RK-Spectro Technical Note 54: Determination of your experimental dead time in fluorescence mode

Introduction

This Technical Note demonstrates how to calculate the experimental dead time of your stopped-flow instrument. The experiment presupposes that the user understands how to design stopped-flow sequences and how to correctly use Bio-kine software. The dead time of a stopped-flow experiment refers to the age of the solution when it reaches the observation point; in other words, it is the time for the mixed solution to pass from the center of the last mixer to the observation point. Besides the total flow rate and cuvette volume, the dead time depends on many different factors. In this technical note, only flow rate and cuvette volume influences will be discussed. There may be a slight difference between the estimated dead time (given by the Biokine software) and the real observed dead time. This is due to the hydrodynamics phenomenon. In this technical note, we will also demonstrate the dependence of the rate constant of the reaction with the NBS concentration. The NBS concentration will range from 2000µM to 200µM. The technique to evaluate dead time in fluorescence mode presented here may be adapted to different instrumental conditions. This experiment can be performed with any optical system allowing total fluorescence detection: MOS-200, MOS250, MOS450 and J-810 and any of our SFM instruments (SFM-20, SFM-300 and SFM-400). We advise using the SFM-300/400 for concentration dependence studies…