46
Bioprocess Engineering
which include steps such as separating the
most plentiful impurities Frst; performing
the most difFcult and costly separations
last; and sequencing processes to exploit
different separation driving forces such
as size, charge, and differences in boiling
points.
Chromatography is deFned as the sep-
aration of substances by the selective
binding of components to porous, solid,
sorptive media. Various types of chromato-
graphic separation are employed includ-
ing adsorption, liquid–liquid partition,
ion exchange, gel Fltration or molec-
ular sieving, afFnity, hydrophobic and
high performance liquid chromatogra-
phy
or
HPLC.
Typically,
a
combina-
tion
of
chromatographic
processes
is
used to purify proteins. ±or example,
an ion exchange column separates neg-
atively charged products from positively
charged and neutral products, where-
upon the desired products are isolated
by means of an afFnity column. AfFn-
ity chromatography is particularly versa-
tile, often displaying exquisite selectivity.
Various afFnity systems are used such
as antigen–antibody, lectin–carbohydrate
moiety, enzyme–substrate, immobilized
metal ion-histidine-containing protein, re-
ceptor–hormone, and so on. In addition,
afFnity chromatography may be used for
environmental remediation using ligands
(chelators or macromolecules) that specif-
ically bind metal ions.
Since chromatography involves the ad-
sorption of species to be puriFed (i.e.
a protein) to a support for separation,
the process is often described by either
±reundlich or Langmuir isotherms. An
empirical formula that describes the ad-
sorption of many antibiotics, hormones,
and steroids is the ±reundlich isotherm:
q
=
Ky
n
(
4
)
where
q
is the amount of solute (product
or contaminant) adsorbed per amount of
adsorbent,
y
is the solute concentration in
solution,
K
is similar to an equilibrium
constant, and
n
is an empirical constant.
The values of
K
and
n
are determined
experimentally. If the adsorption is favor-
able, then
n
<
1; if it is unfavorable,
n
>
1.
The Langmuir isotherm is not empirical;
instead it has a theoretical basis:
q
=
q
0
y
K
+
y
(
5
)
The values of
q
0
and
K
are determined
experimentally. This equation assumes
that there are a speciFc number of ac-
tive sites; the amount of sites for a given
adsorbent is reflected in the value of
q
0
.
At low solute concentrations,
q
=
q
0
.The
Langmuir equation also assumes that an
equilibrium is reached between adsorption
and desorption to the active sites. Other as-
sumptions are that adsorption takes place
monomolecularly and is localized. The
only disadvantage of chromatographic pro-
cesses is that large columns are sometimes
required that result in large pressure drops
over the columns, requiring large auxiliary
equipment like pumps.
Electrophoresis is used for the sepa-
ration of charged molecules based on
differences in both size and charge in
an electric Feld. The separation is ac-
complished by balancing the drag force
of a charged particle with the electrostatic
forces when the particle is moving with
a constant velocity. The net charge,
q
,on
a protein determines its velocity,
V
t
,and
thus its separation:
V
t
=
qE
3
πµ
D
P
(
6
)
where
E
is the electric Feld intensity,
D
P
is
the particle diameter, and
µ
is the viscosity.
The pH of the solution greatly affects the
