Principles of gel filtration chromatography/ size exclusion chromatography

Summary

Gel filtration chromatography separates biomolecules based on their size as they pass through a gel-packed column. Larger molecules, unable to enter the gel pores, remain in the surrounding solution, while smaller molecules penetrate the pores to varying degrees. The extent of penetration depends on molecule size and steric hindrance. If a molecule is smaller than the smallest pore, it can fully access the total pore volume. This process enables effective separation of molecules based on size.

Takeaways

• 🔬 Separation in gel filtration chromatography is based on the size differences of biomolecules.
• 🧪 The process involves passing molecules through a column packed with a gel chromatographic medium.
• ⚖️ Larger molecules are excluded from the gel pores and remain in the solution surrounding the beads.
• 🔍 Smaller molecules can penetrate the gel pores to varying degrees depending on their size.
• 🚫 Molecules too large for the gel pores cannot enter the gel and are confined to the external solution.
• 🌐 Steric hindrance limits access to the pores for medium-sized molecules.
• 🔄 If a molecule is smaller than the smallest pore in the gel, it can enter the total pore volume.
• ⚗️ The separation efficiency is influenced by the molecule's size and its ability to diffuse into the gel.
• 🧬 This method helps distinguish between very large, medium, and small molecules based on their size.
• ⚙️ Gel filtration is an effective technique for separating biomolecules by size in chromatography.

Q & A

What is the primary principle behind gel filtration chromatography?

Gel filtration chromatography separates biomolecules based on differences in their sizes as they pass through a column packed with a gel medium.

How are larger molecules treated in gel filtration chromatography?

Larger molecules, which are too big to enter the pores of the gel, remain in the solution surrounding the beads and are excluded from the gel.

What happens to molecules that are smaller than the gel pores?

Molecules smaller than the pores can enter the gel and penetrate the pores to varying degrees, depending on their size.

Why are molecules of intermediate sizes separated in gel filtration?

Molecules of intermediate sizes are separated because they can partially penetrate the pores, and the extent of their access to the pores depends on their size and steric hindrance.

What role do steric hindrance and molecular size play in gel filtration chromatography?

Steric hindrance limits the ability of molecules to access the pores in the gel. Larger molecules are excluded, while smaller ones penetrate the pores based on their size.

What is the significance of pore size in gel filtration?

Pore size determines which molecules can enter the gel. Larger molecules are excluded, while smaller ones can penetrate the pores and move through the column.

What happens if a molecule is smaller than the smallest pore in the gel?

If a molecule is smaller than the smallest pore, it can enter the total pore volume and fully diffuse through the gel.

What is the chromatographic medium used in gel filtration chromatography?

The chromatographic medium in gel filtration is a gel that contains pores of various sizes, allowing different-sized molecules to be separated based on their ability to enter the pores.

Why is gel filtration chromatography also called size-exclusion chromatography?

Gel filtration is also called size-exclusion chromatography because the separation is based on the size of the molecules, with larger ones being excluded from the gel and smaller ones being allowed to enter.

How does gel filtration chromatography differ from other types of chromatography?

Gel filtration chromatography differs from other chromatography types because it separates molecules solely based on size, whereas others might rely on charge, affinity, or hydrophobicity.