A significant experimental problem when the reaction’s kinetics are fast is ensuring that we rapidly and reproducibly mix the sample and the reagents. For a fast reaction we need to make measurements in a few seconds, or even a few milliseconds. This presents us with both a problem and an important advantage. The problem is that rapidly and reproducibly mixing the sample and the reagent requires a dedicated instrument, which adds an additional expense to the analysis. The advantage is that a rapid, automated analysis allows for a high throughput of samples.
A variety of instruments have been developed to automate the kinetic analysis of fast reactions. One example, shown here, is the stopped-flow analyzer. The sample and reagents are loaded into separate syringes and precisely measured volumes are dispensed into a mixing chamber by the action of a syringe drive. The continued action of the syringe drive pushes the mixture through an observation cell and into a stopping syringe. The back pressure generated when the stopping syringe hits the stopping block completes the mixing, after which the reaction’s progress is monitored spectrophotometrically. With a stopped-flow analyzer it is possible to complete the mixing of sample and reagent, and initiate the kinetic measurements in approximately 0.5 ms. By attaching an autosampler to the sample syringe it is possible to analyze up to several hundred samples per hour.
Another instrumental design is the centrifugal analyzer, a partial cross section of which is shown below. The sample and the reagents are placed in separate wells, which are oriented radially around a circular transfer disk. As the centrifuge spins, the centrifugal force pulls the sample and the reagents into the cuvette where mixing occurs. A single optical source and detector, located below and above the transfer disk’s outer edge, measures the absorbance each time the cuvette passes through the optical beam. When using a transfer disk with 30 cuvettes and rotating at 600 rpm, we can collect 10 data points per second for each sample.
