Determination of the dead time of a stopped-flow fluorometer

This investigation was carried out to develop a convenient alternative method for examining the performance and determining the dead time of a stopped-flow fluorometer. We examined the kinetics for the formation of the fluorescent Mg2+-8-hydroxyquinoline chelate in aqueous solutions. The reversible association of the Mg2+ ion with 8-hydroxyquinoline is a second-order process whose on and off rate constants are dependent on pH. We estimated that the Mg2+ ion chelate has a fluorescence quantum yield of 0.02 in aqueous solutions. Using this reaction we measured the dead time of a stopped-flow fluorometer at different pH values. Measurements of the dead time were found to be reproducible and accurate. The Mg2+-8-hydroxyquinoline reaction fulfills the requirements for a convenient test reaction for dead time measurement of stopped-flow fluorometers. Although the usefulness of the reaction is primarily to determine the dead times of stopped-flow instruments operating in the fluorescence mode, the reaction can also be used for testing an instrument operating in the absorbance mode.