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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 methacrylate 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 non-porous 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 Non-porous 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 Non-porous resin 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

    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 Non-porous 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
    		 Non-porous resin 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, OApak-A Specialty columns for the analysis of organic acids, saccharides and alcohols