Page 251 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioTechnology

P1: GLQ/GLE P2: GPB Final Pages
Encyclopedia of Physical Science and Technology EN014J-683 July 30, 2001 20:3

Separation and Purification of Biochemicals 659

activity occurs. For this reason, HIC is widely employed
in preparative and process scale isolation and purification
of proteins. It should be noted, however, that some slight
changes in the protein structure might occur in HIC due
to a weakening of the hydrophobic forces responsible for
maintaining that structure.

3. Reversed-Phase Chromatography
RPC has become the predominant branch of analyti-
cal chromatography (HPLC) in the life sciences and in
biotechnology. Preparative applications of RPC are the
exception, although some have been reported. Most com-
monly, bonded high performance stationary phases pre-
pared by covalently binding hydrophobic ligands such as
C 4 -, C 8 -, and C 18 -alkyl chains or aromatic functions to
the surface of a rigid siliceous or polymeric support are
FIGURE 6 Hydrophobic interaction chromatography. The mole-
used. Due to the pronounced hydrophobic character of the
cules are bound in presence of high salt as a result of increased
hydrophobic interaction and eluted by decreasing the salt con- stationary phase, proteins and peptides bind tightly from
centration. [From Hydrophobic Interation Chromatography, Princi- a neat aqueous mobile phase and require hydro-organic
ples and Methods. Reproduced by kind permission of Amersham eluents for release. The separation of peptides and pro-
Pharmacia Biotech Limited.]
teins in RPC is therefore typically carried out by gradient
elution with increasing concentration of an organic mod-
ifier such as acetonitrile, methanol, tetrahydrofuran, and
According to the solvophobic theory of HIC, the mag- isopropanol. In addition, the mobile phase usually con-
nitude of retention is mainly determined by the so-called tains low levels of trifluoroacetic or phosphoric acid. The
cavity term, which expresses the free energy change upon role of the acids is to protonate the residual silanol groups
adsorption that is due to the reduction in hydrophobic sur- at the surface of the siliceous support and the carboxyl
face (of the sample molecule or the hydrophobic moiety of groups of the eluates as well as to form ion pairs with
the stationary) exposed to water (i.e., the mobile phase) as the charged amino groups of the substances to be sepa-
the two combine. The cavity term is given roughly by the rated. Ion-pairing agents such as perchlorate can be used
product of the microthermodynamic surface tension of the at neutral pH.
mobile phase and the molecular contact area upon bind- The retention strength increases roughly with the size
ing. Thus retention can be considered a solvent effect and and the hydrophobicity of a given substance. In the case
generally the retention factor decreases with the surface of proteins and larger polypeptides, the prediction of
tension of the mobile phase, i.e., when the salt concentra- the retention behavior becomes difficult, since the three-
tion of a given eluent is lowered. Retention is enhanced by dimensional (3D) structure, i.e., the number and location
a high salt concentration in the aqueous mobile phase and of the hydrophobic patches at the molecule surface, be-
therefore gradient elution with decreasing salt concentra- comes decisive. Moreover, under the conditions of RPC
tion is most commonly used in protein HIC. Reducing the withitsacidic,hydroorganicmobilephases,manyproteins
polarity of the eluent is another way to cause desorption tend to denature and unfold either partially or com-
of the proteins—for example, by using an ethyleneglycol pletely. Oxidation, deamidation, aggregation, and frac-
gradient. The addition of chaotropic substances (guani- tionation are also possible. The use of RPC in prepara-
dine, urea) or detergents as well as a change in temperature tive work commonly requires the refolding of the prod-
or the pH have been used. The typical initial conditions uct into its native configuration after separation. This
(e.g., 1.5 M ammonium sulfate or 4 M sodium chloride), has been shown to be possible for a number of peptides
make HIC an ideal “next step,” e.g., after precipitation and smaller proteins, which are of interest to the phar-
with ammonium sulfate or elution in a high salt (NaCl) maceutical industry such as h-insulin and h-growth hor-
buffer during IEC. mone (hGH). The number of preparative RPC applica-
As in IEC, aqueous mobile phases may be used in HIC. tions in this area remains nevertheless rather small. RPC
Due to the comparatively mild operating conditions of is, on the other hand, often used for the final polish-
HIC, the molecular integrity of the native protein is nor- ing of oligonucleotides and peptides made via chemical
mally well preserved and no significant loss of biological synthesis.