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Between proteins in
solution and
hydrophobic ligands
covalently bonded to
gel matrix of HIC
column
physicochemical method for
separation and analysis of
mixtures, based on
distribution of mixture
components between a
stationary phase and a mobile
(eluent) phase that flows
HIC ligands
Linear alkanes
methyl < ethyl < propyl < butyl < pentyl < hexyl
< heptyl < octyl
Aryl(aromatic groups)
e.g. phenyl
Mixed hydrophobic and aromatic interactions
()
Intermediate hydophobic ligands
e.g. polyethers PEG (polyethylene glycol) < PPG
(polypropylene glycol) < PTMG
(polytetramethylene glycol)
Milder elution conditions
Principle of HIC
Hydrophob
ic surfaces
Lo
w
salt
Highly
Hig
ordere
h
d
salt
water
Less
orde
red
wat
er
The equilibrium of the
Highly ordered water
hydrophobic interaction is controlled
shells surround the
predominantly by the salt concentration.
hydrophobic surfaces
of ligands and proteins
Ref-Hydrophobic Interaction and Reversed Phase
Chromatography
Principles and Methods-handbook by GE healthcare
High
salt
buffer
Hydro
phobic
protein
s
ma
trix
Hydro
phobi
c
ligand
1.Equilibration
HIC medium
equilibrated
with high-salt
start buffer.
Least
Hydroph
obic
proteins
2.Sample application
3.Elution 1
Start buffer causes
Decreasing salt
hydrophobic
content (using
proteins bind to hydrophobic a linear gradient)
ligands on the medium,
causes
becoming
hydrophobic proteins
concentrated on the column. to elute:
Proteins with insufficient
the least hydrophobic
hydrophobicity elute during
proteins
or just
elute first.
Ref-Hydrophobic
Interaction
and
Reversed Phase
after sample application.
Chromatography
6.Wash
5.Elution 3
Final saltfree wash
removes
any
hydrophobica
lly bound
proteins
before reRef-Hydrophobic Interaction and Reversed Phase
equilibration
Chromatography
4.Elution 2
Further decreases in
salt displace
the more
hydrophobic
proteins
(more tightly
bound).
Cytochrome c
-chymotrypsin
RNAseA
Lysozyme
Degree of substitution
Binding capacity of protein
to HIC increases with
increased alkyl chain
length and increased
degree of substitution of
immobilised ligand
Caution: protein can bind
via multipoint attachment,
thus difficult to elute
4.pH
Effect of pH on HIC is not straight forward.
In general an increase in pH weakens
hydrophobic interactions.
Decrease in pH leads to an apparently increase
in hydrophobic Interaction
It is observed that proteins which do not bind
to HIC
adsorbent at neutral pH, bind at acidic pH.
5.Temperature
Visser and Strating (1975): that role of
temperature is a complex issue .
Binding of proteins to HIC adsorbents is
entropy driven (Hjerten, 1976), ie interaction
increases with increase in temperature
This could be due to differential effects by
temperature on the conformational state of
different proteins and solubility in solution
6.Additives
Salts that cause salting-in will weaken
protein-ligand interactions
Alcohols and detergents (non-polar parts)
can compete with protein for HIC absorbent
sites and may displace proteins
Stationar
y phase
bio
po
r
lym
Hydrophobic regi
Advantages of HIC
Large volume of sample can be loaded
Samples with high ionic strength can be
used
Well suited to use before gel filtration,
ion-exchange and affinity chromatography
Sample eluted with low salt