Application of Spectrophotometer

APPLICATION OF
SPECTROPHOTOMETER
Submitted to
Mam Fiza Malik
Submitted by
Qurat ul ain Akhtar
Roll No
BZOF17E086
APPLICATION OF
SPECTROPHOTOMETER:
• There are some major
application of
spectrophotometer.
1. Measurement of concentration
2. Indentification of unknown
molecule
3. Detection of functional group
4. Chemical kinetics
5. Detection of impurities
6. Molecular weight determination
7. Elucidation of structure of
organic compound
TWO MAJOR CATEGORIES OF
SPECTROPHOTOMETER:
There are two major categories on which

basis we utilize it.
Fix wavelength
As a function of wavelength
FIX WAVELENGTH
In the fix wavelength application the wavelength is fixed it is not in
specific range and it is used for quantative purpose.
For example:
measuring the concentration of substance
As function of wavelength
In this the wavelength of light is not specific it is in the specific range
it’s purpose is scanning .
It is used for qualitative purpose
For example: identify unknown molecule in sample.
1. MEASUREMENT OF
CONCENTRATION
Spectrophotometer is used to determine the
concentration of substance in solution.
Prepare the sample
Make series of standard solution of known concentration.
Set spectrophotometer to the maximum light absorption.
Spectrophotometer measures the intensity of light passing

through sample solution in a cuvette and compares it to
intensity of light before it passes through sample.
The advantage of
this representation
is obvious
The height of the

absorption peaks is
directly proportional
to the concentration
of the substance.
CALCULATION OF
CONCENTRATION:
• The calculation of concentration is governed by
the Lambert-Beer Law.
• To calculate the concentration
C=A/e (epsilon)xd
• Where C=The sample concentration in mol
D=Cuvette path length in cm
E=(epsilon)sample specific constant
(describe how much the sample absorb at a given
wavelength)
According to Beer-Lambert
Law:
Absorption Directly
Directly Concentration
proportional
• Graphical representation show:
• Absorption of light in sample is

directly proportional to
concentration.
2- IDENTIFICATION OF
UNKNOWN MOLECULE:
• Each molecule absorb the specific
wavelength of light .
• For example:
• DNA Absorb light 260nm
• Protein Absorb light 280nm
• Thymine Absorb light 260-280nm

• On the basis of absorption of light by
molecule we can identify the unknown
molecule in sample.
• Each molecule in sample can absorb the
specific wavelength of light.
• In this way we can identify the molecule in
sample
Specific Unknow
wavelength of light n
molecule
3:DETECTION OF
FUNCTIONAL GROUP
According to
graphical
representation
Absence of a band at
particular wavelength
regarded as an
evidence for absence
of particular group
4: CHEMICAL KINETICS
• Kinetics of reaction can also be studied using
spectrophotometer
• UV radiation is passed through the reaction cell and the
absorbance changes can be observed.
• Forward reaction:
A+B C+D
reactant product
absorbance absorbance
Reverse reaction:
From spectrophotometer we can observe the direction
reaction either it forward or reverse.
• If the amount reactant increase and absorbance

increase and amount of product is decrease and
absorbance decrease then the reaction is in reverse
reaction.
A+B C+D
reactant product
absorbance absorbance
5: DETECTION OF
IMPURITIES
• UV absorption is one of the best
method for determine of
impurities in organic molecules.
• Additional peaks can be

observed due to impurities in
the sample and it can be
compared with that of standard
raw material.
6:DETERMINATION OF
MOLECULAR WEIGHT:
• The molecular weight of a large variety of amines such as
primary, secondary, tertiary, alkyl, aryl and heterocyclic
amines, have been determined by a study of the
absorption spectra of their picrate and styphnate.
• Molecular weight of compound can be measured
spectrophotometrically by preparing the suitable derivatives
of these compound.
• For example:
• If we want to determine the molecular weight of amine then
it is converted in to amine picrate.
THANK YOU

Application of Spectrophotometer

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Application of Spectrophotometer

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APPLICATION OF

There are two major categories on which

Prepare the sample

Make series of standard solution of known concentration.

Set spectrophotometer to the maximum light absorption.

Spectrophotometer measures the intensity of light passing

The height of the

• Graphical representation show:

• Absorption of light in sample is

• Protein Absorb light 280nm

• Thymine Absorb light 260-280nm

• If the amount reactant increase and absorbance

• Additional peaks can be

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