→ Principle of HIC
→ Advantages of
→ What are the
Source of protein
• Downstream purification
• Separation of biomolecuoles
• Exploits differences in hydrophobicity.
→ Number of hydrophobic aminoacids.
→ Distribution of these aminoacids.
• Separation of substances is based
on their varying strength of
interaction with hydrophobic
groups attached to an uncharged
• Hydrophobic groups on proteins are
sufficiently exposed to bind to the
hydrophobic groups on the matrix.
• How is this achieved?
■ Choice of column → XK colums for
• Column dimensions → Short bed
height (5-15 cm) suitable for HIC
■ Packing of Column: a modern,
highly crosslinked agarose-based gel
such as Sepharose Fast Flow is
however easier than packing a gel
filtration column since the bed
height required is much smaller.
■ Sample preparation
• Sample composition
• Sample volume
• Sample viscosity
■ Sample application
■ Batch Separation
Large volume of sample can be
Samples with high ionic strength
can be used
Well suited to use before gel
filtration, ion-exchange and
Sample eluted with low salt
Purification steps that
generate large sample volume
can be coupled with this
Good for samples after
These techniques may require
pretreatment of samples (e.g.
reducing ionic strength)
Sample can be used in ion
concentration of ligand
Type of base matrix.
concentration of salt
Effects of ligand density-
Degree of substitution
‰ Binding capacity of protein to
HIC increases with increased alkyl
chain length (A) and increased
degree of substitution of
immobilized ligand (B)
‰Caution: protein can bind via
multipoint attachment, thus difficult
O CH2 CH CH2 O ( CH2) 3 CH3
O CH2 CH CH2 O ( CH2) 7 CH3
O CH2 CH CH2 O
‰Important to take note that selectivity will not be exactly
the same even with the same type of ligand if the base matrix
‰Two widely used supports are cross-linked agarose and
synthetic copolymer materials
‰May be necessary to modify adsorption and elution
Type of salt
▪ salt effect follow the
▪ Hydrophobic interaction
increases at increased salt
Increasing salting out effect
Anions: PO43- >SO42- >Cl-
>Br- >NO3- >ClO4- >I- >SCN-
Cations: NH4+ > K+ >Na+
Decreasing surface tension
Increasing chaotropic effect
Effect of pH on HIC is not
‰In general an increase in pH
interactions. It could be due to
increased titration of charged
groups leading to an increase in
hydrophilicity of the proteins
‰Decrease in pH leads to an
apparently increase in
‰Implication: Important factor
to consider for optimization of
HIC interaction. It is observed
that proteins which do not bind
to HIC adsorbent at neutral pH,
bind at acidic pH.
Visser and Strating (1975): that
role of temperature is a
complex issue and differ from
observation of Hierten.
‰ Binding of proteins to HIC
adsorbents is entropy driven
(Hjerten, 1976), i.e. interaction
increases with increase in
‰ Discrepancy in views could
be due to differential effects
by temperature on the
conformational state of
proteins and solubility in
‰ Practical terms: To be aware
that procedure developed
at room temperature may be
different if used in the cold
Salts that cause “salting-
in” will weakenprotein-
parts) can compete with
protein for HIC absorbent
sites and may displace
1.HIC in combination with ammonium sulphate precipitation
Crude purification of human autotaxin
HIC was used for initial purification of autotaxin, a human 125K protein which
stimulates tumour cell motility (49).
2. HIC in combination with ion exchange chromatography
Purification of recombinant HIV reverse transcriptase
Purification of mammalian transcription factors
Micropurification of a GTPase activating protein
3. HIC in combination with gel filtration
A major advantage with adsorption chromatography is the possibility to achieve
decrease in sample volume concomitant with an increase in purity. In a
scheme, HIC and other adsorption chromatography techniques are therefore
frequently used prior to gel filtration, in which sample volume is limited.
4. HIC as a ‘‘single step’’ purification technique
5.Other HIC application areas in the research laboratory
HIC using Phenyl Sepharose CL-4B has been used for exchange of protein-bound detergent
Octyl Sepharose CL-4B has been used for the separation of different forms of dermatan
sulphate proteoglycans (55). HIC of nucleic acids, viruses and cells has also been described.
6. Preparative, large scale applications
Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV-
Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast.
Purification of a monoclonal antibody for in vitro diagnostic use.
Purification of a recombinant Pseudomonas aeruginosa exotoxin produced in E. coli.
M.M. Diogoa, J.A. Queirozb, D.M.F.
Prazeres (2003, March) Assessment of
purity and quantification of plasmid DNA in
process solutions using high-performance
hydrophobic interaction chromatography
Journal: The Journal of Chromatography A
The purpose of the study is to demonstrate the usefulness of HIC for
monitoring the performance of a plasmid DNA isolation process .
Many genetic engineering techniques require a highly pure plasmid DNA
sample , hence it is important to develop a reliable analytical method for
quantification of plasmid DNA and assessing its purity.
Quantification of total plasmid DNA in pure solutions is easily
accomplished , by spectrophotometry at 260 nm . However a
spectrometer cannot be used for the quantification of impure DNA
Techniques like Agarose gel electrophoresis , capillary electrophoresis
and HPLC can be used to measure the purity of the sample , but all these
methods have considerable disadvantages
Agarose gel electrophoresis is not reproducible ,CE and HPLC are
expensive and time consuming .
The researchers used a a column filled with Sepharose CL-6B gel
derivatized with 1,4-butanediol diglycidyl ether.The HIC column was
fitted with a HPLC system , to qualitatively and quantitatively analyze
the plasmid sample.
The column was equilibriated with Tris-Cl buffer (ph 8.0)
The technique takes advantage of the more hydrophobic character of
nucleic acid impurities (RNA, proteins etc )compared to ds DNA
A E coli lysate is injected into the column in two stages :
In the first stage . The sample was eluted with no ammonium sulphate
In the second stage , the sample was eluted with 450 mM and 1050 mM
of ammonium sulphate added to the sample .
The chromatograms of both the stages were obtained from the HPLC
system , the chromatograms were then compared
The above slide shows four chromatograms each representing the
resolved components of the E coli lysate.
The chromatogram A was obtained during the first stage of the
experiment , where the ammonium sulphate concentration was 0 . The
components are not properly resolved , and only a single peak is
The chromatogram B , C , D were obtained when the ammonium
sulphate concentration was 450 mM , 1050 mM and 1.5 M respectively
The above mentioned chromatograms show distinct peaks.The first one
representing the eluted plasmid DNA ,the remaining representing the
impurities eluted out after the ammonium sulphate concentration has
Chromatogram D shows the resolved components of the impurities.
The results obtained showed that that HIC can be used as for
quantification and purity assessment of a plasmid DNA
The method used involved a simple and a rapid (7 Minute
long ) procedure for detecting the purity of a Plasmid
Results obtained from repeating the experiment several
times, showed a standard deviation value of less than
10%,which shows that the results are reproducible.
The technique had the ability to handle highly contaminated
samples ( <5% of plasmid DNA ) without any pre-treatment
such as digestion of high molecular mass RNA
Very useful technique for mAb purification.
Mainly used in the third step as a complementary
technique to protein A and IEC (in-vivo).
HIC can be used in both binding and removal mode.
Can be a useful alternative to SEC for aggregate
HIC is also very useful for purification of antibodies
in 2-step techniques (non-protein A) for in-vitro
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