Spectrofluorimeter A spectrofluorimeter allows one to measure emission and excitation spectra. The components of a Varian Cary® Eclipse spectrofluorimeter and the beam path are shown in the schematic below. ﷯ A Xenon flash lamp generates lights in a broad range of wavelengths (190–1100 nm). To record an emission spectrum (emission intensity vs. wavelength) one needs to select a wavelength that corresponds to a maximum of absorption of the species whose emission is measured. This is done using the excitation monochromator. The monochromatic light that exits the excitation monochromator becomes the excitation light and is sent to a cuvette containing the sample. The sample gets continuously excited and, as a result, starts continuously emitting light. Although the emission propagates in all directions, it is only collected at 900 relative to the direction of the excitation light. The emission generated from the sample contains multiple wavelengths. In order to measure the emission intensity as a function of wavelength, the emission light passes through the emission monochromator before it reaches the detector. The emission monochromator passes through one wavelength at a time. The range of the wavelengths that the emission monochromator passes through is specified by the user. Thus, the detector records the emission intensity (one value for each wavelength) in a specified range of wavelengths. Two Czerny–Turner monochromators are employed in a Varian Cary® Eclipse spectrofluorimeter. Each consists of two motorized slits (entrance and exit ones – the width of both can be controlled in the experiment), two fixed mirrors and a rotating diffraction grating. The first mirror in the beam path collimates the light (makes it travel in parallel beams, i.e. have a focus at infinity). The collimated light reflected from that mirror falls onto the diffraction grating. The diffraction grating separates (disperse) the light by wavelengths. The dispersed light is collected by the second mirror and sent as a bunch of spatially separated wavelengths towards the exit slit. For visible light, this bunch looks like a rainbow. By rotating the diffraction grating, one can control the light of which wavelength passes through the slit. Thus, the monochromator allows separating light spatially based on its wavelength and pass one wavelength at a time through the exit slit.