HPLCHigh Performance Liquid Chromatography

R AJ AT IN D ER IA

D U R G ESH BAGR I
PU N IT R AWAT

Chromatography
It is a technique of separation. In chromatography, the components to be separated are distributed between a stationary bed of large area and a liquid that percolates through or along it. Mass transfer between mobile phase and stationary phase because molecule of mixture absorbed on particle surface. Separation of the components is based on the fact that the rate of travel of an individual solute molecule through a column or thin layer of adsorbent is directly related to the partition of that molecule between the mobile and stationary phase. some time interval they will be distributed in space over the stationary phase and subsequently emerge out of the stationary phase as a single components.

HPLC
Is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component. the method involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analyte. If the interaction is weak, the analyte flow off the column in a short amount of time, and if the interaction is strong, then the elution time is long.

Use
HPLC has been used in medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products).

Process

The detector
There are several ways of detecting when a substance has passed through the column. A common method which is easy to explain uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. If you have a beam of UV light shining through the stream of liquid coming out of the column, and a UV detector on the opposite side of the stream, you can get a direct reading of how much of the light is absorbed. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time.

UV light absorber/detector

A flow scheme of HPLC