HPLC

is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on

pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the

sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the

components as they flow out the column. HPLC has been used for 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) purposes.

Operation

The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating

through the column. The components of the sample move through the column at different velocities, which are function of specific physical interactions with

the sorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and

on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time

measured under particular conditions is considered an identifying characteristic of a given analyte. The choice of mobile phase components, additives (such

as salts or acids) and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the

sample in order to find the HPLC method which gives adequate separation.

يسخدم لفصل مكونات في خليط، لتحديد كل مكون، وتحديد تركيز كل مكون ويدخل في مجالات الادوية،الصيدلانيات وكذلك في الكيمياء التحليلية