High Performance 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 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 made of polar packing material while the mobile 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 materials. 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 together in a mixture of in order to achieve compounds separation. For example, Hexane: IPA (9:1) means the mobile phase consist of a mixture of hexane and IPA at the ratio of 9 to 1. In 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 principle is opposite of normal phase system, where the stationary phase is packed non-polar material and the mobile 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 and reverse phases and some column manufacturers produce two types of cyano column 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 mobile phase will cause high pressure in the HPLC system due to its high density value. In a reverse phase HPLC, the non-polar compounds are retained in the column longer than the polar compounds. In another words, the polar compounds elute faster than the non-polar compounds.