High end Liquid Chromatography (HPLC) is used as an analytical instrument to separate certain compounds in a sample. The HPLC consist of a pump that delivers the mobile phase and sample throughout the system, an auto sampler or injector port for sample introduction, a stationary phase where separation of compounds takes place, a detector in order to detect the compounds and an integrator or a computer system for the visual output.
HPLC first started with normal phase. Normal phase HPLC means the stationary phase is constructed of polar packing material while the cellular phase is of non-polar or low polarity solvents. Commonly used polar stationary phase or column is packed with silica. Silica is relatively the most polar compound compared to all other packing components. Another polar column is cyano column which has a more intermediate polarity. Examples of solvents used to make up an ordinary phase mobile phase are hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol (IPA). Most of the solvents used in the mobile phase are water immiscible and have low polarity. In most cases, these solvents are used jointly in a mixture of in order to achieve compounds splitting up. For example , Hexane: IPA (9: 1) means the mobile phase include a mixture of hexane and IPA on the ratio of 9 to 1. Inside a normal phase application, the non-polar compounds will be eluted faster compared to polar compounds.
In the 1970s, reverse phase HPLC was developed. The basic principle is opposite of normal phase system, where the stationary phase will be packed non-polar material and the mobile phase is polar. Commonly used packaging material in reverse phase columns are silica linked with carbon-18 (C18). Additional columns of more intermediate polarity, such as C8 and cyano. Remember that cyano can be used in both normal plus reverse phases and some column producers produce two types of cyano line to suit each phase. The cellular phase for a reverse phase system usually consists of water or buffer solution, methanol, acetonitrile and IPA. IPA can be used in both reverse plus normal phase as it is miscible along with water as well as water immiscible solvents. However , large amount of IPA in a mobile phase will cause high pressure in the HPLC system due to its high density value.
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In a reverse phase HPLC, the non-polar compounds are retained in the line longer than the polar compounds. In another words, the polar compounds elute faster than the non-polar substances.