Selection guide for biomolecule separation modes

Features and uses of the different 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.

  Ion exchange
BioPro IEX series
Size exclusion
YMC-Pack Diol
For biochromatography
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
Resolution +++ ++ +++
Speed ++ - +++ + +++
Loading +++ ++ ++
Stability +++ +++ + - ++
Usage Proteins
・Charge variant analysis
・Isoform analysis
Analysis of nucleic acids
Impurity analysis of
antibody-drug conjugate
Peptide mapping
Analysis of nucleic acids

Separation mechanism of each separation mode

Ion exchange chromatography

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

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

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.