Download Ion exchange chromatography

Chromatography
Planar
Paper
Column
TLC
(Thin layer
chromatography)
Chromatography is separation method based on different retention of analytes
on the stationary phase. Different chemical of physical phenomena can be used
to retain an analyte. Depending on the phenomenon responsible for separation
several types of chromatography are considered.
Types of chromatography
 Adsorption chromatography
 Ion exchange chromatography
 Size exclusion chromatography
 Ion exclusion chromatography
 Affinity chromatography
Ion exchange chromatography (IEC)
The method is used to separate any biomolecules carrying a charge, such as
proteins, polynucleotides and fragments of both.
Principle of ion exchange
Ion exchange is an equilibrium between an ion attached to a solid support
(ion exchange resin) and an ion in a solution
Res-H+ + Na+ Res-Na+ + H+
Selectivity coefficient
K=
[Res-Na+][H+]
[Res-H+][Na+]
Describes the relative
selectivity of the resins
for H+ and Na+
Example: ion exchange between a sulfocationite and an amino acid
pH2
SO 3
-
Na
+
H 3N
+
COOH
Ion-exchange Resin
SO 3
-
H 3N
Na
+
+
COO
-
pH4.5
Structure of Ion exchange resins
Ion exchange resin
Fixed ion
Matrix
Cross-linked polymer
(polymethacryl,
polydivinylbenzene)
SO 3- Na +
Counter-ion
Anion exchanger: positively charged groups on the solid support
attract solute anion.
Cation exchangers: covalently bound, negatively charged sites
attract solute cations.
Ion Exchange Resins
Examples:
Polystyrene resins. Ion exchange groups
Type
Example of
active group
pH range of
operation
Application example
Strongly acidic
cation exchanger
RSO3-
1-14
Amino acids, inorganic
separations
Weakly acidic
cation exchanger
RCOO-
5-14
Transition elements,
organic bases
Strongly basic
anion exchanger
RN(CH3)3+
1-12
Alkaloids, fatty acids
Weakly basic
anion exchanger
RC2H4N(C2H5)2
1-9
Organic acids,
aminoacids
Requirements to a IE resin for biopolymer separations
1. Mechanical stability
2. Reduced unspecific adsorption
3. Higher saturation capacity
4. Fast mass transfer
5. Sustainability under sanitation conditions
Stationary phases for biopolymer separations
•
•
•
Polysaccharides (cellulose, agarose, dextran)
Synthetic organic polymers (polyacrylamide, polymethacrylate,
polystyrene)
inorganic materials (silica, hydroxyapatite)
Particle size: 2 – 200 mm
analytical chromatography: 2-10 nm
preparative chromatography: 30-200 nm
Pore size: 10 – 100 nm
SEPARATION OF PROTEINS BY IEC
Pros:
 High resolution. A dilute protein or polynucleotide
solution can be rapidly concentrated and simultaneously purified.
 Cheap mobile phases. Simple salt buffers are sufficient
Cons:
 Ion-exchange chromatography is incompatible with mass spectrometry
SEPARATION OF PROTEINS BY IEC
General cases where IEC is applied for protein and biomolecular purification and
separation
1. De novo purification.
2. Purification of a protein with known pI, size, and primary structure
3. Preparative and industrial separation of proteins:
4. High resolution separation of protein variants and/or isoforms
Operation of Ion Exchange Column
1. Loading with salt
2. Equilibration
3. Loading of protein
4. Washout of unbound material
5. Elution
6. Regeneration
7. (Sanitization)
Step 1. Loading with salt
Charged ligands must be entirely saturated with counter-ions.
10 – 100 mM buffer + 1M NaCl or KCl
At least 1 volume of column should be prepared
Step 2. Equilibration
Stable and reproducible separation requires equilibrium to be established between
the mobile phase and the stationary phase
At least 10 column volumes must be eluted through a column to get an
equilibrium conditions
Step 3. Loading of protein
Loading of the protein solution should be performed at the same pH and conductivity
as the equilibration buffer.
Often this is not possible. Then the protein solution can be desalted by size
exclusion chromatography, dialysis and the same ion composition can be obtained
leading to most robust loading conditions.
Step 4. Washout of unbound material
first step of purification
At least 1 column volume of equilibration buffer with
eventually supplements
Step 5. Elution
Separation of the target compound from other bound concomitants
1. Elution by salt gradient
(change salt concentration gradually displacing bound compounds
in an order of increasing affinity )
2. Elution by pH gradient
(change the pH of the mobile phase creating conditions for target
protein to desorb)
Step 6. Regeneration
Preparation a column to new run of separation
Regeneration solution must be strong enough to assure the
desorption of all the sample components from the column
Step 6. Sanitation
Working with biochemical media poses a danger to microorganism
development in a column. To prevent the contamination of target analytes
with pathogenic microorganism a regular sterilization of IEC equipment is
necessary.