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Definition
1850 – British scientists H. Thompson and J. T. Way treated various clays with ammonium sulfate or carbonate in solution to extract the ammonia and release calcium
1927 – the first zeolite mineral column was used to remove interfering calcium and magnesium ions from solution to determine the sulfate content of water
The modern version of IEC was developed during the wartime Manhattan Project for uranium purification
1970s – Dow Chemical perfects IEX |
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Term
| Ion Exchange Chromatography |
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Definition
Ion exchange (IEX) chromatography can be used to separate proteins based on molecular charge
Ion exchange chromatography is a variation of adsorption chromatography (oldest type of chromatography) in which the solid adsorbent has charged groups chemically linked to an inert solid |
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Term
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Definition
Proteins are composed of positively and negatively charged amino acids
The proteins in solution which are to be adsorbed have net charges, which can be balanced by counterions
Ions (immobilized on a gel matrix) can electrostatically bind to the charged groups on the proteins
These ions may then be exchanged for ions in an aqueous solution
Because charged molecules bind to ion exchangers reversibly, molecules can be bound or eluted by changing the ionic strength or pH of the eluting solvent |
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Term
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Definition
Highly pH dependent
Affected by pH, pKa of termini, side chains
pI = pH where all charges are equal
Can be easily calculated (ExPASy/ProtParam)
pH > pI – protein will have a net negative charge
pH < pI – protein will have a net positive charge
Termini – as pH increases, protons dissociate
Amino – NH3+ NH2
Carboxyl – COOH COO-
Side Chains
Acidic – glutamate, aspartate, cysteine, tyrosine
Basic – lysine, arginine, histidine |
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Term
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Definition
There are two types of ion exchangers:
Those which chemically bind negative charges are called anion exchangers
Those that chemically bind positive charges are called cation exchangers
The charges on the exchangers are balanced by counterions
Chloride ions for the anion exchangers
Metal ions for the cation exchangers
Ion exchangers are named for the counterion |
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Term
| Anion Exchange Chromatography |
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Definition
Molecules to be adsorbed from solution have a negative charge (X-), which is counter-balanced by sodium ions (Na+)
An anion exchanger (A+) has chloride ions as the counter-ion to give A+Cl-
When (Na+X-) molecules in solution interact with the ion exchanger, the X- displaces the chloride ion from the exchanger and becomes electrostatically bound to give A+X-, and simultaneously releases sodium ions
X- can then be eluted by changing elution buffer concentrations, pH,
or charge parameters |
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Term
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Definition
AE+ X-(Cl-)
AE+ X-(Cl-)
AE+ Cl- X-
AE+ Cl-
X-X-
Na+(X-) (your sample)
The counter anion Cl- is exchanged for the X- in your sample
AE+ = solid phase or gel matrix with attached ion; Anionic Exchanger (“+”, cation chemically attached to the matrix) |
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Term
| Cation Exchange Chromatography |
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Definition
Molecules to be adsorbed from solution have a positive charge (Y+), which is counter-balanced by chloride ions (Cl-)
A cation exchanger (C-), which has sodium ions as the counter-ion to give C-Na+
When (Cl-Y+) molecules in solution interact with the ion exchanger, the Y+ displaces the sodium ion from the exchanger and becomes electrostatically bound to give C-Y+, and simultaneously releases chloride ions
Y+ can then be eluted by changing elution buffer concentrations, pH, or charge parameters |
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Term
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Definition
CE- Y+(Na+)
CE- Y+(Na+)
CE- Na+ Y+
CE- Na+
Y+Y+
Y+(Cl-) (your sample)
The counter anion Na+ is exchanged for the Y+ in your sample
CE- = solid phase or gel matrix with attached ion; Cationic Exchanger (“-”, anion chemically attached to the matrix) |
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Term
| Ionic Strengths and Selectivity in IEX |
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Definition
Sample components move through the column at different speeds
At low ionic strengths, all components with affinity for the ion exchanger will be tightly adsorbed at the top of the ion exchanger and nothing will remain in the mobile phase
When the ionic strength of the mobile phase is increased by adding a neutral salt, the salt ions will compete with the protein
Increasing the ionic strength even more causes a larger number of the sample components to be desorbed, and the speed of the movement down the column will increase
The higher the net charge of the protein, the higher the ionic strength needed to bring about desorption
At a certain high level of ionic strength, all the sample components are fully desorbed and move down the column with the same speed as the mobile phase |
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Term
| Schematic Illustration of IEX |
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Definition
[image]
Adsorbed proteins are eluted in order of least to most strongly bound molecules by increasing ionic strength or varying the pH of the elution buffer |
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Definition
[image]
As you increase the elution buffer (usually 1M NaCl) the proteins are released due to competition for the beads |
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Term
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Definition
Separate a protein of interest from contaminating proteins if:
Protein mixture is simple (i.e. if prior purification steps have been carried out)
Charge difference between a protein of interest and the contaminating proteins is sufficiently great
Protein recovery from IEX matrices is usually less than 80%
Band broadening
Conductivity |
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Term
| Selecting an IEX Gel Matrix |
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Definition
Proteins carry both (-) and (+) charged groups
The net charge of a protein is dependent on pH
At its isoelectric point, the net charge of a protein is zero and no binding to any type of IEX gel matrix will occur
Choice of an IEX gel depends on the following:
pH range where the protein of interest is stable
Solubility test: aliquot or dialyze sample of protein into various pH and salt concentrations
If the protein is most stable at a pH above its pI, then an anion exchange gel matrix should be used
If it is most stable at a pH below its pI, then a cation exchange gel matrix should be used
Molecular size of the protein being separated
The porosity of an IEX matrix may affect the binding capacity of a gel matrix |
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Term
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Definition
Conditions for adsorption of a protein of interest to an IEX matrix must be found empirically
As a rule of thumb an ion exchanger can be selected for a protein of interest on the basis of its pI:
Protein is neutral at a pH equal to its pI
Positively-charged below pI as hydrogens add to the protein in an acidic environment (NH3 NH4+, COO- COOH)
Negatively-charged above pI as hydrogens dissociate in a basic environment (NH4+ NH3, COOH COO-)
pI = 5.0: cation exchanger pH < 4.0, anion exchanger pH > 6.0
pI = 7.0: cation exchanger pH < 6.0, anion exchanger pH > 8.0
pI = 9.0: cation exchanger pH < 8.0, anion exchanger pH > 9.0 |
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Term
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Definition
Cellulose and cross-linked dextran (Sephadex) are used as the solid supports
Positively charged groups such as diethylaminoethyl (DEAE) and quaternary ammonium (Q) for anion exchange columns
Negatively charged groups such as carboxymethyl (CM) and sulfonic acid (S) for cation exchange columns
The ionic properties of the beads are dependent on pH, but are sufficiently charged to work well as ion exchangers within the pH range 4 to 9 where most protein separations take place |
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