In an SX20 stopped-flow spectrometer, there are two optical pathlengths available for absorbance detection (2mm and 10mm, or 1mm and 5mm depending on cell type). The mixer is an integral part of the observation cell and there is a dedicated observation window for fluorescence detection.
Typically used to gain an understanding of reaction mechanisms including drug-binding processes, or to determine protein structure, stopped-flow spectroscopy enables the study of fast reactions in solution over timescales in the range of 1 millisecond to hundreds of seconds.
A wide range of reactions can be investigated involving, for example, protein-protein interactions, ligand binding, electron transfer, fluorescence resonance energy transfer (FRET), protein folding, as well as enzyme, chemical or coordination reactions.
In most experimental set-ups, two reagents are rapidly mixed together and then ‘stopped’ in an observation cell. The sample cell is irradiated with monochromatic light and as the reaction proceeds the change in the recorded signal, usually a fluorescence signal or absorbance at a specific wavelength, is recorded as a function of time.
Analysis of the resulting kinetic transient can determine reaction rates, complexity of the reaction mechanism, information on short-lived reaction intermediates etc. A series of stopped-flow experiments can be used to show the effect of parameters such as temperature, pH and reagent concentration on the kinetics of a reaction.
Schematic of an SX20 stopped-flow spectrometer - single mixing
The stopping mechanism is a stop-syringe. Reagents are contained in two drive syringes (C and F). A drive ram pushes the syringe pistons such that the reagents are pushed through flow tubing to a mixer and then to the observation cell. This process pushes the ‘old’ contents of the cell towards the stop-syringe. The flow fills the stop-syringe, until the piston hits the trigger-switch. This action simultaneously stops the flow and starts data acquisition. At this instant the age of the reaction of the newly-mixed reagents in the observation cell is about 1 millisecond. The exact age (called the dead-time) is dependent upon the design of the stopped-flow instrument and the observation cell.
Schematic of an SX20 stopped-flow spectrometer - sequential mixing with option SX/SQ
Sequential mixing mode, also referred to as double mixing, is a variation of the stopped-flow technique particularly well-suited to studying reactions between a short-lived reaction intermediate and a third reagent.
In a sequential mixing experiment, two reagents (A and B) are rapidly mixed and held in an aging loop. After a pre-set interval (milliseconds to 10’s of seconds) the loop contents are rapidly mixed with a third reagent (C) in the observation cell and the reaction (between the short-lived-intermediate and C) will be followed in the same manner as for the single mixing mode.