We present a fluorescence excitation-emission-matrix spectrometer with superior data acquisition rates over previous instruments. Light from a white light emitting diode (LED) source is dispersed onto a digital micromirror array (DMA) and encoded using binary n-size Walsh functions ("barcodes"). The encoded excitation light is used to irradiate the liquid sample and its fluorescence is dispersed and detected using a conventional array spectrometer. After exposure to excitation light encoded in n different ways, the 2-dimensional excitation-emission-matrix (EEM) spectrum is obtained by inverse Hadamard transformation. Using this technique we examined the kinetics of the fluorescence of rhodamine B as a function of temperature and the acid-driven demetalation of chlorophyll-a into pheophytin-a. For these experiments, EEM spectra with 31 excitation channels and 2048 emission channels were recorded every 15 s. In total, data from over 3000 EEM spectra were included in this report. It is shown that the increase in data acquisition rate can be as high as [{n(n + 1)}/2]-fold over conventional EEM spectrometers. Spectral acquisition rates of more than two spectra per second were demonstrated.

We present a new rapid-acquisition HPLC detector based on a Hadamard-transform (HT) excitation-emission-matrix (EEM) fluorescence spectrometer allowing the acquisition of twodimensional spectra at a rate faster than 6 spectra per second (<150 ms per spectrum). The instrument uses discrete ultraviolet light emitting diode (UV LED) light sources which are multiplexed using patterns derived from a Hadamard-matrix and affords faster spectral acquisition compared to conventional sequentially scanning EEM spectrometers. This new programmable light source was combined with a commercial fluorescence spectrometer and integrated as a detector into an HPLC system. We characterize the HT-EEM spectrometer by rapidly separating and detecting a mixture of five different coumarin dyes, with all five analytes eluting in under 2 min. A parallel factor analysis (PARAFAC) algorithm was able to readily separate and identify all coumarin fluorophores without any prior knowledge of the system, even decomposing two coeluting analytes into two distinct PARAFAC components. The HT-EEM spectrometer provides a novel and versatile detection technique suited for rapid online analysis and quantification of analytes in separation methods such as HPLC.

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.

A new UV-LED based Hadamard excitation-emission-matrix fluorescence spectrometer was designed and utilized to study the degradation of antioxidants additives to lubricant samples with acquisition times within 2 to 10 seconds.

Hadamard-transform multiplexing has recently been applied to increasingly complex spectroscopic techniques. It had been shown that the data acquisition time for fluorescence Excitation-Emission-Matrix spectroscopy can be reduced by one or two orders of magnitude using Hadamard-Transform multiplexing of the excitation light using a programmable lightsource. In these previous studies, the data acquisition rate had been limited by the time it took to record an EEM, i.e. to complete one cycle of multiplexed excitation spectra. The extraction of chemical information, such as concentration and chemical identity, is then obtained from parallel factor (PARAFAC) analysis of the sequence of EEM spectra. In this contribution we increase the data acquisition rate by another order of magnitude, i.e. to the time it takes to record a single excitation spectrum. Our algorithm is entirely based on improved data processing, i.e. it can be applied to previously recorded Hadamard-Transform multiplexed data sets. The algorithm is based on three previously unexplored approaches: (1) we perform a PARAFAC multivariate analysis on the raw (multiplexed) data set (2) the PARAFAC loading vectors are obtained prior to obtaining the score vectors, (3) when loading vectors are difficult to obtain from the stack of EEM spectra, we instead use a rolling-average approach to considerably increase the number of spectra and the stability of the fit. Analysis of experimental data shows that fluorescence EEM spectra with 7 excitation wavelengths and over 1000 emission wavelengths can be obtained in less than 20 ms.