UV-vis absorption spectroscopy, Chemia, NMR

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Introduction
Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below shows a beam
of monochromatic radiation of radiant power
P
0
, directed at a sample solution. Absorption takes
place and the beam of radiation leaving the sample has radiant power
P
.
The amount of radiation absorbed may be measured
in a number of ways:
Transmittance
,
T = P / P
0
% Transmittance
,
%T = 100 T
Absorbance
,
A = log
10
P
0
/ P
A = log
10
1 / T
A = log
10
100 / %T
A = 2 - log
10
%T
The last equation,
A = 2 - log
10
%T
, is worth remembering because it allows you to easily calculate
absorbance from percentage transmittance data.
The relationship between absorbance and transmittance is illustrated in the following diagram:
So, if all the light passes through a solution
without
any absorption, then absorbance is zero, and
percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and
absorption is infinite.
The Beer-Lambert Law
A=
a
bc
Where A is absorbance (no units, since
A = log
10
P
0
/ P
)
a
is the molar absorbtivity with units of L mol
-1
cm
-1
b
is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in
Now let us look at the Beer-Lambert law and explore it's significance. This is important because
people who use the law often don't understand it - even though the equation representing the law is
so straightforward:
centimetres.
c
is the concentration of the compound in solution, expressed in mol L-1
The reason why we prefer to express the law with this equation is because absorbance is directly
proportional to the other parameters, as long as the law is obeyed. We are not going to deal with
deviations from the law.
Let's have a look at a few questions...
Question :
Why do we prefer to express the Beer-Lambert law using absorbance as a measure of
the absorption rather than %T ?
Answer :
To begin, let's think about the equations...
A=
a
bc
%T = 100 P/P
0
= e
-a
bc
Now, suppose we have a solution of copper sulphate (which appears blue because it has an
absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant
power) changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction
every 0.2 cm as shown in the diagram below.
The Law says that the fraction of the light
absorbed by each layer of solution is the same.
For our illustration, we will suppose that this
fraction is 0
.5 for each 0.2 cm "layer" and calculate the following data:
Path length / cm
0 0.2 0.4 0.6
0.8
1.0
%T
100 50 25 12.5 6.25 3.125
Absorbance
0 0.3 0.6 0.9
1.2
1.5
A=
a
bc
tells us that absorbance depends on the total quantity of the absorbing compound in the light path
through the cuvette. If we plot absorbance against concentration, we get a straight line passing
through the origin (0,0).
Note that the Law is not obeyed
at high concentrations. This
deviation from the Law is not
dealt with here.
The linear relationship between concentration and absorbance is both simple and straightforward,
which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the
absorption rather than %T.
Question :
What is the significance of the molar absorbtivity,
a
?
Answer :
To begin we will rearrange the equation A =
a
bc :
a
= A / bc
In words, this relationship can be stated as "
a
is a measure of the amount of light absorbed per unit
concentration".
Molar absorbtivity is a constant for a particular substance, so if the concentration of the solution is
halved so is the absorbance, which is exactly what you would expect.
Question :
What is the molar absorbtivity of Cu
2+
ions in an aqueous solution of CuSO
4
? It is
either 20 or 100,000 L mol
-1
cm
-1
Answer :
I am guessing that you think the higher value is correct, because copper sulphate
solutions you have seen are usually a beautiful bright blue colour. However, the actual molar
absorbtivity value is 20 L mol
-1
cm
-1
! The bright blue colour is seen because the concentration of
the solution is very high.
-carotene is an organic compound found in vegetables and is responsible for the colour of carrots.
It is found at exceedingly low concentrations. You may not be surprised to learn that the molar
absorbtivity of -carotene is 100,000 L mol
-1
cm
-1
!
Review your learning
You should now have a good understanding of the Beer-Lambert Law; the different ways in which
we can report absorption, and how they relate to each other. You should also understand the
importance of
molar absorbtivity
.
Theoretical principles
Introduction
Different molecules absorb radiation of different wavelengths. An absorption spectrum will show a
number of absorption bands corresponding to structural groups within the molecule. For example,
the absorption that is observed in the UV region for the carbonyl group in acetone is of the same
wavelength as the absorption from the carbonyl group in diethyl ketone.
Electronic transitions
The absorption of UV or visible radiation corresponds to the excitation of outer electrons. There are
three types of electronic transition which can be considered.
When an atom or molecule absorbs energy, electrons are promoted from their ground state to an
excited state. In a molecule, the atoms can rotate and vibrate with respect to each other. These
vibrations and rotations also have discrete energy levels, which can be considered as being packed
on top of each electronic level.
Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional
groups (
chromophores
) that contain valence electrons of low excitation energy. The spectrum of a
molecule containing these chromophores is complex. This is because the superposition of rotational
and vibrational transitions on the electronic transitions gives a combination of overlapping lines.
This appears as a continuous absorption band.
Charge - Transfer Absorption
Many inorganic species show charge-transfer absorption and are called
charge-transfer complexes
.
For a complex to demonstrate charge-transfer behaviour, one of its components must have electron
donating properties and another component must be able to accept electrons. Absorption of
radiation then involves the transfer of an electron from the donor to an orbital associated with the
acceptor.
Molar absorbtivities from charge-transfer absorption are large (greater that 10,000 L mol
-1
cm
-1
).
Review your learning
You should now be aware of why molecules absorb radiation in the UV and visible light regions,
and why absorption spectra look the way they do.
Instrumentation
Introduction
Have a look at this schematic diagram of a double-beam UV-Vis. spectrophotometer;
Instruments for measuring the absorption of U.V. or visible radiation are made up of the following
components;
1. Sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers
4. Detector
5. Signal processor and readout
Each of these components will be considered in turn.
Instrumental components
Sources of UV radiation
It is important that the power of the radiation source does not change abruptly over it's wavelength
range.
The electrical excitation of deuterium or hydrogen at low pressure produces a continuous UV
spectrum. The mechanism for this involves formation of an excited molecular species, which breaks
up to give two atomic species and an ultraviolet photon. This can be shown as;
D
2
+ electrical energy D
2
*
D' + D'' +
hv
Both deuterium and hydrogen lamps emit radiation in the range 160 - 375 nm. Quartz windows
must be used in these lamps, and quartz cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.
Sources of visible radiation
The tungsten filament lamp is commonly employed as a source of visible light. This type of lamp is
used in the wavelength range of 350 - 2500 nm. The energy emitted by a tungsten filament lamp is
proportional to the fourth power of the operating voltage. This means that for the energy output to
be stable, the voltage to the lamp must be
very
stable indeed. Electronic voltage regulators or
constant-voltage transformers are used to ensure this stability.
Tungsten/halogen lamps contain a small amount of iodine in a quartz "envelope" which also
contains the tungsten filament. The iodine reacts with gaseous tungsten, formed by sublimation,
producing the volatile compound WI
2
. When molecules of WI
2
hit the filament they decompose,
redepositing tungsten back on the filament. The lifetime of a tungsten/halogen lamp is
approximately double that of an ordinary tungsten filament lamp. Tungsten/halogen lamps are very
efficient, and their output extends well into the ultra-violet. They are used in many modern
spectrophotometers.
Wavelength selector (monochromator)
All monochromators contain the following component parts;
•
An entrance slit
•
A collimating lens
•
A dispersing device (usually a prism or a grating)
•
A focusing lens
•
An exit slit
Polychromatic radiation (radiation of more than one wavelength) enters the monochromator through
the entrance slit. The beam is collimated, and then strikes the dispersing element at an angle. The
beam is split into its component wavelengths by the grating or prism. By moving the dispersing
element or the exit slit, radiation of only a particular wavelength leaves the monochromator through
the exit slit.
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