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Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

658 Separation and Purification of Biochemicals

FIGURE 5 Ion exchange chromatography. Molecules interact through their net charge, and are eluted by increasing
ionic strength. [From Ion Exchange Chromatography, Principles and Methods. Reproduced with kind permission of
Amersham Pharmacia Biotech Limited.]

of comparatively large sample volumes, in comparison to and pH, provided that the ionic strength of the surrounding
other modes. The recovery of biological activity is usually buffer is low. However, the relationship between retention
excellent.Bothweakandstronganionandcationexchang- and net charge is usually not so straightforward, since the
ers are used. In the case of the weak anion and cations chargedistributionoverthesurfaceoftheproteinmolecule
exchangers, usually carrying diethylaminoethyl (DEAE) is not uniform and steric effects also play an important role
and carboxymethyl (CM) ligands, respectively, the charge in determining the magnitude of interaction. Two models,
density of the stationary phase surface depends on the pH the stoichiometric displacement and the electrostatic in-
of the mobile phase. Strong anion and cation exchang- teraction model are currently used in protein IEC that link
ers carrying sulfonic acid (S) or quaternary ammonium the respective retention factors to the ionic strength of the
(Q) groups are independent of the pH in that sense. Since mobile phase and the number of charged groups involved
the net charge of the protein, and in the case of the weak in the adsorption/desorption process.
ion exchangers, the charge of the chromatographic sur-
face are both pH dependent, control of the mobile phase
2. Hydrophobic Interaction Chromatography
pH is very important in IEC and great attention has to
be paid to the nature of the buffer as well. The sample HIC was developed in the 1970s especially for the
should be at the same pH as the initial mobile phase, and preparative separation of proteins using predominately
of comparable ionic strength, in order to maximize the hydrophilic, agarose-based stationary phases into which
binding. some mildly hydrophobic ligands had been imbedded at
In IEC, the sample components are retained by virtue of fairly low density. Most of these early stationary phases
electrostatic interactions between the charged molecules contained in addition ionic groups, retention was there-
and the oppositely charged chromatographic surface. Dur- fore due to a mixed mode mechanism. More recently,
ing binding, the target molecule is concentrated, and sub- rigid macroporous silica or polymeric supports have been
sequently may be eluted in a purified and concentrated introduced that are covered with a covalently bound hy-
form. Elution with (linear or step) gradients of increasing drophilic surface layer that incorporates appropriate hy-
salt concentration (mostly NaCl) is most widely used in drophobic ligands, such as short alkyl, aryl, or polyether
the IEC of proteins. The increase in the salt concentration chains at a comparatively low concentration. Protein re-
of the eluent results in a “screening” of the charges present tention and selectivity depend on the nature and size of
at the protein and at the stationary phase surface. As a re- the hydrophobic moieties. In practice, the column tem-
sult the attraction is diminished and the proteins elute. A perature, the eluent pH, and the nature of the stationary
pH gradient may also be used for protein elution; however, phase matrix will also have a significant influence on a
due to the technical difficulties in generating smooth and HIC separation. The driving force for retention in HIC is
reproducible pH gradients, this principle is less commonly a hydrophobic effect, i.e., less an attraction between the
employed. protein molecules and the stationary phase but rather the
By a rule of the thumb, retention occurs in IEC when tendency of the surrounding water molecules to avoid con-
the sign of the fixed charges at the surface is the opposite tact with a hydrophobic surface and hence to bring such
of that of the net charge of the protein, which in turn is surfaces into direct contact with each other, as illustrated
proportional to the difference between its isoelectric point in Fig. 6.