Reversed Phase HPLC

Reversed-phase Liquid Chromatography (RP-LC)

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

How Reversed-phase Liquid Chromatography Works?
Reversed-phase liquid chromatography is a dominant analytical technique that separates compounds based on their hydrophobicity using a non-polar stationary phase and a polar mobile phase. It is the most widely used mode of high-performance liquid chromatography (HPLC), especially in biomedical and pharmaceutical applications.

Stationary Phase
The stationary phase consists of hydrophobic materials chemically bonded to porous silica particles. Common bonded hydrocarbons include C18 (octadecylsilane), C8, or C4 chains, with C18 being the most prevalent. The retention of compounds increases with the length of these hydrocarbon chains—longer chains (e.g., C18) retain hydrophobic molecules more effectively than shorter ones (e.g., C8).

Mobile Phase
The mobile phase is polar, typically a mix of water and organic solvents like methanol or acetonitrile. A gradient elution is often employed, starting with a high-water content (polar) and gradually increasing the organic solvent proportion to reduce polarity. This allows less hydrophobic compounds to elute first, followed by more hydrophobic ones. Additives such as buffers adjust pH, influencing solute ionization and retention.

Retention Mechanism
Separation occurs through partitioning between the hydrophobic stationary phase and polar mobile phase:

• Hydrophobic interactions: Non-polar compounds adsorb strongly to the stationary phase, while hydrophilic ones elute quickly.
• Gradient elution: Increasing organic solvent concentration disrupts these interactions, selectively eluting compounds based on hydrophobicity.
• Molecular simulations reveal that C18 chains adopt flexible conformations, creating a dynamic interface where solutes partition based on their affinity for the hydrophobic chains versus the mobile phase.