Ultraviolet-visible

spectrophotometry
 Ultraviolet-visible
spectrophotometry
• Ultraviolet-visible spectroscopy
or ultraviolet-visible
spectrophotometry (UV-Vis or
UV/Vis) refers to absorption
spectroscopy in the UV-visible
spectral region. This means it uses
light in the visible and adjacent
(near-UV and near-infrared (NIR))
ranges.
• This technique is complementary to
 Applications
• UV/Vis spectroscopy is routinely used
in the quantitative determination of
solutions of transition metal ions
and highly conjugated organic
compounds.
• Solutions of transition metal ions can
be coloured (i.e., absorb visible
light) because d electrons within
the metal atoms can be excited
from one electronic state to
• Organic compounds, especially those
with a high degree of conjugation,
also absorb light in the UV or visible
regions of the electromagnetic
spectrum. The solvents for these
determinations are often water for
water soluble compounds, or
ethanol for organic-soluble
compounds.
• While charge transfer complexes also
give rise to colours, the colours are
often too intense to be used for
quantitative measurement.
 Beer-Lambert law
• states that the absorbance of a solution
is directly proportional to the
concentration of the absorbing
species in the solution and the path
length. Thus, for a fixed path length,
UV/VIS spectroscopy can be used to
determine the concentration of the
absorber in a solution. It is necessary
to know how quickly the absorbance
changes with concentration. This can
be taken from references (tables of
molar extinction coefficients), or more
accurately, determined from a
calibration curve.
• A UV/Vis spectrophotometer may be
used as a detector for HPLC. The
presence of an analyte gives a
response which can be assumed to
be proportional to the
concentration
•
• The method is most often used in a
quantitative way to determine
concentrations of an absorbing
species in solution, using the Beer-
Lambert law:
• where A is the measured absorbance,
I0 is the intensity of the incident
light at a given wavelength, I is the
transmitted intensity, L the
pathlength through the sample,
and c the concentration of the
absorbing species. For each species
and wavelength, ε is a constant
known as the molar absorptivity or
extinction coefficient. This constant
is a fundamental molecular
property in a given solvent, at a
particular temperature and
pressure, and has units of 1 / M *
 UV spectrophotometer
• The most common spectrophotometers are used in the UV and
visible regions of the spectrum, and some of these instruments
also operate into the near-infrared region as well.
• The instrument used in ultraviolet-visible spectroscopy is called a
UV/vis spectrophotometer. It measures the intensity of light
passing through a sample (I), and compares it to the intensity
of light before it passes through the sample (Io). The ratio I / Io
is called the transmittance, and is usually expressed as a
percentage (%T). The absorbance, A, is based on the
transmittance:
• A = − log(%T / 100%) The basic parts of a spectrophotometer are
a light source, a holder for the sample, a diffraction grating or
monochromator to separate the different wavelengths of light,
and a detector. The radiation source is often a Tungsten
filament (300-2500 nm), a deuterium arc lamp which is
continuous over the ultraviolet region (190-400 nm), and more
recently light emitting diodes (LED) and Xenon Arc Lamps for
the visible wavelengths. The detector is typically a photodiode
or a CCD. Photodiodes are used with monochromators, which
filter the light so that only light of a single wavelength reaches
the detector. Diffraction gratings are used with CCDs, which
collects light of different wavelengths on different pixels.
• A = − log(%T / 100%) The basic parts of a
spectrophotometer are a light source, a
holder for the sample, a diffraction grating
or monochromator to separate the
different wavelengths of light, and a
detector. The radiation source is often a
Tungsten filament (300-2500 nm), a
deuterium arc lamp which is continuous
over the ultraviolet region (190-400 nm),
and more recently light emitting diodes
(LED) and Xenon Arc Lamps for the visible
wavelengths. The detector is typically a
photodiode or a CCD. Photodiodes are
used with monochromators, which filter
the light so that only light of a single
wavelength reaches the detector.
Diffraction gratings are used with CCDs,
which collects light of different
wavelengths on different pixels.
   

Diagram of a single-beam UV/vis spectrophotometer.