Fluorescence spectra, fluorescence quantum yield, and fluorescence lifetime are very important characteristic parameters in fluorescence analysis. After excitation, all molecules do not emit photons. Many pathways other than fluorescence emission contribute to fluorophore de-excitation. The fraction of the excited molecules that return to the ground state (S1→S0 transition) with emission of photon is known as the "fluorescence quantum yield". The fluorescence quantum yield is an intrinsic property of a fluorophore. In the following, we discuss about the quantum yield and the relationship among fluorescence lifetime and how the quantum yield can be determined for a flurophore molecule. Measurements of the absolute quantum yields require sophisticated instrumentation. However, one can easily determine the relative quantum yield of a fluorophore with respect to a reference fluorophore for which the precise quantum yield has been known. Two methods for relative quantum yield determinations are available: single-point method and comparative method. The single-point method uses the integrated emission intensities from a single sample and the reference at identical concentration. This method is fast and easy but is not always reliable due to the inaccurate measurement of the fluorophore's absorbance. The comparative method involves the use of multiple well characterized references with known fluorescence quantum yields and the calculations of the slopes of the line generated by plotting the integrated fluorescence intensity against the absorption for multiple concentrations of fluorophore as well as the reference. The comparative method is time consuming but provides higher accuracy.

In fluorescence one gets two kinds of spectra: excitation and emission spectra. The spectrum observed by measuring the variation of the fluorescence intensity from a fluorophore as a function of the emission wavelength is termed a measured emission spectrum. The corrected emission spectrum is obtained after correcting for instrumental and sample effects. The spectrum obtained by measuring the variation of the fluorescence intensity from a fluorophore as a function of the excitation wavelength is termed a measured fluorescence excitation spectrum. A corrected excitation spectrum is obtained if the photon flux incident on the sample is held constant. For a sufficiently dilute solution when the fraction of the exciting radiation absorbed is proportional to the absorption coefficient of the fluorophore and the quantum yield is independent of the exciting wavelength, the corrected excitation spectrum will be identical in shape to the absorption spectrum. Corrected spectra are important in the determination of quantum yield of fluorophores and in quantitative analysis. In this experiment, the single point method is applied to determine the fluorescence quantum yield of Rhodamine B by comparing with Rhodamine 6G which has fluorescence quantum yield of 0.95. Rhodamine dyes and their derivatives are commonly used as fluorescence probes for biological assays.