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1. INTRODUCTION

Chromatography is an analytical chemistry technique used

in separating mixtures.

It enables the separation, identification, and purification of the

components of a mixture for qualitative and quantitative analysis.

TYPES OF CHROMATOGRAPHY It is derived from the Greek word

“Chroma”- Color and “Graphien” – Write
Facilitator: Dr. Thaimuta Z.

Chromatography was first devised in Russia by

OUTLINE the Italian-born scientist Mikhail Tsvet in 1900.

1. Introduction
He developed the technique and coined the
2. Classification term chromatography primarily for the
3. Mechanism of separation separation of plant pigments such as chlorophyll,
carotenes, and xanthophylls.

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Mobile phase is a gas or liquid that

carries the components of a mixture past
the stationery phase
Stationery phase is a solid or a liquid
phase that interacts with components of the
mobile phase while remaining fixed

Chromatography involves a mixture (sample) being dissolved in a

mobile phase which may be a gas or a liquid. 2. CLASSIFICATION
The mobile phase is then forced through an immobile, immiscible
stationary phase.
Chromatographic methods can be classified into three
Components of a mixture will have differing solubilities at each
different ways:
phase i.e. a component that is more soluble in the stationery phase
will take longer to go through it than a component that is less 1. Based on shape of chromatographic bed:
soluble in the stationery phase.  Two dimensional(Planar) – Thin layer and paper
chromatography
The inverse is true. This is what is termed “Differential distribution”  Three dimensional - Column Chromatography
and this is the basis of Chromatography
2. Based on physical state of mobile or stationery
Proteins can be purified based on characteristics such as size and phase:
shape, ionic charge, hydrophobic groups present on the surface, and  Gas chromatography
binding capacity with the stationary phase.  Liquid chromatography

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3. Based on mechanism of separation:

 Ion exchange chromatography
 Partition chromatography
 Affinity chromatography
 Adsorption chromatography
 Size Exclusion chromatography

A. PLANAR CHROMATOGRAPHY
In planar chromatography, the stationary phase is coated on a
sheet of paper (paper chromatography) or is bound to a solid
surface (thin-layer chromatography)
In TLC, the sample is applied as a small spot or band near the
bottom edge of the plate.
Many stationary phases are available for TLC,
including
Then, the plate is placed in a closed tank containing the mobile
phase, which migrates up the plate by capillary action.
(1)silica,
(2) cellulose,
After the mobile phase travels a desired distance, the plate is
removed from the tank and is dried . Solutes are visualized directly (3) alumina, and
if they are colored or fluorescent by eliciting a chemical reaction to (4) alkyl-bonded silica particles
yield a visible product.

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B. COLUMN CHROMATOGRAPHY

In column chromatography, the stationary phase is either;

coated onto or is chemically bonded to support
particles that are then “packed” into a tube,
or the stationary phase is coated onto the inner
surface of a tube.
Gas Chromatography and Liquid Chromatography
are two categories of column chromatography

II. LIQUID CHROMATOGRAPHY

I. GAS CHROMATOGRAPHY
GC usually is the method of choice for volatile Separation by LC is based on the differential distribution of the
solutes between a liquid mobile phase and a stationary phase
components.
In GC, a gas mobile phase, or carrier gas, is used to
carry a mixture of volatile solutes through a column In the clinical laboratory, HPLC is the most widely used form of LC.
containing the stationary phase, which usually is a
nonvolatile liquid coated or bonded to particles or the High-performance liquid chromatography (HPLC), also called "High
inner surface of a capillary. pressure liquid chromatography", is an example of Liquid
The mobile phase, or carrier gas, is typically an inert chromatography that uses columns containing small particles of
stationary phase and requiring high pressure to push the mobile
gas, such as (1) nitrogen, (2) helium, or (3) argon phase past the stationary phase.

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I. ION EXCHANGE CHROMATOGRAPHY

Ion-exchange chromatography is based on an exchange
of ions between a charged stationary phase and ions on
the opposite charge in the mobile phase
Cation-exchange particles contain negatively charged
functional groups such as (1) carboxy , (2) phosphate, or
(3) sulfonate ions.
 These groups bind positively charged components
(cations) in the mobile phase
Anion-exchange particles contain positively charged
groups such as primary, tertiary, or quaternary amines
that bind negatively charged components (anions) in the
mobile phase

3. MECHANISM OF SEPARATION

As mentioned earlier, there are 5 major

mechanisms employed in chromatography:
ion-exchange,
partition,
adsorption,
size-exclusion, and
affinity mechanisms.

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II. PARTITION CHROMATOGRAPHY

The differential distribution of solutes between two
immiscible liquids is the basis for separation by partition
chromatography.

Operationally, one of the immiscible liquids serves as the

stationary phase.

To prepare this phase, a thin film of the liquid is chemically

bonded onto the surface of support particles or onto the
inner walls of a capillary column

Partition chromatography is classified as either 1) gas- III. SIZE EXCLUSION CHROMATOGRAPHY

liquid chromatography (GLC) or as; 2) Liquid-liquid
chromatography (LLC). Size-exclusion chromatography, (also known as gel-
filtration/ gel-permeation/ molecular- sieve
chromatography), separates solutes on the basis of their
molecular size in solution.
LLC is further categorized as a) Normal phase or b)
Reverse phase. A variety of materials are used as stationary phases for
size-exclusion chromatography, including:
(1)cross-linked dextran,
For normal-phase LLC, a polar liquid is used as the (2) polyacrylamide,
stationary phase and a relatively nonpolar solvent is (3) agarose,
used as the mobile phase. (4) polystyrene- divinylbenzene, and
(5) porous glass.
Beads of these materials have pores that allow small
In reverse-phase chromatography, the stationary phase molecules to enter and to be retained to a greater extent
is nonpolar, an the mobile phase is relatively polar than large molecules that are excluded from entering
pores

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IN SUMMARY
IV. AFFINITY CHROMATOGRAPHY

In affinity chromatography, one component of

highly specific molecular interaction pairs, such as
(1) enzyme-inhibitor, (2) hormone-receptor, or
(3) antigen-antibody, is immobilized in a
stationary phase and is used to capture
molecules from the mobile phase

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REFERENCES
1. Tietz fundamentals of Clinical chemistry and Molecular diagnostics
– 7TH Edition
2. https://www.slideshare.net/nadeemakhter7374/chromatography
-34247423
3. https://www.slideshare.net/khadeejaikram56/classification-of-
chromatography