Ultraviolet (UV) Spectroscopy – Use and Analysis

Of all the forms of radiation that go to make up the electromagnetic spectrum UV is

probably the most familiar to the general public (after the radiation associated with visible
light which is, for the most part, taken for granted).
UV radiation is widely known as something to be aware of in hot weather in having a
satisfactory effect of tanning the skin but which also has the capacity to damage skin cells
to the extent that skin cancer is a direct consequence of overexposure to UV radiation. This
damage is associated with the high energy of UV radiation which is directly related to its
high frequency and its low wavelength (see the equations below).
This self guided tutorial is designed to introduce you to UV spectroscopy and give you
enough information to ensure that you understand how you can use the technique as a
quantitative as appose to a qualitative method. The basic knowledge presented here will
help in understanding the problem involved in Experiment 4 of CH199.

E = energy; c = speed of light; λ = wavelength;

υ = frequency; h = Planck’s constant

c=λ υ E = hυ E = (hc)/ λ E ∝ 1/ λ

JDHinks 2002
 Ultraviolet (UV) Spectroscopy – Use and Analysis
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When continuous wave radiation is passed through a When continuous wave radiation passes through a
prism a diffraction pattern is produced (called a transparent material (solid or liquid) some of the
spectrum) made up of all the wavelengths associated radiation might be absorbed by that material.
with the incident radiation.
Spectrum with ‘gaps’ in it

Spectrum

Transparent material that

Diffraction prism
absorbs some radiation
Radiation source

If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light
spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is
passed).
The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the
ground state) to a higher energy state (called the excited state).
The difference between all the spectroscopic techniques is that they use different wavelength radiation that has
different associated energy which can cause different modes of excitation in a molecule.
For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a
molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding
electrons from a low energy orbital to a higher energy orbital.
The energy of the ‘missing’ parts of the spectrum corresponds exactly to the energy difference between the orbitals
involved in the transition. JDHinks 2002
 Ultraviolet (UV) Spectroscopy – Use and Analysis
The bonding orbitals with which you are familiar are the σ -bonding orbitals
σ* typified by simple alkanes. These are low energy (that is, stable).
Unoccupied
Next (in terms of increasing energy) are the π -bonding orbitals present in all
Energy Levels
π * functional groups that contain double and triple bonds (e.g. carbonyl groups and

Increasing energy
alkenes).
Higher energy still are the non-bonding orbitals present on atoms that have lone
pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
n All of the above 3 kinds of orbitals may be occupied in the ground state.

Two other sort of orbitals, called antibonding orbitals, can only be occupied by an
Occupied electron in an excited state (having absorbed UV for instance). These are the π *
π and σ * orbitals (the * denotes antibonding). Although you are not too familiar
Energy with the concept of an antibonding orbital just remember the following – whilst
Levels electron density in a bonding orbital is a stabilising influence it is a destabilising
σ influence (bond weakening) in an antibonding orbital.
Antibonding orbitals are unoccupied in the ground state

A transition of an electron from occupied to an unoccupied energy level can be

UV

caused by UV radiation. Not all transitions are allowed but the definition of which
are and which are not are beyond the scope of this tutorial. For the time being be
aware that commonly seen transitions are π to π * which correctly implies that UV
is useful with compounds containing double bonds.
A schematic of the transition of an electron from π to π * is shown on the left.

JDHinks 2002
 Ultraviolet (UV) Spectroscopy – The Instrumentation
The instrumentation used to run a UV is shown below. It involves two lamps (one for visible light and one for UV
light) and a series of mirrors and prisms as well as an appropriate detector. The spectrometer effectively varies the
wavelength of the light directed through a sample from high wavelength (low energy) to low wavelength (high
energy).
As it does so any chemical dissolved in a sample cell through which the light is passing may undergo electronic
transitions from the ground state to the excited state when the incident radiation energy is exactly the same as the
energy difference between these two states. A recorder is then used to record, on a suitable scale, the absorption of
energy that occurs at each of the wavelengths through which the spectrometer scans.

The recorder assembly

The spectrometer itself – this houses the lamps, mirrors,

prisms and detector. The spectrometer splits the beam of
radiation into two and passes one through a sample and
one through a reference solution (that is always made up
of the solvent in which you have dissolved the sample).
The detector measures the difference between the sample
and reference readings and communicates this to the
recorder.

The samples are dissolved in a solvent which is transparent to UV light and put into sample cells called cuvettes.
The cells themselves also have to be transparent to UV light and are accurately made in all dimensions. They are
normally designed to allow the radiation to pass through the sample over a distance of 1cm.

JDHinks 2002
 Ultraviolet (UV) Spectroscopy – The Output
The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series
of broad humps of varying height. An example is shown below.

*Absorbance has no units

– it is actually the
logarithm of the ratio of

Increasing absorbance *
light intensity incident on
the sample divided by the
Beer Lambert Law light intensity leaving the
sample.

A = ε .c.l

Decreasing wavelength in nm

There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of
a known compound in a solution of unknown concentration. It is not so useful in determining structure although
it has been used in this way in the past.
The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described
above.
A = absorbance; c = concentration in moles l-1 ; l = pathlength in cm ; ε = molar absorptivity (also known as
extinction coefficient) which has units of moles-1 L cm -1 .
JDHinks 2002
 Ultraviolet (UV) Spectroscopy – Analysing the Output
Absorbance
Beer Lambert Handling samples of known concentration
1.0
If you know the structure of your compound X and you
Law wish to acquire UV data you would do the following.
A = ε .c.l Prepare a known concentration solution of your sample.
Run a UV spectrum (typically from 500 down to 220 nm).
0.5 From the spectrum read off the wavelength values for each
of the maxima of the spectra (see left)
Read off the absorbance values of each of the maxima (see
left).
0.0 Then using the known concentration (in moles L-1 ) and the
350 400 450
known pathlength (1 cm) calculate the molar absorptivity
wavelength (nm)
(ε ) for each of the maxima.
Determining concentration of samples with Finally quote the data as follows (for instance for the largest
known molar absorptivity (ε ). peak in the spectrum to the left and assuming a concentration
Having used the calculation in the yellow box to of 0.0001 moles L-1 ).
work out the molar absorptivity of a compound you λ max = 487nm A= 0.75
can now use UV to determine the concentration of ε = 0.75 /(0.001 x 1.0) = 7500 moles-1 L cm -1
compound X in other samples (provided that these
sample only contain pure X).
Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value
of ε . In the case above this is at the peak with the highest wavelength (see above).
Having found the absorbance value and knowing ε and l you can calculate c.
This is the basis of your calculation in Experiment 4 of CH199 and also the principle used in many experiments to
determine the concentration of a known compound in a particular test sample – for instance monitoring of drug
metabolites in the urine of drug takers; monitoring biomolecules produced in the body during particular disease states
JDHinks 2002
 and finally…… the use of UV in CH199: Experiment 4
Absorbance
You are using UV to determine the concentration of carotene in
2.0 a vegetable extract. This is possible because you are being
provided with the molar absorptivity (ε ) of the compound
which has been accurately determined in the past.
Bear in mind that during any exercise where you are trying to
acquire quantitative data your accuracy throughout is essential.
You must prepare solutions with accurately known dilutions to
1.0 ensure that your absorbance reading can be converted
accurately into a concentration using the Beer Lambert Law
(A = ε .c.l)

0.0
350 450 550
wavelength (nm)
The normal settings for the spectrometer are as follows throughout CH199. Always check with a
demonstrator that these are correct – never assume!
Start of scan at 550 nm Absorbance at bottom of graph-paper = 0 Path length = 1 cm
End of scan at 300 nm Absorbance at top of graph-paper = 2
Chart divisions on graph paper (X axis) = 20 nm per cm.
You will need to collect the data for the largest peak (highest absorbance) only and use this to work out the
quantity of carotene in your sample.
JDHinks 2002