1. Presented by
Dr. A. N. M. Al-Razee M. Sc., Ph. D.
Email: al_razee8@yahoo.co.uk
Deputy Chief Chemist, ACESD, TICI (08 years)
• Previous experience:
12 years in ammonia plant of Urea Fertilizer Factory
2 years in Quality Control officer in Incepta Pharmaceuticals Ltd.
2. Spectroscopy
• Light is a type of energy made of electromagnetic
waves, a blend of magnetism and electricity.
Visible light is only one kind of light, or
electromagnetic radiation.
Spectroscopy = interactions between light & matter
E = hn = hc/l
10. UV-Visible Spectrophotometer
• The wavelength range of UV radiation is 200 nm-
400 nm. There are mainly two types of UV region.
1. 200 nm- 400 nm that is called near ultraviolet
region.
2. Below 200 nm that is called far ultraviolet region.
• The wavelength of visible radiation is 400 nm- 800
nm.
• ΔE= E1- E0 = hv = hc/λ
• ΔE depends upon how tightly the electrons are bound in the bonds and
accordingly, absorption will occur in UV or visible range.
12. Theory of Atomic
Spectroscopy.
Atomic spectroscopy is based
upon
• absorption,
• fluorescence or
• emission of electromagnetic
radiation by atoms or ions
14. Fundamentals
• Absorption and emission of light by compounds is
generally associated with transitions of electrons
between different energy levels
14
E2
E1
E0
DE = hn = hc/l
Emission: Sample (in an excited state)
produces light/looses energy
Absorption: sample takes up energy
Consumes light of appropriate wavelength
http://physics.nist.gov/PhysRefData/ASD/lines_form.html
Atomic spectra: line spectra provide specificity: each element has its own pattern,
as each element has its own electronic configuration
ground state
excited states
DE2
DE1
E2
E1
E0
DE2
DE1
15. Light and the perception of color
Light is a form of electromagnetic radiation. When it
falls on a substance, three things can happen:
• the light can be reflected by the substance
• it can be absorbed by the substance
• certain wavelengths can be absorbed and the
remainder transmitted or reflected Since reflection of
light is of minimal interest in spectrophotometry, we
will ignore it and turn to the absorbance and
transmittance of light
16. Beer-Lambert's law of absorption
• According to Beer’s law, when a beam of monochromatic radiation
passes through a solution of an absorbing substance, absorption of
radiation by the solution is proportional to the concentration of the
solution.
i.e., A C
• According to Lambert’s law, when a beam of monochromatic light
passes through a homogeneous absorbing medium, the absorption
of radiation by the medium is proportional to the length of the
absorbing medium.
i.e., A l
• Mathematically A C×l
•
i.e. A = . ×C×l Where, = molar absorptivity ( liter mol-1,cm-1)
• This is the fundamental equation of spectrometry, and is often
spoken of as the ‘Beer-Lambert’ law.
17. • Again if Io is the intensity of the incident light and It is the
intensity of the transmitted light, then the ratio It / Io is
the fraction of the incident light transmitted by the
medium and is termed the transmittance T.
• i.e., T= It / Io
• Its reciprocal is Io / It , and the absorbance A of the
medium is given by
• A = log Io / It
• There is a relationship between the absorbance A, the
transmittance T, and molar absorption coefficient,
• A = .C.l = log Io / It = - log It / Io = - logT
18. Limitations of Beer-Lamber's law
• The electromagnetic radiation should be
monochromatic
• The light beam should not be scattered
• No association or dissociation reaction
occurs
• The solution should be diluted.
20. 20
1- Sources of light
Continuous Sources
Visible and near IR
radiation
Ultraviolet
radiation
Deuterium Lamp
200-400 nm
Tungsten Lamp
320-2500 nm
2- Filters
Filters permit certain bands of wavelength (bandwidth of ~ 50 nm)
to pass through.
The simplest kind of filter is absorption filters , the most common of
this type of filters is colored glass filters.
They are used in the visible region.
21. 21
• Filters permit certain bands of wavelength (bandwidth of ~ 50 nm) to pass
through.
• The simplest kind of filter is absorption filters , the most common of this
type of filters is colored glass filters.
• They are used in the visible region.
• The colored glass absorbs a broad portion of the spectrum
(complementary color) and transmits other portions (its color).
Disadvantage
• They are not very good wavelength selectors and can’t be used in
instruments utilized in research.
• This is because they allow the passage of a broad bandwidth which
gives a chance for deviations from Beer’s law.
• They absorb a significant fraction of the desired radiation.
i- Filters
22. 22
3- Sample compartment (cells)
For Visible and UV spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy
1 cm 1 cm
Opaque
Face
Transparent
Face
Long pathlength
Short pathlength (b)
1 cm pathlength cuvet
23. Diffraction gratings
• Early diffraction gratings were made of glass through
which the radiation passed and became diffracted these
known as transmission gratings. To achieve the
diffraction of ultraviolet radiation however modern grating
spectrophtometers employ metal reflection gratings with
the radiation is reflected from the surfaces of a series of
parallel grooves. These are often known as echelette.
24. 24
1. The reflection grating
is ruled with a series of closely
spaced, parallel grooves with
repeated distance d.
2. The grating is covered with
Al to make it reflective.
3. When polychromatic light is
reflected from the grating, each
groove behaves as a new point
source of radiation.
4. When adjacent light rays are in
phase, they reinforce one another
(constructive interference).
5. When adjacent light rays are not
in phase, they partially or
completely canceled one another
(destructive interference).
Reflection followed by either constructive
or destructive interferences
Echellette Reflection Grating
1 2
25. Photomultiplier tube
• The photomultiplier tube is a commonly used
detector in UV-Vis spectroscopy.
• There are three main electrodes within a
Photomultiplier.
o 1. Photocathode.
o 2. Dynodes.
o 3. Anode.
26. 26
Photomultiplier tube
It is a very sensitive device in which electrons emitted from the photosensitive
cathode strike a second surface called dynode which is positive with respect to
the original cathode.
Electrons are thus accelerated and can knock out more than one electrons from
the dynode.
If the above process is repeated several times, so more than 106 electrons are
finally collected for each photon striking the first cathode.
photochathode
anode
high voltage
voltage divider network
dynodes
light
electrons
e-
29. Atomic spectra vs molecular spectra:
• Lines Bands
29
(nm)
Typical atomic spectrum Two typical molecular spectra
Y axes: intensity of absorbed light. Under ideal conditions proportional
to analyte concentration (I c; Beer’s law).
e.g. acquired by AAS Acquired by UV-Vis spectroscopy
30. 30
Applications of Ultraviolet/Visible
Molecular Absorption Spectrophotometry
Molecular spectroscopy based upon UV-Vis radiation is used
for identification and estimation of inorganic, organic and
biomedical species.
Molecular UV-Vis absorption spectrophotometry is employed
primarily for quantitative analysis.
UV/Vis spectrophotometry is probably more widely used in
chemical and clinical laboratories throughout the world than any
other single method.
31.
32. 32
Selection of wavelength
Absorbance measurements are always carried out at fixed
wavelength (using monochromatic light). When a wavelength is
chosen for quantitative analysis, three factors should be considered
1. Wavelength should be chosen to give the highest possible sensitivity.
This can be achieved by selecting lmax or in general the wavelengths at
which the absorptivity is relatively high.
λmax
λmax - wavelength where maximum
absorbance occurs
33. 33
By performing the analysis at
such wavelengths, it will be
sure that the lowest sample
concentration can be
measured with fair accuracy.
For example, the lowest
sample concentration (10-5 M)
can be measured with good
accuracy at lmax, while at other
wavelength (l1), it may not be
detected at all.
Absorbance
lmax l1
wavelength
10-2 M
10-3 M
10-4 M
10-5 M
5x10-5 M
35. Question
1. Use Beer’s Law to determine molar
absorptivity and specific absorbtivity.
2. Function of grating
3. Parts of monocromator
4. Basic steps for determination of unknown
conc.
