High-Performance Liquid Chromatography (HPLC)
is a separation technique that involves
• the injection of a small volume of liquid sample
• into a tube packed with tiny particles (3 to 5 micron (μm) in diameter called the(stationary phase)
• where individual components of the sample are moved down the packed tube (column) with a liquid (mobile phase) forced through the column by high pressure delivered by a pump.
• These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.
• These separated components are detected at the exit of this tube (column) by a flow-through device (detector) that measures their amount. An output from this detector is called a “liquid chromatogram”.
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the 5 major HPLC components and their functions
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1. Pump
• The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate, expressed in milliliters per min (mL/min).
• Normal flow rates in HPLC are in the 1-to 2-mL/min range.
• Typical pumps can reach pressures in the range of 6000-9000 psi (400-to 600-bar).
• During the chromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).
2. Injector
• The injector serves to introduce the liquid sample into the flow stream of the mobile phase.
• Typical sample volumes are 5-to 20-microliters (μL).
• The injector must also be able to withstand the high pressures of the liquid system.
• An autosampler is the automatic version for when the user has many samples to analyze or when manual injection is not practical.
3. Column
• Considered the “heart of the chromatograph” the column’s stationary phase separates the sample components of interest using various physical and chemical parameters.
• The small particles inside the column are what cause the high backpressure at normal flow rates.
• The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph.
4. Detector
• The detector can see (detect) the individual molecules that come out (elute) from the column.
• A detector serves to measure the amount of those molecules so that the chemist can quantitatively analyze the sample components.
• The detector provides an output to a recorder or computer that results in the liquid chromatogram (i.e., the graph of the detector response).
5. Computer
• Frequently called the data system, the computer not only controls all the modules of the HPLC instrument but it takes the signal from the detector and uses it to determine the time of elution (retention time) of the sample components (qualitative analysis) and the amount of sample (quantitative analysis).
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Types of Chromatography
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1)Adsorption Chromatography :-
Chromatography in which separation is based mainly on differences between the adsorption affinities of the sample components for the surface of an active solid.
2)Partition Chromatography :-
Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography)
, or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography).
3)Ion Exchange Chromatography :-
Chromatography in which separation is based mainly on differences in the ion-exchange affinities of the sample components.
Anions like SO3- or cations like N(CH3)3+ are covalently attached to stationary phase, usually a resin
4)Molecular Exclusion Chromatography :-
A separation technique in which separation mainly according to the hydrodynamic volume of
the molecules or particles takes place in a porous
non-adsorbing material with pores of approximately
the same size as the effective dimensions in solution of the molecules to be separated.
5) Affinity chromatography :-
The particular variant of chromatography in which the unique biological specificity of the analyte and ligand interaction is utilized for the separation
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