HPLC
HPLC
HPLC
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Limitation of classical LC
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High performance liquid chromatography (HPLC)
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Instrumentation of HPLC
Gradientelution: continuous
change of solvent composition to
increase MP strength
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Sample preparation
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4. The Column
It is made of stainless-steel tubing
Column length is: 10 – 30 cm and internal
diameter is: 4 – 10 mm
Column packing typically have particle
sizes 5 or 10 µm. Column of this type often
contain 40000-60000 plates/m
It is expensive and easily damaged by
dust or particles in the sample or solvent.
Protection:
1. Periodically renew guard columns
containing the same SP like main
column.
2. Pass solvents and samples through filters 11
van Deemter for HPLC
H ∞ A + C
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5.1 UV detector
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5.2. Fluorescence detector
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Partition Chromatography
The separation can be performed into two ways based on
stationary phase (normal phase or reverse phase)
In normal-phase chromatography, the least polar component is
eluted first; increasing the polarity of the mobile phase then
decreases the elution time. In contrast, with reversed-phase
chromatography, the most polar component elutes first, and
increasing the mobile phase polarity increases the elution time.
Bonded-phase packing
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Problem
Consider the following three compounds
HOCH2CH2OH CH3CH2CH2CH2CH2CH3 C6H5CH3
(ethylene glycol) (hexane) (toluene)
where toluene has a polarity that is in between the two other
compounds.
Olefins<Ethers<Esters<Lactones<Aldehydes<Amines<Phenols<Acids.
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Applications in separation of natural products
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Selection of the solid support
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Size-exclusion or gel permeation chromatography
Porous polymeric matrix: formed of spongy particles,
with pores completely filled with the liquid mobile
phase (gel).
Separation mechanism
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Applications of GPC to natural products
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Ion Exchange Chromatography
Process by which ions of an electrolyte solution are brought
into contact with an ion exchange resin.
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Cation Exchangers
Active ions (counter ions) are cations.
The polar groups attached to the matrix are acidic
(sulphonic acids, carboxylic acids, phenols, phosphoric acids)
Example: X-COO- H+
X = Frame work (matrix)
-COO- = fixed charge (anionic), non-exchangeable
H+ = counter ion (cation), Exchangeable
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Regeneration of the resin
Ion exchange process is generally reversible e.g in the
following:
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Anion Exchangers
Active ions (counter ions) are anions.
The polar groups attached to the matrix are tertiary or
quaternary ammonium groups (basic).
X = Framework (matrix)
-NR3+ = Fixed charge (cationic), non-exchangeable
-OH– = counter ion (anion), Exchangeable
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Applications of Ion Exchange Chromatography
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