Selection guide for biomolecule separation modes
YMC offers three different ranges of products with different separation modes for applications involving nucleic acids, peptides, and proteins: reversed-phase, ion exchange, and size exclusion chromatography. Ion exchange chromatography is used for analysing proteins with high molecular weights including antibodies. Rapid, high resolution reversed-phase chromatography is suitable for peptide mapping, an analytical method for proteins, while size exclusion chromatography is preferable for separation of molecules with large difference in molecular weights such as aggregates of antibodies.
As a result, specific separations are possible by selecting the optimal mode depending on use and purpose of the separation.
BioPro IEX series
|Principle of separation||Electric charge||Molecular weight||Hydrophobicity|
|Usable molecular weight||Up to several millions||Up to about 1,000,000||Up to about 150,000|
|Speed||++ - +++||+||+++|
|Stability||+++||+++||+ - ++|
・Charge variant analysis
Analysis of nucleic acids
|Impurity analysis of
Analysis of nucleic acids
Ion exchange chromatography is a technique used to separate ionic molecules by differences in their net charge. Generally, resins (media), which have cationic or anionic groups, are used as stationary phases and the counter-ion added buffers are used as mobile phases.
In the sample application step, molecules with opposite charge to the media bind to them by ionic interaction. Next, in the elution step, by increasing the concentration of the counter-ions in the mobile phase, molecules with the lowest net charge are eluted first and those with higher charge are eluted later.
Size exclusion chromatography is a technique used to separate molecules by their size. As a stationary phase, silica gels and polymers which have pores with a network structure are used and as a mobile phase, buffers which have high solubility and pH stability towards the compounds are used. Smaller molecules that can penetrate deeper into the pores are eluted later from the column while larger molecules which cannot enter the pores are eluted earlier from the column.
Reversed-phase chromatography is a technique used to separate molecules by hydrophobic interactions between compounds and a stationary phase and between compounds and a mobile phase. Stationary phases include silica gels and hybrid silica to which hydrophobic functional groups such as C18, C8, and C4 are bonded are used, with mobile phases being mixtures of organic solvents and aqueous acid solutions or buffers. As the concentration of organic solvents in the mobile phase is increased, the molecules with lower hydrophobicity are eluted first.