An Introduction to
Chromatography
What is Chromatography?
Chromatography is a technique for separating mixtures
into their components in order to analyze, identify, purify,
and/or quantify the mixture or components.
separation methods that combine separation and analysis
• Analyze
Separate • Identify
• Purify
• Quantify
Mixture Components
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Chromatography
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient of sample components
between 2 different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
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Distribution Coefficient
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.
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Definition of Chromatography
Simplified Definition:
Chromatography separates the components of a mixture by
their distinctive attraction to the mobile phase and the
stationary phase.
Explanation:
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a certain
distance through the stationary phase, depending on their
attraction to both of the phases
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Illustration of Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture Components
Affinity to Stationary Affinity to Mobile
Components
Phase Phase
Blue ---------------- Insoluble in Mobile Phase
Black
Red
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Yellow
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Chromatography
Chromatogram - Detector signal vs.
retention time or volume
Detector Signal 1 2
time or volume
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Milestones in Chromatography
• 1903 Tswett - plant pigments separated on chalk
columns
• 1931 Lederer & Kuhn - LC of carotenoids
• 1938 TLC and ion exchange
• 1950 reverse phase LC
• 1954 Martin & Synge (Nobel Prize)
• 1959 Gel permeation
• 1965 instrumental LC (Waters)
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Classification of Methods
• There are two classification schemes:
– mobile phase
– attractive forces (separation mechanism)
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Mobile Phase
• gas (GC)
• water (LC)
• organic solvent (LC)
• supercritical fluid (SCFC)
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Classification based on Mobile Phase
• Liquid chromatography (includes column
chromatography, thin-layer, and HPLC)
– Stationary phase: silica, alumina, etc.
– Mobile phase (moving phase): organic solvents
– Important properties: polarity
• Gas chromatography
– Stationary phase: a film of a polymer or a wax. The film
must have a high boiling point
– Mobile phase: gas (Helium is the usual carrier gas)
– Important properties: boiling point
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Classification based on Mobile
Phase
Liquid chromatography (LC)
Column High performance Thin layer
(gravity flow) (pressure flow) (adsorption)
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FIVE BASIC LIQUID CHROMATOGRAPHY
The 5 basic liquid chromatography modes are named according to the mechanism
involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
5. Affinity Chromatography
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What’s that ?
• Chromatography
• Distribution Coefficient
• Stationary phase
• Mobile phase
• Classification based on mobile phase
• Classification based on attractive forces
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What’s that ?
• Normal Phase Chromatography
• Reverse Phase Chromatography
• Adsorption Chromatography
• Partition Chromatography
• Ion Exchange Chromatography
• Gel Permeation Chromatography (exclusion
chromatography)
• Affinity Chromatography
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Thank You for
Your Kind Attention
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