High performance liquid chromatography (HPLC) is a fast column liquid chromatography method where a solvent is passed through a column under high pressure (of up to 400atms). Like every other form of chromatography, HPLC separates a liquid sample into its constituent parts on the basis of the differences between molecules of the subject mixture and the molecules mobile and stationary phases.
For chromatography to occur, there must be a stationary phase and a mobile phase. The adsorbent normally is a solid (or a liquid) in nature and the mobile-phase normally a liquid or a gaseous matter.
The solvent is responsible for carrying the constituents of the subject mixture through the stationary phase. More lagging is experienced in components which interact more with the stationary phase.
In HPLC, a pressure pump forces the solvent (mobile phase) together with the subject mixture through a column with the stationary phase material (normally a solid). In the column, each component of the mixture will interact differently with the stationary phase.
Due to the interaction with the stationary phase, these components in the mixture will separate, each exiting the column on its own. It is important that the temperature of both the phases be kept constant.
Components of an HPLC Instrument: Any HPLC instrument generally comprises of:
Columns: This is where the stationary-phase material is placed. It is about 5 mm in diameter and can be as long as 300m.
Pumps: These supply high pressure of up to 400 atms that forces the mixture and solvent through the column.
Sampler Injector: This delivers the mixture in the subject to the mobile phase.
Detector: This device is located at the and of the column. It facilitates quantitative analysis of the different components of the mixture. The device detects the components as they flow out of the column. UV-spectroscopy is a commonly used detector.
Related: Different Types of HPLC Detectors
Different Types Of High Performance Liquid Chromatography
The different types of HPLC exist on the basis of the stationary phase system. Different materials used in the stationary phase have different methods of interaction with components. The following are the different types of HPLC.
Size-exclusion HPLC: The material used in the stationary phase in this type operates on the basis of components' molecular size. The material has pores of specific sizes. The larger molecules are eluted faster than the smaller ones.
Ion-exchange HPLC: This type of HPLC operates on the basis of ionic charges. The adsorbent has ionic charges that are opposite to the subject constituents' ionic charges. Constituents with a higher ionic charge will experience more attraction and so they will lag through the column. Those with a lower ionic charge will experience lesser attraction and they will be eluted fast.
Related: Steps for HPLC Method Development
Normal phase HPLC: The basis for the operation of normal phase HPLC is polarity. The solvent is non-polar (normally an organic compound) and adsorbent polar. Polar materials will interact more with each other as opposed to polar and non-polar interactions. The less polar components of a mixture will be eluted faster than the more polar components.
Reverse Phase HPLC: In this type, the solvent polar (hydrophilic) and adsorbent non-polar. It is the opposite of the normal phase HPLC. The non-polar components will take longer to exit the column.
Uses Of HPLC In Pharmaceuticals: In the pharmaceutical industry, it is mainly used for analytical studies. Manufactured drugs are always under constant analysis to check for compliance with the required standards and determine their dosage.
I need Operation of Agilent HPLC model 2100 series
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