Liquid Chromatography Lab

High performance liquid chromatography (HPLC) is a separation technique utilizing differences in distribution of compounds in two phases; called the stationary phase and the mobile phase. The stationary phase designates a thin layer created on the surface of fine particles and the mobile phase designates the liquid flowing over the particles. Under a certain dynamic condition, each component in a sample has a different distribution equilibrium depending upon the solubility in the phases and or molecular size. As a result, the components move at different speeds over the stationary phase and are thereby separated from each other. These components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles. These separated components are …show more content…
Mobile phase is constantly fed into the column inlet at a constant rate by a pump. The sample is injected from a sample injector, located near the column inlet. The injected sample enters the column with the mobile phase and the components in the sample migrate through it, passing between the stationary and the mobile phases. Compounds move in the column only when they are in the mobile phase. Compounds that tend to be distributed in the mobile phase therefore migrate faster through the column while compounds that tend to be distributed in the stationary phase migrate slower. In this way, each component is separated on the column and sequentially elutes from the outlet. Each compound eluting from the column is detected by a detector connected to the outlet of the column.
PRINCIPLE OF RP-HPLC
In RP-HPLC compounds are separated based on their hydrophobic character.retension is the result of interaction of then nonpolar components of the solutes and the non-polar stationay phase. Figure 2 High Performance Liquid Chromatography