using a spectrophotometer as a fluorometer

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drdee's picture
using a spectrophotometer as a fluorometer

Our UV-VIs is a Shimadzu UV-2550.  I need to do some fluorescense experiments.  It seems to me that a UV that can both absorb and transmit should be able to do fluorescence experiments.  Please help me to understand the physics of this and how to set up the experiment if it is possible.  Thanks

Dr. Analytical
Dr. Analytical's picture
In a typical absorbance

In a typical absorbance instrument, the light passes through the sample (in the sample cell), and the amount of light at any wavelength that comes through is measured.  In a double beam instrument, you have solvent only in your "reference" cell.  The amount of light that goes through the reference cell is also measured.  The percent transmittance (%T) is then calculated from the ratio of the two light intensities.  Absorbance is calculated from the following equation:
A = 2 - log %T
If you have a single beam instrument, then you have to measure a solvent "blank" before you measure your sample.  The calculations are the same, it's just a different sequence.
Fluorescence also involves the absorbance of light, you are correct, but what happens next is the important difference.  If a molecule does not fluoresce, then it loses the energy from absorbing light through vibrations and other processes.  But if the molecule fluoresces, then it loses energy through emission of light (at a longer wavelength) that we can measure.  This is the "glow" that we see when we shine an ultraviolet light on objects - they absorb the UV light and release visible light.
A fluorescence spectrophotometer will typically have a monochromator to control the wavelength of light going into the cell (excitation wavelength) and a second monochromator to monitor the fluorescence (emission wavelength).  The fluorescence is measured at right angles to the emission source, to minimze interferences.
So, getting back to your question, you would need a way to excite the sample at one wavelength and monitor the fluorescence at a different wavelength.  This is usually not possible with a normal spectrophotometer.  You might get some hints about fluorescence behavior from an absorbance instrument, but only because of interference.  To make quantitative measurements, you will need to have the correct instrumentation.