Ultraviolet-visible spectroscopy or ultraviolet-visible
spectrophotometry refers to absorption spectroscopy or reflectance
spectroscopy in the ultraviolet-visible spectral region.
It analyses many substances on how they absorb wavelengths of light
which can then be used to identify what compound they are make up
of. When a substance absorbs light is appears a colour. The colour
absorbed is the complementary colour of what it appears (it absorbs
every other wavelength than the one it appears) . For example:
chlorophyll- the pigment in green plants appears green since it
absorbs violet and red light and not green which is reflected off the
It works by a light passing through a solution. The higher the
concentration of the solution the more light is absorbed. Light of a
certain wavelength and energy is reflected onto the sample, which
absorbs an amount of energy from the light. The energy of the light
transmitted from the sample is measured, which calculates the
absorbance of the sample. After the light source is a monochromator
which filters the light and selects a specific wavelength by using
either a prism or a diffraction grating. After the monochromator is a
series of lenses, slits, mirrors, and filters that concentrate, increase
spectral purity and direct the light towards the sample containing the
solutions to be tested. A detector calculates the amount of light
being absorbed in the sample and the amount of light that goes
through then helps analyse concentration of the sample.
UV-VIS Spectrometers have either a single or double beam in the
instrument. A single beam will only have a place for one sample to be
run at a time, whereas, a double beam has two beams where an
unknown and reference sample can be run at the same time.
Usually in a UV Spectrometer there are two light sources: one gives out
visible light and the other gives out UV light. Tungsten is usually used as
the visible light and deuterium lamp is usually used as the UV light. A
mirror opposite these light sources directs like from the source to the
monochromator. The monochromator splits the light into wavelengths.
This scans through the sample and the wavelengths- this is done by a
grater which rotates. The wanted wavelength is then split into two partsone part goes through the sample and the other through the reference
cell. Both wavelengths once pasted through each sample is reflected
onto a detector, then this data is sent to the computer where it is
graphed. The instrument has a lid that is shut once the sample and blank
are placed in so that no light from the outside can interfere with the
experiment. A computer controls the whole instrument.
Diagram of a UV-VIS Spectrometer
Sample is dissolved in a solvent
Sample is placed in a test cell
Pure blank= reference cell
The cells are made form a special kind of glass which is able to
transmit UV light
A spectrum is obtained by measuring the absorbance against a
range of frequencies
This spectrum can then be compare to a known spectra for the
substances in the sample.
Simple diagram of how a UV-VIS
Spectrometer works.
The UV-Vis spectra arise because photons in this region of the
electromagnetic spectrum have sufficient energy to make electrons from
low energy levels to higher levels. This can occur in atoms, ions or
molecules. Different substances have different energy levels, the energy
and wavelength of light needed to “jump” the electrons vary. The
spectrum is there to identify the substance. When the sample absorbs
visible light a colour appears. The colour seen is the colour that is
reflected off the sample, all the complementary colours are therefore
The method is used in a quantitative way to determine the
concentrations of an absorbing species in a solution. The relationship
between absorbance and concentration is described in the Beer Lambert
A= εcl
Where A is the measured absorbance, c is concentration in mol-L, l is the
length of the cell in cm and ε is the constant. If the same cell size is used,
the absorbance can be used to measure the concentration of the
analyte. For each species and wavelength, ε is the constant known
(compound used as a reference).
The Visible Spectrum from the
UV Region to the IR Region.
Clinical analysis- measuring bodily fluid concentration such as
blood and urine. Also the sugar and haemoglobin content in
blood. In pathology labs in hospitals.
Determining the amount of coloured dye in plastics
Identifying a metal ions presence in a substance, even if the
metal ion itself is colourless.
Determining the amounts of nutrients, additives and
contaminants in water and foods. In food and drink
manufacturing companies.
In quantitative analysis of DNA and proteins in molecular biology.
• A light source shutter controls the level of light from a lamp that
passes through the sample. The shutter is the only moving part of a
UV-VIS spectrometer. The advantage of this system is the simple
design of the instrument.
• The sample analysis using UV-VIS is a very quick process compared to
other methods of sample detection analysis.
• The UV-VIS technique is non-destructive to the sample and has a
high sensitivity for detecting organic compounds.
• Less likely to suffer interference than other analytical techniques.
• No single lamp emits all the light wavelengths necessary for analysis.
Changing the lamp is a time-consuming process.
• UV-VIS spectrometers need regular calibrations to maintain the accuracy
and precision of the instrument. Choosing what type of material to use as
the calibrator, requires the knowledge of the type of sample that is being
• Stray light can be a problem for UV-VIS spectrometers. This can be caused
by the person using it and trying to detect the sample using too much
wavelength range or by poor instrument design.
The data from the experiment is sent directly to the computer which
controls the instrument. Here it is graphed on a calibration graph and
depending on if it was a sample with a control sample, it will graph then
against each other to show the differences in wavelengths and
absorbance rates. It is interpreted by the colour on the spectrum which
has a dip in the graph or no absorbance, it is that colour. It can also be
assessed by seeing the colours that were absorbed by the sample and
then matching then with their complementary colour which was
reflected. By measuring the absorbance of light in the unknown solution,
you can also find its concentration simply through the graph.
Model of the colours in the UV-Vis
Finding the concentration of an
unknown solution is made easy by
simply graphing the absorbance
rates on a calibration graph.
Spectrometer calibration is a process where a scientific instrument known
as a spectrometer is calibrated to confirm that it is working properly. This
is important, as it ensures that the measurements obtained with the
instrument are accurate.
A UV-VIS Spectrometer is calibrated because the performance of
Spectrometers includes testing the resolution, wavelength accuracy and
stray light affect needs to be performing right to get precise results.
The calibration of UV-VIS Spectrometers are performed with a
combination of liquid filters and solid filters. Liquid and solid-state filters
are calibrated by the filter manufacturers.
Solid-state filters maintain their performance for many years when
handled properly but the performance of liquid filters decreases much
quicker due to slow irreversible chemical reactions, photonic-promoted
chemical reactions, and other reasons.
Wavelength accuracy is achieved through calibrating the peaks of several
wavelengths in the UV, visible, and sometimes the IR spectra.
A UV-VIS Spectrometer is capable of analysing:
• Blood
• Urine
• Food
• Drink
• Dye in plastics
• Metal ions
• Proteins
All of these substances above are capable of being analysed in everyday
life, such as in hospitals and food manufacturing places. They need to be
analysed for medical reasons, food safety reasons, colours of substances
and also for compounds present in substances. It analyses these because
it is able to detect substances which only absorb in the UV region, so it
isn’t seen by the naked eye and could cause harm if not detected.
Very selective- less likely to suffer interference from similarly coloured
Uses light of a specific frequency.
Similar to IR, NMR, GC and HPLC in terms of their analytes; organic
Like IR, NMR, GC, Gravimetric analysis, Volumetric analysis, HPLC and
gravimetric analysis in the substances they sample; liquids and gases,
food and water.
Advantages: Simple to operate (if you are a trained professional) and
readily automated.
Like Gravimetric analysis and volumetric analysis, they are only suited to
be run on larger concentrations.
As a detector for GC and HPLC can readily determine ppm levels of
TLC which needs to be coloured or visible under UV light.
Refer to page 87 of your text books to worked example 7.4 which is an
example of how a UV-VIS calibration graph is interpreted and how it can
simply help you to find the concentration of the unknown sample if you
already have its absorbance rate graphed.
UV-VIS is the oldest form of spectroscopy.
There are two types of UV-VIS Spectrometers: Single-beam UV/VIS
spectrophotometer the light only passes through the sample and
double-beam UV/VIS spectrophotometer where the light passes through
a “beam chopper” which directs the beam through the sample.
Some substances such as the transition metals and some organic dyes
are coloured and can be seen by the human eye which means they
absorbs colours in both the ultra-violet and visible regions. Substances
that are colourless to the human eye mean they only absorb in the ultraviolet region.
The source of light produces white light which contains all wavelengths
and all colours. Different wavelengths mean different colours.
Less than 300nm is in the UV region and isn’t visible to human eyes.
UV-VIS is usually run on solutions since light doesn’t pass through solids.
Glass and plastic test tubes can not be used to hold the sample during
the experiment since they absorb UV light.
• http://www.ehow.com/list_6466475_advantages-disadvantages-uv_visspectrometer.html
• http://answers.yahoo.com/question/index?qid=20061019181021AAeIE5X
• http://en.wikipedia.org/wiki/Ultraviolet%E2%80%93visible_spectroscopy
• Heinemann Chemistry 2 VCE Units 3 & 4
• Ms Shipp-Tink

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