Ion-exchange Phases Selectivity
Ion-exchange phases separate solutes on the basis of differences in ionic charge. Retention in ion-exchange chromatography is determined by the pH of the eluent, the nature and ionic strength of the buffer and temperature. Column efficiencies are lower than in reversed-phase HPLC. Eluents are normally aqueous but can contain some organic components.
Both silica-based and polymer-based ion-exchangers are available. For the former, ionic species are attached to the silica surface, whereas for the latter the ion-exchange groups are distributed throughout the matrix. Silica-based materials maintain mechanical strength and higher efficiency advantage, whereas the polymer-based materials have greater pH stability.
Ion-exchange is used for the analysis of small ions but the key application area of the technique is the separation of biomolecules such as peptides, proteins, and oligonucleotides. Weak ion-exchangers are used for the analysis of inorganic ions, a technique more specifically termed ion chromatography.
The exchange capacity of an ion-exchanger is an important measure of its retentivity (typically measured in milliequivalents per gram material). For any column, the packing density of the phase must also be taken into account. Wide pore materials will typically have lower ion-exchange capacities.
Cation-exchange phases contain negatively charged functional groups and retain positively charged cations. Conversely, anion-exchange phases retain negatively charged analytes by their positively charged functional groups. In the schematics below, the ion strength of the counterions can be adjusted to shift the equilibrium position and thus the retention times of the analytes.
|Type||Strength||Nomenclature||Typical Functionality||pH Ionisation Range|
|Anion||Weak||WAX||Amine||Ionised at specific pH|
|Strong||SAX||Quaternary Ammonium||Ionised over complete pH range|
|Weak||WCX||Carboxylic Acid||Ionised at specific pH|
|Phase||Base Material||Classification||Particle Size||Capacity||pH Range|
|Bettsep® SP300/700||Polymer||SCX||30um, 70um||≥130 mg Lysozyme||2-12|
|Bettsep® Q300/700||SAX||30um, 70um||≥130 mg Lysozyme||2-12|
|Sepromax® S40||SCX||40μm||≥ 60 mg/ml hlgG||2-12|
|Sepromax® CM40||WCX||40μm||≥80 mg Lysozyme||6-12|
|Sepromax® Q40||SAX||40μm||≥ 100 mg BSA||2-12|
|Sepromax® D40||WAX||40μm||≥ 100 mg BSA||2-9|