Transcript Ion Exchange Chromatography

Ion Exchange Chromatography
• Some ion exchangers are regarded as weak, that is
functioning best over a comparatively narrow pH
range, while others are strong, that is functioning over
a wider pH range. Exchangers with quaternary amino
or sulfonic acid groups behave as strong anion and
cation exchangers, respectively, while
aromatic/aliphatic amino and carboxylic acid groups
are weak anion and cation exchangers, respectively.
• Choice of ion exchange stationary phase is heavily
influenced by knowledge of the pI of the protein of
interest.
• In an ion exchange chromatography experiment (Figure),
sample is applied to a stationary phase which has been
charged with the ion to be exchanged; the counterion (e.g.
Cl−).
• The protein (and any other species of like charge in the
sample) may exchange with this counterion, binding to the
ion exchanger.
• It is important that the sample is free of components of
like charge since these are usually present in large molar
excess relative to the concentration of protein.
• The ion exchanger will not distinguish between proteins
possessing a single positive charge and, for example, a Na+
ion also present in the sample.
• Desalting is carried out before ion exchange either by gel filtration
chromatography , by dialysis or by centrifugal filtration.
• Elution of bound proteins is achieved by reversing the process of
binding and, again, exchanging a counterion for protein.
• This is usually carried out by applying a large excess of a salt (e.g.
NaCl) containing the counterion in the mobile phase.
• Because proteins have different net charge, they may bind to an
ion exchanger at a given pH with a variety of strengths, that is some
proteins may bind strongly whilst others bind weakly or not at all.
• We can take advantage of this to separate proteins by applying salt
in a continuous gradient. Weakly bound proteins will elute first
from such a system while strongly bound proteins elute later.
Selection of an Ion Exchanger
Selection of an Ion Exchanger
Selection of an Ion Exchanger
• Below the isoelectric pH the protein has a
positive charge and binds to a cation
exchanger.
• Above the isoelectric pH the protein has a
negative charge and binds to an anion
exchanger.
Choice of Buffer
• Cationic buffer should be used with anion
exchanger.
• Anionic buffer should be used with cation
exchanger.
• Ion exchangers are commonly used in column
form.