Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate

1. CHROMATOGRAPHY
BY
OMKAR ABADHESH
MISHRA
MSC ( BIOTECHNOLOGY)

2. INTRODUCTION
• The substances in a mixture are not chemically
combined, so therefore they can be separated through
some physical process.
• chromatography, technique for separating the
components, or solutes, of a mixture on the basis of the
relative amounts of each solute distributed between a
moving fluid stream, called the mobile phase, and a
contiguous stationary phase.
• The mobile phase may be either a liquid or a gas, while
the stationary phase is either a solid or a liquid.
• Chromatography is the ability to separate molecules using
partitioning characteristics of molecule to remain in a
stationary phase versus a mobile phase.
• Once a molecule is separated from the mixture, it can be
isolated and quantified

3. HISTORY
• Mikhail Tswett, Russian, 1872-1919 Botanist In
1906 Tswett used to chromatography to
separate plant pigments
 He called the new technique chromatography
because the result of the analysis was 'written in
color' along the length of the adsorbent column
• Chroma means “color” and graphein means to
“write”
• Chromatography has application in every
branch of the physical and biological sciences
• 12 Nobel prizes were awarded between 1937
and 1972 alone for work in which
chromatography played a vital role

4. DIFFERENT CHROMATOGRAPHIC TECHNIQUES

5. 1.THIN LAYER CHROMATOGRAPHY
 TLC is a method for identifying substances and testing
the purity of compounds.
 TLC is a useful technique because it is relatively quick
and requires small quantities of material.
 Separations in TLC involve distributing a mixture of two or
more substances between a stationary phase and a
mobile phase.
 The stationary phase: is a thin layer of adsorbent (usually
silica gel or alumina) coated on a plate.
 The mobile phase: is a developing liquid which travels up
the stationary phase, carrying the samples with it.
 Components of the samples will separate on the
stationary phase according to how much they adsorb on
the stationary phase versus how much they dissolve in
the mobile phase

7. 2.PAPER CHROMATOGRAPHY
 A method of partition chromatography using
filter paper strips as carrier or inert support.
 The factor governing separation of mixtures of
solutes on filter paper is the partition between
two immiscible phases.
 One is usually water adsorbed on cellulose
fibres in the paper (stationary phase).
 The second is the organic solvent flows past the
sample on the paper (stationary phase).
 Paper

8.  A method of partition chromatography using
filter paper strips as carrier or inert support.
 The factor governing separation of mixtures of
solutes on filter paper is the partition between
two immiscible phases.
 One is usually water adsorbed on cellulose
fibres in the paper (stationary phase).
 The second is the organic solvent flows past the
sample on the paper (stationary phase).
 Partition occurs between the mobile phase and
the stationary aqueous phase bound by the
cellulose.
 The isolation depends on partition coefficient of
the solute.

10. 3.COLUMN CHROMATOGRAPHY
 Stationary phase is held in a narrow tube through
which the mobile phase is forced under pressure or
under the effect of gravity.
 Adsorbents: The most common are Alumina & Silica
gel in which the interactions with solute molecules is
due to OH groups present on their surface.
 More polar molecules are adsorbed more strongly &
thus, will elute more slowly Strength of adsorption of
polar groups (solutes) on polar support is in the
following order:
-C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2
< -OH < - COOH Olefins < Ethers < Esters <
Lactones <Aldehydes < Amines < Phenols < Acids.

12. PARTITION CHROMATOGRAPHY
 In partition chromatography a solid support with
a high surface area such as crushed firebrick or
keiselguhr is coated with a high boiling liquid
which acts as the stationary phase. Separation
occurs because of the differences in solubility
for the analytes in the stationary and mobile
phases.
 The partition coefficient is defined as:
 K = Concn. in stationary phase /Concn. in
mobile phase

14. TYPES PARTITION CHROMATOGRAPHY
 There are two types of partition chromatography
normal phase and reversed phase, they are
defined by the relative polarities of the mobile
and stationary phases
 For this reason, the use of silica (a polar
molecule) as the stationary phase (as in
adsorption chromatography) is also considered
to be a normal phase separation method.
 Because of its versatility and wide range of
applicability, reversed-phased chromatography
is the most frequently used hplc method.

15. GEL PERMEATION CHROMATOGRAPHY (GPC)
 Gel permeation chromatography is atype of
size exclusion chromatography that separates
analytes on the basis of size.
 This type is also known as:
1. Size Exclusion Chromatography (SEC)
2. Molecular Exclusion Chromatography (MEC)
3. Molecular Sieve Chromatography (MSC)
4. Gel Filtration Chromatography (GFC)
5. Gel Chromatography

16. STATIONARY PHASE
 Porous polymeric matrix: formed of spongy particles,
with pores completely filled with the liquid mobile
phase (gel). The gels (polymers) consist of open,
three-dimensional networks formed by cross-linking
of long polymeric chains.
 The pore size varies with the degree of cross-linking.
The diameter of the pores is critical as separation is
based on that molecules above certain size are totally
excluded from the pores because they can not enter
the gel.
 The interior of the pores is reached, partially or
wholly, by smaller molecules.

18. MOBILE PHASE
 Mobile phase Mobile phase is a liquid as
water or dilute acohol
 Separation mechanism Based on difference
between the solutes molecular weights.
Molecules will distribute themselves outside
& inside the pores according to their size.
 Larger are excluded, medium sized enter
half-way & smallest permeate all the way.

19. APPLICATIONS
 Determination of M. wt. of peptides, proteins &
polysaccharides. Desalting of colloids e.g. desalting
of albumin prepared with 2% (NH4)2SO4.
 Separation of mixture of mono- & polysaccharides.
 Separation of amino acids from peptides & proteins.
 Separation of proteins of different molecular weights.
 Separation of mucopolysaccharides & soluble RNA.
 Separation of myoglobin & haemoglobin.
 Separation of alkaloids & purification of enzymes

20.  McMurry, John (2011). Organic chemistry: with biological applications (2nd ed.).
Belmont, CA: Brooks/Cole. p. 395. ISBN 9780495391470.
 ^ Hostettmann, K; Marston, A; Hostettmann, M (1998). Preparative
Chromatography Techniques Applications in Natural Product Isolation (Second
ed.). Berlin, Heidelberg: Springer Berlin Heidelberg.
p. 50. ISBN 9783662036310.
 ^ "chromatography". Online Etymology Dictionary.
 ^ Ettre LS, Zlatkis A, eds. (26 August 2011). 75 Years of Chromatography: A
Historical Dialogue. Elsevier. ISBN 978-0-08-085817-3.
 ^ Ettre, L. S.; Sakodynskii, K. I. (March 1993). "M. S. Tswett and the discovery of
chromatography II: Completion of the development of chromatography (1903–
1910)". Chromatographia. 35 (5–6): 329–338. doi:10.1007/BF02277520.
 ^ "The Nobel Prize in Chemistry 1952". nobelprize.org. Retrieved 25
August 2016.
 ^ "The Importance of Laboratory Testing for Cannabis Products". 25 October
2018. Archived from the original on 14 November 2018. Retrieved 14
November 2018.
 ^ Ettre, L. S. (1993). "Nomenclature for chromatography (IUPAC
Recommendations 1993)". Pure and Applied Chemistry. 65 (4): 819–
872. doi:10.1351/pac199365040819.
BIBLIOGRAPHY

21. THANK YOU
OMKAR ABADHESH MISHRA
MSC ( BIOTECHNOLOGY)