Simultaneous Double-Pass Absorption and Fluorescence Excitation-Emission Matrix Spectroscopy for Measurements of Reaction Kinetics.

A novel Hadamard-transform excitation-emission matrix (EEM) spectrometer generates two-dimensional (2D) fluorescence matrices at a data acquisition rate of over 6 EEMs per minute and with a spectral resolution of 5.3 nm. Using Fresnel reflections from the sample cell, we could record optical transmission spectra synchronously with the 2D EEMs. The spectrometer was integrated into a custom-designed stopped-flow injection device to collect visible absorption and fluorescence EEM spectra of reacting solutions. Two different kinetic studies on two rapidly evolving chemical reactions with multiple overlapping spectral components were conducted by collecting over 8400 absorption spectra and EEMs. The third-order rate constant for the demetalation of chlorophyll-a to pheophytin-a was experimentally determined to be 450 ± 20 M-2·s-1 as derived from a parallel factor (PARAFAC) analysis where absorption and fluorescence data were combined. A PARAFAC analysis of data collected from the insertion of a copper atom into pheophytin-a resulted in several absorbing components and only a single fluorescing component. A reaction model with an association complex and a sitting-a-top (SAT) complex as intermediates explained the absorption data, resulting in a sequence of second-order reactions with rate constants of 4.0 ± 0.4, 2.7 ± 0.3, and 0.28 ± 0.02 M-1·s-1, respectively. The rate constant of the fluorescence decrease was determined to be 1.7 ± 0.2 M-1·s-1, which is consistent with the fluorescence component being attributed to both the pheophytin-a and the association complex.