Liquid chromatography is a technique used to separate a sample into its individual parts. This separation occurs based on the chemical or physical interactions of the sample with the mobile and stationary phases.
How does liquid chromatography work?
Because there are many stationary/mobile phase combinations that can be employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. Liquid-solid column chromatography, the most popular chromatography technique, features a liquid mobile phase that slowly filters down through the solid stationary phase, bringing the separated components with it.
It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different transportation rates for the different components and leading to the separation of the components as they flow out of the column.
High performance liquid chromatography
There are many different types of chromatography techniques and systems available for a wide range of applications all of which are defined as High Performance Liquid Chromatography (HPLC).
HPLC analysis focuses on macromolecule isolation through chemical interaction, affinity or hydrodynamic volume. SEC-HPLC works by physical interaction with the chromatography columns porous media – this is a noteworthy difference between SEC and many other liquid chromatography techniques. Chemical interaction of the sample with the column is not required or wanted as the separation should be based only on the molecular size (by a particle's Stokes radius). SEC is used primarily for the analysis of large molecules such as proteins, polymers and polysaccharides.
Click here to view our UPLC vs HPLC comparison.
GPC / SEC Columns
In SEC columns, smaller molecules in the sample will be able to enter the pores of the porous media, reside there longer, or enter more pores more often. On the other hand, larger molecules in the sample are more restricted in the size of pores they can enter, enter less often, or simply bypass the pores if they are too large to enter the pores.
Larger molecules will therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. In SEC / GPC remember that Big Ones Come Out First.
GPC / SEC detector solutions
Malvern Panalytical offer a range of systems and advanced detectors for Size-exclusion chromatography (SEC), also known as Gel permeation chromatography (GPC) or gel filtration chromatography, for the measurement of absolute molecular weight, molecular size, intrinsic viscosity, branching and other physical parameters.
