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

Abstract

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.

Rapid Fluorescence EEM spectra acquisition using super-cycle Hadamard-Transform Multiplexing

“…This programmable light source has been described previously. 19 The center of Figure 1 shows the 77 S-matrix used for the construction of the Walsh In an experiment, the corresponding fluorescence emission is recorded with a CCD spectrometer. The resulting matrix contains fluorescence intensity as a function of emission wavelength, and excitation mask number (Figure 2 left where each excitation spectrum is given in Figure 1 right).…”

“…This multiplex advantage is apparent in the brighter timeintegrated emission spectrum (Figure 1 right), and has been demonstrated experimentally in several studies involving kinetics of fast reactions, and in separation science. [19][20][21]…”

“…Recently, our group introduced the Hadamard Transform (HT) as a simple method to multiplex the excitation light and thereby considerably accelerate the data acquisition rate to 6-7 EEM spectra per second (<150 ms per spectrum). [19][20][21] In this report, we describe a mathematical method to further accelerate the acquisition rate by one order of magnitude or more. Interestingly, the highest spectrally-resolved instruments, i.e.…”

“…Since then, these masks have been replaced with digital-micro-mirror (DMD) devices for the multiplexing of light 20,21,25 , but it is also possible to multiplex by turning discrete wavelengths on and off should the light source consist of a battery of lasers or LED, for instance. 19 Multiplexing of spectroscopic data offers a superior signal-to-noise ratio (SNR) compared to direct measurements 26,27 . This so-called Fellgett's advantage has been demonstrated in our previous Hadamard-Transform Excitation-Emission-Matrix (HT-EEM) instruments.…”

“…HT-EEM spectroscopy had been applied to time-dependent EEM spectroscopic measurements of e.g., chemical kinetics, or integration of EEM detection into an HPLC instrument. [19][20][21] Here, we present a new algorithm for improving the time resolution of HT multiplexed data by another order of magnitude.…”