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Ion-exchange chromatography separates molecules according to type and strength of their charge. For this, a column is used which consists of separation resin beads that are either positively or negatively charged. A positively charged bead, known as an anionic-exchanger, will tend to bind to biomolecules with a net negative charge, and a negatively charged bead, known as a cationic-exchanger, will tend to bind to biomolecules with a net positive charge. The complex surface of biomolecules consists mostly of both anions and cations whose charge is neutralized at the isoelectric point (i.e., where pH = IP). Thus, by carefully choosing the buffer pH value, a suitable range above the IP of the target molecule will permit exchange of anions with quaternary ammonium groups (“Q” Resins) or below the IP will permit exchange of cations with sulfonic acid groups (“S” Resins). The binding of the biomolecules to the beads is fully reversible and their removal (elution) is usually achieved through the flow of increasing amounts of salts across the resin. The cation and anion salt ions compete with the binding of the biomolecules to the charged beads causing the biomolecule to release from the resin and elute from the column. The order in which biomolecules elute is dependant on their net charge, with the weakest charged molecules eluting first.
The chromatographic separation of biomolecules is influenced by four parameters; resolution, resin capacity, process speed (flow rate) and recovery. Process flow rates and resolution are influenced by the resin bead size. Resins of larger bead sizes, >40 µm, typically permit high flow rates but have reduced resolution where as resins with smaller bead sizes, <40 µm, have increased resolution but require slower process flows due to backpressure. Ion-exchange resins often have high binding capacities, high flow characteristics and potentially excellent resolution. Early in a purification process, process speed is important and IEC is often used for the separation of biomolecules from large volumes of sample at the primary capture step of a purification scheme. In subsequent steps were resolution becomes more important, process speed is sacrificed in favor of increased resolution. Again, IEC resins with small bead sizes are often used in the final polishing steps of a purification scheme. The typical means of increasing resolution is to increase the bed height of the column. However, this leads to increased backpressure, which can physically damage the resin, and thus reduces process time. High resolution and fast process times are therefore tradeoffs made in designing a purification scheme.
Biofox™ IEC resins eliminate this tradeoff by virtue of their ability to withstand high pressures. This property allows for the design of purification schemes using columns with high resolution and high process flow rates. Where commonly used IEC resins will fail at pressures above 100 psi, Biofox™ IEC resins maintain their physical integrity at pressures of up to 580 psi. Biofox™ resins are produced from agarose beads using a proprietary cross-linking method that results in a highly porous and physically stable resin bead with high capacity and excellent recovery properties. By no sacrificing resolution for process speed, more efficient and cost-effect purifications schemes are possible.
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