SPECTROSCOPY

• Interaction of Radiation with a sample

• The study of molecular or atomic structure of a

substance by observation of its interaction with
electromagnetic radiation

• QUANTITATIVELY - For determining the amount

of material in a sample

• QUALITATIVELY – For identifying the chemical

structure of a sample
https://www.youtube.com/watch?v=MKFjGIrRKt4
 THE ELECTROMAGNETIC
SPECTRUM
• Most of us are aware of many different ways of
transmitting energy and these phenomena come
together in one physical entity called the
ELECTROMAGNETIC SPECTRUM
• The difference between these sources of radiation is
the amount of energy they radiate.
• The radiation from these and other sources covers a
range of energies
 
 The Electromagnetic Spectrum
Radio waves

Microwave

Infra-red

Visible

Ultraviolet

X-rays

Gamma rays
Long Wavelength Shor
t
Low Energy
High
Low Frequency
High
RADIATION IS TRANSMITTED IN
A WAVEFORM
• LOW ENERGY RADIATION has a LONG
WAVELENGTH

• HIGH ENERGY RADIATION has a SHORT

WAVELENGTH
 Radiation Energy

• The strength of the radiation energy will interect with the

molecules in different ways:
– High energy sources produce breaking of bonds
• X-Ray, γ Rays, …

– Medium energy sources excite electrons

• UV / VISIBLE Spectroscopy

– Low energy sources produce vibrations in chemical bonds

• Infrared Energy

– Very low energy sources produce rotation of the chemical bonds

• Microwaves and Radio waves
 EFFECT OF ENERGY ON A MOLECULE

ELECTROMAGNETIC SPECTRUM
ENERGY
1.2 x105 1.2 x107 12000 310 150 0.12 0.0012
( kJ/mol)

Electronic excitation
e-

FREQUENCY
(Hz) 1020 1018 1016 1014 1012 108

Cosmic γ x Ultra visible Infrared Microwave

Radio
rays rays violet waves
rays
WAVELENGTH
(m) 10-12 10-11 10-9 10-6 10-3 10-1
 VISIBLE SPECTROSCOPY
WHAT IS COLOUR?

Colour is a sensation which occurs when light enters the eye and focuses on
the retina at the back of the eye. The light actually initiates a photochemical
reaction in the nerve cells attached to the retina. These transmit impulses to
the brain and stimulate our sense of colour

RETINA
 
CONES - Give colour and three
types which pick up red, blue BRAIN
and green 

RODS - Give grey/black and = CONES

= RODS
also used for night vision.

All the colours we actually sense are made up of these three colours
together with white and grey and black.
 VISIBLE SPECTROSCOPY
COMPOSITION OF WHITE LIGHT
• Sunlight is white light and covers a wavelength range of 380-750nm. A simple
physics experiment shows that white light is actually a composition of a range
of colours i.e., light of different energies and hence wavelengths.

When white light falls on an

object the colour detected by Red
the eye will depend upon the O range
WHITE
ABSORPTION/REFLECTION Yellow
LIGHT
properties of the material G reen
in the object;  B lue
Indigo
Violet

• If the material completely REFLECTS all light it appears WHITE

• If the material absorbs a constant fraction of the light across the spectrum it
appears GREY.
• If the material completely ABSORBS all the light it appears BLACK
 VISIBLE SPECTROSCOPY
When a sample only absorbs light of a single wavelength the eye sees
COMPLEMENTARY colours.

Wavelength Range Absorbed Colour Absorbed Colour Seen By Eye

380 - 430 Violet Yellow - Green
  430 - 480 Blue Yellow
480 - 490 Green - Blue Orange
 
490 - 500 Blue - Green Red
500 - 560 Green Purple
560 - 580 Yellow - Green Violet
580 - 590 Yellow Blue
590 - 610 Orange Green - Blue
610 - 750 Red Blue - Green

LOW HIGH
 Vibrational Energy Levels
Vibrational levels

rotational levels
S1
Effects of the energy levels
depending on the nature of
the energy received
absorption
Energy

Vibrational levels

rotational levels
S0
Ground state
UV-Vis IR mW
 UV / VISIBLE SPECTROSCOPY

UV Radiation – Wavelength range 220 - 380nm

VISIBLE Radiation – Wavelength range 380 - 780nm

Substances can absorb varying amounts of UV and/or Visible radiation at

particular wavelengths – Coloured compounds absorb energy in both UV
and visible region of the electromagnetic spectrum.

Substances can be liquids or solids and measurements are made with

instruments called SPECTROPHOTOMETERS or SPECTROMETERS.

Modern instruments can be coupled to microscopes which allow solid

samples and very small samples of solids and liquids to be analysed both
qualitatively and quantitatively.
 UV / VISIBLE SPECTROSCOPY - THEORY
INCIDENT LIGHT TRANSMITTED LIGHT
254nm 254nm
SAMPLE
Intensity (I o ) Intensity (I t )

• If a particular wavelength of UV or Visible radiation can be isolated from the source and
passed through a sample which can ABSORB some of the radiation then the
TRANSMITTED light intensity (I t ) will less than the INCIDENT light intensity (I o).

• The amount of light transmitted with respect to the incident light is called
TRANSMITTANCE (T) ie.,

T=
It

Io
• Sample can absorb all or none of the incident light and therefore
• transmittance often quoted as a percentage eg.,
It
%T= X 100
Io
 UV / VISIBLE SPECTROSCOPY - THEORY

ABSORBANCE A = - log10 T
It 2
A = - log10
B
Io
A
A = log10 Io 0
It 220 Wavelength(nm) 380

For of %T = 0 and 100 the corresponding absorbance

values will be 0 and 2 respectively

By plotting Absorbance vs wavelength an ABSORBANCE SPECTRUM is

generated. The absorbance spectra for the same compounds A and B are
shown.

With the advantage that absorbance measurements are usually linear with
Concentration, absorbance spectra are now used
 THE LAWS OF SPECTROPHOTOMETRY

There are two very important basic laws and a third one which is a
combination of the two.

LAMBERTS LAW – ABSORBANCE (A) proportional to the PATHLENGTH (l)

of the absorbing medium.

BEERS LAW - ABSORBANCE (A) proportional to the CONCENTRATION (c)

of the sample.

BEER- LAMBERT LAW - ABSORBANCE (A) proportional to c x l

A  cl
A = Ecl (A is a ratio and therefore has no units)
The constant E is called the MOLAR EXTINCTION COEFFICIENT

Link to “Beer-Lambert law” video

 UV / VISIBLE SPECTROSCOPY - THEORY
UNITS OF THE MOLAR EXTINCTION COEFFICIENT
• CONCENTRATION (c) - Moles litre-1
• PATHLENGTH (l) - cm

A = Ecl Hence E= A
cl
E = 1 ˛

mole litre-1 x cm
E = mole-1 litre x cm -1
But 1 litre = 1000cm3
E = 1000 mole -1 cm3 x cm -1
Hence Units of E = 1000 cm2 mole -1
 UV / VISIBLE SPECTROSCOPY - THEORY

IMPORTANCE OF THE BEER LAMBERT LAW

A = Ecl but if E and l are constant

ABSORBANCE  CONCENTRATION and should be linear relationship

Prepare standards of the analyte to be quantified at known concentrations
and measure absorbance at a specified wavelength.

Prepare calibration curve.

ABSORBANCE AT 300nm
From measuring absorbance of sample x
x
Concentration of analyte in sample
can be obtained from the calibration curve x
x
E can be obtained from the slope of the
calibration curve for a given wavelength () x
CONCENTRATION (moles litre-1 )
 UV / VISIBLE SPECTROSCOPY - THEORY
RULES FOR QUANTITATIVE ANALYSES
x

ABSORBANCE AT 300nm
At high concentrations the calibration curve may deviate
from linearity – Always ensure your concentration of the
sample falls within the linear range – if necessary dilute x
sample x
Absorbance not to exceed 1 to reduce error* x

CHOOSE CORRECT WAVELENGTH x

CONCENTRATION (moles litre-1 )
An analyte may give more than one absorbance maxima
(max) value. A
C max
0.6
B
Many compounds absorb at 220-230nm hence do not use A

Need to choose wavelength more specific

to compound (SELECTIVITY) and if more
than one select one with highest absorbance
as this gives less error – hence use C
0
220 Wavelength (nm) 380
 Let’s play a bit !
A couple of things to take into account…

The intensity of incident light from the light source is always 110.0 photons/sec

You have to calculate Transmittance T= It / Io and Absorbance A=–Log T by

yourself and supply the website with the values you obtain

Now you can play with the virtual spectrophotometer changing the path length,
concentration, calculate the Molar Absorptivity (or Molar Extinction Coefficient)
And run a calibration curve….

Enjoy!!
 Example of calculations for
photometry
Given the following set of data for a compound C:
Can you give the least square equation better fitting the curve?
(Conc=X, Abs=Y)

0 .7
Conc (M) Abs 0 .6 y = 1.0 137x + 0 .1378
R2 = 0 .997
0.1 0.2322 0 .5
0 .4
0.2 0.3456
Abs
0 .3
0.3 0.4532 0 .2
0.4 0.5331 0 .1
0.5 0.6453 0
0 0 .2 conc 0 .4 0 .6

22
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Is the fitting of the curve to the equation
acceptable? How can you tell?

What is the concentration of C when we

obtain an Absorbance of 0.3321?

The concentration is: Abs= 1.0137 * Conc + 0.1378

Abs= 0.3321 – Abs blank= 0.3321- 0.13800 = 0.1941

Conc= Abs – 0.1378 = 0.1941 – 0.1378 = 0.055 M

1.0137 1.0137

23
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 Acknowledgements
• JISC
• HEA
• Centre for Educational Research and Development
• School of natural and applied sciences
• School of Journalism
• SirenFM
• http://tango.freedesktop.org

This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 2.0 UK: England & Wales License