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Topic 3a Chromatography PDF
Topic 3a Chromatography PDF
CHROMATOGRAPHY
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Principles of chromatography
• Chromatography is a physical method of separation in which the components
to be separated are distributed between two phases
• One of which is stationary (stationary phase) while the other (the mobile
phase) moves through it in a definite direction.
• Molecules that spend most of their time in the mobile phase are carried along
faster.
Principles of chromatography
• In a chromatographic separation of any type, different
components of a sample are transported in a mobile phase (a gas,
a liquid, or a supercritical fluid).
• The mobile phase penetrates or passes through a solid or
immiscible stationary phase.
• Solutes (eluates) in the sample usually have differential
partitioning or interactions with the mobile and stationary phases.
• Since the stationary phase is the fixed one then those solutes
which have stronger interactions with the stationary phase will
tend to move slower (have higher retention times) than others
which have lower or no interactions with the stationary phase will
tend to move faster.
Principle of Chromatography
• However, when their properties are so different, a solute will not tend to
stay
and interact with the stationary phase and will thus prefer to stay in the
mobile phase and move faster; a polar solvent and a non polar stationary
phase is a good example.
Principle of Chromatography
• Distances between solute bands become greater with time and as solutes start to leave the
column they are sequentially detected.
Principle of Chromatography
• Paper chromatography
• Thin Layer Chromatography (TLC)
• Column Chromatography
• Ion Exchange Chromatography
• Gas chromatography
• HPLC Chromatography
• Gel permeation/Filtration/Size Exclusion Chromatography
• Affinity Chromatography
Types of chromatography
Paper chromatography
• Paper chromatography is a techniques that involves placing a small dot or line
of sample on to a strip of chromatography paper.
• The paper is placed in a jar containing a
shallow layer of solvent and sealed.
• Molecules larger than pores can not permeate into gel particles, and
they are retained between particles within a restricted area.
• Molecules smaller than the pores are diffused into pores, and as
molecules get smaller, they leave the column with proportionally
longer retention times
Types of chromatography
• AFFINITY CHROMATOGRAPHY