Hydrophobic Interaction Chromatography (HIC)
Reversed-Phase Chromatography (RPC)
Amelie Eriksson Karlström
KTH
amelie@biotech.kth.se
Hydrophobic interaction chromatography
(HIC)
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” (Hjertén, 1973)
”Salt-promoted adsorption chromatography” (Porath, 1986)
1
Adsorption chromatography
Binding of the biomolecule to be purified to the matrix by multiple
weak, non-covalent van der Waals forces and hydrogen bonds
Protein surface properties
Electrostatic surface potential
Negative potential
Neutral
Positive potential
Proteins have different molecular
surfaces depending on the
surface-exposed amino acids:
charged
polar
non-polar
adsorption
chromatography
Acetylcholine esterase
ion-exchange
chromatography
2
Hydrophobicity scale
Kyte-Doolittle
Arginine
Lysine
Asparagine
Aspartic acid
Glutamine
Glutamic acid
Histidine
Proline
Tyrosine
Tryptophan
Serine
Threonine
Glycine
Alanine
Methionine
Cysteine
Phenylalanine
Leucine
Valine
Isoleucine
-4.5
-3.9
-3.5
-3.5
-3.5
-3.5
-3.2
-1.6
-1.3
-0.9
-0.8
-0.7
-0.4
1.8
1.9
2.5
2.8
3.8
4.2
4.5
Protein hydrophobicity
Increased ordering of water molecules surrounding the
hydrophobic regions of proteins ⇒ decrease in entropy
ΔG = ΔH - TΔS
H2O
H2O
H2O
H2O
H2O
H2O
3
Hydrophobic effect
Non-polar compounds spontaneously associate in water due to
hydrophobic interactions.
The ordered water molecules around the hydrophobic groups are
displaced to the more unstructured water.
ΔS > 0, ΔG < 0
Protein-protein
interactions
Protein folding
Protein-matrix
interactions
Principle of HIC
ligand
protein
ligand
protein
Water molecules are ordered
around the hydrophobic
patches of the protein and the
immobilized ligand
The ordered water molecules
are released to the bulk water
upon binding of the protein to
the stationary phase
4
Parameters affecting HIC
• Stationary phase
– Matrix (type)
– Ligand (type, density)
• Mobile phase
–
–
–
–
ligand
Salt (type, concentration)
pH
Temperature
mobile
Additives
matrix
phase
HIC stationary phases
Hydrophobic ligands are immobilized on solid supports (= matrix)
The degree of substitution can affect the separation
Types of matrices used:
• Carbohydrates
• cross-linked agarose (Sepharose)
• dextran (Sephadex)
• cellulose
• Silica
• Synthetic copolymers
5
HIC ligands
• Linear alkanes
methyl < ethyl < propyl < butyl < pentyl < hexyl < heptyl < octyl
Increased hydrophobicity increases the strength of the interaction,
but may also reduce the adsorption selectivity
• Aryl (aromatic) groups
e.g. phenyl
Mixed hydrophobic and aromatic interactions (π−π)
• Intermediate hydrophobic ligands
e.g. polyethers
PEG (polyethylene glycol) < PPG (polypropylene glycol) < PTMG
(polytetramethylene glycol)
Milder elution conditions
Use of salts in HIC
Binding at high salt concentration
Retention on the stationary
phase is favored
Elution at low salt concentration
6
Salt effects on protein precipitation
Anti-chaotropic
(water-structuring)
Increasing salting-out effect
PO43-, SO42-, CH3COO-, Cl-, Br-, NO3-, ClO4-, I-, SCNNH4+, Rb+, K+, Na+, Li+, Mg2+, Ca2+, Ba2+
Chaotropic
(water-destructuring)
Hofmeister series for precipitation of proteins from aqueous solutions:
Increasing salting-in effect
Increased structuring of water ⇒ increased strength of hydrophobic interactions
Salt effects on the surface tension of water
Increasing molal surface tension of water
MgCl2, Na2SO4, K2SO4, (NH4)2SO4, MgSO4, Na2HPO4, NaCl, LiCl, KSCN
Increased surface tension ⇒ increased retention of proteins in HIC
Retention depends on preferential interactions of the salts with the
stationary phase and the proteins
7
Effects of temperature and pH in HIC
Protein retention
Temp
Protein elution
pH
3
4
5
6
7
8
9
10
The retention is dependent on the pI of the protein
Elution can be performed by increasing the pH
Use of additives in HIC
• To increase the solubility of the protein
• To promote elution of the protein
• Column cleaning
• water-miscible alcohols (ethanol, ethyleneglycol)
• aliphatic amines (butylamine)
• detergents (Triton X-100, Tween 20)
• chaotropic salts
8
Application of HIC
HIC is typically used in combination with other purification methods
– First step in purification from biological medium
– After salt (ammonium sulfate) precipitation
– After ion-exchange chromatography
Salt concentration
already high in the
sample
Example. Process for large-scale purification of mouse IgG1
Cell
culture
Filtration
Addition
of
(NH4)2SO4
HIC
Gel
filtration
Concentration
Reversed phase chromatography (RPC)
Loading
ligand
Binding
ligand
Elution
ligand
Binding of the protein in a polar mobile phase
Elution by changing the composition of the mobile phase to become
more non-polar
9
Partition chromatography
Partition between a stationary phase and a mobile phase
Normal phase
Polar stationary phase ⇔ non-polar mobile phase
Separation of small, organic analytes
Reversed phase
Non-polar stationary phase ⇔ polar mobile phase
Separation of proteins and nucleic acids
HIC vs RPC
HIC
– Less substituted matrix
– Less hydrophobic ligands
– Weaker binding
– Elution with water/dilute buffers
– Native protein
– Adsorption chromatography
– Low pressure chromatography
RPC
– More substituted matrix
– More hydrophobic ligands
– Stronger binding
– Elution with non-polar solvents
– Denatured protein
– Partition chromatography
– High performance liquid
chromatography (HPLC)
10
RPC stationary phase
Hydrophobic ligands immobilized on porous solid supports
Parameters affecting the separation:
– Type of matrix
⇒ typically rigid, to withstand high pressure
– Bead size
⇒ smaller particles give higher resolution
– Pore size
⇒ wide pores give efficient transfer of molecules
– Type of ligand
⇒ more hydrophobic molecule requires less
hydrophobic ligand
– Ligand density
⇒ aging of columns releases ligand and leaves
exposed silanol groups
RPC matrix
Types of matrices used:
• Silica
• Synthetic polymers (e.g. polystyrene)
Typical bead size: 3-10 µm
Typical pore size: 100-500 Å
Polystyrene/divinylbenzene
5 µm beads
Important factors:
• Pressure stability (high degree of cross-linking is necessary)
• Chemical stability (silica gels are base-sensitive!)
11
RPC ligands
• Linear alkanes
C18
C8
C4
• Aryl (aromatic) groups,
e.g. phenyl
Coupling of ligands
Si
Surface-exposed silanol groups on the silica gel
OH
Coupling of C18 ligand
CH3
CH3
Si
OH
+
Cl
Si
(CH2)17
Si
CH3
O
Si
(CH2)17
CH3
CH3
CH3
Capping of residual silanol groups
CH3
CH3
Si
OH
+
Cl
Si
CH3
CH3
Si
O
Si
CH3
CH3
12
Column length
Generally, the resolution of chromatographic separations increases
with increasing column length.
For RPC of peptides and proteins, the effect of column length is
quite small, due to the ”on/off” partition between the stationary
phase and the mobile phase (adsorption/desorption)
RPC mobile phase
Typically mixtures of water/aqueous buffers and watermiscible organic solvents at low pH are used
Organic solvents:
- acetonitrile
- methanol
H3 C
C
H3 C
N
OH
O
- tetrahydrofuran
Relative retention of a polypeptide:
MeOH < EtOH < CH3CN < 1-propanol, 2-propanol
13
Flow rate
A lower flow rate can lead to:
increased resolution
Equilibrium between
stationary and mobile
phase
or decreased resolution
Longitudinal diffusion
Temperature
The resolution generally increases with increasing temperature:
The viscosity of the solvents
decreases with increasing
temperature
⇒ more efficient transport of
solute between the mobile phase
and the stationary phase
14
Buffer and pH
• Typically organic buffers are used, which can be removed
from the sample by lyophilization
• For separation of proteins: typically pH < 7.5, often pH 2-3
- column stability
- separation
- low ionic strength
• At low pH the protein constitutes a homogeneous population:
- COO- ⇒ COOH
- NH2 ⇒ NH3+
Ion-pairing
The mobile phase contains hydrophobic counter ions to the
charged analyte ⇒ increased retention of hydrophilic compounds
++ +
+
+
++ ++ +
-
Example: TFA (trifluoroacetic acid)
peptide-NH3+ + CF3COO- → peptide-NH3+ CF3COOExample: TEA (triethylamine)
nucleic acid-PO4- + (CH3CH2)3NH+ → nucleic acid-PO4-(CH3CH2)3NH+
15
Elution
%B
Isocratic elution
Time
%B
Gradient elution
Time
Example of typical RPC run
Solvent A: 0.1% TFA - H2O
Solvent B: 0.1% TFA - CH3CN
Column: C18
Sample: mixture of peptides
Gradient:
0%B
0 min
0%B
5 min
50%B 30 min
100%B 31 min
100%B 36 min
0%B
37 min
(equilibration)
(flow-through)
(elution)
(wash)
(wash)
(re-equilibration)
%B
0
5
30 31
36 37
Time
(min)
16
High performance liquid chromatography
(HPLC)
The resolution is increased with smaller particle size
(increased surface area ⇒ increased number of theoretical plates)
Smaller particle size ⇒ increased back-pressure
In HPLC systems, columns, stationary phases, tubings etc designed to
withstand high pressure are used
HPLC system
17
HPLC system overview
Solvent A
Solvent B
Sample
Mixing
vessel
High
pressure
pump
Sample
injection
port
Guard
column
Column
Detector
Integrator
Printer
Computer
Sample outlet
Fraction collector
HPLC pumps and solvents
The solvents used need to be of high purity and degassed
- HPLC grade solvents
- filters
- vacuum
- sonication
- helium bubbling
18
HPLC sample injection
Manual injection using
a microsyringe
Auto-injector
Sample preparation:
- high purity
- mobile phase solvents
- filtering
HPLC columns and matrices
HPLC columns are made of stainless steel to withstand high
pressure
Diameter:
Ø = 1-2 mm
Ø = 4.6 mm
Ø = 10-25 mm
Ø = 10-25 mm
Column type:
microcolumn
analytical column
semi-preparative column
preparative column
Flow rate:
50-200 µl/min
0.5-2.0 ml/min
5-10 ml/min
10-100 ml/min
length: 10-50 cm
The matrix is typically formed by rigid, porous, spherical particles
19
HPLC detectors
• UV detector (variable wavelength)
• Diode array detector (scanning wavelength)
• Fluorescence detector
• Electrochemical detector
• Refractive index detector
UV detection
Peptides, proteins
210-220 nm - peptide bond
280 nm
- aromatic side chains
Oligonucleotides
260 nm
20
Applications for RP-HPLC
Analysis and purification of:
• Naturally occurring peptides and proteins
• Recombinant peptides and proteins
• Chemically synthesized peptides
• Peptide fragments from enzyme digests
• Chemically synthesized oligonucleotides
RP-HPLC of recombinant proteins
Recombinant Aβ1-42
His-tag fusion purified by IMAC and
cleaved by factor Xa
Vydac polystyrene/divinylbenzene column
Solvent A: 5 mM potassium acetate, pH 8.0
/ 5% acetonitrile
Solvent B: 5 mM potassium acetate, pH 8.0
/ 90% acetonitrile
Gradient: 0-20% B over 8 min, 20-26% B
over 12 min
Aβ1-42
Aβ6-42
21
RP-HPLC of synthetic peptides
58 aa peptide synthesized by solid phase peptide synthesis
Amersham Biosciences, Source polystyrene/divinylbenzene column
Solvent A: 0.1% TFA-H2O
Solvent B: 0.1% TFA-acetonitrile
Gradient: 20-40% B from 5-25 min
RP-HPLC of tryptic digest
Abs
215 nm
Time (min)
Transferrin digested with trypsin
HAISIL C18 column
22
RP-HPLC of oligonucleotides
135-mer, 5’-protected with
dimethoxytrityl group
Vydac C4 column
Solvent A: trimethylammonium
acetate, pH 7.0
Solvent B: acetonitrile
Gradient: 0-60% B from 5-40 min
Purified, deprotected 135-mer
Vydac C4 column
Solvent A: trimethylammonium
acetate, pH 7.0
Solvent B: acetonitrile
Gradient: 0-20% B from 5-25 min
Liquid Chromatography - Mass
Spectrometry (LC-MS)
Injection loop
200 µl/min
15 µl/min
200 nl/min
I
LC
MS
23