Displacement chromatography
The result is that the components are resolved into consecutive "rectangular" zones of highly concentrated pure substances rather than solvent-separated "peaks".
[n 2] A molecule with a high affinity for the matrix (the displacer) will compete more effectively for binding sites, leaving the mobile phase enriched in the lower-affinity solute.
At the beginning of the run, a mixture of solutes to be separated is applied to the column, under conditions selected to promote high retention.
The other components also begin to form zones, but the continued supply of the mixed feed at head of the column prevents full resolution.
For some matrices, reactive groups on the stationary phase can be titrated to temporarily eliminate the binding sites, for instance weak-acid ion exchangers or chelating resins can be converted to the protonated form.
To achieve separation in either elution or displacement chromatography, there must be appreciable differences in the affinity of the respective solutes for the stationary phase.
In contrast to elution chromatography, solutes separated in displacement mode form sharp-edged zones rather than spreading peaks.
Furthermore, because displacement chromatography takes advantage of the non-linearity of the isotherms, loadings are deliberately high; more material can be separated on a given column, in a given time, with the purified components recovered at significantly higher concentrations.
High-retention conditions can be employed without gradient operation, because the displacer ensures removal of all solutes of interest in the designed run time.
[6][7][8] Because of the concentrating effect of loading the column under high-retention conditions, displacement chromatography is well suited to purify components from dilute feed streams.
Another disadvantage is that the raw chromatogram, for instance a plot of absorbance or refractive index vs elution volume, can be difficult to interpret for contiguous zones, especially if the displacement train is not fully developed.
In these cases, displacement chromatography is an efficient technique for the purification of proteins from complex mixtures at high column loadings in a variety of applications.
[13][14][15] This research was significant in that it represented a major departure from the conventional wisdom that large polyelectrolyte polymers are required to displace proteins in ion exchange systems.
[16][17][18][19][20][21][22] In addition, the utility of displacement chromatography for the purification of recombinant growth factors,[23] antigenic vaccine proteins[24] and antisense oligonucleotides[25] has also been demonstrated.
Displacement chromatography can be readily carried out using a variety of resin systems including, ion exchange, HIC and RPLC.
