FAQs on Purification with Ion Exchange Chromatography

What is the difference between a strong ion exchanger and a weak ion exchanger?

The terms “strong” and “weak” refer to the acid/base properties of the functional group. If the ligand is derived from a strong acid or a strong base it is referred to as a strong ion exchange resin. If the ligand is derived from a weak acid or a weak base it is referred to as a weak ion exchange resin.

How do I know if I should use a strong or weak ion exchanger to use for purifying my target molecule?

Strong ion exchanger are always the best starting point. If the selectivity is not sufficient, switch to a weak ion exchanger. The capacity of strong ion exchangers (Q, SP, Sulfate, or S) do not vary over a wide pH range, while the capacity of weak ion exchangers (DEAE, NH2 , or CM) varies with pH.

You can use both cations and anions for most applications, but one of the two types is often more suitable for your molecules of interest. If the isoelectric point (pI) of your protein is known, start with a cation exchanger if pI >7, and an anion exchanger if pI <7. 

How can I maximize resolution between my target molecule and impurities when using Ion Exchange?

There are many ways in which better separation can be achieved in ion exchange chromatography and these guidelines can be applied to other modes of chromatography as well. First, resolution can be maximized by increasing bed height. Typical column bed heights range from 15 cm to 30 cm L. Going any higher than 30 cm will result in higher back pressure and reduced throughput if the linear velocity needs to be reduced. A benefit of greater bed heights is to increase sample residence time in the column at the same flow rate, resulting in an increase in binding capacity. Second, a gradient may be used instead of step elutions to resolve impurities. The slope of the gradient can be adjusted to provide either a sharp or gradual change in conductivity depending on how close the impurities elute to the target molecule.

Be aware that shallow gradients result in broad elution peaks. Lastly, pH can be adjusted to affect how tightly molecules bind and subsequently elute from the column. Small changes in pH can cause retention time shifts for elution peaks and may allow for better separation. Furthermore, if the pKa of the target molecule is vastly different from contaminants, either anion or cation exchange may be appropriate if the pH is adjusted above or below the pKa of the target molecule.

How do I know which ion exchanger to use for my purification?

Start with a strong ion exchanger. If the selectivity is not good enough, try a weak ion exchanger. Strong ion exchangers (Q, SP, and S) have constant capacity over a wide pH range, while the capacity of weak ion exchangers (DEAE, ANX, and CM) varies with pH.

Proteins can be purified with both cations and anions but one may be more efficient for your particular protein. If you know the isoelectric point (pI) of your protein, start with a cation exchanger for your capture step if pI>7, and with an anion exchanger if pI <7.

What are typical buffers used for ion exchange chromatography?

* not recommended for resin with NH2 ligand.