Column chromatography - gel filtration chromatography lecture

Summary

This tutorial video from 'Somos Fallacy' delves into the principles of Size Exclusion Chromatography, also known as Gel Filtration Chromatography. It explains how proteins and nucleotides are separated based on size and hydrodynamic volume, using agarose as the stationary phase to create a porous network. The video clarifies misconceptions about molecular weight separation and highlights the technique's advantages for identifying protein states and its limitations in resolution and separating closely sized molecules.

Takeaways

• 🔍 Size-exclusion chromatography, also known as gel filtration chromatography, is a technique used to separate molecules based on their size and molecular weight.
• 🧬 The stationary phase in size-exclusion chromatography is typically composed of agarose, a polymer that forms a network with pores of varying sizes.
• 🚫 Unlike other chromatographic techniques that may involve charge-based separation, size-exclusion chromatography relies solely on the gravitational force for the movement of molecules.
• 🕰 Large molecules cannot enter the small pores of the agarose network and thus elute faster, while smaller molecules get trapped and take longer to elute.
• 📊 The elution profile of a size-exclusion chromatography run typically shows larger molecules coming out first, followed by smaller ones, creating a characteristic curve.
• 🔬 Hydrodynamic volume, the volume a molecule occupies in solution, is a key factor in separation, with folded proteins having a smaller hydrodynamic volume compared to unfolded proteins.
• 🔬 The technique can be used to differentiate between folded and unfolded proteins based on their hydrodynamic volumes, which are approximately 14 angstrom for folded and 36 angstrom for unfolded proteins.
• 👍 A significant advantage of size-exclusion chromatography is its ability to separate large molecules and identify the state of protein folding.
• 🚧 The resolution of size-exclusion chromatography is limited, meaning it may not effectively separate molecules with very similar sizes or hydrodynamic volumes.
• 🚫 It is a common misconception that the technique separates molecules strictly by molecular weight, but it actually separates based on size and hydrodynamic volume.
• 📉 The technique has disadvantages including low resolution and difficulty in separating molecules of very close sizes, making it less suitable for high-resolution separations.

Q & A

What is size exclusion chromatography also known as?

Size exclusion chromatography is also known as molecular exclusion chromatography, molecular permeation chromatography, gel filtration chromatography, and sometimes just molecular exclusion chromatography.

What is the basis for the separation of molecules in size exclusion chromatography?

In size exclusion chromatography, molecules are separated based on their size and molecular weight, with larger molecules typically eluting before smaller ones due to their inability to enter the pores of the stationary phase.

What is the stationary phase in size exclusion chromatography?

The stationary phase in size exclusion chromatography is typically agarose, a polymer network that creates a structure with small pores through which molecules can pass.

How does the process of size exclusion chromatography differ from gel electrophoresis?

Size exclusion chromatography relies on gravity as the driving force for the separation of molecules, whereas gel electrophoresis uses an electric current to drive the movement of charged molecules through a gel matrix.

What is the concept of void volume in size exclusion chromatography?

Void volume in size exclusion chromatography refers to the volume of the column that is not occupied by the stationary phase, allowing larger molecules to pass through quickly without entering the pores.

Why is size exclusion chromatography particularly useful for protein separation?

Size exclusion chromatography is useful for protein separation because it allows for the separation of proteins based on their size and hydrodynamic volume, which can be indicative of whether the protein is folded or unfolded.

What is the hydrodynamic volume and why is it significant in size exclusion chromatography?

The hydrodynamic volume is the volume or area occupied by a molecule in a solution, which influences how it is separated in size exclusion chromatography. It is significant because it can indicate the folded or unfolded state of a protein.

What are some disadvantages of size exclusion chromatography?

Some disadvantages of size exclusion chromatography include its low resolution, meaning it is not very effective at separating molecules of very similar sizes, and its reliance on size rather than molecular weight, which can sometimes lead to inaccurate separations.

How does the percentage of agarose in the stationary phase affect the separation process?

The percentage of agarose in the stationary phase affects the pore size of the agarose network. A higher percentage of agarose results in smaller pores, which can influence the separation of molecules based on their size.

What is the principle behind the entrapment of smaller molecules in the pores of the stationary phase?

The principle behind the entrapment of smaller molecules is that they can enter and pass through the pores of the stationary phase, taking a longer path and thus eluting later than larger molecules, which cannot enter the pores and therefore travel a shorter distance through the void volume.

How can size exclusion chromatography help in identifying the native state of a protein?

Size exclusion chromatography can help identify the native state of a protein by separating it based on its hydrodynamic volume. A folded protein has a smaller hydrodynamic volume (14 angstroms) compared to an unfolded protein (36 angstroms), allowing for the determination of its state.