What is HPLC?
High‑performance liquid chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a liquid sample.
It works by pumping a liquid mobile phase at high pressure through a column packed with a solid stationary phase; different compounds interact differently with the stationary phase and therefore elute at different times (retention times)
The Basic Idea
- The mobile phase (solvent or solvent mixture) is pumped at high pressure through the column.
- A small volume of sample is injected into this flowing mobile phase.
- As the sample travels through the column, its components partition between the mobile phase and the stationary phase.
- Components that interact strongly with the stationary phase move more slowly and have longer retention times; weakly interacting components elute faster.
- A detector at the end of the column measures the concentration of each component as it elutes, producing a chromatogram (signal vs. time)
Main Components of an HPLC system
- Solvent reservoir(s)
Hold the mobile phase(s), often with gas‑tight caps and sometimes an in‑line degasser to remove dissolved gases. - Pump
Delivers the mobile phase at a controlled flow rate and high pressure (typically up to hundreds of bar).
In gradient systems, it mixes two or more solvents in changing proportions over time. - Injector / autosampler
Introduces a defined volume of sample into the mobile‑phase stream.
Manual injectors use a loop and valve; autosamplers can handle many samples automatically. - Column
A steel or PEEK tube packed with small particles (e.g. silica‑based), coated with a stationary phase.
Separation takes place here as analytes distribute between stationary and mobile phases. - Column oven (optional)
Controls the temperature of the column, improving reproducibility and sometimes resolution. - Detector
Converts the concentration of eluting analytes into an electrical signal (e.g. UV‑Vis, diode‑array, fluorescence, refractive index).
The output vs. time is the chromatogram. - Data system
Software that records, processes, and displays chromatograms, and calculates peak areas, retention times, and results.
Key Equations
- Retention factor, �k