Spectrofluorometer

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The spectrofluorometer is an instrument which takes advantage of fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. A certain excitation wavelength is selected, and the emission is observed either at a single wavelength or a scan is performed to record the intensity versus wavelength also called an emission spectra. See Fluorescence spectroscopy

schematic of a spectrofluorometer

Generally spectrofluorometers use high intensity light sources to bombard a sample with as many photons as possible. This allows for the maximum number of molecules to be in the excited state at any one point in time. The light is either passed through a filter, selecting a fixed wavelength, or monochromator, which allows you to select a wavelength of interest to use as the exciting light. The emission is collected at 90 degrees to the exciting light. The emission too is either passed through a filter or a monochromator before being detected by a PMT, photodiode, or CCD detector. The signal can either be processed as a digital or analog output. Systems vary greatly and a few things must be considered when choosing. First is signal to noise, there are many ways to look at the signal to noise of a given system but the accepted standard is by using water Raman. Sensitivity or detection limit is another spec to look at, that is how little light they can measure. The standard for this would be fluorescein in NaOH, typical values for a high end instrument are in the femtomolar range. Stray light is another big issue in these instruments. Stray light is basically how monochromatic the light is. This matters when you have a highly scattering sample, however one can always use an excitation wavelength further away from the emission band to negate this issue or use a laser or interference filter.

These systems come with many options now here are a few:

• Polarizers
• Peltier temperature controllers
• Cryostats
• Cold Finger Dewars
• pulsed Lasers for lifetime measurements
• LEDs for lifetimes
• Filter holders
• Adjustable optics (very important)
• Solid sample holders
• Slide holders
• Integrating spheres
• NIR detectors
• Bilateral slits
• Manual slits
• Computer controlled slits
• Fast switching monochromators
• Filter Wheels