Chemistry - UV - visible spectroscopy
UV - visible spectroscopy
UV-visible spectroscopy operates on principles similar to those previously
discussed for colorimetry.
In UV-visible spectroscopy, the sample, usually in solution, is placed in
the instrument in a special cell that allows visible and ultraviolet light
to pass through it.
There are usually two light sources in a UV-visible spectrophotometer: a
tungsten lamp for wavelengths in the visible region and a hydrogen lamp for
wavelengths in the ultraviolet region of the electromagnetic spectrum.
Calibration of a UV-visible spectrophotometer also involves determining
the wavelength of light at which the component for which the sample is
being analysed absorbs strongly but other components don't.
This wavelength is then used for a series of standards, i.e. solutions of
known concentration, and their measured absorbances form the basis of a
calibration curve establishing the relationship between absorbance and
A monochromator is used to select the required wavelength. The absorption
of this light by a standard (or the sample being analysed) in the
(photo)cell is determined by comparing the intensity of the light leaving
the cell with that of the light entering the cell. Via a calibration curve,
the measured absorbance of the unknown can be converted to a concentration
During calibration, the absorbance of a reference sample of pure solvent
(a blank) is measured to check for any reflection, scattering or absorption
of light by the cell used to hold the standards or sample for analysis.
The diagram below shows the key components of a UV-visible spectrometer
By exposing a sample in a UV-visible spectrometer to a range of
wavelengths, a characteristic absorption spectrum can be established. The
spectrum shown here indicates that the substance being analysed absorbs
most strongly at wavelengths of 450 nm (_blue_) and 650 nm (_red_). Such
spectra can be useful in identifying substances. The UV spectra of many
organic compounds can be used as chemical fingerprints.
Benzene-based (aromatic) organic compounds absorb strongly in the UV
spectrum because of the delocalised electrons in the benzene ring.
Common uses of UV-visible spectroscopy include analysis of body fluids
such as urine and blood. It is a versatile method of analysis used for
measuring the concentration of metal ions in solution as well as organic
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