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labsep
Education Centre
About the purification of biomolecules
Purpose of purification
Developing purification protocols
How to combine purification steps
Purification development - summary
LC techniques
Affinity Chromatography
Desalting & Gel Filtration
Hydrophobic interaction chromatography
Ion exchange chromatography
Animation
Basic Principles
The Separation Mechanism
Type of ion exchangers
Elution modes
The typical Ion exchange experiment
Charge properties of proteins and peptides
Effect of running pH
Resolution in IEX
Optimisation of IEX experiments
Ion Exchange in practice
Technique Profile
What is Ion Exchange?
Reversed phase chromatography
Protein Purifier software
BioProcess™ Glossary
Effect of running pH

Titration curves and resolution
Consider three proteins (red (R), blue (B), and green (G) curves in Fig 6.1), each with its own unique titration curve

pH is by far the most effective parameter to control selectivity in IEX.
.
At low pH all the proteins are positively charged in the order R>G>B.
Cation exchange will elute them in the order
B, G, R.
Anion exchange. Being positively charged they will all elute unseparated in the wash-through fraction.


At less acidic pH R and G are still positively charged (R>G), while B is negatively charged.
Cation exchange will elute B in the flow-through fraction, while R and G elute in the order: G, R but are better separated.
Anion exchange will elute B by the gradient, while R and G, being positively charged are found in the flow-through fraction being positively charged.		Fig 6.1. The effect of varying pH in anion exchange and cation exchange chromatography.At high pH all the proteins carry negative net charges, B>G>R.
Cation exchange will elute them all in the flow-through fraction.
Anion exchange will elute all the proteins by the gradient and in the order: R, G, B.



At slightly alkaline pH G too has switched to negative net charge. R, however, is still positively charged.
Cation exchange will elute B and G in the wash-through fraction and R by the gradient.
Anion exchange will elute B and G by the gradient, while R is found in the wash-through fraction.
.
    Finding the best pH
    The test tube method (Fig 6.2) is a simple method for finding the pH value at which the target protein binds to an ion exchanger. Once determined, this pH value can then be used as running pH for the first separation.

    Fig 6.2. Test tube method for estimating the pH at
    which the target protein binds to an anion exchanger.

    The example in Figure 6.2 used an anion exchanger and the target protein was found to bind completely at pH 7.5.

    The test tubes contain a small quantity of an anion exchanger suspended in buffers of different pH values and a small amount of the sample. After agitation and centrifugation the supernatants are assayed for target protein content. In the example in Figure 6.2 the protein binds completely from pH 7.5 and upwards.

    Electrophoretic titration curves (Fig 6.3) provide much more detailed information than the test tube method, since all proteins in the sample are analyzed in the same run and at a broad pH span.

    Fig 6.3. Electrophoretic titration curves of proteins from
    a meat extract showing their net charges as a function of pH.

    With access to a chromatography system equipped for automatic buffer preparation, automated pH scouting is a very effective way to determine the optimum running pH .

    A practical example of pH scouting is the separation of pancreatin with ÄKTAexplorer, shown in Figure 6.4. The change in the separation by altering the buffer pH is clear.

    Fig 6.4. Automatic pH scouting performed on ÄKTAexplorer 100.
    Sample: 2 mg crude pancreatin.
    Column: RESOURCE Q; 6 ml