HPLC-Detector Based on Hadamard-Transform Fluorescence Excitation-Emission-Matrix Spectroscopy

We present a new rapid-acquisition HPLC detector based on a Hadamard-transform (HT) excitation-emission-matrix (EEM) fluorescence spectrometer allowing the acquisition of two-dimensional 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.