Lecture III

1) Absorption Spectrum .

2) Chromophores and Auxochromes .

3) Bathochromic shift and Hypsochromic shift .

 1) Absorption spectrum
according to the electronic transition that occur in
each organic molecule, absorption spectrum is
obtained by plotting Absorbance (A) as a function of
wavelength (λ ).
It has characteristic
shape with the λ of
maximum absorbance
(λ max ).
It is characteristic for
each molecule according
to its structure and the
type of transitional
energy

Therefore it is used for identification of a chemical

substance
(qualitative analysis). Also λ max is used for quantitative
measurement, in order to increase sensitivity and to
minimize error of the analytical method.
 2) Chromophores and Auxochromes
Chromophres :
re unsaturated groups responsible for π - π * and n→ π * electron
transitions. e.g. C=C , C=O , N=N and N=O ( 200nm-800nm)

-Auxochromes
Are saturated groups posses unshared electrons, and
does
not absorb in near UV or visible radiations e.g.
3)Bathochromic & Hypsochromic shift
OH,NH2.
-Bathochromic shift (or red hift)
It is the shift of λ max to a longer wavelength due to
substitution with certain functional groups (e.g. –OH and –
NH2), when two or more chromophores are present in
conjugation, change in pH and effect of the medium
- Hypsochromic
(solvent). shift (or blue shift)
It is the shift of λ max to a shorter wavelength due to
removal
 - Hyperchromic effect

an increase in the intensity of absorption usually due to

introduction of an auxochrome

- Hypochromic effect
It involves a decrease in the intensity of absorption

-Auxochromes

when attached to chomophoric molecule, increase both its

wave length and intensity of absorption maximum .

Because auxochrome inters into resonance interaction

with the chromophore , thus increase the extent of
conjugation, shift the absorption maximum to longer
wave length
 Changes in
Absorption
spectrum

Example 1): effect of

conjugation on
absorption spectrum

Increase in
conjugation, increas
absorbance of light to
higher λ ,
bathochromic shift
with hyperchromic
effect.
 Lecture IV

1)Effect of pH on absorption spectrum.

2)Polychromatic and Monochromatic light.

3)Theory of light absorption

Example 2) Effect of pH
The spectra of compounds containing acidic (phenolic-OH)
or
basic (-NH2) groups are dependent on the pH of the
medium. The U.V spectrum of phenol
..
:OH
..
:O - in acid medium, benzenoid
form while in alkaline
-
OH + H
+ medium is the phenate
+
H anion ,quinonoid
The free pair of form
of
electrons of O2 increasing
the elocalization of the π -
O electrons, leading to the
formation of conjugated
system.
So , electrons become more
- energetic and need less energy to
be excited, therefore absorb
Phenol longer λ bathochromic shift ; red
shift) with hyperchromic effect
 Its spectrum
exhibits
bathochromic shift Aniline
and hyperchromic :NH2
+ NH3
effect in alkaline
-
medium due to its OH
+
H
conversion to the
quinonoid species
While in acid
medium (anilinium ) + NH2
lost the free pair
electrons of N
decrease the -

conjugation .
s spectrum in acid medium exhibit hypsochromic shif
and hypochromic effect due to its conversion to
he benzenoid species.
 Polychromatic light

A beam of light containing several wavelengths , e.g. white ligh

Monochromatic light

beam of light containing radiation of only one discrete wavelen

 Lecture VI

1-Isosbestic point
2-Colorimetry .
 Isosbestic point

-At different pH, the spectrum will be shifted to different

λ max
but all spectra intersect at certain λ which is known as
isosbestic point
- At isosbestic point, the same absorbance is given for
the same concentration at different pH,i.e. absorbance is
not pH dependent but concentration dependent
-Thus solution ; its λ max affected by pH , must be buffered
at specific pH or measurements are carried out at the
isosbestic point.
 Colorimetry
When white light passes through a colored
substance, a characteristic portion of the mixed
wavelengths is absorbed.

The remaining light will then assume the

complementary color to the wavelength(s)
absorbed.
Complementary colors are diametrically opposite
each other. Thus, absorption of 420-430 nm light
renders a substance yellow, and absorption of
500-520 nm light makes it red.
 wavelength region, nm color complementary color

400-435 Violet Yellow-green

435-480 Blue Yellow
480-490 Blue-green Orange
490-500 Green-blue Red
500-560 Green Purple
560-580 Yellow-green Violet
580-595 Yellow Blue
595-650 Orange Blue-green
650-750 Red Green-blue

Colored substances appear colored because they selectively

absorbed some of wavelengths of visible light and
transmitted other wavelengths or colors (apparent color),
Red substances absorb the blue- green wavelengths
from the visible region, so the transmitted light appears
red
Blue substances absorb the yellow wavelengths, so the
transmitted light appears blue.
 Requirements for substances to be measured
colorimetricaly:

Substance must be coloured e.g CuSO4, organic dyes,…

2-If the substance to be analysed is colourless, it

must react with certain reagent (known as
chromogen) to produce equivalent coloured
product.

3-If there is no suitable chromogen, the

substance must be converted to a certain
derivative which has a suitable chromogen.
2-If the substance to be analysed is colourless,
it must react with certain reagent (known as
chromogen) to produce equivalent coloured
product.
1.Orthophenanthrolene reacts with ferrous
(Fe2+ )in buffered medium (acidic pH) to
produce intense red color.

2+

N N
2+
3 + Fe Fe
N N

3
3-If the sample is colorless and there is no
suitable chromogen, the substance must be
converted to a certain derivative which can be
react with suitable reagent producing color .

-Esters are first converted to hydroxamic acid

derivative through the reaction with
hydroxylamine. Hydroxamic acid derivative
gives purple color on addition of ferric (Fe3+ )
due to the formation of iron chelate .

O
O
+ H2N – OH
R –C– O Et R –C– NH – OH + Et OH
Hydroxamic acid derivative + Fe3+
then measuring the absorbance at 520nm.
Chromogen is
a compound containing chromophoric group

Requirements for ideal chromogen

1-Should be colorless or easily separated from
the colored product

2-It Should be selective.

3-Its reaction to produce colored product,
should be of known mechanism and proceed
stoichiometrically.
4-The full development of color must be rapid.

5-It must produce only one color of specified λ max .

 Requirements for coloured product

1-Should be of intense color, to increase the sensitivity

2-Should be unaffected by pH or the pH must be

specified and maintained by suitable buffer or the
measurement is carried out at λ of isosbestic

3-Should be stable with time

4.The reaction of its formation, must be rapid and quantitative.

5-The colored product, should obey Beer-lambert’s

law, i.e on plotting A versus C at fixed b, we obtain
straight line passing through the origin.
 Quiz
I)Discuss shortly

-Interaction of a substance with EMR

-Factors affecting absorption spectrum

II) Solve the following problem

1-A 5.00x10-4 M sample solution is measured in a
cell with 1 cm bath length ; its absorbance at
592nm equals 0.446 .
a-What is the molar absorptivity at 582nm.
If a solution of unknown concentration of the
same sample has an absorbance 0.125 at the
same wave length.
b-What is its concentration
2-Calculate the wave legnth in um , and
in Angestron

3-Calculate the frequancy and energy of

this wave length

III) Complete the answer in Exercise

1