HPLC
HPLC
HPLC
2. Pumping systems
4. Columns
5. Detectors
1. Mobile phase reservoirs and solvent treatment systems
• Gases and dust are present in the solvents and they are to be
removed. Gases produce bubbles in the column and thereby cause
band spreading
Advantages
• The resulting particles are often coated with thin organic films,
which are chemically or physically bonded to the surface
Detectors
• The detector should have low dead volume to minimize extra
column band broadening
• It has been estimated that more than three quarters of all HPLC
separations are currently performed with reversed-phase, bonded,
octyl- or octyldecyl-siloxane packings.
• With such preparations, the long chain hydrocarbon groups are aligned
parallel to one another and perpendicular to the surface of the
particle, giving a brush like non-polar, hydro carbon surface
Increasing polarity
Applications of Partition Chromatography
• Disadvantage
High electrolyte concentrations are required to elute most
analyte ions in a reasonable time. As a consequence, the
conductivity of the mobile phase components tend to swamp
that from the analyte ions, thus greatly reducing the detector
sensitivity
➢ Column Packings
• Gel Permeation:
Type of size-exclusion chromatography in which the packing
is hydrophobic. It is used to separate nonpolar species
Affinity Chromatography
• Affinity Chromatography involves covalently bonding a reagent,
called an affinity ligand, to a solid support.
• Procedure
(1) When sample passes through the column, only the molecules
that selectively bind to the affinity ligand are retained
(2) Molecules that do not bind pass through the column with the
mobile phase
(3) After the desired molecules are removed, the retained analytes
can be eluted by changing the mobile phase conditions
➢ Stationary Phase
➢ Applications