dimensions of the flow cell, but this approach has limitations due to the decreased signal-to-noise ratio caused by smaller observation windows and shorter pathlengths.
The fluorescence quenching reaction between N-acetyltryptophanamide (NAT) and N-bromosuccinimide (NBS), as described by Peterman, is a commonly used method for measuring the dead time of a stopped-flow instrument.
Stopped-flow spectrophotometers may function as stand-alone instruments, but they are often integrated into systems for circular dichroism (CD), absorbance, and/or fluorescence measurements, or equipped with various accessories to support specialized applications.
The stopped-flow method evolved from the continuous-flow technique developed by Hamilton Hartridge and Francis Roughton[7] to study the binding of oxygen to hemoglobin.
By moving the colorimeter along the tube and knowing the flow rate, Hartridge and Roughton were able to measure reaction progress at specific time intervals.
This innovation was groundbreaking for its time, demonstrating that processes occurring within milliseconds could be studied using relatively simple equipment, despite the limitations of instruments requiring seconds for each measurement.
However, the method had significant practical constraints, particularly the need for large quantities of reactants, making it suitable mainly for studies on abundant proteins like hemoglobin.
For example, in studies of nitrogenase catalysis from Klebsiella pneumoniae[10], the agreement in half-times showed that absorbance at 420 nm corresponded to Pi release, but obtaining this result through quenched-flow required 11 individual data points, highlighting the method's demanding nature.
For a broader perspective, Zheng et al. (2015) review various analytical methods for investigating biological interactions, including stopped-flow analysis, surface plasmon resonance spectroscopy, affinity chromatography, and capillary electrophoresis.
