Reversed Phase HPLC

Being the most common principle of HPLC/UHPLC separation mode, reversed phase chromatography offers dynamic retention of compounds with hydrophobic and organic functionality. Retention of these compounds by reversed-phase HPLC involves a combination of hydrophobic and van der Waals-type interactions between each target compound and both the stationary phase and mobile phase.

Stationary phases used in reversed phase chromatography typically consist of varying lengths of hydrocarbons such as C18, C8, and C4 or strongly hydrophobic polymers such as styrene divinylbenzene

• C18 HPLC columns are the most preferred as they offer an excellent range of hydrophobic separation power along with high surface area coverage
• C8 HPLC columns are used when less retention compared to a C18 is needed
• C4 and C5 HPLC columns are commonly used to separate large macromolecules such as proteins