Introduction

Gel filtration chromatography, also known as size exclusion chromatography (SEC), is a widely used technique in biochemistry and molecular biology to separate proteins and other macromolecules based on their size. Unlike other chromatography techniques that separate molecules based on their charge or affinity, gel filtration chromatography primarily works by differentiating molecules based on their hydrodynamic volume.

In this article, we will dive into the mechanism of gel filtration chromatography, explain its principle, and understand how it is used for protein separation and purification.

What is Gel Filtration Chromatography?

Gel filtration chromatography is a type of size exclusion chromatography that separates proteins and other biomolecules by their size. The process involves passing a mixture of proteins through a column filled with porous beads. These beads allow smaller molecules to enter their pores, causing them to take a longer path through the column. Larger molecules, on the other hand, cannot enter the pores and thus move through the column more quickly.

The hydrodynamic volume of the protein is the key factor in determining how fast it moves through the column:

• Large proteins have a larger hydrodynamic volume and cannot enter the pores of the beads. As a result, they elute from the column earlier.

• Smaller proteins have a smaller hydrodynamic volume, which allows them to enter the pores of the beads. They take longer to pass through the column and thus elute later.

This size-dependent separation helps to purify proteins or other macromolecules in the sample.

Principle of Gel Filtration Chromatography

The principle behind gel filtration chromatography is based on the size exclusion of molecules:

• The column is filled with porous beads, typically made of materials like dextran, agarose, or polyacrylamide.

• When a mixture of proteins is applied to the column, larger molecules are excluded from entering the pores and thus move through the column faster.

• Smaller molecules, which can enter the pores, take longer to pass through as they are temporarily trapped within the beads.

The separation of proteins is achieved as they elute from the column at different times based on their size or hydrodynamic volume.

Answer to the Question

The question asks:
Gel filtration chromatography separates proteins on the basis of:

• (a) Affinity tag
  This is incorrect. Affinity chromatography separates proteins based on specific interactions with an affinity tag, not their size.

• (b) Charge
  This is incorrect. Charge-based separation occurs in techniques like ion exchange chromatography, not gel filtration.

• (c) Hydrodynamic volume
  This is correct. Gel filtration chromatography separates proteins based on their hydrodynamic volume, which is related to their size.

• (d) Hydrophobicity
  This is incorrect. Hydrophobicity is the basis of hydrophobic interaction chromatography, not gel filtration.

Therefore, the correct answer is:

(c) Hydrodynamic volume

Conclusion

Gel filtration chromatography is a powerful and widely used technique for protein separation and purification. It works on the principle of size exclusion, where molecules are separated based on their hydrodynamic volume. This technique is particularly useful for separating proteins of different sizes and purifying them based on their molecular size, without the need for additional chemical interactions like charge or hydrophobicity. Understanding the principles behind gel filtration is essential for researchers working in protein biochemistry and molecular biology.

Answer:

The correct answer is:
(c) Hydrodynamic volume