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

Area of Science:

• Analytical Chemistry
• Spectroscopy
• Chromatography

Background:

• Conventional excitation-emission-matrix (EEM) fluorescence spectrometers have limitations in spectral acquisition speed.
• Rapid analysis is crucial for complex samples in techniques like High-Performance Liquid Chromatography (HPLC).

Purpose of the Study:

• To develop and characterize a new rapid-acquisition HPLC detector using Hadamard-transform (HT) EEM fluorescence spectroscopy.
• To demonstrate the instrument's capability for fast and accurate online analysis of analytes.

Main Methods:

• Integration of a programmable ultraviolet light-emitting diode (UV LED) light source, multiplexed using Hadamard-matrix patterns, with a commercial fluorescence spectrometer.
• Utilizing the HT-EEM spectrometer as a detector in an HPLC system for rapid separation and detection.
• Application of a parallel factor analysis (PARAFAC) algorithm for data processing and analyte identification.

Main Results:

• The HT-EEM spectrometer achieved spectral acquisition rates exceeding 6 spectra per second (<150 ms per spectrum).
• Successful rapid separation and detection of a five-coumarin dye mixture within 2 minutes.
• PARAFAC successfully resolved and identified all coumarin fluorophores, including decomposing two coeluting analytes into distinct components.

Conclusions:

• The developed HT-EEM fluorescence spectrometer offers a significant advancement in spectral acquisition speed for HPLC detection.
• This novel detector provides a versatile platform for rapid online analysis and quantification of analytes in chromatographic separations.
• The system demonstrates robust performance in resolving complex mixtures and coeluting compounds.