1) The absorption of light by organic compounds involves the promotion of electrons from ground state to excited state molecular orbitals. Sigma electrons undergo σ-σ* transitions at shorter wavelengths while pi and non-bonding electrons undergo π-π* and n-π* transitions at longer wavelengths. 2) Chromophores are functional groups responsible for electronic transitions, imparting color. Auxochromes enhance absorption by chromophores through resonance. Conjugation and pH can shift absorption to longer wavelengths while dilution, solvents, and temperature can affect absorption spectra. 3) Spectrophotometry is widely used for quantitative analysis due to its sensitivity, selectivity, accuracy and ease. Both absorbing and non-absorbing

1. 1
Interaction of Radiation and Matter-Spectrophotometry
The electronic transitions that take place in the visible and ultraviolet regions of the spectrum are due
to the absorption of radiation by specific types of bonds, and functional groups within the molecule.
The wavelength and extent of absorption depend on the precise molecular structure.
The intensity of absorption is dependent on the probability of the transition occurring when the
electronic system and the radiation interact.
H
H
C O
Consider formaldehyde: three types
of molecular orbitals
The wavelength of absorption is a measure of the energy required for the transition.

2. 2
Electronic Excitation
The absorption of light energy by
organic compounds in the visible and
ultraviolet region involves the
promotion of electrons in , , and n-
orbitals from the ground state to higher
energy states. This is also called
energy transition. These higher energy
states are molecular orbitals called
antibonding.
The higher energy transitions ( *) occur a
shorter wavelength and the low energy
transitions (*, n *) occur at longer
wavelength.
Energy

*
*
n




*

*
n

*
n

*
Antibonding
Antibonding
Nonbonding
Bonding
Bonding

3. The outer electrons in an organic molecule may occupy one of three different energy levels:
1- Sigma () electrons (single covalent bond (σ-bond): They are bonding electrons posses the
lowest energy level ( it is the most stable).
2- Pi () electrons: They are bonding electrons of higher energy than sigma electrons.
3- Non-bonding (n) electrons: They are of atomic orbital of hetero atoms (N,O, halogen or S)
which don’t participate in bonding, they usually occupy the highest level of ground state.
In excited state:
 electrons under goes σ-σ* transition [high energy].
-electrons under goes  -* transition, while,
n electrons under goes n- * or n-* transitions.
3

4. Interaction of Radiation and Matter-Spectrophotometry
 Most σ-σ* absorption for individual bonds takes
place below 200 nm in the vacuum UV-region
and compound containing just σ-bonds are
transparent in the UV-VIS region.
 π-π* and n- π* the absorption occurs in the UV-
VIS region.
 π-π* and n- π* transitions involve important
functional groups that are characteristic of many
analyte.
ground
state
excited
state
4
Compounds containing only -electrons are the
saturated hydrocarbons which absorbs <200nm
(i.e.) in the far UV. They are transparent in the near
UV (200-300nm) making them ideal solvents for
other compounds to be studied in this region.

5.  The absorbance of EMR in the UV-VIS regions depends on the structure of organic
molecules.
 Absorbance of EMR by organic molecule is achieved by chromophoric groups
(chromophores) assisted by auxochromes, where the electrons of absorbing molecule
is excited, i.e it undergo transition from the ground state to the excited state.
 CHROMOPHORE: This term was previously used to denote a functional group of some other
structural feature of which gives a color to compound. For example- Nitro group is a
chromophore because its presence in a compound gives yellow color to the compound. But
these days the term chromophore is used in a much broader sense which may be defined as
“any group which exhibit absorption of electromagnetic radiation in a visible or ultra-visible
region “It may or may not impart any color to the compound
Chromophores are unsaturated groups responsible of π-π* and n-π* electronic
transitions and imparts color to the molecules e.g C=C, C=O, N=N, N=O.
AUXOCHROMES: It is a group which itself does not act as a chromophore but when attached to
a chromophore, it shifts the absorption towards longer wavelength along with an increase in the
intensity of absorption.
Auxochromes act by entering into resonance interaction and shift the λmax to longer
5

6. 6
The absorbing groups in a molecule are called chromophores. Amolecule containing a
chromophore is called a chromogen. An auxochrome does not itself absorb radiation,
but, if present in a molecule, it can enhance the absorption by a chromophore

7. Interaction of Radiation and Matter-Spectrophotometry
Bathochromic shift (Red-shift): is the shift of λmax to longer wavelength.
Hypsochromic Shift (blue-shift): is the shift of λmax to shorter wavelength.
Hyperchromic effect: is an increase in absorption intensity (absorptivity).
Hypochromic effect is a decrease in absorption intensity (absorptivity).
7

8. Spectrophotometry
Conjugation of double bonds lowers
the energy required for the transition. In
molecules containing a series of
alternating double bonds (conjugated
systems), the π-electrons are delocalized
and require less energy for excitation, so
that the absorption shifts to longer
wavelengths.
8
1. Conjugation Effect
Generally, extending conjugation leads to red shift

9. 2. Effect of pH
The spectra of compounds containing acidic (phenolic-OH) or basic (-NH2) groups are
dependent on the pH of the medium.
9
1. Phenol
The spectrum of phenol in alkaline
medium exhibits bathochromic shift (red
shift), due to delocalization of electrons
that required lower energy for excitation so
appear at longer wavelength
O
O
OH
un-dissociated form
Acid-medium
dissociated form
Alkaline-medium
2. Aniline
The absorption spectrum of aniline in acid medium
shows hypsochromic shift (blue-shift) and
hypochromic effect.
This blue-shift is due to the protonation of the
amino-group, hence the pair of electrons is no longer
available for the quinonoid conjugated structure
which is formed in alkaline medium.
NH3
NH2
NH2
Acid-medium
Alkaline-medium

10. 2. Effect of pH
When running the UV/VIS absorption spectra of a
know concentration of phenol as a function of pH of
the medium, all the spectra interact at certain
wavelength which is called "The ISOBESTIC Point".
10
ISOBESTIC Point
At this wavelength the two absorbing
species, quinonoid and benzenoid for
phenol, have the same asbsorptivity
(absorbance).
At isosbestic point, absorbance is not pH
dependent.

11. 3. Dilution Effect
An example of this effect is the change of color of dichromate solution upon dilution
with water as given by the following equilibrium.
11
4. Solvent Effect
 The same substance usually has different spectrum in different solvent.
 The spectrum in polar solvent varies than in non-polar solvents. Polar solvent tend to
interact with functional groups present in the molecules by hydrogen-bond and Vander-
Wal-forces.
Cr2O7
2-
+ H2O H2CrO4 2H+
+ 2CrO4
2-
Orange
Ymax= 440 nm
Yellow
Ymax= 390 nm
5. Temperature Effect
Change in temperature may shift the ionic equilibrium, so the temperature must be the
same for all measurements.

12. Spectrophotometry Choice of solvent
12
 The requirements for a solvent to be used in colorimetric or spectrophotometric
determinations are:
1. must dissolve the substance under determination.
2. dose not interact with the solute.
3. must not show significant absorption at the wavelength to be employed in the
determination.
• Water is an excellent solvent, because it is transparent throughout the visible region
and down to a wavelength of a bout 200 nm.
•Aliphatic hydrocarbons, methanol, ethanol and diethyl ether are transparent to
ultraviolet radiation and are frequently employed as solvents for organic compounds.

13. 13
Quantitative Applications
The important characteristics of
spectrophotometric and photometric methods are:
1- Wide applicability.
2- High sensitivity.
3- Moderate to high selectivity.
4- Good accuracy.
5- Ease and convenience.
Application to Absorbing Species
Spectrophotometric determination of organic
compounds containing chromophores is thus
potentially feasible.
A number of inorganic species also absorb. We
have noted that many ions of the transition metals
are colored in solution and can thus be determined
by spectrophotometric measurement. In addition,
a number of other species show characteristic
absorption peaks, including nitrite, nitrate, and
chromate ions, the oxides of nitrogen, the
elemental halogens, and ozone.

14. 14
Quantitative Applications
Many non absorbing analytes can be
determined photometrically by causing them
to react with chromophoric reagents to give
products that absorb strongly in the
ultraviolet and visible regions.
The successful application of these color-
forming reagents usually requires that
their reaction with the analyte be forced to
near completion.
Typical inorganic reagents include the
following: thiocyanate ion for iron(III).
Applications to Nonabsorbing Species :

15. 15
wave Length A
430 0.08
450 0.22
470 0.29
490 0.44
510 0.62
520 0.85
540 0.87
570 0.42
600 0.16
630 0.06

16. • Draw the calibration curve then determine the conc. of sample
1 and 2 Concentration
Of standards
Absorbance A
ug/L *
0 0.00
2 0.13
4 0.25
6 0.38
8 0.52
10 0.64
12 0.77
Sample 1 0.34
Sample 2 0.68

17. • Draw the standard addition curve then determine the conc. of
the unknown sample.
Concentration
Added to sample
Absorbance A
ug/L *
0 0.35
2 0.48
4 0.60
6 0.73
8 0.87
10 0.99