25 Questions
What tool is used to fill the column?
Syringe
What is the purpose of filling the column?
To fill the column with buffer to just above the bed support screen
Where is the GF buffer injected into?
Column outlet tubing
What is the final level of buffer in the column?
Just above the bed support screen
What is filled with GF buffer?
The empty column
What is the primary factor in selecting a gel filtration matrix?
The range of size of molecules to be separated
What is the main purpose of gel filtration?
To separate molecules based on size
What is the goal of selecting a gel filtration matrix?
To separate molecules based on their size and goal of separation
What is gel filtration used for?
To separate molecules of different sizes
What is the basis of separation in gel filtration?
Molecular size
What is the minimum molecular weight of proteins that are completely excluded from Sephadex G-75?
70,000
What is the term for the volume of buffer required to elute a given substance?
Elution volume
Which of the following is a characteristic of Sephadex G-75?
It can separate proteins based on their molecular weights
What happens to proteins with molecular weights greater than 70,000 when they are passed through Sephadex G-75?
They are completely excluded
What is the purpose of measuring the elution volume of a compound?
To determine its separation characteristics
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
What is the purpose of collecting the column effluent?
To analyze the separated molecules
What is the benefit of using an automated fraction collector?
It improves the efficiency of the collection process
What is the role of the column effluent in the separation process?
It contains the separated molecules
What is the relationship between the hydrostatic pressure or flow rate and the separation process?
It affects the separation of molecules
What happens to Cyt09rorne c during the process?
It is completely included and elutes last
Which of the following proteins elute in order of decreasing molecular weight?
The other proteins, excluding Cyt09rorne c
What is true about the elution of the other proteins?
They are partially included and elute in order of decreasing molecular weight
Why does Cyt09rorne c elute last?
Because it is below the lower fractionation limit
What is the relationship between the molecular weight of the proteins and their elution order?
Higher molecular weight proteins elute last
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.
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.
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