High Performance Liquid Chromatography (HPLC) is used being an analytical instrument to separate certain substances in a sample. The HPLC include a pump that delivers the 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 in order to detect the compounds and a good integrator or a computer system for the visible output.
HPLC first started with normal phase.
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Normal phase HPLC means the stationary phase is made of polar packing material while the cellular phase is of non-polar or reduced polarity solvents. Commonly used polar fixed 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 a normal 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 collectively in a mixture of in order to achieve compounds separation. For example , Hexane: IPA (9: 1) means the mobile phase include a mixture of hexane and IPA at the ratio of 9 to 1. Inside a normal phase application, the non-polar compounds will be eluted faster than the polar compounds.
In the 1970s, reverse phase HPLC was developed. The rule is opposite of normal phase system, where the stationary phase can be packed non-polar material and the cellular 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. Note that cyano can be used in both normal and reverse phases and some column manufacturers 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 plus normal phase as it is miscible along 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. Within a reverse phase HPLC, the non-polar compounds are retained in the column longer than the polar compounds. Within another words, the polar substances elute faster than the non-polar compounds.