Download Ion exchange chromatography File

Ion exchange chromatography
What is ion exchange
chromatography ?
• IEC is a technique for separating proteins
according to their charge
• Its easy of use and scale up capabilities
• Large volumes be applied
• High resolution and high capacity method
Principle
Packing material
• Resin are charged molecules
• Securely bound to column by covalent bonds
• Negatively or positively charged groups
Anion exchanger
• Positively charged beads “exchange” with negatively
charged counter ions
• Negatively charged molecules - “anions”
Cation exchangers
• Negatively charged beads exchange positive
counter-ions (cations)
Functional Group Counter-ion
Anion Exchanger
-O-CH2-CH2-N +
Diethylaminoethyl
H (CH2CH3)2
(DEAE)
Cation Exchanger
-O-CH2-COO Carboxymethyl (CM)
Cl Na +
Separation of protein
• Proteins are charged molecules
• Interaction with the packing material depends on
– Overall charge
– Distribution of that charge over the protein surface
• They displace mobile counter ions bound to the resin
• Mobile counter ions: When the packing material is
suspended in buffer containing NaCl, the charged
groups become loosely associated with Na+ and Clions of the opposite charge. These loosely bound ions
are called mobile counter-ions.
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Net charge on protein will depend on
– Composition of amino acids in the protein
– pH of the buffering solution.
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Charge distribution will depend on
– How the charges are distributed on the folded protein
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Isoelectric point ( pI) - The pH at which a particular protein is overall
electrically neutral ie. the number of positive charges is equal to the
number of negative charges.
< pI - A protein has more positively charged amino acids and
therefore an overall positive charge. It will bind to cation exchangers
> pI - A a protein has more negatively charged amino acids and an
overall negative charge. It will bind to anion exchangers
At its pI, a protein will not bind to either a cationic or an anionic
exchanger.
Column materials - Polystyrene
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Ion-exchangers made by co-polymerisation of styrene with divinyl
benzene. Polystyrene itself is a linear polymer.
Divinyl benzene, is a cross-linker
Resins with low degree of cross-linking are more permeable to high
molecular weight compounds, but they are less rigid and swell
more when placed in buffer
Sulphonation of cross-linked polystyrene results in sulphonated
polystyrene resin such as Dowex 50- strong acidic exchanger
Basic exchangers are prepared by reacting cross-linked
polystyrene with chlormethyl ether and then by the chlorogroup
with tertiary amines – CH2 N+ (CH3)3 Cl- groups are ionized.
Modified cellulose and Sepharose
• Modified cellulose is an alternative to polystyrene
based exchangers
• Cellulose is a high molecular weight i.e.
carboxymethyl cellulose (CM-cellulose) where the –
CH2OH group is converted to –CH2OCH2COOH and
DEAE-cellulose [CH2OCH2CH2N(CH2CH3)2]
• Commercially available in gel and bead form
• Sepharose type is derived from cross-linked agarose
• Sephadex and Sepharose are used for the separation
of high molecular weight proteins and nucleic acids
Total exchange capacity
• Defined as number of milliequivalents of
exchangeable ions available either per gram of dried
exchanger or per unit volume of hydrated resins
– Bio Rad AG1-X4 = 1.2 meq cm-3
– DEAE-Sephadex A-25 = 0.5 meq cm-3
– CM-Sepharose CL-6B = 0.2 meq cm-3
• Polystyrene exchangers are obtainable in a number
of mesh size. All exchangers are supplied with
counter ions i.e Na+ or Cl-.
Classification of ion exchange
media
Media
(X = matrix)
Anion exchangers
X-CH2N+(CH3)3
X-CH2NH+(CH3)2
(CH3CH2)2
X-CH2CH2NH+
diethylamino-ethyl (DEAE)
Cation exchangers
X-SO3X-COOX-CH2COOcarboxymethyl (CM)
Nature
pH
range
Applications
strong
intermediate
weak
2 - 11
2-7
3-6
nucleotides
organic acids
proteins
strong
intermediate
weak
2 - 11
6 - 10
7 - 10
amino acids
peptides
proteins
Buffer
• The pH of the buffer used should be one pH unit
above or below isoionic point of the compounds.
• Cationic buffers are tris, pyridine and alkyl amines
and they are used with anion exchangers.
• Anionic buffers are acetate, barbiturate and
phosphate and they are used with cationic
exchangers.
Sample application
• Amount dependent upon size of the column
and capacity of exchanger.
• For the isocratic elution, sample volume is 15% of bed volume.
• For the gradient elution, sample volume is not
important.
• Large volumes of dilute solution can be
applied as they get bound at the top of
column.
Elution
• Gradient elution is most common and
gives better results.
• With anion exchanger, pH gradient
decreases and ionic strength increases.
• With cation exchangers, both pH and
ionic gradients increase.
Applications
• Separation of amino acids achieved by strong acid cation
exchanger – Dowex 50-PS and SP-Sephadex-cellulose
• Separation of proteins by weakly acidic and basic
exchangers derived from cellulose and agarose
– Proteins with isoionic point < 7 – DEAE-cellulose using low ionic
strength
– Proteins with isoionic point > 7 – CM-cellulose using buffer pH 4-5
– Proteins with isoionic point  7 – Either
• Determination of base composition of nucleic acids
• RNA hydrolysed by both enzymatic and base hydrolysis.
DNA resisted to base hydrolysis but cleaved by DNAases.
• Separation of amino acids achieved by strong
acid cation exchanger – Dowex 50-PS and SPSephadex-cellulose
– Introduction of sample at pH 1-2 to ensure complete
binding
– Gradient elution by increasing pH and ionic
concentration
– Acidic amino acids, aspartic and glutamic acids elute
first followed by neutral amino acids, glycine and
valine. Basic amino acids retain their net negative
charge up to pH values 6-11 and elute last.