Stopped-Flow techniques

1. Determining the Rate of a Chemical Reaction
Stopped-Flow Techniques
Halavath Ramesh
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2.  Kinetic methods are the measurement of the analytical signal is made under
dynamic conditions in which the concentrations of reactants and products are
changing as a function of time.
 Fast reactions are the chemical reactions which take place at a very fast rate.
 These reactions can take place in seconds or in minutes. In general the
reactions between ionic compounds are fast.
 Chemical kinetics, also known as reaction kinetics, is the branch of physical
chemistry that is concerned with understanding the rates of chemical reactions.

3. Determining the rate of a chemical reaction, experimentally
•Chemical methods
•Physical methods
•Stopped-flow techniques
•Flash photolysis techniques
•Perturbation-relaxation methods

4. Introduction
 Stopped-flow is a lab technique for studying fast chemical reactions.
 The stopped-flow technique was developed in the 1940s by modification of
the continuous flow method with the principal advantage of more economic
use of reagents.
 A stopped-flow instrument is a rapid mixing device used to study the
chemical kinetics of fast reactions in solutions.
 The stopped-flow technique allows for the evaluation of solution based
kinetics on a milliseconds timescale with a very small volume of reactants
used.
 Many methods for kinetic studies are based on the measurement of a
change in the concentration of a reagent or product as a function of time after
the reagents have been mixed.

5. This approach requires that the process of interest be slow enough to give a reaction
time that is longer than the time needed for reagent mixing and instrument activation.
However, many biological interactions can occur within seconds (s) or milliseconds
(ms), a fact which has limited the application of many traditional kinetic methods to
such systems.
Detection in stopped-flow analysis can be accomplished by using various
methods that are able to selectively monitor a reagent or product in the reaction.
Absorbance and fluorescence are two common detection methods that are employed
for this type of experiment.

6. Mechanism
 This technique involves two reactants hold in separate reservoirs that are prevented
from freely flowing by syringe pumps.
 The reaction is initiated by depressing the reactant syringes, and thus releasing the
reactants into the connecting “ mixing chamber” where the solutions are mixed.
 The reaction is monitored by observing the change in absorbance of the reaction
solution as a function of time.
 As the reaction progresses it fills the “ stop syringe “ which then expands until it hits
a block at the point when the reaction has reached a continuous flow rate, there by
stopping the flow and the reaction, and thus allowing the researcher to calculate the
exact initial rate of reaction.
 Stopped-flow technique works because within millisecond of combining the two
reactants the absorbance can be read.

7.  In addition the stop syringe assures for a steady rate of flow pass the
spectrophotometer so that reactants are being added to solution and forming
products at a consistent rate.
 After perhaps a few milliseconds the observation cell is filled by a piston
linked to a sensing switch that triggers the measuring device and the flow is
stopped suddenly.
 For studied different forms of spectroscopy and scattering of radiation are
common methods used.
 The dead time is the time between the end of mixing the two solutions and
the beginning of observation of the kinetics of the reaction.
 The usual dead time of a stopped flow apparatus is 1-2 milli seconds.
 A stopped- flow instrument coupled to either a circular dichroism
spectrometer or a fluorescence spectrometer is often used in the field of
protein folding , to observe rapid unfolding and or refolding of proteins.

8. Model: SFM-20
 Stopped-flow is one of a number of techniques used to study
the kinetics of reactions of in solution.
 It is extensively used to study biomolecular interactions,
kinetics and fast reaction mechanisms typical of many reactions
in chemical and biological systems.
It is also used in the field of protein folding to observe rapid
unfolding and/or refolding of proteins.
 Two reactant solutions are rapidly mixed by being forced into a
mixing chamber, and then through an observation cell.

9.  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.
 The SFM-20 is a high performance, modular stopped-flow system, designed for
single mixing rapid kinetics applications.
 Its mixing ratio ranges from 1:1 to 1:40 , dead time 0.6ms with the micro volume
cuvette.
 It is compatible with MOS-200/M,MOS-450/AF-CD,MOS-DA.
 The signal detection is performed by a photomultiplier directly mounted on the
stopped-flow and connected to its control unit.
 The photomultiplier can be attached at 180A° of the light source or at 90A° allowing
absorbance or fluorescence measurements(both at the same time using an optional
additional detection channel).
 For fluorescence measurements standard filters can be installed in front the
photomultiplier tube inside the holder.

14. Figure 36.3 Schematic of a stopped-flow experiment. Two reactants are rapidly introduced
into the mixing chamber by syringes. After mixing chamber, the reaction kinetics are
monitored by observing the change in sample concentration versus time, in this example by
measuring the absorption of light as a function of time after mixing.

22. Advantages of stopped flow method:
1. Volume of reagent used is very little.
2. A permanent recorder can record the progress of the reactions
as a wide range.
3. This method is not affected by the rate and characters of the
flow.