You are on page 1of 22

Water repelling

In this case it refers


to the hydrophobic
regions on protein
surface

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

May be explained on basis of entropy


Under the condition of H <<TS, the Gibbs free energy is
negative (-GTS) thus constituting an entropy driven
reaction
Dissolution of non polar solute in water: Thermodynamically
unfavorable process: here change in entropy is negative
because of the development of an ordered structure of water
molecules around the solute molecule. The change in
enthalpy is also negative as many new hydrogen bonds are
formed between water molecules as an ordered structure
develops
In aq solutions of high salt conc aggregation of the non-polar
solute molecules by hydrophobic interaction, as in salting out
of proteins is accompanied by positive change in entropy as
the water molecules of ordered structure around the solute
molecule are released in the bulk phase. Therefore it is
thermodynamically favorable

hydrophobic substance as protein or hydrophobic


ligand immersed in water- analogous to the surface
tension phenomenon happens.
water molecules cannot wet the surface of the
hydrophobic substance. Instead they form a highly
ordered shell around the substance, due to inability to
form hydrogen bonds in all directions.
To minimize the extent of this shell, decrease in the
number of ordered water molecules, that
thermodynamically more favourable situation --in
which entropy increases.
to gain entropy, hydrophobic substances are forced to
merge to minimize the total area of such shells.
Thus hydrophobic interaction depends on behaviour of
water molecules than on direct attraction between
hydrophobic molecules
Reference-J.A. Queiroz et al. : Journal of
Biotechnology 87 (2001)

Adsorption of protein through non-covalent


interactions between nonpolar regions on the
protein surface and a hydrophobic matrix
Typically performed at high concentrations of salts
elution by decreasing the salt concentration

Salting-out chromatography (Tiselius, 1948)


Hydrophobic affinity chromatography (Shaltiel & Er-el,
1973)
Hydrophobic interaction chromatography (Hjertn, 1973)
Salt-promoted adsorption chromatography (Porath, 1986)

HIC stationary phases


Hydrophobic ligands are immobilized on solid
supports (= matrix)
Types of matrices used:
Carbohydrates
cross-linked agarose (Sepharose)
dextran (Sephadex)
cellulose
Silica
Synthetic copolymers

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

Proteins separated in order of increasing surface


hydrophobicity

Factors affecting HIC


The main parameters to consider when
selecting HIC media and optimizing
separation processes on HIC media are:
Ligand type and degree of substitution
Type of base matrix
Type and concentration of salt
pH
Temperature
Additives

1.Ligand type affects


protein absorption
because interactions
may not be strictly
hydrophobic
Straight chain alkyl
ligands show pure
hydrophobic character
whereas with aryl ligands
both aromatic and
hydrophobic
interactions are possible.
Choice of ligand is
empirical and must be
established by

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

2.Type of base matrix


Important to take note that selectivity will
not be exactly the same even with the same
type of ligand if the base matrix is different
Two widely used supports are cross-linked
agarose and synthetic copolymer materials
May be necessary to modify adsorption
and elution conditions

3.Type and concentration of


salt
Salts that produce relatively higher salting out (eg
Na, K or ammonium sulfates) effectively promote ligandprotein interactions in HIC

Amounts of protein bound increases almost linearly


with increase salt concentration
Bound proteins are desorbed by washing the HIC
column with dilute buffer solutions (near neutral pH) or
water.
Influence of different salts on HI predicted by
Hofmeister series
ANION
sulphate>chloride>bromide>nitrate>perchlorate>iodide

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

Biopolymer (phenyl agarose Binding Surface)


Driving force for hydrophobic adsorption
Water molecules surround the analyte and
the binding surface.
When a hydrophobic region of a
biopolymer binds to the surface of a
mildly hydrophobic stationary phase,
hydrophilic water molecules are
effectively released from the surrounding
hydrophobic areas causing a
thermodynamically favorable change in
entropy.
Temperature plays a strong role
Ammonium sulfate, by virtue of its good
salting-out properties and high solubility
in water is used as an eluting buffer

Stationar
y phase
bio
po
r
lym

Hydrophobic regi

Main steps in HIC


1. Preparation of HIC GEL
2. Column packing and
equilibration
3. Bind protein to HIC media at high
salt concentration (0.5 M
ammonium sulfate)-high salt
solution increases the
hydrophobicity of protein by
exposing hydrophobic regions
4. Wash column with buffer
of similar salt concentration--allows
proteins with various levels of
hydrophobicity to gradually unbind
from the stationary phase and be
collected in a test tube
Batch wash: 0.5M, 0.3M, 0M
5. Elute column with decreasing salt
concentration
Gradient type: For example, 0.5 to 0M

Elution with decreasing salt concentration


used in purification of acid phosphatase
from bovine cortical bones

Elution with decreasing polarity of eluent


used in purification of human pituitary
prolactin

Elution using detergent used for


separation/purification of proteins

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