3. Scattering & Absorption
• When light impinges on solutions
and crystals two distinct processes
occur:occur:
1. Light scattering and
2. Light absorption
Based on these developed fundamental techniques for
characterization and analysis of molecules
4. Absorption & Fluorescence
• Absorption in UV regions is specially valuable
for molecular structure elucidation.
• In some molecules process of absorption is
followed by emission of light of a differentfollowed by emission of light of a different
wavelength called FLUORESCENCE.
• Fluorescence assists in characterization and
analysis of biologically significant molecules.
• Other techniques used: NMR (sec, ter structure)
and MS (mol. Wt.).
5. • Composed of a continuum of waves with
different properties.
• Several regions are of importance in
biochemical studies:
Electromagnetic Spectrum
biochemical studies:
1. X-ray- For x-ray crystallography up to 7nm.
2. Ultraviolet: 180-340nm.
3. Visible: 340-800nm.
4. InfraRed:1000 to 100000nm and
5. Radio waves: NMR 106 to 1010 nm.
8. Light wave or Particle?
Wavelength
λ=λ=λ=λ= C////ννννC νννν
9. As particle
Light also behaves as though it were
composed of energetic particles. The
amount of energy associated with these
particles or photons is given by
Where h is Plank’s constant
E=hνννν
10. Rayleigh Scattering
• Photon of specified energy interacts with a
molecule either
1. Gets scattered
2. Transfers energy producing excited state.
Rayleigh scattering occurs when a photon
collides with a molecule and is diffracted
or scattered with unchanged frequency.
• Used in techniques like x-ray diffraction,
Electron microscopy, laser/neutron
scattering etc.
11. Absorption
• Transfer of energy from a photon to a
molecule.
• Electrons promoted from ground state to
excited state.—electronic transition.excited state.—electronic transition.
• UV and visible energy sufficient for
electronic transition basis for
spectroscopy.
12.
13. Electronic Spectroscopy
• Ultraviolet (UV) and visible (VIS)
spectroscopy.
• Earliest method of molecular
spectroscopy.spectroscopy.
• A phenomenon of interaction of
molecules with ultraviolet and
visible lights.
14. UV & VIS Spectroscopy
• In structure determination : UV-VIS
spectroscopy is used to detect the
presence of chromophores like
1. Dienes,
2. Aromatics,
3. Polyenes, and
4. Conjugated ketones, etc.
15. Terms: UV absorptions
1. Chromophores: functional groups that
cause electronic transitions.
2. Auxochromes: substituents with unshared pair
e's like OH, NH, SH ..., when attached to π-
chromophore they generally move the absorptionchromophore they generally move the absorption
max. to longer λ.
3. Bathochromic shift/ red shift. shift to longer λ.
4. Hysochromic shift/ blue shift.: shift to shorter λ.
5. Hyperchromism: increase in ε of a band.
6. Hypochromism: decrease in ε of a band.
16.
17. Why should we learn?
Organic molecules have chromophores that
absorb UV.
UV absorbance is 1000 x easier to detect per
mole than NMR.mole than NMR.
Still used in following reactions where the
chromophore changes.
Useful because timescale is so fast, and
sensitivity so high.
18. Uses for UV
Knowing UV can help you know when to
be skeptical of quantitative results.
Assessing purity of a major peak in
HPLC. Sensitivity makes HPLC sensitive.HPLC. Sensitivity makes HPLC sensitive.
One of the best ways for identifying the
presence of acidic/basic groups, due to big
shifts in λ for a chromophore containing a
phenol, carboxylic acid, etc.
20. Components
1. Light source
2. Monochromator
3. Sample Chamber3. Sample Chamber
4. Detector: Photo multiplier tube
5. Printers & Recorders : mostly
is a computer now a days.
21. 1) Light Source
• Hydrogen/Deuterium Lamps－a truly
continuous spectrum in the ultraviolet
region is produced by electrical
excitation of deuterium at lowexcitation of deuterium at low
pressure. (160nm~375nm).
• Tungsten Filament Lamps－the most
common source of visible and near
infrared radiation (340nm~800nm)..
22. 2) Monochromator
• Prism or Diffraction grating used.
• Used as a filter: the monochromator
will select a narrow portion of the
spectrum (the bandpass) of a given
sourcesource
• Used in analysis: the monochromator
will sequentially select for the
detector to record the different
components (spectrum) of any source
or sample emitting light.
29. Chamber varieties
Two types:
1. Single beam: hold one cuvette
2. Double beam: hold two: 1 for
reference (solvent) and one forreference (solvent) and one for
sample.
In double beam instruments the sample
spectrum is continuously corrected by
substraction of the reference spectrum
30. 4) Detector: Photovoltaic
Detector
• Measures the intensity of photons by means
of the voltage developed across the
semiconductor layer.semiconductor layer.
• Electrons, ejected by photons from the
semiconductor, are collected by silver layer.
• The potential depends on the number of
photons hitting the detector.
34. Principle of Phototube
Detector
• This detector is a vacuum tube with a
cesium-coated photocathode.
• Photons of sufficiently high energy
hitting the cathode can dislodge electrons,
which are collected at the anode.
• Photon flux is measured by the current
flow in the system.
36. Principle of
Photomultiplier Detector
• The detector consists of a photo-emissive
cathode coupled with a series of electron-
multiplying dynode stages, and usuallymultiplying dynode stages, and usually
called a photomultiplier.
• The primary electrons ejected from the
photo-cathode are accelerated by an
electric field so as to strike a small area
on the first dynode.
37. • The impinging electrons strike with
enough energy to eject 2-5 secondary
electrons, which are accelerated to the
Principle of
Photomultiplier Detector
electrons, which are accelerated to the
second dynode to eject still more electrons.
• A photomultiplier may have 9 to 16 stages,
and overall gain of 106~109 electrons per
incident photon.
39. Single and Double
Beam Spectrometer
• Single-Beam: There is only one light
beam or optical path from the source
through to the detector.through to the detector.
• Double-Beam: The light from the source,
after passing through the
monochromator, is split into two
separate beams-one for the sample and
the other for the reference.
40. Rotates, to achieve scan
Two photomultiplier inputs,
differential voltage drives amplifier.
41. Double beam in space configuration
Double beam in time configurationDouble beam in time configuration
42. Quantitative Analysis
Beer-Lambert Law
Quantitative measurements in spectrophotometry
are evaluated using this law. Absorbance
A = -log I/I = εεεεLCA = -log I/Io= εεεεLC
ε = molar absorptivity (L mol-1 cm-1)
L= Path length of the sample
C=Concentration of the compound in
solution, expressed in mol L-1
45. Limits to Beer’s Law
• Chemical Deviations
－absorbing undergo association,
dissociation or reaction with the
solventsolvent
• Instrumental Deviations
－non-monochromatic radiation
－stray light
