Advanced Analytical

Techniques
Code: CPS213

Lecture 3
In Lecture 2
• Radiation sources
• Ways of absorbing energy

• Which electrons get excited?

•Types of Electronic Transition

• Absorption Spectrum and its types

In Lecture 3
• Spectra-structure correlation
• Types of λmax shifting

• Factors affecting absorption curve

Spectra-structure correlation
• The absorption of electromagnetic radiation in UV-visible regions
depends primarily upon the number and arrangement of the electrons
in the absorbing molecules.
• It is necessary to define certain terms:
1.Chromophore
2.Auxochrome
Chromophores: (Chrom = color, phore = carrier)
• These are functional groups that capable of absorbing UV and
/or visible light.
• They give color to the substances.
• They have unsaturated bonds such as C=C, -C=O, -N=N, and -
C≡N (π electrons).
Auxochromes:
• These are functional groups which can not give colors to
substances alone but have the ability to increase
the coloring power of chromophores

• They do not absorb radiations longer than 200 nm, but when
attached to a given chromophore, cause a shift to a
longer wavelength with increase in absorption intensity, e.g., -
OH, -NH2.
Examples of Auxochromes:
Chromophores
In Lecture 3
• Spectra-structure correlation
• Types of λmax shifting

• Factors affecting absorption curve

Types of λmax shifting
Types of λmax shifting
Types of λmax shifting
Bathochromic shift (red shift): it is the shift of λmax to a longer
wavelength.

Hypsochromic shift (blue shift): it is the shift of λmax to a shorter

wavelength.

Hyperchromic effect “Up shift”: Enhancement of molecule

absorptivity (or absorption intensity).

Hypochromic effect “Down shift”: Decrease of molecule

absorptivity (or absorption intensity).
Types of λmax shifting
Bathochromic shift (red shift): Due to substitution or solvent effects,
eg:
1- Substitution with -OH or -NH2.
2- Decreasing polarity of the solvent causes red shift in n-π* transition of
the carbonyl compounds.
3- Conjugation of two or more chromophores.
All are causes that increase electron conjugation

Hypsochromic shift (blue shift):

Due to substitution or solvent effects, eg:
1- Removal of conjugation by increasing the polarity of the solvent
All are causes that decrease electron conjugation
Hyperchromic effect “Up shift”: Addition of auxochrome

Hypochromic effect “Down shift”: Addition of group that changes

molecule geometry
In Lecture 3
• Spectra-structure correlation
• Types of λmax shifting

• Factors affecting absorption curve

Factors affecting the absorption curve:
I. pH effect
The spectra of compounds containing acidic or basic groups are
dependent on the pH.
Ex 1:
Phenol and phenolic compounds are good examples.
In acid medium the predominant species is the undissociated form,
while in alkaline medium the predominant species is the phenate
form.
OH O O

+ H+

acid medium alkaline medium

Factors affecting the absorption curve:
I. pH effect
Ex 1:
Why?????
The spectrum in alkaline medium exhibits bathochromic shift with
hyperchromic effect.

This shift is due to the participation of the pair of electrons in

resonance with the π -electrons of the benzene ring, thus increasing
the delocalization of the π -electrons.
Factors affecting the absorption curve:
I. pH effect
Ex 2:
The UV spectrum of Aniline in acid medium shows hypsochromic
shift with hypochromic effect.

This blue shift is due to the protonation of the amino group, hence
the pair of electrons is no longer available and the spectrum in this
case is similar to that of benzene.
+NH 2 NH 2 NH 3

+ H+

- H+
alkaline acid medium
medium
Factors affecting the absorption curve:
I. pH effect
Important note:
When running the UV/VIS absorption spectra of a known
concentration of phenol as function of pH of the medium, all the
spectra intersect at certain wavelength which is called the
isosbestic point at which, the two absorbing species, quinonoid and
benzenoid, have the same absorptivity.

Absorption curves of equimolar

solutions of Entacapone in 0.1 N
NaOH (—) and 0.1 N HCl (- -).
Factors affecting the absorption curve:
II. Effect of Redox Reaction
Oxidation of diphenylamine will convert the benzenoid spectrum to
the quinonoid spectrum of the oxidized form which exhibits a
hyperchromic shift due to increase in conjugation and increase in
the absorbance of light by a compound which appears colored and
exhibits a hyperchromic effect.
H
2 N

Red Ox

H H
N N + 2H+ + 2e

Red Ox

N N + 2H+ + 2e
Factors affecting the absorption curve:
III. Solvent effect
“A good solvent should not absorb UV or visible radiation in the
same region as the substance whose spectrum is being
determined”.
transparent solvent in the UV / visible spectroscopy.

Solvents may affect absorption of the compound by:

1• Solvents can absorb the light and prevent it from reaching the
molecule.

Usually solvents which do not contain conjugated systems are most suitable
for these purposes. The most commonly used solvents are water ethanol
and n-hexane, each of them is transparent in the regions of the ultraviolet
spectrum.
- Choose a solvent that, at the least, will not interfere by absorbing
much of the light you want to strike your analyte molecules.
- Remember that the solvent is usually more than the analyte.

Solvent UV Cutoff
Factors affecting the absorption curve:
III. Solvent effect
Solvents may affect absorption of the compound by:
2• Solvents may have a strong effect on the position of max.
wavelength, due to its effect on the energy of transition.

Due to the effect of the polarity of the solvent. Polar solvent may
form hydrogen bonding with some solutes which may affect their
absorption spectrum while non-polar solvent do not form hydrogen
bonds with solutes.
Factors affecting the absorption curve:
III. Solvent effect

Solvents may affect absorption

of the compound by:

3• Solvents can interact with

molecules and often alter their
absorbance spectra.
Factors affecting the absorption curve:
IV. Dilution effect
• An example of this effect is the change of color of dichromate
solution, upon dilution with water from orange to yellow

• Also in case of weak acids e.g. benzoic acid, the unionized and
ionized forms have different ʎ max, upon dilution the ionized form
increase on the expense of the unionized form and shift in ʎ max
occurs. For measurement dilution must be carried out with acid or
alkali or solution with definite ionic strength.
Factors affecting the absorption curve:
V. Temperature effect
• Changes in temperature may shift ionic equilibria. In addition an
increase in temperature may cause bathochromic effect on some ions
e.g. solution of ferric chloride in HCl on heating changes from
yellow to red. The temperature should be the same for all
measurements.
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