Ion Exchange Chromatography
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.
IEC 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.
Applications
Being a non-denaturing technique, IEC is one of the most frequently used modes in the separation of biomolecules. Proteins have numerous functional groups that can have both positive and negative charges. IEC separates proteins according to their net surface charge, which is dependent on the pH and ionic strength of the mobile phase.
| Typical applications are |
| Separation of peptides and proteins |
| Analysis of charge isomers of proteins |
| Quality control of recombinant proteins, such as monoclonal antibodies (mAbs) |
| Separation of oligonucleotides, siRNA, PCR fragments and related nucleic acids |
| Analysis of sugars, amino acids, nucleic acid bases, and small drug candidates |
| TSKgel Ion Exchange Columns |
Tosoh Bioscience offers a broad line of high efficiency columns for anion as well as cation exchange chromatography.
The product line contains polymethacrylate- and silica-based columns for the analysis of proteins, peptides, and nucleic acids. Polystyrene divinylbenzene-based ion exchange columns are available for analyzing low molecular weight sugars and drug candidates, as well as amino acids. Hydrophilic polymer-based columns packed with nonporous resin particles are available for the separation of protein, protein aggregates, PEGylated proteins and charge isomers of mAbs.