App Note: How does the analysis of biomolecules benefit from UHPLC?

Ultra high performance liquid chromatography (UHPLC) provides improved separation speed, throughput, and sensitivity by employing stationary phase particles of around 2μm or smaller. UHPLC has found widespread use in the analysis of small molecules in pharmaceutical, food, and environmental areas. Are these analytical benefits also applicable to the separation of larger molecules, such as proteins and peptides? Biomolecules have, apart from their size, other differences to small molecules (e.g. charges, complex structures) that make the application of UHPLC less straightforward. This article discusses the rationale of using UHPLC in life sciences and demonstrate where the potential is. It also discusses the requirements needed for a true bio UHPLC system and shows examples of relevant biomolecule separations…

Ultra high performance liquid chromatography (UHPLC) provides improved separation speed, throughput, and sensitivity by employing stationary phase particles of around 2μm or smaller.

UHPLC has found widespread use in the analysis of small molecules in pharmaceutical, food, and environmental areas. Are these analytical benefits also applicable to the separation of larger molecules, such as proteins and peptides?

Biomolecules have, apart from their size, other differences to small molecules (e.g. charges, complex structures) that make the application of UHPLC less straightforward. This article discusses the rationale of using UHPLC in life sciences and demonstrate where the potential is. It also discusses the requirements needed for a true bio UHPLC system and shows examples of relevant biomolecule separations.

An important physical parameter in the discussion of chromatographic performance of large molecules is the diffusion coefficient. As a consequence, chromatography, being a diffusion-controlled process, is highly influenced by the molecular size of the analyte. The increase of flow rates typically applied to increase throughput in UHPLC methods works for small molecules, but appears counter intuitive for the analysis of slower-diffusing molecules, such as proteins.