01

BEER- LAMBERT LAW

AND
APPLICATIONS

PRESENTED BY :
ROLL NO :
COURSE CODE :
COURSE TITLE : Basic Analytical chemistry (I) Theory
DEGREE PROGRAM : MSc (I) Chemistry
PRESENTED TO : Dr. Muhammad Waseem Mumtaz

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CONTENTS

 Introduction …………………………………………….04
 Lambert’s law ……………………………………...........05
 Beer’s law ……………………………………………...06
 Beer Lambert law …………………………………….....07
 Working curve ……………………………………….....08
 Deviations ……………………………………………....09
 Reason of deviations …………………………………....10
 Applications …………………………………………….14
 References ……………………………………………....20

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 INTRODUCTION
 Combination of two laws
 Lambert in 1758, said that absorbance of sample depend upon the path
length.
 August Beer in 1852, discovered that absorbance of sample depend upon
the concentration.

August Beer Johann Lambert

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Lambert’s law

 Absorbance and path length is directly proportional to each other

A ∝ l
𝐼°
A = ∝ l
𝐼
𝐼°
 = 𝜀
𝐼
A =𝜀l

Fig 01 : Lambert’s law

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 Beer’s Law

 Absorbance and concentration are directly proportional to each other

A ∝ c
𝑰°
A = ∝𝒄
𝑰
𝑰°
 = 𝜺
𝑰
A = 𝜺c

Fig 02 : Transmittance and absorbance

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 Beer-Lambert law

 An relationship among the absorbance, concentration and path length

 A = εcl
 A is the absorbance
 ε is the extinction coefficient
 c is the concentration of species
 l is the path length

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Working curve
• Working curve is
produced by plotting
the absorbance against
concentration.
• From this, determine
the concentration of an
unknown sample by
knowing the absorption

Fig 03: Working curve

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Deviations
 The action of departing from an accepted standard

Types:
 Positive deviation
 Negative deviation

Fig 04 : Graph for deviation

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Reason for deviations

Instrumental deviation:
These are occur due to how the absorbance measurement are made.

Chemical deviations:
These are due to specific chemical species of sample which is being
analyzed.

 Real deviations:
These are due to the limitation of law itself.

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Instrumental Deviations
 This law deviate when
polychromatic light is used.
 If a band is chosen such that
the molar absorbtivity of
analyte at these wavelengths
changes a lot, the absorbance
of the analyte will not follow
beer Lambert law.
 Due to the presence of Stray
Radiation
 Due to mismatched cuvettes.

Fig05: the graph obtain due to change in wavelength

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 Chemical Deviations
 This type of deviation is occur due to
chemical phenomenon involving the
analyte molecules due to association,
dissociation and interaction with the
solvent to produce a product with
different absorption characteristics.
 For Example:
 Phenol red undergoes a resonance
transformation, moving from the
acidic(yellow ) to basic form (red).
 Absorption of sample changes with
the change in pH of the solvent.
Fig 06:graph showing deviation

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Real Deviations

 This law is capable of describing absorption behaviour of solution

containing relatively low amounts of solute dissolved in it(<
10𝑚𝑀)
 When the concentration of the analyte in the solution is high
(>10mM), analyte begin to behave differently due to interaction
with the solvent and other solute molecules and at same time even
due to hydrogen bonding interactions.
Method Deviations
 This type of deviation occur due to personal error.

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APPLICATIONS

In Analytical chemistry

In Bacterial growth kinetics
ELISA
Nucleic acid concentration
In atmosphere

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In Analytical
chemistry
 Separation, quantification
and identification of
matter by
spectrophotometry
 No involvement of pre-
processing
 Example
 Bilirubin in blood
 concentration of various
compound in different
food sample
 Carbonyl group can be
detected
Fig 07: Calibration curve

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In Bacterial growth kinetics
 Grow bacterial culture all over the night and take small amount in
sample cuvette
 Placed the sample cuvette in spectrophotometry

Fig o8 : Bacterial growth kinetics 16

ELISA
 ELISA strands for Enzymes Linked Immunosorbent Assay
 It is commonly used laboratory test to detect the antibodies in blood
 This test is used to detect infection with HIV. It has high sensitivity
 It is a plate based assay designed for detecting and quantifying
substances such as proteins, peptides, hormones

Fig 09 : Principle of ELISA

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Nucleic acid concentration
 By using the spectrophotometry, we quantitatively analyze the
concentration of nucleic acid DNA and RNA
 If any impurity in it, is also show

Fig10 : Nucleic acid spectrum Fig 11 : Spectrum with impurities

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In Atmosphere

 Beer- Lambert law is used to describe the attenuation of solar

radiation as it travels through the atmosphere. In this case scattering
as well as absorption occur.
 The attenuator considered depend on the wavelength range.

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REFERENCES

 https://www.edinst.com/blog/the-beer-lambert-law/
 http://elchem.kaist.ac.kr/vt/chemed/spec/beerslaw.htm
 https://www.bio-rad-antibodies.com/elisa-types-direct-indirect-sandwich-
competition-elisa-formats.html
 https://chem.libretexts.org/Bookshelves/Analytical_Chemistry/Book%3A_
Analytical_Chemistry_2.0_(Harvey)/10_Spectroscopic_Methods/10.2%3
A_Spectroscopy_Based_on_Absorption

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