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CHROMATOGRAPHY
AND ITS TYPES

• Chromatography (from Greek chroma “color”
and graphein “to write”) is the collective term for
a set of laboratory techniques for the separation
of mixture
...

• The various constituents of the mixture travel at
different speeds, causing them to separate
...

• He continued to work with chromatography in the first
decade of the 20th century, primarily for the separation of
plant pigments such as Chlorophyll, Carotenes, and
Xanthophylls
...


HISTORY

• Chromatography usually consist of mobile phase and
stationary phase
...

• Stationary phase This phase is always composed of a
“solid” phase or “a layer of a liquid adsorbed on the
surface a solid support”
...


PRINCIPLE

• Eluent- Fluid entering column/solvent that carries the
analyte
• Eluate- Mobile phase leaving the column
• Stationary phase – Immobilized phase
• Immobilized on the support particles or on the inner wall
of the column tubing e
...

• It is collected as the time from injection to detection
• Sample
(Analyte):
substance
analyzed
in
chromatography
• A chromatogram
is
the
visual
output
of
the chromatograph
...


•
•
•
•
•
•
•
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Paper Chromatography
...

Gel filtration Chromatography
...

Ion exchange Chromatography
...

Affinity Chromatography
...


TYPES OF
CHROMATOGRAPHY

• Technique for separating dissolved chemical substances
by taking advantage of their different rates of migration
across sheets of paper
...

The paper is initially impregnated with some
suitable solvent to create a stationary liquid phase
• The solvent penetrates the paper by capillary action and,
in passing over the sample spot, carries along with it the
various components of the sample
...


• Thin Layer Chromatography is a technique used to isolate
non-volatile mixtures
...

• The material usually used is aluminium oxide, cellulose,
or silica gel
...

• The separation relies on the relative affinity of
compounds towards both the phases
...

• 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
...


Principle

• Size-exclusion chromatography is also known as Gel
filtration chromatography and separates molecules
according to size ,shape, molecular weight
...

• It is generally a low-resolution chromatography technique
and thus used for purification
...


GEL FILTRATION
CHROMATOGRAPHY

• The stationary phase used is a porous polymer matrix
whose pores are completely filled with the solvent to be
used as the mobile phase
...

• The flow of the mobile phase hence will cause larger
molecules to pass through the column unhindered,
without penetrating the gel matrix, whereas smaller
molecules will be retarded according to their penetration
of the gel
...
Adsorbents are packed into a column in a
glass tube
...

• Adsorbents such as silica gel, alumina, charcoal powder
& calcium hydroxyl apatite are used
...


Principle

• Ion exchange chromatography uses an ion exchange
mechanism to separate molecules on the basis of their
electrical charges
...

• Cation exchangers & Anion exchangers are used as ion
exchange resins
...

• It is the method of choice for the separation of volatile
substances or the volatile derivates of certain nonvolatile substances
...

• The mixture of volatile material is injected into the
column along with mobile phase

GAS LIQUID
CHROMATOGRAPHY

• Affinity chromatography is a separation method based on
a specific binding interaction between an immobilized
ligand and its binding partner
...

• The degree of purification can be quite high depending on
the specificity of the interaction and, consequently, it is
generally the first step, if not the only step, in a
purification strategy
...


High Performance Liquid
Chromatography

• The sample is carried by a moving carrier gas stream of
helium or nitrogen
...

• Because of this versatility, HPLC is used in a variety of
industrial and scientific applications, such as
pharmaceutical, environmental, forensics, and chemicals
...

• It is also useful for the analysis of drugs, hormones &
vitamins
...

• The technique is also useful for the determination of
molecular weight of proteins
...
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