Objectives

High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture.

The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy. HPLC is thus basically a highly improved form of column liquid chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures.

People

Prof. Rintu Banerjee

Facilitator

Agricultural and Food Engineering

rb@agfe.iitkgp.ac.in

+91-3222-283104

Dr. Samiran Sona Gauri

Technical Staff

Central Research Facility

asmi04@gmail.com

+91-3222-282499

Equipment Details

HPLC Agilent 1100

Utility and Working Principal

The separation principle of HPLC is based on the distribution of the analyte (sample) between a mobile phase (eluent) and a stationary phase (packing material of the column). Depending on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. The specific intermolecular interactions between the molecules of a sample and the packing material define their time “on-column”. Hence, different constituents of a sample are eluted at different times. Thereby, the separation of the sample ingredients is achieved. A detection unit (e.g. UV detector, PDA etc) recognizes the analytes after leaving the column. The signals are converted and recorded by a data management system (computer software) and then shown in a chromatogram. After passing the detector unit, the mobile phase can be subjected to additional detector units, a fraction collection unit or to the waste. In general, a HPLC system contains the following modules: a solvent reservoir, a pump, an injection valve, a column, a detector unit and a data processing unit.

Applications of HPLC
The HPLC has developed into a universally applicable method so that it finds its use in almost all areas of chemistry, biochemistry, and pharmacy.

• Analysis of drugs
• Analysis of synthetic polymers
• Analysis of pollutants in environmental analytics, pesticides etc.
• Determination of drugs in biological matrices
• Isolation of valuable products
• Product purity and quality control of industrial products and fine chemicals
• Separation and purification of biopolymers such as enzymes or nucleic acids
• Water purification
• Pre-concentration of trace components
• Ligand-exchange chromatography
• Ion-exchange chromatography of proteins
• High-pH anion-exchange chromatography of carbohydrates and oligosaccharides.