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non-specific interactions, such as hydrogen bonding and hydrophobic interaction,
in governing affinity in ion exchange materials (16). Most of the time,
it is those nonspecific interactions that can be exploited to generate the unique
displacer–stationary phase interaction leading to high affinity displacers. Studies
done using a homologous set of displacers (17–19) have shed light into the structural
components that would increase the affinity of a displacer on a particular
stationary phase. For example, increased displacer affinity with increasing
flexibility and number of aromatic rings was observed on polymethacrylatebased
Waters strong cation exchange resin (17,18). Similarly, on hydrophilic
resins, such as agarose-based SP Sepharose XL (from GE healthcare), displacer
affinity was shown to be dominated by the electrostatic interactions (charge of
the displacer), whereas hydrophobicity was the key component for displacer
affinity on polystyrene-divinylbenzene-based supports (19).
In the early years (1978–1995), high molecular weight displacers were
utilized for displacement chromatography for purification of many proteins,
such as the use of carboxymethyldextrans for purification of -lactoglobulins,
ovalbumin, -lactalbumin and soy-bean tripsin (20–23). Other examples of
such displacers are chondroitin sulfate (24) and Nalcolyte 7105 (25). Nalcolyte
7105 was utilized as a displacer for the purification of a four-component
protein mixture composed of ribonuclease, -chymotripsinogen, cytochrome
A and lysozyme resulting in successful purification at preparative scale on
a cation exchange support (26). Nontoxic displacers, such as protamine and
heparin sulfate, were reported by Gerstner et al. (27–29) for use in anion
exchange systems. Protamine sulfate was later utilized by Barnthouse et al.
(30) for purification of recombinant human brain-derived neurotrophic factor,
rHuBDNF, using cation exchange displacement chromatography.
One of the advances in displacement chromatography came with the introduction
of low molecular weight displacers (