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  Section: Practical Skills in Chemistry » Instrumental techniques
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Fluorescence spectrophotometry

Instrumental techniques
  Basic spectroscopy
    Introduction to spectroscopy
    UV Ivisible spectrophotometry
    Fluorescence spectrophotometry
    Phosphorescence and luminescence
    Atomic spectroscopy
  Atomic spectroscopy
    Atomic Absorption Spectroscopy
    Atomic Emission Spectroscopy
    Inductively coupled plasma
    Decomposition techniques for solid inorganic samples
  Infrared spectroscopy
  Nuclear magnetic resonance spectrometry
    1H-NMR spectra
    13C-NMR spectra
  Mass spectrometry
    Interfacing mass spectrometry
  Chromatography ~ introduction
    The chromatogram
  Gas and liquid chromatography
    Gas chromatography
    Liquid chromatography
    High-performance liquid chromatography
    Interpreting chromatograms
    Optimizing chromatographic separations
    Quantitative analysis
    The supporting medium
    Capillary electrophoresis
    Capillary zone electrophoresis (CZE)
    Micellar electrokinetic chromatography (MEKC)
  Electroanalytical techniques
    Potentiometry and ion-selective electrodes
    Voltammetric methods
    Oxygen electrodes
    Coulometric methods
    Cyclic voltammetry
  Radioactive isotopes and their uses
    Radioactive decay
    Measuring radioactivity
    Chemical applications for radioactive isotopes
    Working practices when using radioactive isotopes
  Thermal analysis

The principal components of a fluorescence spectrophotometer (fluorimeter) are shown in Fig. 26.4. The instrument contains two monochromators, one to select the excitation wavelength and the other to monitor the light emitted, usually at 90° to the incident beam (though light is actually emitted in all directions). As an example, the wavelengths used to measure the highly fluorescent compound naphthalene are 270 nm (excitation) and 340 nm (emission). Some examples of molecules with intrinsic fluorescence are given in Table 26.1.

Compared with UV/visible spectrophotometry, fluorescence spectroscopy has certain advantages, including:
  • Enhanced sensitivity (up to IOOO-fold),since the emitted light is detected against a background of zero, in contrast to spectrophotometry where small changes in signal are measured against a large 'background' (see eqn [26.5]).
  • Increased specificity, because not one, but two, specific wavelengths are required for a particular compound.
However, there are also certain drawbacks:
  • Not all compounds show intrinsic fluorescence, limiting its application. However, some non-fluorescent compounds may be coupled to fluorescent dyes, or fluorophores (e.g. alcohol ethoxylates may be coupled to naphthoyl chloride).
  • The light emitted can be less than expected owing to quenching, i.e. when substances in the sample (e.g. oxygen) either interfere with energy transfer, or absorb the emitted light (in some instances, the sample molecules may self-quench if they are present at high concentration).
Components of a fluorimeter
Fig. 26.4 Components of a fluorimeter
(fluorescence spectrophotometer). Note that
sample cells for fluorimetry must have clear
sides all round.

Examples of compounds with intrinsic fluorescence
Table 26.1 Examples of compounds with intrinsic fluorescence

The sensitivity of fluorescence has made it invaluable in techniques in which specific chemicals, e.g. polycyclic aromatic hydrocarbons and alcohol ethoxylates, are linked to a fluorescent dye for detection in high-performance liquid chromatography.


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