What affects ion exchange chromatography?
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What affects ion exchange chromatography?
The factors that affect separation during ion exchange chromatography include the surface area of the stationary phase (resin bead size); the density of exchange sites on the stationary phase surface (cross-linkage); the flow rate of the mobile phase (resin bead size and column geometry; system pressure in high- …
What are the limitations of ion exchange chromatography?
One of the main disadvantages of ion exchange chromatography is its buffer requirement: because binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the stationary phase, IEX columns must be loaded in low-salt buffers.
How does salt affect ion exchange chromatography?
An impure protein sample is loaded into the ion exchange chromatography column at a particular pH. A salt gradient is used to elute separated proteins. At low salt concentrations, proteins having few charged groups are eluted and at higher salt concentrations, proteins with several charged groups are eluted.
How does temperature affect ion exchange chromatography?
(2008) studied the effect of temperature on ion exchange equilibrium for uni-univalent systems and , and uni-divalent systems and using Duolite ion exchange resin A-102 D, and they showed that the affinity of the resin for the metal ions increases with increasing temperature.
How can the resolution of chromatography be improved?
How to Improve Resolution in HPLC
- Increasing column length.
- Decreasing particle size.
- Reducing peak tailing.
- Increasing temperature.
- Reducing system extra-column volume.
How does pH affect ion exchange?
An increase in pH increases the negatively charged nature of the sorbent surface. This leads to an increase in the electrostatic attraction between positively charged metal ions and negatively charged sorbent and results in increased sorption of metal ions.
Why is ion exchange chromatography so effective?
Ion exchange chromatography can be used to separate proteins because they contain charged functional groups. The ions of interest (in this case charged proteins) are exchanged for another ions (usually H+) on a charged solid support. The solutes are most commonly in a liquid phase, which tends to be water.
How does pH affect ion exchange chromatography?
Many chromatographers also use changes in pH to affect a separation. In cation exchange chromatography, raising the pH of the mobile phase buffer will cause the molecule to become less protonated and hence less positively charged.
Why is NaCl important for ion exchange chromatography?
NaCl dissociates in Na+ and Cl- in solution. In anion-exchange chromatography, Cl- competes with negatively charged analytes to bind with positively charged stationary phase, thereby eluting the analytes.
How can chromatography be improved?
In liquid chromatography, the easiest way to increase a solute’s retention factor is to use a mobile phase that is a weaker solvent. When the mobile phase has a lower solvent strength, solutes spend proportionally more time in the stationary phase and take longer to elute.
How can separation of chromatography be improved?
Depending on the situation, separations can sometimes be improved by increasing the column plate number, by using smaller particles or by increasing column length. The disadvantages of these approaches are higher operating pressures and increased separation times for longer columns.
Should I use anion or cation-exchange?
Based on the acidic theoretical pI, if you do the purification at a neutral pH (7-8), the net charge will be negative, so you should use an anion exchange column (Anion exchange columns have positive charge). Cation exchange might work.