High end Liquid Chromatography (HPLC) is used as an analytical instrument to separate certain substances in a sample. The HPLC contain a pump that delivers the particular mobile phase and sample through the entire system, an auto sampler or injector port for sample introduction, a stationary phase where separation of compounds takes place, a detector to detect the compounds and a good integrator or a computer system for the visual output.
HPLC first started along with normal phase. Normal phase HPLC means the stationary phase is constructed of polar packing material while the mobile phase is of non-polar or low polarity solvents. Commonly used polar stationary phase or column is filled 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 separation. For example , Hexane: IPA (9: 1) means the mobile phase contain a mixture of hexane and IPA at the ratio of 9 to 1.
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In the normal phase application, the non-polar compounds will be eluted faster than the polar compounds.
In the 1970s, invert phase HPLC was developed. The theory is opposite of normal stage system, where the stationary phase can be packed non-polar material and the cellular phase is polar. Commonly used packing material in reverse phase columns are silica linked with carbon-18 (C18). There are other columns of more intermediate polarity, such as C8 and cyano. Note 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 mobile phase for a reverse phase system usually consists of water or buffer solution, methanol, acetonitrile and IPA. IPA can be used in both reverse and normal phase as it is miscible with water as well as water immiscible solvents. However , large amount of IPA in a cellular phase will cause high pressure in the HPLC system due to its high density value. In the reverse phase HPLC, the non-polar compounds are retained in the column longer than the polar compounds. In another words, the polar substances elute faster than the non-polar compounds.