HPLC is a chromatographic method used to separate the components of a mixture. The typical design of an HPLC experiment usually involves these elements.
- A filtered solvent reservoir for mobile phase.
- Degassing system to prevent bubbles in mobile phase.
- A pump to maintain constant flow of mobile phase despite the back pressure applied by the resistance of flow through the packed column.
- An injector to inject samples. This can be manual or automatic.
- A column (solid phase). Length, internal diameter and stationary phases are to be chosen.
- A detector/data system to plot the chromatogram. includes UV, diode and fluorescent detectors.
Samples are forced to flow under high pressure through the column (solid phase). Solvents (mobile phase) allow the flow of the sample into the column, where it is separated into its components according to their interaction with the solid phase. components are detected and recorded on a chromatogram. The resolution of various components is determined by the extent of interaction between the solute components and the stationary phase.
In normal phase chromatography, the mobile phase is non polar while the stationary phase is polar. In reversed phase chromatography, the mobile phase is polar while the stationary phase is non polar.