We describe a new method, phase-resolved subnanosecond spectroscopy (PRS), for the spectral differentiation of fluorophores in a mixture. The technique required adding a phase-variable rectifying detector to the SLM 4800S phase-spectrofluorometer. The theory of PRS is based on the sinusoidal fluorescence emission of a population of molecules in response to sinusoidally modulated excitation light. The total a–c fluorescence signal is passed through the phase-variable detector which nulls the emission signal of any component in quadrature with the reference angle. The emission characteristics of the remaining component, or components, are more readily and accurately revealed. We investigated the sensitivity and selectivity of PRS. The sensitivity of PRS was demonstrated by nulling the contribution of the Raman scatter band of a nanomolar solution of quinine bisulfate to the real-time emission spectrum resolved at 8-nm bandpass. We demonstrated the selectivity of PRS by resolving the emission spectrum of anthracene and perylene from a 1 : 1 mixture with a lifetime differential of only 600 ps. Also, the emission spectra of 2,2-phenylene bis-(5-phenyloxazole) and dimethyl 2,2-phenylene bis-(5-phenyloxazole) were also resolved from a 1:1 mixture in ethanol. The lifetime differential here was only 200 ps.

We also applied the PRS technique to investigate the heterogeneous emission due to tyrosine and tryptophan residues in the protein bovine serum albumin (BSA). Suppression of the fluorescence emission of tryosine at 300 nm allowed us to resolve the emission spectrum of tryptophan. Suppression of the fluorescence emission of tryptophan at 400 nm allowed us to resolve the emission spectrum of tryosine. The PRS spectra of the two components suggested the presence of an additional species that may add to the heterogeneous emission of the protein.