a short presentation on chromatography.

1. CHROMATOGRAPHY
PRESENTING TO:
NISHAT RAYHANA
LECTURER
UNIVRSITY OF
DEVELOPMENT ALTERNATIVE

2. MEMBERS
1. TOWHIDUR RAHMAN TUHIN 032182016
2. TOFAYAL AHMED 032182017
3. NUSRAT TAIFA 032182018
4. ESHITA ANAM SHANTA 032182019
5. HEERA BARUA 032182020
6. EITIKA ROY 032182006
7. SHAMIMA NASRIN BITHI 032182011

3. INTRODUCTION
 Chromatography is derived from two Greek
words chroma meaning “color” and graphein
meaning “to write “ that is chromatography
means “color writing”
 Chromatography may be defined as the
separation technique based on the differential
distribution of a mixture of substances
between two phases one of which is
percolated through the others.
 According to IUPAC definition it is a technique
of separation, distribution and determination
of individual component from mixture by
using two phases

4. History of chromatography
 Chromatography was first developed and
defined by the Russian botanist M. Tsweet in
1906.
 He produced a colorful separation of plant
pigments using a column of calcium
carbonate.
 The paper and thin layer chromatography
was first discovered in 1938.
 The GLC was first developed in 1952.
 The HPLC was first developed in 1968.

5. PHASES IN CHROMATOGRAPHY
There are two phases in
chromatography. Stationary phase and
Mobile phase.
1) Stationary Phase: It is a kind of fixed
bed of large surface area. The
stationary phase is a solid, a liquid, or
a gel that remains static when a gas
or liquid moves over its surface and
separates out into its various
components.
2) Mobile Phase: The liquid or gas that
flows through a chromatography
system, moving the materials to be
separated at different rates over the
stationary phase.

6. IMPORTANCE OF
CHROMATOGRAPHY
 It is one of the most important
analytic tool.
 It serves as a means for resolution
of mixtures and for the isolation
and partial description of the
separated substances.
 It permits the separation and
partial description of unsuspected
and unknown substances.
 It is an indispensable laboratory
method in all sciences dealing with
chemical substances and their
chemical reactions.

7. Theory of Chromatography
 There are two theories to explain
chromatography.
 They are :
• Plate theory : - older
- described by Martin and Synge.
• Rate Theory: - currently in use
- discussed by Grindings

8. Plate Theory
The plate theory considers that,
The chromatographic column contains a large
number of separate layers, called theoretical
plates.
Separate equilibrations of the sample
between the stationary and mobile phase
occur in these plates.
The solute moves down the column which is
considered as a series of stepwise transfer from
one plate to the next.

9. Rate Theory
 The rate theory considers the
dynamics of the solute particle as it
passes through the void spaces
between the stationary phase
particles in the system.
 It also considers the kinetics of
solutes as it is transferred to and
from the stationary phase.
The chromatogram may be evaluated
qualitatively by determining the Rf
value.
The Rf value can be defined as the
ratio of the distance moved by the
solute and the distance moved by the
solvent.

10. Classification of chromatography
Chromatography may be four types-
1. Adsorption chromatography
2. Partition chromatography
3. Ion exchange chromatography
4. Size exclusion chromatography

11. Adsorption Chromatography
 If the stationary phase is a solid, the process is called
adsorption chromatography.
 In this method, the sample is dissolved in the mobile phase
and by means of electrostatic forces, the stationary surface
absorbs the dissolved solute from the mobile phase.

12. Partition Chromatography
 If the stationary phase is a liquid, the process is
called partition chromatography.
 Here the stationary phase contains an inert solid material,
which support the thin layer of liquid. The retention and
separation of solutes of the mixture depend on their partition
co-efficient.

13. Ion-exchange chromatography
 In this method, the stationary phase consists of a polymeric
matrix which contains some ionic fractional group on its surface.
 Here the mobile phase is always liquid. As the mobile phase
passes over this surface, components of sample are retained by
forming electrostatic chemical bond.

14. Size exclusion chromatography
 It is a chromatographic method in which molecules in
solution are separated by their size and in some cases
molecular weight.
 In this method small molecules leave the mobile phase to
diffuse into the pores and large molecules which will not fit into
the pores remain in the mobile phase and are not retained.

15. PAPER CHROMATOGRAPHY
Paper chromatography was first
introduced by German scientist
Christian Friedrich Schonbein
(1865).
It is a liquid partition
chromatography.
It is an analytic technique for the
separation and identifying
mixtures that are either colored
or can be made colored.
Used for separation of amino
acids, sugars, sugar derivatives
and peptides.

16. PAPER CHROMATOGRAPHY
PHASES
 Stationary Phase: Filter paper
serves here as stationary
phase. Which is made of
cellulose.
 Mobile Phase: Mixture of
immiscible solvents. Mixture
of alcohols such as butyl, iso-
propyl, and H2O commonly
are employed with NH3 or
CH3COOH.

17. DIFFERENT TYPES OR MODES OF
PAPER CHROMATOGRAPHY
1) Ascending Chromatography:
Here the development of paper
occurs due the solvent
movement or travel in upward
direction on the paper.
2) Descending Chromatography:
Here development of paper
occurs due to solvent travel
downwards on the paper.

18. 3) Ascending-descending
Chromatography: Here solvent first
travels upwards and then
downwards on the paper.
4) Circular Chromatography:
Here the solvent travels from
the center(mid point) towards
periphery or circular
chromatography paper.
5) Two dimensional
Chromatography: Here the
chromatogram development
occurs in two directions at right
angles.

19. WHAT NEED IN PAPER
CHROMATOGRAPHY
1. Chromatography paper
(stationary phase)
2. Chromatography
chamber
3. Paper clip
4. Skewer or pencil
5. Water soluble black
marker
6. Scissors
7. Solvents (mobile phase)

20. APPLICATION OF PAPER
CHROMATOGRAPHY
1. Determination of vitamin-C content of orange
juice.
2. Separation and identification of a mixture of free
amino acids.
3. Clarification of identity of powered cascara and
powdered Rhubarb.
4. Identification of barbiturates.
5. Chromatographic separation of Dichlophene and
Hexachlorophene.

21. THIN LAYER CHROMATOGRAPHY
 Thin layer chromatography was first
introduced by Izamailov and
Shraiber in 1938. They both used it
for separation of plant extracts.
 TLC is a Chromatography
technique used to separate
mixtures.
 Thin layer chromatography is
performed on a sheet of glass,
plastic, or aluminum foil, which is
coated with a thin layer of
adsorbent material, usually silica
gel, aluminum oxide, or cellulose
(blotter paper. This layer of
adsorbent is known as the
stationary phase.

22. THIN LAYER CHROMATOGRAPHY
PHASES
Stationary phase:
 Silica is commonly used as stationary
phase.
 The separation of sample mixture will
be dependent on the polarity of
sample.
 Some modified silica is also used in
certain purposes.
Mobile phase:
 The ability of mobile phase to move
up is dependent on the polarity itself
 Volatile organic solvents is preferably
used as mobile phase.

23. MATERIALS
• TLC plate
• Developing container
- chamber/ jar/ glass beaker
• Pencil
• Ruler
• Capillary pipe
• Solvents / mobile phase
- organic solvents
• UV lamp

24. METHOD
Developing container preparation
TLC plate preparation
Spotting TLC plates
Develop the plate
Detection of spot

25. USES OF THIN LAYER
CHROMATOGRAPHY
 Monitor the progress of a reaction
 Identify compounds present in a
given substance
 Determine the purity of a substance

37. Purpose of
chromatography
 Analytical: determine the chemical
composition of a sample.
 it is done normally with smaller amounts of
material and is for establishing the presence
or measuring the relative proportions of
analyte in a mixture.
 Preparative: it’s purpose is to separate the
components of a mixture for later use. It
 purify and collect one or more components
of a sample.

38. Use of chromatography
• Pharmaceutical company
• Hospital
• Environmental agency
• Separating mixtures of compounds
• Identifying unknown compounds etc.

39.  Column chromatography: it is generally used as a
purification technique. It isolates desired compounds from
a mixture.
 Thin layer chromatography (TLC): it is use to separate the
non volatile mixtures. In food and cosmetic industry TLC is
used for separation and identification of colors,
preservatives, sweetening agents.
 Paper chromatography: it is used in the sequencing of
DNA and RNA. It is also used in scientific studies to identify
the unknown organic and inorganic compounds from a
mixture.

40. Gas chromatography: it is use to test the purity of
a particular substance or separating the different
components of a mixture.
HPLC: it is both used in a laboratory and clinical
science. in medical industry it is use in drug analysis.
in labs to determine the amount of drug present in
blood or urine sample.
In forensic lab it is use to determine which fluids
and components are present in human body after
death .