High-Performance Liquid Chromatography (HPLC) is an analytical technique used extensively to separate the individual components within a complex mixture. The technique forces a liquid solvent, known as the mobile phase, through a column packed with a solid material, called the stationary phase, at high pressure. As the mixture travels through the column, the different compounds separate based on their chemical properties. The time it takes for a component to exit the column is called its retention time or elution, and the order of exit is determined by their interaction with the two phases.
The Competition: Stationary Phase Versus Mobile Phase
The separation of compounds in HPLC is a continuous competition between the two phases for the solute molecules. The stationary phase is the solid packing material inside the column, and the mobile phase is the liquid solvent mixture that flows through it. Compounds in the sample constantly partition between these two phases as they move through the column.
A compound that interacts more strongly with the stationary phase will spend more time attached to the column material, resulting in a longer retention time and eluting later. Conversely, a compound that has a greater affinity for the mobile phase will be quickly swept along with the solvent flow. The physical and chemical properties of both phases dictate which compounds are retained longer and which elute first.
Reversed-Phase Chromatography: The Most Common Scenario
Reversed-Phase Chromatography (RPC) is the most common mode of HPLC, dictating elution order based on hydrophobicity. In this setup, the stationary phase is non-polar, typically silica bonded with long hydrocarbon chains (C18 or C8). The mobile phase is polar, often a mixture of water or an aqueous buffer combined with an organic solvent like acetonitrile or methanol.
The rule in RPC is that the most polar compound elutes first. Polar compounds are repelled by the non-polar stationary phase and are quickly carried through the column by the polar mobile phase. Retention is driven by the hydrophobic effect, where non-polar molecules associate strongly with the non-polar stationary phase. Consequently, non-polar compounds are retained longer and elute later.
Normal-Phase Chromatography: The Opposite Rule
Normal-Phase Chromatography (NPC) represents the opposite polarity arrangement from RPC, resulting in an inverse elution order. In NPC, the stationary phase is polar, most commonly bare silica, while the mobile phase is non-polar, such as hexane or a mixture of hexane and a less polar solvent like ethyl acetate. This setup is less common than RPC but is valuable for separating compounds that are highly soluble in organic solvents.
The rule here is that the non-polar compound elutes first. Non-polar compounds do not interact strongly with the polar stationary phase and are rapidly carried away by the non-polar mobile phase. Conversely, polar compounds are strongly retained because they form strong dipole-dipole or hydrogen-bonding interactions with the polar silica surface, causing them to elute much later.
Controlling Elution Order with Mobile Phase Adjustments
Once the separation mode is chosen, the elution order can be fine-tuned by adjusting the mobile phase strength. Elution strength refers to the solvent’s ability to move compounds off the stationary phase. In RPC, increasing the percentage of the organic solvent (like acetonitrile or methanol) makes the mobile phase less polar and stronger, which reduces the retention time for all compounds.
A common technique for handling samples with a wide range of polarities is Gradient Elution. This involves gradually changing the mobile phase composition during the run by increasing the amount of the strong solvent. Starting with a weak mobile phase allows polar compounds to separate well. Gradually increasing the strength accelerates the elution of the more strongly retained, non-polar compounds, resulting in sharper, more defined peaks.