Gel Filtration Principles

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Questions and Answers

What tool is used to fill the column?

Pipette

Micropipette

Microscope

Syringe (correct)

What is the purpose of filling the column?

To sterilize the column

To fill the column with buffer to just above the bed support screen (correct)

To wash the column

To equilibrate the column

Where is the GF buffer injected into?

Column outlet tubing (correct)

Column body

Column inlet tubing

Column support screen

What is the final level of buffer in the column?

Just above the bed support screen Signup and view all the answers

What is filled with GF buffer?

The empty column Signup and view all the answers

What is the primary factor in selecting a gel filtration matrix?

The range of size of molecules to be separated Signup and view all the answers

What is the main purpose of gel filtration?

To separate molecules based on size Signup and view all the answers

What is the goal of selecting a gel filtration matrix?

To separate molecules based on their size and goal of separation Signup and view all the answers

What is gel filtration used for?

To separate molecules of different sizes Signup and view all the answers

What is the basis of separation in gel filtration?

Molecular size Signup and view all the answers

What is the minimum molecular weight of proteins that are completely excluded from Sephadex G-75?

70,000 Signup and view all the answers

What is the term for the volume of buffer required to elute a given substance?

Elution volume Signup and view all the answers

Which of the following is a characteristic of Sephadex G-75?

It can separate proteins based on their molecular weights Signup and view all the answers

What happens to proteins with molecular weights greater than 70,000 when they are passed through Sephadex G-75?

They are completely excluded Signup and view all the answers

What is the purpose of measuring the elution volume of a compound?

To determine its separation characteristics Signup and view all the answers

What is the purpose of allowing the elution to proceed at an appropriate hydrostatic pressure or flow rate?

To ensure a consistent separation of molecules Signup and view all the answers

What is the purpose of collecting the column effluent?

To analyze the separated molecules Signup and view all the answers

What is the benefit of using an automated fraction collector?

It improves the efficiency of the collection process Signup and view all the answers

What is the role of the column effluent in the separation process?

It contains the separated molecules Signup and view all the answers

What is the relationship between the hydrostatic pressure or flow rate and the separation process?

It affects the separation of molecules Signup and view all the answers

What happens to Cyt09rorne c during the process?

It is completely included and elutes last Signup and view all the answers

Which of the following proteins elute in order of decreasing molecular weight?

The other proteins, excluding Cyt09rorne c Signup and view all the answers

What is true about the elution of the other proteins?

They are partially included and elute in order of decreasing molecular weight Signup and view all the answers

Why does Cyt09rorne c elute last?

Because it is below the lower fractionation limit Signup and view all the answers

What is the relationship between the molecular weight of the proteins and their elution order?

Higher molecular weight proteins elute last Signup and view all the answers

Study Notes

Gel Filtration (GF) Gel Matrix

The choice of matrix depends on the range of size of molecules to be separated and the goal of the separation.
Filling the column with GF buffer is done using a syringe, injecting until the column is filled to just above the bed support screen.

Characteristics of Sephadex G-75

Proteins with molecular weights greater than 70,000 are completely excluded.
Volume of buffer required to elute a substance is known as the elution volume, Ve.

Elution Process

Elution proceeds at an appropriate hydrostatic pressure or flow rate.
Column effluent is collected using an automated fraction collector.
Elution order: proteins are partially included and elute in order of decreasing molecular weight, with the smallest (Cytochrome c) eluting last.

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Description

Learn about the principles of gel filtration, including the choice of matrix, and its applications in separating molecules based on size. Understand the importance of gel filtration in various scientific fields.