High Pressure Liquid Chromatography

• High-performance liquid chromatography (HPLC),  is a chromatography technique used to separate the components in a mixture to identify each component and to qualify each component.
• This method involves a liquid sample being passed over a  solid adsorbent material packed into a column using a flow of a liquid solvent. Each analyte in the sample interacts slightly differently with the adsorbent material, thus retarding the flow of the analytes. If the interaction is weak, the analytes flow of the column in a short amount of time, and if the interaction is strong, then the elution time is long.

Application:-

• Medical (e.g. detection of vitamin D level in blood serum)
• Legal (e.g.detecting performance enhancement drug in urine)
• Research (e.g. Separating the components of a complex biological sample or of synthetic chemical for each other)
• Manufacturing (e.g. during the production process of pharmaceutical and biological products
• HPLC has many uses such as drug testing, testing for vitamins in food and growth promoters in meat. In each case components of the mixture are separated and detected.

Basic Components of HPLC:-

1. Solvent Reservior
2. The Pump system controls the flow and measures the volume of solvent (the mobile phase). The flow rates of HPLC columns are slow – often in the range of 0.5 - 5 cm3 min-1.
3. The Injector System: The sample to be separated is injected into the liquid phase at this point.
4. The Column is made of steel and packed usually with porous silica particles (the stationary phase). Different materials can be used depending on the nature of the liquid. A long column is not needed because separation in HPLC is very efficient. Columns are usually 10 –30 cm long, with an internal diameter of 4 mm. Different components of the sample are carried forward at different rates by the moving liquid phase, due to their differing interactions with the stationary and mobile phases.
5. The Detector: When the components reach the end of the column they are analysed by a detector. The amounts passing through the column are small, so solutes are analysed as they leave the column. Therefore HPLC is usually linked to a spectrometer (e.g. ultra violet or mass spectrometry). The length of time it takes for a compound to reach the detector allows the component to be identified. Like the GC, once the retention time of a solute has been established for a column using a particular set of operating conditions, the solute can be identified in a mixture. A chromatogram is obtained for the sample.