Application of the techniques

In a real-time analysis the composition of the system is analysed while the reaction is in progress. Either a small sample is withdrawn or the bulk solution is monitored. In the flow method the reactants are mixed as they flow together in a chamber (Fig. 22.1). The reaction continues as the thoroughly mixed solutions flow through the outlet tube, and observation of the composition at different positions along the tube is equivalent to the observation of the reaction mixture at different times after mixing. The dis advantage of conventional flow techniques is that a large volume of reactant solution is necessary. This makes the study of fast reactions particularly difficult because to spread the reaction over a length of tube the flow must be rapid. This disadvantage is avoided by the stopped-flow technique, in which the reagents are mixed very quickly in a small chamber fitted with a syringe instead of an outlet tube (Fig. 22.2). The flow ceases when the plunger of the syringe reaches a stop, and the reaction continues in the mixed solutions. Observations, commonly using spectroscopic techniques such as ultraviolet–visible absorption, circular dichroism, and fluorescence emission, are made on the sample as a function of time. The technique allows for the study of reactions that occur on the millisecond to second timescale. The suitability of the stopped f low method to the study of small samples means that it is appropriate for many biochemical reactions, and it has been widely used to study the kinetics of protein folding and enzyme action (see Impact I22.1 later in the chapter). Very fast reactions can be studied by flash photolysis, in which the sample is exposed to a brief flash of light that initiates the reaction and then the contents of the reaction chamber are monitored. Most work is now done with lasers with photo lysis pulse widths that range from femtoseconds to nanoseconds (Section 14.5). The apparatus used for flash photolysis studies is based on the experimental design for time-resolved spectroscopy (Section 14.6). Reactions occurring on a picosecond or femtosecond timescale may be monitored by using electronic absorption or emission, infrared absorption, or Raman scattering. The spectra are recorded at a series of times following laser excitation. The laser pulse can initiate the reaction by forming a reactive species, such as an excited electronic state of a molecule, a radical, or an ion. We discuss examples of excited state reactions in Chapter 23. An example of radical generation is the light-induced dissociation of Cl₂(g) to yield Cl atoms that react with HBr to make HCl and Br according to the following sequence:

Cl2 + hν→Cl+Cl

Cl +HBr→HCl*+Br

HCl*+M→HCl+M

Here HCl* denotes a vibrationally excited HCl molecule and M is a body (an unreact ive molecule or the wall of the container) that removes the excess energy stored in HCl. A so-called ‘third body’ (M) is not always necessary for heteronuclear diatomic mole cules because they can discard energy radiatively, but homonuclear diatomic molecules are vibrationally and rotationally inactive, and can discard energy only by collision. In contrast to real-time analysis, quenching methods are based on stopping, or quenching, the reaction after it has been allowed to proceed for a certain time. In this way the composition is analysed at leisure and reaction intermediates may be trapped. These methods are suitable only for reactions that are slow enough for there to be little reaction during the time it takes to quench the mixture. In the chemical quench f low method, the reactants are mixed in much the same way as in the flow method but the reaction is quenched by another reagent, such as solution of acid or base, after the mixture has travelled along a fixed length of the outlet tube. Different reaction times can be selected by varying the flow rate along the outlet tube. An advantage of the chemical quench flow method over the stopped-flow method is that spectroscopic fingerprints are not needed in order to measure the concentration of reactants and products. Once the reaction has been quenched, the solution may be examined by ‘slow’ techniques, such as gel electrophoresis, mass spectrometry, and chromatography. In the freeze quench method, the reaction is quenched by cooling the mixture within milliseconds and the concentrations of reactants, intermediates, and products are measured spectroscopically.

Fig. 22.1 The arrangement used in the flow technique for studying reaction rates. The reactants are injected into the mixing chamber at a steady rate. The location of the spectrometer corresponds to different times after initiation.

Fig. 22.2 In the stopped-flow technique the reagents are driven quickly into the mixing chamber by the driving syringes and then the time dependence of the concentrations is monitored.

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مواضيع ذات صلة

The primary quantum yield

Timescales of photophysical processes

The catalytic efficiency of enzymes

The Michaelis–Menten mechanism of enzyme catalysis

Enzymes

Monitoring the progress of a reaction

The measurement of diffusion coefficients

Solutions of the diffusion equation

Diffusion with convection

Conductivities and ion–ion interactions

The drift speed

Strong electrolytes