Cation Exchange Chromatography

Summary

The transcript explains the process of cation exchange chromatography, a technique used to separate and concentrate biomolecules based on their charge. It involves using a matrix of charged polymers to bind the target molecule, cytochrome C, which has a positive charge at pH 7. The process includes resin equilibration with a sodium phosphate buffer, applying the sample to the resin, collecting the non-bound substances, and finally eluting cytochrome C with a sodium chloride solution, which displaces the bound protein by increasing ionic strength.

Takeaways

• 😀 Ion exchange chromatography separates or concentrates biomolecules based on their charge.
• 😀 The stationary phase in ion exchange chromatography is a matrix containing insoluble charge polymers with exchangeable ions.
• 😀 The target molecule's charge is pH-dependent, which affects the choice of matrix used in the chromatography process.
• 😀 Cytochrome C has an overall positive charge at pH 7, making cation exchange chromatography suitable for its separation.
• 😀 The cation exchange chromatography procedure involves resin equilibration, sample application, resin collection, and elution of Cytochrome C.
• 😀 Resin equilibration is done using two washes of 1 M sodium phosphate buffer at pH 7.
• 😀 During sample application, the negatively charged resin binds Cytochrome C, displacing sodium counter ions.
• 😀 The resin is collected in a syringe column, and non-bound substances are drained out.
• 😀 To elute Cytochrome C, a buffer solution with a higher sodium chloride concentration (8 M) is used.
• 😀 The increased ionic strength of the elution buffer results in sodium ions replacing Cytochrome C on the column.

Q & A

What is ion exchange chromatography used for?

Ion exchange chromatography is used to separate or concentrate biomolecules based on their charge.

What is the role of the stationary phase in ion exchange chromatography?

The stationary phase in ion exchange chromatography is a matrix made of insoluble charged polymers that contain loosely held ions, which can be exchanged with the target molecule.

How does the charge of the target molecule influence the choice of matrix?

The charge of the target molecule, which is pH-dependent, determines the type of matrix used in the ion exchange process. At pH 7, cytochrome C has an overall positive charge, so a cation exchange chromatography matrix is used.

What are the main steps involved in the cation exchange chromatography procedure for cytochrome C?

The main steps include resin equilibration, sample application to the resin, resin collection, and finally, elution of cytochrome C.

What is the purpose of resin equilibration in the cation exchange chromatography process?

Resin equilibration involves washing the cation exchange resin beads with sodium phosphate buffer to prepare the resin for binding with the target molecule, cytochrome C.

How does the sample application work in the cation exchange chromatography procedure?

During sample application, the negatively charged resin binds to cytochrome C, while sodium counter ions are displaced, concentrating the cytochrome C on the resin.

What happens during the resin collection step?

During resin collection, the resin is gathered into a syringe column, and the non-bound substances are drained out, leaving the cytochrome C bound to the resin.

What is used to elute cytochrome C from the column?

To elute cytochrome C from the column, a buffer solution with a higher concentration of sodium ions (8 M sodium chloride) is used.

Why does increasing the ionic strength of the buffer help elute cytochrome C?

Increasing the ionic strength of the buffer causes sodium ions to replace cytochrome C on the column, allowing the cytochrome C to be eluted.

At which pH does cytochrome C have a positive charge, requiring cation exchange chromatography?

At pH 7, cytochrome C has an overall positive charge, making cation exchange chromatography the appropriate method for targeting the protein.