High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) has become a very versatile and powerful
separation and analytical method over the years. It is an advanced form of liquid
chromatography (LC).
Instead of introducing the solvent into the column and allowing it to drip down under the
influence of gravity, in HPLC the sample is forced through the column under high pressures of
nearly 400 atm , resulting in faster and more efficient separation.
This technique is also called high pressure liquid chromatography.
The Principle of HPLC
HPLC follows the same basic principle as chromatography. Different components in the sample
have varying affinities to the adsorbent material. This causes a difference in the flow rate for
each component which leads to their separation as they come out of the column. The only
difference is that the speed and sensitivity of HPLC is much higher than that of LC due to the
application of a high pressure.
The magnitude of pressure applied depends on several factors such as the length and diameter
of the column, flow rate, size of particles in the stationary phase, and mobile phase
composition.
The Components of HPLC
Columns: HPLC columns are normally made of stainless steel and are 50 - 300mm long with an
internal diameter of 2 - 5mm. They are filled with the adsorbents (stationary phase) of particle
size 3 – 10µm.
The Components of HPLC
Columns:
HPLC columns are normally made of stainless steel and are 50 - 300mm long with an internal
diameter of 2 - 5mm. They are filled with the adsorbents (stationary phase) of particle size 3 –
10µm.
Sample Injector: The sample is injected into the column by an injector which is capable of
handling sample volumes in the range of 0.1 - 100mL under high pressures of up to 4000psi.
Reservoir:
The solvent or the mobile phase is placed in a glass reservoir. It is usually a blend of polar and
non-polar liquids whose concentrations depend on the sample composition.
Pump: The solvent in the mobile phase is aspirated by a pump from the reservoir and forced
through the HPLC column and then the detector.
Detector:
The detector in a HPLC system is located at the end of the column and it detects the
components of the sample that elute from the column. Different types of detectors such as
fluorescence, mass-spectrometric, UV-spectroscopic, and electrochemical detectors are used.
Data collection systems:
The signal from the detector is received by recorders which are used to process, store, and
reproduce chromatographic data. The data is interpreted and integrated by a computer which
produces a user-friendly chromatograph.

The Technique of HPLC

The key steps in the HPLC separation technique are as follows:

 Injection of the liquid sample into the column containing the stationary phase.
 Individual sample components are forced down the tube by high pressure from the
pump.
 Components are separated under the influence of various chemical/physical
interactions with the particles in the stationary phase.
 The separated analytes are identified by the detector present at the end of the column.
 The detector measures the concentration of the components.
 Data from the detector is processed and a chromatogram is produced.

Block Diagram of HPLC