Chromatography is a laboratory technique used to separate mixtures into individual components. It works by distributing the components between two phases, usually a stationary phase and a mobile phase. There are several types of chromatography defined by the stationary and mobile phases used, including gas chromatography which uses an inert gas as the mobile phase, and high performance liquid chromatography which uses high pressure to force a liquid mobile phase through a column. Chromatography has many applications in fields like chemistry and biochemistry for analyzing and purifying compounds.

1. CHROMATOGRAPHY
Presented by
Savidha Sam
Msc I zoology
St.Gregorios College, Kottarakkara

2. WHAT IS CHROMATOGRAPHY?
 Chromatography is a laboratory technique for the separation of mixture into their
components.
The word chromatography is derived from two Greek words: chroma means “colour” and
graphein means “to write”.

3. INVENTION OF CHROMATOGRAPHY
Column chromatography was developed by the American Chemist D T Day in 1900.
M S Tswett ,Russian Botanist (referred to as the father of chromatography )is credited for the
development of chromatography.
In 1906,he separated chlorophyll and other pigments in plant extract using
an adsorbent column packed with powered calcium carbonate and
petroleum ether as the solvent.
M S Tswett 1872-1919

4. PRINCIPLE AND DEFINITION
The basic principle behind is the differential distribution of sample components between two mutually
immiscible phases- a stationary phase and a mobile phase.
Stationary phase remains fixed while the mobile phase percolates through the intensities over the surface of
stationary phase.
 Stationary phase : A phase that is fixed in a place either in a column or in a planar surface. The stationary
phase can be solid or liquid.
Mobile phase : A phase that moves over or through the stationary phase, carrying with the analyte
mixture. It is also called the eluting fluid. The mobile phase can be gas or a liquid.

5. In Chromatography the compounds are physically separated by distributing themselves
between two phases
Stationary Phase
Mobile Phase

6. CHROMATOGRAPHY TERMS
Analyte : the substance to be separated during chromatography.
Eluent : solvent that carries the analyte
Elution : motion of the mobile phase through the stationary phase.
Stationary phase : The part of the chromatography system that is fixed in place.
Chromatograph : An instrument that enables a sophisticated separation,
Ex: Gas Chromatography, Liquid Chromatography
 Rf Value = distance travelled by solute
distance travelled by solvent
Rf – Retardation factor. The Rf value has to be between 0 and 1

7. CHROMATOGRAM
 Visual Output of the chromatograph
 A chromatogram is essentially the output of a chromatography run. It is an electronic file or hardcopy containing
the information generated during the chromatography run.

8. DIFFERENT TYPES OF CHROMATOGRAPHY

9. CLASSIFICATION
On the basis of interaction of solute
to the Stationary phase
On the basis of chromatographic
bed shape
Techniques by physical state of
mobile phase

10. Adsorption Chromatography
Partition Chromatography
Ion Exchange Chromatography
Molecular exclusion Chromatography

11. On the basis of chromatographic
bed shape
Column chromatography
TLC (Thin Layer Chromatography)
Paper Chromatography

12. Techniques by physical state of
mobile phase
Gas Chromatography
Liquid Chromatography
Affinity Chromatography
Super critical chromatography

15. CLASSIFICATION OF CHROMATOGRAPHY

16. ADSORPTION CHROMATOGRAPHY
 Adsorption Chromatography is probably one of the oldest type of chromatography around.
It utilizes a mobile liquid or gaseous phase that is absorbed onto the surface of a stationary
solid phase
The Equilibrium between the mobile and stationary phase accounts for the separation for the
separation of different solutes.
Definition

17. COLUMN CHROMATOGRAPHY
Column Chromatography is one of the most useful methods for the separation and
purification of both solids and liquids.
It is a Liquid – Solid chromatography. Here Liquid is mobile phase and solid is stationary
phase.
Principle
• Adsorption
• Mixture of components dissolved in the mobile
phase is introduced into the column. Components
moves depending upon their relative affinities.

18. Fig: Column chromatography

19. Column Chromatography is a preparative technique used to purify
compounds depending on their polarity or hydrophobicity.
 In column chromatography, a mixture of molecules is separated based on
their differentials partitioning between a mobile phase and a stationary
phase
Column chromatography is suitable for the physical preparation of gram
quantities of material. A solvent act as the mobile phase while finely
divided solid surface act as the stationary phase.
Usually a glass tube with diameter from 1cm to 10 cm and a height of 20
cm to 50 cm with a tap at the bottom is used for this purpose.

20. Column Chromatography Procedures
• Place a layer of mineral wool at the bottom of the column
and then fill the column with silica powder(absorbent –that
may be alumina ,silica,calcium carbonate etc); that’s the
stationary phase.
• Saturate the silica powder with solvent; this is your mobile
phase.
• Pour your sample mixture into the top of the column.
• Open the tap at the bottom of the column whilst continuously
pouring solvent into the top of the column. Allow the solvent
to carry the sample through the silica powder and collect the
effluent that flows out of the bottom.

21. TYPES OF COLUMN CHROMATOGRAPHY
Based on the difference of the flow rates of the mobile phase; column
chromatography can be several type
Gravity Column Chromatography
The mobile phase moves through the stationary phase by the influence of
gravitational force.
Flash Column Chromatography
The mobile phase is pushed by stream of air or nitrogen using special valves
(adaptor)

23. ION EXCHANGE CHROMATOGRAPHY

24. ION EXCHANGE CHROMATOGRAPHY
Ion exchange chromatography (or ion chromatography) is a process that allows the
separation of ions and polar molecules based on their affinity to ion exchangers.
It is a technique for separation of charged macromolecules such as
protiens,nucleotides,amino acids and sugars based on their affinity of the ion exchanger
Principle
It is based on the reversible electrostatic interaction of ions with the separation matrix. ie,In
the separation occurs by reversible exchange of ions between the ions present in the solution
and those present in the ion exchange resin.

25. • Electrostatic force is the force of attraction or repulsion between two charged particles. It's
also called Coulomb's force

26. • In this process two types of exchangers i.e., cationic and anionic exchangers can be
used.
• 1. Cationic exchangers possess negatively charged group, and these will attract
positively charged cations. These exchangers are also called “Acidic ion exchange”
materials, because their negative charges result from the ionization of acidic group.
• 2. Anionic exchangers have positively charged groups that will attract negatively
charged anions. These are also called “Basic ion exchange” materials

27. TYPES OF ION EXCHANGE CHROMATOGRAPHY
• Cation exchange chromatography
positively charged molecule are attached to a negatively charged solid support.
• Anion exchange chromatography
Negatively charged molecule is attaracted to a positive charged solid support

29. INSTRUMENTATION OF ION EXCHANGE CHROMATOGRAPHY

30. APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY
An important use of ion-exchange chromatography is in the routine analysis of amino acid
mixtures.
The 20 principal amino acids from blood serum or from the hydrolysis of proteins are
separated and used in clinical diagnosis.
 This is most effective method for water purification. Complete deionization of water (or) a
non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and
solute anions for hydroxyl ions. This is usually achieved by method is used for softening of
drinking water.
 In the analysis of products of hydrolysis of nucleic acids. In this way, information is gained
about the structure of these molecules and how it relates to their biological function as
carriers of hereditary information.

31. ADVANTAGES OF ION EXCHANGE CHROMATOGRAPHY
• It is one of the most efficient methods for the separation of charged particles.
• It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.
• Ion exchange is used for both analytical and preparative purposes in the laboratory, the
analytical uses being the more common.
• 4. Inorganic ions also can be separated by ion-exchange chromatography.
Limitations of ion exchange chromatography
• Only charged molecules can be separated.

32. HPLC (HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

33. INTRODUCTION
HPLC stands for “High Performance Liquid Chromatography” (sometimes referred
to as the High Pressure Liquid Chromatography)
High Performance Liquid Chromatography is a powerful tool in analysis, it yields
High Performance and high speed compared to traditional column chromatography
because of the forcibly pumped mobile phase
High Performance Liquid Chromatography is a technique that can separate a
mixture of components.

34. PRINCIPLE
• High Performance Liquid Chromatography is a type of liquid
chromatography where the sample is forced through a column that is
packed with a stationary phase irregularly or spherically shaped particles at
high pressure
• High Performance Liquid Chromatography is a Chromatographic
technique used to separate a mixture of compounds in analytical chemistry
and biochemistry with the purpose of identifying, quantifying or purifying
the components in a mixture

35. FEATURES
 High Performance Liquid Chromatography
 High Pressure Liquid Chromatography
 it is column Chromatography
 it is Liquid Chromatography
 it can mainly divided by two types :-
• Normal phase HPLC
• Reversed Phase HPLC
 It is used as qualitative as well as quantitative analysis

36. HPLC SYSTEM

38. HPLC COLUMN

39. APPLICATIONS

40. GAS CHROMATOGRAPHY

41. GAS CHROMATOGRAPHY

42. INSTRUMENTATION

43. PRINCIPLE

44. TYPES OF GAS CHROMATOGRAPHY

45. FACTORS WHICH INFLUENCING GAS CHROMATOGRAPHY
volatility of
components
polarity of
components
column
temperature

46. USES OF GAS CHROMATOGRAPHY
 widely used for the qualitative and quantitative analysis of a large number of compounds
This technique provides a high speed and resolution
 very good reproducibility and high sensitivity
 non volatile substance can also separated if converted into volatile one by oxidation,
acylation, alkylation etc.
 alcohols, esters, fatty acids and amines present in biological samples are often separated by
gas chromatography

47. THANK YOU