Analytical method · Separation
High-Performance Liquid Chromatography (HPLC)
A separation technique that resolves a mixture into its components so each can be detected and measured.
What is it?
HPLC pushes a liquid sample through a packed column under high pressure. Components interact with the column differently and therefore emerge (elute) at different times, producing a separated signal at a detector.
What does it measure?
The separation of components over time and the detector response for each, most commonly by UV absorbance.
What type of result does it produce?
A chromatogram (signal vs. time) from which relative peak areas and retention times are read.
What can the result support?
- Relative detected composition under the stated method
- Comparison of retention against a reference under the same method
- A basis for a method-specific purity figure
What can it not establish?
- Identity on its own
- Absolute quantity without appropriate calibration
- Sterility, endotoxin level, biological activity, or fitness for use
What variables affect the result?
- Column chemistry and condition
- Mobile-phase composition and gradient
- Flow rate and temperature
- Detection wavelength
- How peaks are integrated
Example output
A single dominant peak with small satellite peaks, reported as a percentage of total detected area under the specified conditions.
Common interpretation mistake
Reading a high purity percentage as proof of identity or of fitness for use — it is neither.