Fluorescence spectroscopy explained
Fluorescence spectroscopy uses the fluorescence properties of different sample materials to identify them, making it an effective method to spot different drugs, chemicals and metabolites.
It works by detecting the wavelength of energy emitted by fluorescent samples when their electrons are excited and then return to the ground state.
Because some energy is lost in the process, the sample emits light that is of a longer wavelength than that used to excite the electrons, and the difference between the two – called the Stokes shift after Irish physicist George Gabriel Stokes – gives each material its own distinct absorption and emission spectra.
The entire process takes place in around 10 nanoseconds, or one one-hundred-millionth of a second, although the ‘fluorescence lifetime’ of different materials can vary by several orders of magnitude.
Fluorescence spectroscopy equipment
The instrumentation used in fluorescence spectroscopy needs to excite the sample using photons and then detect the wavelength of the energy emitted when the excited electrons return to the ground state.
Typical equipment used for this includes:
- A light source (often xenon or mercury arc lamps at 300-800 nm).
- A monochromator (a high-precision optical filter that ensures the incident beam is at a very precise wavelength).
- A detector (a sensitive photomultiplier that measures the wavelength of emission from the sample).
In some cases a chemical fluorescent probe may also be needed, as described below, if the sample does not contain a substance that is naturally fluorescent.
Helping extrinsic fluorophores to glow
Not all materials are naturally fluorescent – or ‘intrinsic fluorophores’, to use the scientific term.
However, those that do not naturally display fluorescent properties, known as ‘extrinsic fluorophores’, can still be analysed using fluorescence spectroscopy.
In order to do this, the sample is dosed with a substance that acts as a fluorescent probe by attaching to the extrinsic fluorophore compound.
Factors that prevent fluorescence
There are a number of factors that can reduce the fluorescence of the sample and affect the quality of fluorescence spectroscopy results as a consequence.
Some examples include:
- Absorption of fluorescence by the solution itself.
- Extremes of pH for certain fluorophores.
- Oxidation of the fluorophore due to dissolved oxygen.
- Solvent polarity and presence of heavy atoms.
- Low molecular rigidity in the fluorophore.
Any of these factors can reduce the level of fluorescence achieved from the sample – and in some cases can prevent fluorescence completely.
Benefits of fluorescence spectroscopy
As long as the conditions are right, fluorescence spectroscopy is a fast, easy and accurate way to identify known substances based on their emission spectra.
With high-precision optical filters determining the wavelength of the incident beam to within a very narrow range, the results are reliable and easily repeatable.
This makes for a powerful and convenient method to analyse all kinds of samples, with good confidence in being able to identify their contents.
Contact us today to learn about bespoke fluorescent filters we design.