The Advancement of Hadamard-Transform Excitation-Emission-Matrix Spectroscopy

Abstract

A multiplexed approach to multidimensional fluorescence spectroscopy is presented. Numerous modifications were made to a home-built Hadamard-Transform Excitation-Emission Matrix (EEM) spectrometer resulting in a greatly improved spectral resolution of 5.3 nm, at a data acquisition rate of over 6 EEM spectra per minute. In addition, provisions were made to simultaneously record optical transmission spectra with the 3D EEM spectra. Integration of the spectrometer into a custom-designed stopped-flow injection device allowed for the simultaneous collection of UV/Vis absorbance and fluorescence EEM spectra of reacting solutions. With this optimized configuration, over 8400 EEM spectra were collected while performing two kinetic studies on two rapidly evolving chemical reactions with multiple overlapping spectral components. Improved reproducibility reduced the uncertainty of kinetic rate constants from 40% to 4%. The third-order rate constant for the demetallation of chlorophyll-a to pheophytin-a was experimentally determined to be 611±20 ×102 M-2∙s-1 as derived from a parallel factor (PARAFAC) analysis where absorbance 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 fluorescence component. A reaction model with a pre-association complex and sitting-a-top (SAT) complex as intermediates explained the absorbance data resulting in a sequence of second-order reactions with rate constants of 4.03 ± 0.2 M-1 s-1, 2.71 ± 0.2 M-1 s-1, and 0.28±0.02 s-1∙M-1, respectively. The rate constant of fluorescence decay was determined to be 1.7±0.1 M-1∙s-1 which is consistent with the fluorescence signal being a combination of the pheophytin-a and the pre-association complex.