High Performance Liquid Chromatography (HPLC)
Description of method
High-resolution liquid chromatography is a method of analysis for non-charged molecules. For separation it makes use of the differently strong interactions between substances and the filler material (stationary phase).
The stationary phases used are mainly silica gel (normal phase) and reversed phases (RP8 / RP18). In the recent past, the separation of enantiomers on chiral (optically active) phases has been added.
Nearly all organic solvents from nonpolar, such as hexane, to strongly polar, such as methanol and even water, can be used as mobile phases. The choice of solvent is only limited by the type of detector and the choice of temperature.
The detectors normally used are UV probes, so that the mobile phase should not absorb. Due to the different interactions between analytical substance, stationary and mobile phase, the substances are separated and provide a characteristic retention time for these conditions.
Features of HPLC
- rapid simultaneous determination of organic substances in solution (no homogeneity problems) in the trace range up to the preparative scale
- analysis times of generally a few minutes per sample; difficult samples may take up to one hour or more per analysis
- high accuracy and reproducibility of the analysis results using standard solutions for the calibration of the method
The substances must be completely dissolved.
HPLC analysis examples
- analysis of environmental samples
- separation of low-molecular cell constituents
production control in organic synthesis