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Polarization-based fluorescent method for enhanced analytical determination of mixed fluorophores in fluid

Real-time mixture analysis is widely needed in environmental monitoring, kinetics analysis, and industrial process monitoring etc. Conventional methods for mixture analysis such as HPLC, HPLC/MS, or GC/MS, are not good for real-time purposes. Recently, we have demonstrated fluorescence polarization could be used as an additional dimension with which to distinguish fluorescent molecules by the introduction of some specific micelles. In this report, a novel method, the polarized fluorescence spectrometry including polarization information, has been proposed and used for simultaneous determination of fluorescein (FLR) and rhodamine 123 hydrate (R123H) without spectral dispersion. This could also be used for enhanced analytical determination of mixed fluorophores with 2-D fluorescence (excitation-emission matrix). At this time, conventional 2-D fluorescence spectra already provide enough information for principal component regression (PCR) or parallel factor analysis (PARAFAC) to distinguish four fluorophores: FLR, R123H, rhodamine 6G (R6G), and rhodamine B (RB), even FLR and R123H have a very similar 2-D fluorescence spectra.