Question

Separation of proteins on the basis of molecular size is achieved by:

• A. Chromatography on a carboxymethyl (CM) cellulose column.
• B. Iso-electric focusing.
• C. Gel filtration chromatography.
• D. Chromatography on a diethylaminoethyl (DEAE) cellulose column.

Hint:

Size exclusion through porous gel beads, large molecules elute first, is the basis of gel filtration chromatography.

Answer 1

The Correct Option is C

This question is testing whether you know which protein separation method depends on the size of the molecule rather than its electrical charge. Let's go through the four techniques listed.

1. CM cellulose chromatography: Carboxymethyl cellulose carries a negative charge on the column matrix, so it binds and separates positively charged proteins, this is a cation exchange method, based on charge, not size.
2. Iso-electric focusing: This technique separates proteins according to their isoelectric point, the pH value at which each protein has zero net charge. Proteins migrate through a pH gradient until they stop at their own isoelectric point, again this is a charge based separation, not a size based one.
3. Gel filtration chromatography: Also known as size exclusion chromatography, this method uses porous beads packed into a column. Large proteins are excluded from the small pores and pass quickly around the beads, while small proteins wander into the pores and are delayed. Molecules therefore come out of the column strictly in order of size, largest first, smallest last. This is exactly the size based method the question is asking about.
4. DEAE cellulose chromatography: Diethylaminoethyl cellulose carries a positive charge on the matrix, so it binds and separates negatively charged proteins, an anion exchange method, again based on charge, not size.

Only gel filtration chromatography sorts proteins purely by their physical size as they pass through the porous gel beads.

Let's summarize:

• CM cellulose and DEAE cellulose chromatography are ion exchange methods, based on charge.
• Iso-electric focusing is based on isoelectric point, also a charge property.
• Gel filtration chromatography alone separates strictly by molecular size.

So the technique that separates proteins on the basis of molecular size is gel filtration chromatography.