Question

What is Chromatofocusing?

Answer 1

Hint: This is a technique that was developed by Sluyterman and his colleagues between the years 1977-1981. It is actually a technique used for protein isolation. Chromatofocusing combines the advantage of high-capacity ion-exchange procedures with the high resolution of isoelectric focusing into a single chromatographic focusing procedure.

Complete answer:
The chromatofocusing method is actually a variant of the primitive ion-exchange chromatographic method which was modified to give faster results with higher resolution and selectivity.
This technique is used selectively for proteins, often for the final polishing of proteins. The basic principle of this technique is separation of proteins based on their isoelectric points.
We may discuss the procedure of chromatofocusing in a brief way:
The chromatofocusing medium is equilibrated with a start buffer at a pH slightly above the highest pH required. There is a range of buffers that can be selected based on our needs.
Sample is applied to the chromatographic column by mixing it with the start buffer.
Then, the elution buffer (polybuffer) is passed through the column and begins to titrate the amines on the medium and the proteins.
Thus, a gradient pH is developed.
Proteins in the sample that are at a pH above their pI are negatively charged and retained near the top of the column.
The ones having their pH below pI begin to migrate down and bind to that part of the column where the pH is above their pI.
(pI: The pI of each protein is the pH at which the protein has zero surface charge.)

Note:
The chromatofocusing method of protein isolation is a very advanced technique but still it has a major drawback. It is less suitable for the isolation of proteins that precipitate irreversibly at or near their isoelectric points because these proteins are likely to precipitate on the column if they reach a high enough concentration.
Some learners may get confused as the principle of chromatofocusing is very much similar to that of isoelectric focusing. But the point to be noted here is that in chromatofocusing no electric field is involved. Instead a pH gradient is made to propagate inside an ion-exchange chromatography.