This is a widely used technique for measuring the absorption of radiation in
the visible and UV regions of the spectrum. A spectrophotometer is an
instrument designed to allow precise measurement at a particular wavelength,
while a colorimeter is a simpler instrument, using filters to measure broader
wavebands (e.g. light in the green, red or blue regions of the visible
Principles of light absorption
Two fundamental principles govern the absorption of light passing through a
- The absorption of light is exponentially related to the number of
molecules of the absorbing solute that are encountered, i.e. the solute
- The absorption of light is exponentially related to the length of the light
path through the absorbing solution, l.
These two principles are combined in the Beer-Lambert relationship, which is
usually expressed in terms of the intensity of the incident light (I0
) and the
emergent light (I
|⇒ Equation [26.5]
where ε is a constant for the absorbing substance at the wavelength the
measurement is made and is termed the absorption coefficient or absorptivity, [C]
is expressed as either mol L−1
or g L−1
and I is given in cm. This
relationship is extremely useful, since most spectrophotometers are constructed
to give a direct measurement of log10 (I0/I)
, termed the absorbance (A)
, of a solution (older texts may use the outdated term optical
density). Note that for substances obeying the Beer-Lambert relationship, A is
linearly related to [C]
. Absorbance at a particular wavelength is often shown as
a subscript, e.g. A550
represents the absorbance at 550 nm. The proportion of
light passing through the solution is known as the transmittance (T),
calculated as the ratio of the emergent and incident light intensities.
Some instruments have two scales:
- An exponential scale from zero to infinity, measuring absorbance.
- A linear scale from 0 to 100, measuring (per cent) transmittance.
For most practical purposes, the Beer-Lambert relationship will apply and
you should use the absorbance scale.
The principal components of a UV/visible spectrophotometer are shown in
Fig. 26.2. High-intensity tungsten bulbs are used as the light source in basic
instruments, capable of operating in the visible region (i.e. 400-700nm).
Deuterium lamps are used for DV spectrophotometry (200-400 nm); these lamps are fitted with quartz envelopes, since glass does not transmit UV
The spectrophotometer is a major improvement over the simple colorimeter
since it uses a diffraction grating to produce a parallel
|Fig.26.2 Components of a UV/visible
beam of monochromatic
light from the (polychromatic) light source. In practice the light emerging from
such a monochromator is not of a single wavelength, but is a narrow band of
wavelengths. This bandwidth is an important characteristic, since it determines
the wavelengths used in absorption measurements - the bandwidth of basic
spectrophotometers is around 5-10 nm while research instruments have
bandwidths of less than 1nm.
Bandwidth is affected by the width of the exit slit (the slit width), since the
bandwidth will be reduced by decreasing the slit width. To obtain accurate
data at a particular wavelength setting, the narrowest possible slit width
should be used. However, decreasing the slit width also reduces the amount
of light reaching the detector, decreasing the signal-to-noise ratio. The extent
to which the slit width can be reduced depends upon the sensitivity and
stability of the detection/amplification system and the presence of stray light.
Most UV/visible spectrophotometers are designed to take cells (cuvettes) with an optical path length of 10mm. Disposable plastic cells are suitable for
routine work in the visible range using aqueous and alcohol-based solvents,
while glass cells must be used for most other organic solvents. Glass cells are
manufactured to more exacting standards, so you should use optically
matched glass cells for accurate work, especially at low absorbances < 0.1),
where any differences in the optical properties of cells for reference and test
samples will be pronounced. Glass and plastic absorb UV light, so quartz
cells must be used at wavelengths below 300nm.
Before taking a measurement, make sure that cells are clean, unscratched,
dry on the outside, filled to the correct level and in the correct position in
their sample holders. Unwanted material can accumulate on the inside faces
of glass/quartz cells, so remove any deposits using acetone on a cotton bud,
or soak overnight in 1 mol L−1
nitric acid. Corrosive and hazardous solutions
must be used in cells with tightly fitting lids or Teflon®
stoppers, to prevent
damage to the instrument and to reduce the risk of accidental spillage.
Basic instruments use photocells similar to those used in simple
colorimeters or photodiode detectors. In many cases, a different photocell
must be used at wavelengths above and below 550-600nm, owing to
differences in the sensitivity of such detectors over the visible waveband. The
detectors used in more sophisticated instruments, give increased sensitivity
and stability when compared with photocells.
Digital displays are increasingly used in preference to needle-type meters,
as they are not prone to parallax errors and misreading of the absorbance
scale. Some digital instruments can be calibrated to give a direct readout of
the concentration of the test substance.
Types of U'Vlvisible spectrophotometer
Basic instruments are single-beam spectrophotometers in which there is only
one light path. The instrument is set to zero absorbance using a blank
solution, which is then replaced by the test solution, to obtain an absorbance
reading. An alternative approach is used in double-beam spectrophotometers,
where the light beam from the monochromator is split into two separate
beams, one beam passing through the test solution and the other through a
reference blank. Absorbance is then measured by an electronic circuit which
compares the outputs from the reference (blank) and sample cells. Double-beam spectrophotometry reduces measurement errors caused by fluctuations
in output from the light source or changes in the sensitivity of the detection
system, since reference and test solutions are measured at the same time
(Box 26.1). Recording spectrophotometers are double-beam instruments,
designed for use with a chart recorder or computer, either by recording the
difference in absorbance between reference and test solutions across a
predetermined waveband to give an absorption spectrum, or by recording the
change in absorbance at a particular wavelength as a function of time (e.g. in
a kinetic determination.
Quantitative spectrophotometric analysis
A single (purified) substance in solution can be quantified using the Beer-
Lambert relationship (eqn [26.5]), provided its absorptivity is known at a
particular wavelength (usually at the absorption maximum for the substance,
since this will give the greatest sensitivity). The molar absorptivity is the
absorbance given by a solution with a concentration of 1 mol L−1
(= 1 kmol m−3
) of the compound in a light path of 1cm. The appropriate
value may be available from tabulated spectral data (e.g. Anon., 1963), or it
can be determined experimentally by measuring the absorbance of known
concentrations of the substance (Box 26.1) and plotting a standard curve.
This should confirm that the relationship is linear over the desired
concentration range and the slope of the line will give the molar absorptivity.