Size Exclusion Chromatography (SEC) separates molecules based on their size, or more precisely, their hydrodynamic volume. It is based on the discrimination of individual sample components by the pores of the packing material. Large sample molecules cannot or can only partially penetrate the pores and elute from the column first, whereas smaller molecules can access all or a larger number of pores and elute later. SEC is the only mode of chromatography that does not involve interaction with a stationary phase by means of adsorption or partitioning of the solutes.
The terms SEC, GFC (gel filtration chromatography) and GPC (gel permeation chromatography) all refer to the same chromatographic technique. In GFC an aqueous mobile phase is used, while an organic mobile phase is employed in GPC. The general term SEC covers both uses.
Separation in Ion Exchange chromatography (IEC or IEX) is based on reversible adsorption of charged solute molecules to immobilized functional groups of opposite charge. Biomolecules generally have charged groups on their surfaces, which change with the buffer pH. Elution can be accomplished by changing the ionic strength or the pH, of which changing the ionic strength by increasing the salt concentration is most common.
IEX is further subdivided into cation exchange and anion exchange chromatography. Anion and cation exchange phases are classified as strong or weak, depending on how much the ionization state of the functional groups vary with pH. A strong ion exchange phase has the same charge density on its surface over a broad pH range, whereas the charge density of a weak ion exchange phase changes with pH, affecting its selectivity, which differs at different pH values.
Reversed Phase Liquid Chromatography (RPC) has become an accepted tool for the separation of peptides, proteins and other biopolymers in the pharmaceutical, chemical and biochemical industries.
Reversed Phase Liquid Chromatography (RPC) separates molecules based on nonpolar interactions and requires a nonpolar stationary phase and a polar mobile phase. Typically the mobile phase consists of a mixture of water (buffer) and acetonitrile, methanol, THF, or 2-propanol.
Hydrophobic interaction chromatography (HIC) is based on non-polar interactions that are induced by high salt mobile phases. Stationary phases are similar to reversed phase chromatography (RPC) but the density of functional groups is lower. A weakly non-polar stationary phase is used with an aqueous mobile phase containing a high concentration of a chaotropic salt.
The technique is mainly applied to the separation of proteins, which are eluted by decreasing the salt concentration or by adding a low percentage of organic solvent. Although also based on hydrophobic interactions, selectivity in HIC separations is distinctly different from that in reversed phase chromatography. Despite the lower peak capacity in HIC compared to RPC, HIC has the advantage that the mobile phase conditions (primarily aqueous) do not usually disrupt higher-order protein structures.
TSKgel FcR-IIIA columns are based on a recombinant Fc gamma IIIa receptor which is immobilized as a ligand on polymer particles. They separate monoclonal antibodies according their affinity to the receptor and enable a fast analysis of biological activity as well as fractionation along with biological activity.
Aqueous size exclusion chromatography (SEC) is an established method for the analysis of proteins and monoclonal antibodies, including fragments, monomers, and aggregates under non-denaturing conditions.
TSKgel SW series columns have been the industry’s workhorses for the SEC analysis of biotherapeutics for decades. TSKgel UP-SW3000-LS columns are the latest addition to this renowned column family.
TSKgel UP-SW3000-LS columns offer high resolution, sharp peak shape and high efficiency yielding methods that are robust, reproducible and easily transferable between UHPLC and HPLC systems.
These U/HPLC columns provide significantly lower noise levels than other columns when coupled with advanced detectors, shortening the time for equilibration and improving data quality. For multi-angle light scattering (MALS) applications, columns with low noise levels yield high signal-to-noise ratios and this will improve sensitivity of detection.
For mass spectrometry applications, low shedding columns will increase electrospray ionization efficiency and enhance overall MS performance and instrument uptime.