High Performance Liquid Chromatography HPLC is used as an analytical tool to separate specific compounds in a sample. The HPLC include a pump which provides the mobile phase and sample through the system, an auto sampler or injector interface for sample introduction, a stationary phase where separation of substances occurs, a sensor to detect the compounds and an integrator or a computer system for the visual output. HPLC first began with normal phase. Normal phase HPLC means the stationary phase is made from polar packaging material while the mobile phase is of non-polar or low polarity solvents.
Commonly used polar stationary phase or column is full of silica. Silica is comparatively the most polar compound in comparison to all other packaging materials. Another polar column is cyano column that has a more intermediate polarity. Examples of solvents used to compose a regular phase mobile phase are hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol IPA. The majority of the solvents used in the mobile phase is water chromatogram and have low polarity. Generally, these compounds are used together in a mix of in order to attain compounds separation. By way of instance, Hexane: IPA 9:1 signifies the mobile phase include a combination of hexane and IPA in the ratio of 9 to 1. In a standard phase program, the non-polar compounds will be eluted faster than the polar chemicals.
From the 1970s, reverse phase HPLC was developed. The principle is reverse of regular phase system, in which the stationary phase is packed non-polar material and the mobile phase. Commonly used packing material in reverse phase columns are silica linked with carbon-18 C18. There are additional columns of more intermediate polarity, such as C8 and cyano. Notice that cyano can be used in both regular and inverse phases and some column manufacturers create two different types of cyano column to match each stage. The mobile phase for a reverse phase system usually includes buffer or water Alternative, methanol, acetonitrile and IPA. IPA can be used in both opposite and Normal phase since it is miscible with water in addition to water immiscible solvents. However, large amount of IPA at a mobile stage will cause high pressure in the HPLC System because of the high density value. In a reverse phase HPLC, the non-polar Chemicals are retained in the column more than the polar chemicals. In another words, the polar compounds elute faster than the non-polar compounds.