prof.

aza
 Affinity chromatography is the most
specific chromatographic method.
The interaction is biochemical in
nature, e.g.
 The highly specific nature of these
interactions is due to the fact that
the two participating compounds are
ideally suited to each other both
spatially and electrostatically.

prof. aza


prof. aza
Fig. 17.1 Principle of affinity
chromatography. L, ligand; S, sample. δ+
and δ - represent partial charges (less
than one elementary charge).
prof. aza
If other sample components are present in the solution, e.g.

then they do not match the ligand and are not adsorbed.

The sample

is specifically retained by the stationary phase,

prof. aza
whereas the other molecules (proteins,
enzymes. etc.) are removed by the
mobile phase.
The sample is now freed from all other
impurities. It cannot
be isolated until it has been separated
from the stationary phase, this being
done by elution with a solution
containing a product with a great
affinity to the ligand or even by a
change in pit or ionic strength.
prof. aza
 As any biochemical activity can take
place in just one specifically defined
medium, it is clear that a p14 or
concentration gradient may break the
specific intact ion.

prof. aza
prof. aza
prof. aza
prof. aza
 Affinity chromatography differs from other
chromatographic modes in that a suitable stationary
phase can specifically ‘catch’ either a single or several
components out of a random mix of products owing
to a naturally occurring biospecific bond. A suitable
elution process then provides the pure compound (s).
 No column is required for isolation purposes. hence
affinity chromatography is frequently carried out in
open Systems. e.g. suction filtering. Classical columns
with a hydrostatic eluent feed offer a further
possibility. This chapter. however, is confined to a
description of separations with high- performance
stationary phases (10 mm and below ) with high rapid
chromatography can be achieved. Very small
columns may be used.

prof. aza
 No column is required for isolation
purposes. Hence affinity
chromatography is frequently carried
out in open Systems. e.g. suction
filtering.
 Classical columns with a hydrostatic
eluent feed offer a further possibility.

prof. aza
 This chapter however, is confined to a
description of separations with high-
performance stationary phases (10 mm
and below ) with high, rapid
chromatography can be achieved.
 Very small columns may be used.

prof. aza
 Stationary phases for affinity
chromatography can he prepared by
the user himself, often starting from
diol- or amino-silica.
 However, it is much simpler to buy an
‘activated’ gel which allows the
desired ligand to be bound according
to well known methods. For separation
problems which require common
ligands, ready-to-use stationary
phases are commercially available.

prof. aza
 Most phases are built up with a long-
chain group between the silica and the
ligand, the so-called spacer to
guarantee free accessibility of the
sample molecules to the bonding site at
the ligand.

prof. aza
 The bonded sample can be eluted by a
gradient (pH, ionic strength or
competing sample) or by a ‘pulse’ &. the
latter being a typical affinity
chromatography characteristic.
 The bond-breaking compound is
injected whilst the mobile phase
passes through the column unchanged.

prof. aza
 The sample size is restricted only
by the bonding capacity of the
column. The yields of biologically
active protein frequently reach
100%, which means that
denaturation and irreversible
adsorption are negligibly low in
many cases.
prof. aza
 Anti-IgG3 ligands bond all antibodies
of the lgG class specifically.
 The stationary phase synthesis
required for the separation of lgG
shown in Fig. 17.3, was described by
the authors in the reference cited in
the caption. The mobile phase starts
off with a pH of 7.4: a switch to pH
2.2 breaks the antigen-
immunoadsorbent bond and the IgG is
eluted.
prof. aza
 As the characteristic lgG fluorescence
is quenched at pH 2.2. the pH of the
column effluent had to be raised by
adding a buffer of pH 8. The yield of
active lgG was in excess of 97% . The
stationary phase remains active for a
long time provided that the column is
stored at 4 0C when not in use.

prof. aza
 Lactins are plant proteins which can
specifically bond hexoses and
hexosamines.
 Concanavalin A is a lectin widely used in
the affinity chromatography and can
isolate glvcopmoteins, glvcopeptides
and glycolipids. Elution is triggered by
a pulse of sugar solution.

prof. aza
 Separation of the enzyme
peroxidase (a lycoprotein) is shown
in Fig. 17.4.

prof. aza
prof. aza
prof. aza
prof. aza
 Detection is effected at two
wavelengths simultaneously:
280 nm (non-specific for all
proteins) and 405 nm
(peroxidase-specific owing to
the protohaemin group).

prof. aza
 Cibacron Blue is a popular ligand as it
can hind a large number of enzymes
and also some blood proteins. It is
often referred to as a pseudo-affinity
ligand’ because it is a synthetic
triazine dye and not a naturally
occurring biomolecule.

prof. aza
 This greatly reduces the activity of the
eluted enzyme in most cases. One
advantage of Cibacron Blue is its low
price. The separation of isoenzymes H4
and M4 from lactate dehydrogenase
(LDH). eluted in sequence by a gradient of
reduced nicotinamide adenine dinucleotide
(NADH). is shown in Fig.17.5. A post-
column reactor for measuring enzyme
activity was used as a detector (see
Section 20.5).
prof. aza
Retention due to biospecific interaction
using a ligand molecule chemically coupled
to a dextran or cellulose matrix - (see 3.4
above for diagram).
Hence may be able to isolate analyte from
complex mixture.
Examples

prof. aza
 Analyte Bound ligand
species
enzyme substrate ??, coenzyme,
reversible competitive
inhibitor
antigen antibody
antibody antigen, cell fragment
hormone receptor, binding protein
receptor effector molecule, eg
hormone, neurotoxin
prof. aza
prof. aza
 Elution can be by displacement with
ligand molecules in free solution. But
analyte then eluted as complex with
ligand. Better to elute by change of pH
to weaken binding.
 Chemistry of ligand coupling to matrix
using cyanogen bromide.

prof. aza