SOLUTIONS

Ion Exchange Chromatography

 

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.


Anion Exchange Chromatography

Anion exchange chromatography is practiced with either a strong or a weak anion exchange column, containing a quarternary ammonium ion, or with a weak anion exchanger, having either a tertiary or secondairy amine functional group, such as DEAE (diethylaminoethyl). A counter ion, often Cl-, maintains electroneutrality.

In ion exchange chromatography the pH of the mobile phase buffer must be between the pI or pKa of the charged molecule and the pKa of the charged groups on the solid support. For instance, in anion exchange chromatography a molecule with a pI of 6.8 is run in a mobile phase buffer at pH 8.0 with the solid support pKa at 10.3.


TSKgel Column Type Benefit
Q-STAT, DNA-STAT  Nonporous with high surface density of quaternary ammonium groups
DEAE-5PW, SuperQ-5PW  Polymethacrylate resin derivatized with diethylaminoethyl (DEAE) and trimethylamino (SuperQ) ligands 
BioAssist Q Contain very large pores (400 nm), resulting in high binding capacity and improved recovery of activity; available exclusively in PEEK housing
DEAE-NPR, DNA-NPR Nonporous with 2.5 µm particles; fast analysis; high protein recovery
DEAE-2SW, DEAE-3SW, QAE-2SW  Silica-based with diethylaminoethyl (DEAE), and trimethylamino (QAE) functional groups
Sugar AXG, Suger AXI, SAX   Specialty columns for the analysis of mono and disaccharides, as well as organic acids and sugar alcohols 

More Information on TSKgel Anion Exchange Columns


Cation Exchange Chromatography

Cation exchange chromatography is practiced with either a strong or a weak cation exchange column, containing a sulfonium ion, or with a weak cation exchanger, having usually a carboxymethyl (CM) functional group. A counter ion, often Na+, maintains electroneutrality.

In ion exchange chromatography the pH of the mobile phase buffer must be between the pI or pKa of the charged molecule and the pKa of the charged groups on the solid support. For example, a molecule with a pI of 8.2 is run in a mobile phase buffer at pH 6.0 with the solid support pKa at 1.2 in cation exchange chromatography.


TSKgel Column Type 
Benefit 
CM-STAT, SP-STAT
Nonporous with high surface density of carboxymethyl (CM) and sulfopropyl (SP) groups
CM-5PW, SP-5PW
Polymethacrylate resin derivatized with carboxymethyl (CM) and sulfopropyl (SP) ligands
BioAssist S  Contain very large pores (130 nm), resulting in high binding capacity and improved recovery of activity; available exclusively in PEEK housing
SP-NPR Nonporous with 2.5 µm particles; fast analysis; high protein recovery
CM-2SW, CM-3SW,
SP-2SW 
Silica-based with carboxymethyl (CM) and sulfopropyl (SP) functional groups
SCX
Specialty column for the analysis of organic acids, saccharides and alcohols

More Information on TSKgel Cation Exchange Columns