High Performance Liquid Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture or analyte in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).


All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.

Instrumentation of HPLC: 

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HPLC instrumentation includes a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.

1.   Solvent Reservoir         

Mobile phase contents are contained in a glass reservoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample.

2. Pump A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated. 3. Sample Injector: The injector can be a single injection or an automated injection system.

An injector for an HPLC system should provide injection of the liquid sample within the range of

0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi).

4. Columns: Columns are usually made of polished stainless steel, are between 50 and

300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 ┬Ám.

5.     Detector: The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UVspectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.

6.     Data Collection Devices: Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.


The reservoir holds the solvent, which is referred to as the mobile phase because it moves. There are usually a minimum of two reservoirs in a system, fitted with a gas diffuser through which helium can be bubbled. A pump is used to generate a specified flow of the mobile phase. Most HPLCs are now fully automated and controlled by computer. The injector, or auto sampler, introduces the solvent into a phase stream that carries the sample into the high pressure (up to400 bar) column, which is referred to as stationary phase. A detector is needed to see the separated compound bands as they elute from the high pressure column. The information is sent from the detector to a computer which generates the chromatogram.



Applications of HPLC:

 The information that can be obtained by HPLC includes resolution, identification and quantification of a compound. It also aids in chemical separation and purification. The other applications of HPLC include

v  Pharmaceutical Applications

1.  To control drug stability.

2.  Tablet dissolution study of pharmaceutical dosages form.

3.  Pharmaceutical quality control.


v  Environmental Applications

1.  Detection of phenolic compounds in drinking water.

2.  Bio-monitoring of pollutants.


v  Applications in Forensics

1.  Quantification of drugs in biological samples.

2.  Identification of steroids in blood, urine etc.

3.  Forensic analysis of textile dyes 

4.  Determination of cocaine and other drugs of abuse in blood, urine etc.


v  Food and Flavor

1.  Measurement of Quality of soft drinks and water.

2.  Sugar analysis in fruit juices.

3.  Analysis of polycyclic compounds in vegetables.

4.  Preservative analysis.


v  Applications in Clinical Tests

1.  Urine analysis, antibiotics analysis in blood.

2.  Analysis of bilirubin, biliverdin in hepatic disorders.

Detection of endogenous Neuropeptides in extracellular fluid of brain etc.

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