BiT

FACULTY OF CHEMICAL AND FOOD

ENGINEERING
DEPARTMENT : CHEMICAL
ENGINEERING
ANALAYTICAL CHEMISTRY GROUP
ASSIGNEMNT
Group members Id
1. Abirham Tadesse 1200780
2. Ali Kedir 1200869
3. Amare Gossa 1200917
4. Eleni Kefelew 1201509
5. Fanaye Gashaw 1207832
6.Kaleab Tadele 1202084
7. Mahilet Awrares 1202254
8. Nathan Hailu 1202616
9. Yonas Niguse 1203431
10.Zelalem Birhanu 1203493
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INTRODUCTION
 What is chromatography method of analysis ?
 Basic class of chromatograpy
1) planar chromatography
2) column chromatography
 Application of chromatography
 limitation of chromatograpy
What is chromatograpy
chromatograpy is a physical process method
of separation in which the components to be
separated are distributed between two
phases, one which is stationary phase and
other is mobile phase move in a definite
direction.
This method is used to separate and identify
the components of complex mixtures like
organic molecule,hormones, pigments etc.
History
The word chromatograpy is derived from
two greek word-chroma means -'colour'
and graphein means 'to write'.

 It was the russian botanist mikhail tsvet

who invented the first chromatography
technique in 1901.
Principle of Chromatography
Chromatography is based on the principle
where molecules in mixture applied onto the
surface or into the solid, and fluid stationary
phase (stable phase) is separating from each
other while moving with the aid of a mobile
phase.
The factors effective on this separation
process include molecular characteristics
related to adsorption, partition , and affinity or
differences among their molecular weights .
Because of these differences, some
components of the mixture stay longer in
the stationary phase, and they move
slowly in the chromatography system,
while others pass rapidly into the mobile
phase, and leave the system faster.
Three components thus form the basis of the
chromatography technique.
Stationary phase: This phase is always
composed of a “solid” phase or “a layer of a
liquid adsorbed on the surface solid support”.
Mobile phase: This phase is always composed of
“liquid” or a “gaseous component.”
Separated molecules
The type of interaction between the stationary
phase, mobile phase, and substances
contained in the mixture is the basic
component effective on the separation of
molecules from each other.
Terms in chromatography
1) Eluate: Combination of mobile phase and solute exiting the column.
2) Eluent: The mobile phase used to carry out a separation.
3) Elution: The process of passing mobile
phase through the column to transport
solutes down the column.
4) Chromatogram: A plot of detector signal output versus time or
elution volume during the chromatographic process.
5) Chromatograph: A device used to
implement a chromatographic separation.
6) Retention time (RT): is a measure of the
time taken for a solute to pass through a
chromatography column.
7) Analyte : the substance to be separated
during chromatography.
1. Planar chromatography
Planar chromatography is a liquid
chromatography in which the stationary
phase is arranged in the form of a planar or
flat bed and the mobile phase moves by
capillary action.
 There are two main branches of planar
chromatography:
A) Paper chromatography, and
B) Thin-layer chromatography (TLC).
 A. paper chromatography
paper chromatography,
in analytical chemistry,
technique for
separating dissolved
chemical substances
by taking advantage of
their different rates of
migration across sheets
of paper.
principle
principle involved can be partition chromatography
or adsorption chromatography. Partition
chromatography because the substances are
partitioned or distributed between liquid phases .
The two phases are water held in pores of the filter
paper and the other phase is a mobile phase which
passes through the paper. When the mobile phase
moves, the separation of the mixture takes place.
The compounds in the mixture separate themselves
based on the differences in their affinity towards
stationary and mobile phase solvents under the
capillary action of pores in the paper.
Applications
 To study the process of fermentation and ripening.
 To check the purity of pharmaceuticals.
 To inspect cosmetics.
 To detect the contaminants in drinks and foods.
 To examine the reaction mixtures in biochemical
laboratories.
 To determine dopes and drugs in humans and animals.
limitations
Paper chromatography cannot handle
large amounts of sample.
Paper chromatography is ineffective in
quantitative analysis.
Paper chromatography cannot separate
complex mixtures.
Less Accurate than HPLC or HPTLC
 B. Thin layer chromatography(TLC)
TLC is a method of
separation or
identification of a
mixture of components
into individual
components by using
finely divided adsorbent
solid / (liquid) spread
over a glass plate and
liquid as a mobile phase.
Principle
The separation relies on the relative affinity of
compounds towards both the phases. The
compounds in the mobile phase move over the
surface of the stationary phase. The movement
occurs in such a way that the compounds which
have a higher affinity to the stationary phase
move slowly while the other compounds travel
fast. Therefore, the separation of the mixture is
attained. On completion of the separation
process, the individual components from the
mixture appear as spots at respective levels on
the plates. Their character and nature are
Disadvantages
Thin Layer Chromatography plates do not
have longer stationary phase.
When compared to other chromatographic
techniques the length of separation is limited.
The results generated from TLC are difficult
to reproduce.
The detection limit is high and therefore if you
want a lower detection limit, you cannot use
TLC.
It is only a qualitative analysis technique and
not quantitative.
2. Column chromatography
Column chromatography separates
substances based on differential adsorption
of compounds to the adsorbent as the
compounds move through the column at
different rates which allows them to get
separated in fractions.
Column chromatography is a technique
which is used to separate a single chemical
compound from a mixture dissolved in a
fluid.
Principle
When the mobile phase along with the
mixture that needs to be separated is
introduced from the top of the column, the
movement of the individual components
of the mixture is at different rates. The
components with lower adsorption and
affinity to the stationary phase travel faster
when compared to the greater adsorption
and affinity with the stationary phase.
 The components that move fast are removed first whereas
the components that move slowly are eluted out last.
 The rate of the movement of the components is expressed
as:
 Rf = the distance travelled by solute/ the distance travelled
by the solvent
 Rf is the retardation factor.
Applications of Column
chromatography
 This technique is used for the separation
of diastereomers.
 It is used to isolate racemates.
 It is used to separate geometrical isomers.
 Tautomeric mixtures can be separated
from this process.
 To purify homogeneity of various
coloured compounds.
 Disadvantages Of Column Chromatography

 It is a time-consuming process for the

separation of compounds.
 It is expensive as higher quantities of
solvents are required.
 The automated process becomes
complicated and therefore costly.
 It has a low separation power.
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Pharmaceutical sector
To identify and analyze samples for the
presence of trace elements or chemicals.
Separation of compounds based on their
molecular weight and element
composition.
Detects the unknown compounds and
purity of mixture.
In drug development.
Chemical industry
In testing water samples and also checks
air quality.
HPLC and GC are very much used for
detecting various contaminants such as
polychlorinated biphenyl (PCBs) in
pesticides and oils.
In various life sciences applications.
Food Industry
In food spoilage and additive detection
Determining the nutritional quality of food

Forensic Science
In forensic pathology and crime
scene testing like analyzing blood
and hair samples of crime place.
Reference
https://microbenotes.com/
chromatography-principle-types-and-
applications/
https://
chromatography.conferenceseries.com/
events-list/applications-of-
chromatography
https://www.researchgate.net/
publication/
310994699_Fundamentals_and_Techniqu
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