Chromatography: Chromatography is defined as the technique of separating substances according to their partition coefficients between two immiscible phases.

One of these phases is a mobile phase and the other is a stationary phase (stationary phase is the phase that is in place. Act as filters for each substance from the compound and Mobile phase is the phase that is a carrier for compounds move through the separation on the stationary phase). Although originally intended to separate and recover ( isolate and purify ) the components of a sample, today, complete chromatography systems are often used to both separate and quantify sample components. The word chromatography comes from two Greek word chorma for colour and graphe for writing. The term chromatography was originally applied by a Russian botanist Michel Semonovich Tswett in 1906 to a procedure where a mixture of different colored pigments (chlorophylls and xanthophylls) is separated from each other. Classification: The chromatography can be divided into two groups i) Gas chromatography ii) Solution Chromatography Solution chromatography can be divided as follow: Adsorption chromatography Column chromatography Partition chromatography Ion Exchange chromatography Reserved phase chromatography Electrophoresis chromatography It can also be classified as 1) Paper chromatography 2) Thin Layer chromatography 3) Gas chromatography 4) HPLC (High pressure liquid ) chromatography 5) Radio Chromatography . Principle of Chromatography: In this method, the separation of components of a mixture depends on the different affinities for a fixed or stationary phase (such as solid or liquid) and their differential solubility in a moving or mobile phase.

Separation starts to occur when one component is held more firmly by the stationary phase than the other which tends to move on faster in the mobile phase .Thus, the underlying principle of chromatography is first to absorb the component of a mixture on an insoluble material and then to differentially remove (or elute) these components one by one with suitable liquid solvents. The adsorbent can be packed in the form of column or paper or ion exchange resins. The principle of chromatography can be explain by the following figure

Figure: Chromatography In the above figure a liquid is pumped through a bed of particles. The liquid is called the mobile phase and the particles the stationary phase. A mixture of the molecules that shall be separated is introduced into the mobile phase.

In the figure the blue molecules shall be separated from the red molecules and a mixture containing these two types of molecules is introduced into the mobile phase in front of the stationary phase. The mixture of the red and blue molecules is then transported by the mobile phase through the stationary phase.

The molecules in the mixture that adsorbs the most to the stationary phase, in this particular case the red molecules, is moving slowest through the particle bed. The red molecules become separated from the blue! Reference: http://www.studyhplc.com/ http://hplcmethodanalysis.org/how-to-choose-the-optimum-hplc-column/