Factors interfering with fluorescence intensity

If the concentration of a solution prepared for fluorescence measurement is too high, some of the light emitted by the sample as fluorescence will be reabsorbed by other unexcited molecules in solution. For this reason, fluorescence measurements are best made on solutions with an absorbance of less than 0.02 at their maximum, i.e. solutions of a sample 10-100 weaker than those which would be used for measurement by UV spectrophotometry. 

Heavy atoms in solution quench fluorescence by colliding with excited molecules so that their energy is dissipated, e.g. chloride or bromide ions in solution cause collisional quenching. 

Formation of a chemical complex with other molecules in solution can change fluorescence behaviour, e.g. the presence of caffeine in solution reduces the fluorescence of riboflavin. This alteration of fluorescence upon binding is used to advantage when examining binding of fluorescent molecules to proteins or other constituents of cells. 

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Fluorescence is generally more sensitive to environmental factors than absorbance measurements. Signal intensity may be affected by pH, temperature, quenching, interfering substances, solvent, or interference from Rayleigh and Raman scattering. Many fluorescent species contain ionisable groups whose fluorescent properties are sensitive to pH. In some cases only one of the ionised species may be fluorescent. An example is the barbiturates which only fluoresce at elevated pH in the di-anionic form. The relationship of fluorescence intensity with pH should always be examined as part of the development of the method. 

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