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ULTRAVIOLET/VISIBLE
ABSORPTION SPECTROSCOPY
Widely used in chemistry. Perhaps the most widely used in Biological
Chemistry. Easy to do. Very easy to do wrong.
MUJEEB KHAN
BASIS SEMINAR
29th April 2009
OUTLINE
™INTRODUCTION
™ABSORPTION MECHANISM
™TERMINOLOGY
™ABSORPTION EFFECTING FACTORS
™INSTRUMENTATION
™APPLICATIONS
Introduction
Electronic Excitation by UV/VIS Spectroscopy:
X-ray:
core electron
excitation
UV: valence
electronic
excitation
IR: molecular
vibrations
Radio waves:
Nuclear spin states
(in a magnetic field)
Introduction
¾Used to study molecules and their electronic transitions.
¾Principle: The energy absorbed corresponds to the amount
necessary to promote an electron from one orbital to another.
¾Commonly used to determine the concentration of an
absorbing species in solution (Quantitative Analysis) using
Beer-Lambert law:
ABSORBANCE
1,0
where:
350 nm
0,8
0,6
0,4
0,2
0,0
A = Absorbance
200 250 300 350 400 450
WAVELENGTH(nm)
I0 = Intensity of the incident light
I = Intensity of the light transmitted through the sample
ε = Molar absorptivity (L mol-1cm-1)
l = sample path length (cm)
c = Concentration of the solution (mol/L)
500
UV/VIS Electronic Transitions
¾Molecules have quantized energy levels.
¾Bonding orbitals are lower in energy than antibonding orbitals.
¾Non-bonding orbitals contains lone pair of electrons.
¾As light absorbs electrons „jumps“ from bonding or nonbonding orbital to the anti-bonding orbitals.
UV/VIS Electronic Transitions
The Important Transitions are:
¾from pi bonding orbitals to pi anti-bonding orbitals.
¾from non-bonding orbitals to pi anti-bonding orbitals.
¾from non-bonding orbitals to sigma anti-bonding orbitals.
¾Groups in a molecule which absorb light are known as chromophores.
Absorption mechanism: A Case study of 1,3-Butadiene
¾Has four π molecular orbitals
¾Bonding orbitals are occupied
¾Anti-bonding orbitals are unoccupied
1,3-Butadiene
¾The interaction of the two double bonds with each other to
produce a delocalised system of pi electrons over all four atoms
is known as conjugation.
Absorption mechanism: A Case study of 1,3-Butadiene
LUMO
π*
Energy
hν
(UV Irradiation)
Four p atomic
orbitals
HOMO
Ground state electronic
configuration
π
„Excited“ state electronic
configuration
Terminology: Chromophore
¾Chromophore: A covalently unsaturated group responsible
for electronic absorption. or Any group of atoms that absorbs
light whether or not a color is thereby produced. e.g. C=C, C=O,
NO2 etc.
¾A compound containing chromophore is called chromogen.
¾There are two types of chromophore:
I. Independent chromophore: single chromophore is
sufficient to import color to the compound e.g. Azo group
II. Dependent chromophore: When more than one
chromophore is required to produce color. e.g. acetone
having one ketone group is colorless where as diacetyl
having two ketone group is yellow.
Terminology: Auxochrome
¾Auxochrome: A saturated group with non-bonding electron when attached
to chromophore alters both wavelengths as well as intensity of absorption.
e.g. OH, NH2, NHR etc.
¾Bathochromic shift: (Red shift) shift of lambda max (λmax) to
longer side or less energy is called bathochromic shift or read shift.
This is due to substitution or solvent effect.
Hypsochromic
¾Hyperchromic effect: an increase in absorption intensity
¾Hypochromic effect: a decrease in absorption intensity
200
Bathochromic
Hypochromic
¾Hypsochromic shift: (Blue shift) shift of lambda max
(λmax) to shorter side and higher energy is called
hypsochromic or blue shift. e.g solvent effect.
Hyperchromic
¾Bathochromic group: The group which deepens the color of chromophore
is called bathochromic group. e.g. Primary, secondary and tertiary amino
groups.
nm
800
Solvent Effects
¾Different compounds may have very different absorption maxima and
absorbances.
¾Intensely absorbing compounds must be examined in dilute solution, so
that significant light energy is received by the detector, and this requires the
use of completely transparent (non-absorbing) solvents.
¾Typical solvents are water, ethanol, hexane and cyclohexane.
¾Solvents having double or triple bonds, or heavy atoms (e.g. S, Br & I)
are generally avoided.
¾ Because the absorbance of a sample will be proportional to its molar
concentration in the sample cuvette, a corrected absorption value known as
the molar absorptivity is used when comparing the spectra of different
compounds.
Solvent Effects
UV ABSORPTION
SPECTRA of 1,2,4-Triazine
Instrumentation
Spectrometric instruments have a common set of general
features. often, one technique is distinguished from
another by differences in these features. Some specific
features for the UV/VIS Experiment.
Light Sources: Deuterium lamp, W Filament (halogen
lamp) and Xe arc lamp.
Wavelength Selectors: Filters and Monochromators.
Sample Container: Fused silica, quartz and glass.
Detectors: Phototube, PMT, photodiode, photodiode
array, and CCD array.
Instrumentation
Beckman DU640 UV/Vis spectrophotometer.
Instrumentation
Software
Source: Deuterium Lamp
Source: Tungsten Filament
Source: Xenon Lamp
Tube filled with Xe (or sometime a mixture of Hg and Xe), invented in
1940, commercialized in 1961 by Osram. Pass a low voltage DC current
to excite Xe. The broad spectral output closely resembles a natural day
light, and is often used in projection system (e.g. 15 kW IMAX system)
Optical Filters
¾Filters can absorb light with dye molecules incorporated into the glass or gel
¾They can also pass or reject bands of light becouse of interference effects with
multiple layers of materials.
¾They can select a rather narrow region of light to allow through to a detector
¾They are useful when a specific, known band of radiation needs to be monitored.
Monochromator
¾A monochromator disperses the light in order to select a narrow
bandwidth.
¾Both gratings and prisms can be used for this dispersion.
¾Numerous instrumental designs are available to account for various
optical requirements
Applications
UV/VIS spectroscopy is routinely used for the quantitative
determination of analytes solutions in transition metal ions and
highly conjugated organic compounds.
¾Solutions of transition metal ions can be coloured (i.e.,
absorb visible light) because d electrons within the metal
atoms can be excited from one electronic state to another.
¾While charge transfer complexes also give rise to
colors, the colors are often too intense to be used for
quantitative measurement.
¾The absorbance of a solution is directly proportional to
the concentration of the absorbing species in the solution
and the path length. Thus, for a fixed path length,
UV/VIS spectroscopy can be used to determine the
concentration of the absorber in a solution.
Linl for the video
¾http://www.youtube.com/watch?v=O39avevqndU