High Performance Liquid Chromatography

HPLC is a technique used to separate the different components in a mixture, to identify and to quantify each component. It separates the components of complex biological samples or of similar synthetic chemicals from each other. HPLC instruments typically includes a sampler, pumps and a detector.

Measurement theory

Common methods include the combination of water with various organic solvents – the most common are acetonitrile and methanol. Some HPLC techniques also work water-free.

Pumps pass pressurized organic solvents through a column filled with a solid adsorbent material (granular material made of solid particles, e.g. silica or polymers, 2-50 micrometers in size). Each component in the sample interacts slightly different with the adsorbent material, which results in different flow rates. These different rates separate one component from the others as it flows out the column. Composition and temperature play a major role in the separation process. The detector analyses the amount of sample component emerging from the column. Common detectors are UV-Vis, photodiode array (PDA) or are based on mass spectroscopy. A digital microprocessor and user software control the HPLC instrument and provide data for further analysis.

Very high operational pressures (50-350 bar) distinguishes HPLC is from the traditional, low pressure, liquid chromatography. The sample amounts separated in analytical HPLC are very small: typical column dimensions are 2.1-4.6 mm diameter, and 30-250 mm length. The columns are also made with smaller sorbent particles (2-50 micrometer in average particle size). This gives these modern technique superior resolving power when separating mixtures.

Examples of use
  • Detection of vitamin D levels in blood serum or performance enhancement drugs in urine
  • Measurement of components during the production process of pharmaceutical and biological products
  • Analysis of substances processed in agriculture and food manufacture