Rapid scanning stopped-flow instrumentation

Measurements of absorption as a function of time can provide a wide range of useful information. Brzovic and Dunn [8] describe instrumentation for measuring the time dependence of absorption spectra after rapid mixing of reactants. Several rapid-scanning stopped-flow instruments are commercially available reactions that take place in a millisecond or longer can be studied. Enzyme-catalyzed reactions with natural chromophores, such as NADH, are discussed, and the substitution of a colored metal center [Co(II)] for a colorless one [Zn(II)] are also described. Detailed mechanistic conclusions for horse liver alcohol dehydrogenase (LADH) are given. 

Before any instrument can be used with confidence its operating characteristics must be well understood. The exceptional versatility of the vidicon rapid scanning stopped-flow system allowed a large variety of characterization experiments to be easily performed. A few of the more important experiments designed to help characterize the vidicon detector will be presented before the actual application of the instrument is discussed. 

Today a stopped-flow instrument consists of oifly the unit itself combined with a diode array detector and a computer allowing fast kinetic measurements of time-resolved UV-vis spectra under anaerobic, high pressure and/or low temperature conditions. Improvements have been made as well, for example, syringes are installed vertically instead of horizontally (to avoid problems with gas bubbles) and polyetheretherketone (PEEK) is used instead of Teflon for valves and flow tubes to improve the anaerobic capabilities of the instrument. Further, the syringe drives setup was optimized. Application of rapid-scan devices (usually, but not exclusively, diode arrays) allows complete spectra to be collected at very short time intervals during a reaction. 

In Fig. 8, the UV-Vis spectral changes that occurred upon mixing aqueous solutions of 1 and H2O2 in the chamber of the stopped-flow instrument equipped with a rapid scan spectroscopic attachment are reported as a ftmction of time. The final spectrum of the reaction solution is in good agreement with the spectrum of [Ru (edta)0)] Umax=391nm, emax=8000M cm )... 

Options for multichannel LC-CD detection do exist.f " Stopped-flow accessories for commercial instruments are available that allow part of an eluted fraction to be taken off-line into a microcell placed in the regular sample compartment where data are measured in the normal way. The method still requires rapid scanning capabilities. Repeated injections and multiple scans can be averaged to improve the quality of the signal. A major deterrent to the progress in the early development of HPLC-CD detection was the lack of a dedicated instrument at a reasonable cost, the only option being a fully equipped CD instrument. 

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