Rapid reaction techniques following mixing

The flow techniques just described are limited by the speed with which it is possible to mix solutions. There is no difficulty, using optical or other techniques, in following the course of a very rapid reaction, but for hydrodynamic reasons it is impossible to mix two solutions in less than about 10 s. If the half-life is less than this, the reaction will be largely completed by the time that it takes for mixing to be achieved, and any rate measurement made will be of the rate of mixing, not the rate of reaction. The neutralization of an acid by a base, i.e, the reaction... 

When the rate of a reaction is faster than the time it takes us to introduce and mix reagents, we must turn to a method that achieves rapid mixing and has the ability to monitor the extent of reaction at various times. The most common methods involve flow tubes (Figure 7.16). The reactants are mixed at the point where the two tubes intersect, and then theex-tent of reaction is followed by an analysis of the mixture at various points along the observation tube. The flow rate of the tube is known, so that the time from the point of mixing is known at each analysis point along the tube. In the stopped-flow method, the flow is suddenly stopped at various times, and the analysis is performed at the same point in the observation tube at each different time. Several commercial versions of this apparatus are available for stopped-flow analyses, and it is a particularly common method for the analysis of enzyme-catalyzed reactions. With this technique reactions with half-lives as short as milliseconds can be measured. 

Let us examine some batch results. In trials in which 5 mL of a dye solution was added by pipet (with pressure) to 10 mL of water in a 25-mL flask, which was shaken to mix (as determined visually), and the mixed solution was delivered into a 3-mL rectangular cuvette, it was found that = 3-5 s, 2-4 s, and /obs 3-5 s. This is characteristic of conventional batch operation. Simple modifications can reduce this dead time. Reaction vessels designed for photometric titrations - may be useful kinetic tools. For reactions that are followed spectrophotometrically this technique is valuable Make a flat button on the end of a 4-in. length of glass rod. Deliver 3 mL of reaction medium into the rectangular cuvette in the spectrophotometer cell compartment. Transfer 10-100 p.L of a reactant stock solution to the button on the rod. Lower this into the cuvette, mix the solution with a few rapid vertical movements of the rod, and begin recording the dead time will be 3-8 s. A commercial version of the stirrer is available. 

The initial evidence for the formation of an acyl-enzyme ester intermediate came from studies of the kinetics with which chymotrypsin hydrolyzed analogs of its normal polypeptide substrates. The enzyme turned out to hydrolyze esters as well as peptides and simpler amides. Of particular interest was the reaction with the ester p-nitrophenyl acetate. This substrate is well suited for kinetic studies because one of the products of its hydrolysis, p-nitrophenol, has a yellow color in aqueous solution, whereas p-nitrophenyl acetate itself is colorless. The change in the absorption spectrum makes it easy to follow the progress of the reaction. When rapid-mixing techniques are used to add the substrate to the enzyme, an initial burst of p-nitrophenol is detected within the first few seconds, before the reaction settles down to a constant rate (fig. 8.8). The amount of p-nitrophe-... 

The combination of rapid mixing and fast detection systems allows cationic polymerisations to be followed on an even shorter time scale than with adiabatic calorimetry. Recent commercial stop-flow spectrophotometers have a dead time of about 15 msec, an improvement of more than one order of magnitude over previous home-made models. This implies that reactions with half lives of less than 100 msec can be analysed kinetically with a good degree of accuracy. Hi -vacuum techniques are not compatible with these instruments and all operations are therefore carried out in an inert atmosphere. 

As discussed further in the following sections, there are other variations of rapid mixing/quench methods in which the enzymatic reaction is terminated by freezing the reaction mixture with liquid isopropane. The frozen sample is then analyzed hy electron paramagnetic resonance (EPR), solid-state NMR, or other spectroscopic techniques such as resonance Raman spectroscopy that can accommodate a solid sample. Perhaps the major limitation for implementation of this methodology is the sensitivity of the spectroscopic method and the requirement for large amounts of enzyme. ... 

---