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.
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
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|
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|
|Silica-based with carboxymethyl (CM) and sulfopropyl (SP) functional groups|
|SCX||Specialty column for the analysis of organic acids, saccharides and alcohols|
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