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Reverse Phase HPLC
Reverse phase HPLC is more commonly used compared to NP HPLC. This technique can be used to separate, identify and or quantitate components in mixtures of soluble organic components based on their hydrophobicity.
The stationary phase is nonpolar, like C18 bonded silica. The mobile phase is polar, usually being water and polar organic solvent. Compounds with the most hydrophobicity elute later in the chromatogram and those with the least hydrophobicity elute earlier. Isocratic and gradient modes can be used to optimize separation of analytes of interest from matrix components that potentially can interfere with quantitation.
Normal Phase HPLC
Although this type of chromatography isn’t used as often, there are many good reasons to choose Normal Phase HPLC. It is great when a compound is too hydrophobic or hydrophilic for reverse-phase HPLC. It can also be used for isomer separation, if the sample injection solvent is non-polar, or if recovery in non-polar solvents is desirable.
In normal phase chromatography, the stationary phase is polar, usually using silica. The mobile phase is nonpolar, using hexane or chloroform. Compounds with greater polarity elute later in the chromatogram and those with the least polarity elute earlier.