The basic principle of High-Performance Liquid Chromatography (HPLC) is the separation of components in a mixture based on their different interactions with two phases:
- Stationary phase – a solid or liquid supported on a solid inside a column.
- Mobile phase – a liquid that flows through the column under high pressure.
How it works:
- The sample mixture is injected into the mobile phase (solvent) and pumped through the stationary phase (the column).
- Each component in the sample interacts differently with the stationary phase — some are attracted more strongly, while others pass through faster.
- Because of these different interaction strengths (based on polarity, size, or charge), the components separate as they travel through the column.
- The separated components exit the column at different times (called retention times) and are detected by a detector.
In simple terms:
HPLC separates compounds because each one moves through the column at a different speed depending on how strongly it sticks to the stationary phase versus how easily it dissolves in the mobile phase.