This set of Biophysics Multiple Choice Questions & Answers (MCQs) focuses on “Molecular Exclusion Chromatography – Set 2”.
1. Consider the following image as a hypothetical demonstration of gel exclusion chromatography. The size of the protein A, B, C and D are 10kd, 6kd, 2kd, 8kd respectively. What will be the elution flow of these proteins from last eluted to the first?
a) A<C<B<D
b) C<D<B<A
c) C<B<A<D
d) C<B<D<A
View Answer
Explanation: In a gel exclusion chromatography, the proteins get separated only on the basis of their size. The smaller protein will pass through the porous beads thereby will be eluted last. So the correct order of elution is C<B<D<A.
2. Gel exclusion chromatography doesn’t completely depend on which of the following property?
a) Size of the protein
b) The shape of the protein
c) Length of the side chain
d) Charge of the protein
View Answer
Explanation: Gel exclusion chromatography completely depends on those properties which are related to size. But this chromatography does not separate proteins on the basis of their charge.
3. You have a 102cc column of gel exclusion chromatography. Suppose no buffer is given to you. If it’s completely filled with bead, what will be the approximate free space volume present in the column?
a) 20cc
b) 102cc
c) 50cc
d) 36cc
View Answer
Explanation: Generally, the void volume is 35% of the total column volume. Therefore, 35% of 102cc is 35.7 that is almost equals to 36 cc.
4. Which of the following protein can surely be used for the determination of void volume?
a) Human serum albumin
b) Bovine serum albumin
c) Blue dextran
d) Heat shock protein
View Answer
Explanation: A comparatively larger protein is used to measure the void volume of the column because we can surely tell that the protein bypasses the porous beads. Blue dextran’s molecular weight is 66Kd which is very large, larger than all the commonly known proteins.
5. In a gel exclusion chromatography, if the void volume is 22cc and the total bead volume is 55cc what is total column volume of that gel exclusion column?
a) 102cc
b) 77cc
c) 33cc
d) 150cc
View Answer
Explanation: Total column volume of a gel exclusion column = void volume + bead volume. Therefore, the total column volume of that gel exclusion column is 22+55 = 77cc.
6. The following image is the demonstration of gel exclusion chromatography. Which of the following statement is true?
a) Protein A is bigger in size and present in a larger amount
b) Protein B is smaller in size and present in a larger amount
c) Protein A is smaller in size and present in a smaller amount
d) Protein B is bigger in size and present in a larger amount
View Answer
Explanation: In case of gel exclusion chromatography, because of the presence of porous beads, smaller beads gets diffused into this and takes longer time to elute so be is the smaller in size. Secondly, more absorbance means more protein is present therefore, B is present in a larger amount.
7. For a gel exclusion chromatography, if the void volume is given as 70cc then what is the approximate total volume of the column?
a) 25cc
b) 50cc
c) 200cc
d) 140cc
View Answer
Explanation: Void volume is typically 35% of the total column volume. Therefore, the total volume of the column is 70*100/35 = 200cc.
8. You have two proteins – one protein is 6kd and positively charge while another protein is 30kd and negative in charge. You performed the following chromatography experiment to separate them. What is the technique you have used and identify both the proteins?
a) Protein A is 30kb, protein B is 6Kb and the technique is absorption chromatography
b) Protein B is 30kb, protein A is 6Kb and the technique is absorption chromatography
c) Protein B is 30kb, protein A is 6Kb and the technique is Gel filtration chromatography
d) Protein A is 30kb, protein B is 6Kb and the technique is Gel filtration chromatography
View Answer
Explanation: Due to having size greater than the pore size of the beads, the larger protein will be eluted faster. Therefore protein A is 30kd as it is less traveled protein.
9. The following image is the demonstration of protein movement paths inside a gel exclusion chromatography column. Arrange the following proteins according to their increasing size.
a) Protein B < Protein D < Protein A < Protein C
b) Protein B < Protein D < Protein C < Protein A
c) Protein B < Protein C < Protein D < Protein A
d) Protein D < Protein B < Protein C < Protein A
View Answer
Explanation: Due to having size greater than the pore size of the beads, the larger protein will bypass the beads and will be eluted faster. Whereas, the smaller protein, due to diffusion through the beads, will take a lot of time to be eluted.
10. Which of the following molecule will be eluted first in a size exclusion chromatography?
a) Blue Dextran
b) HSA
c) BSA
d) Lactate dehydrogenase
View Answer
Explanation: Blue dextran is the largest protein with size 66kd. As in size exclusion chromatography, the larger protein elutes faster therefore, blue dextran will be eluted first.
11. The following diagram is the demonstration of a size exclusion chromatography where 4 proteins were separated. What will be the order of diffusibility of proteins inside the column?
a) Protein D > Protein B > Protein C > Protein A
b) Protein C > Protein D > Protein B > Protein A
c) Protein B > Protein D > Protein C > Protein A
d) Protein B > Protein D > Protein A > Protein C
View Answer
Explanation: In a size exclusion chromatography, the smaller proteins due to lesser size can diffuse through the porous beads. So, smaller the size of the protein greater its diffusibility rate.
12. Which of the following material will you use to separate protein size ranging 1000kd to 5000kd?
a) Sephacryl S-400
b) Sephacryl S-200
c) Sephadex G-50
d) Sephadex G-10
View Answer
Explanation: Sephacryl S-400 beads have comparatively greater pore size, so it can be perfectly used to separate proteins size ranging 1000kd to 5000kd. The rest of the beads are used for the separation of smaller sized proteins.
13. Which of the following property is independent of the size of the column?
a) Void volume
b) Elution volume / Void volume
c) Elution volume
d) Void volume / Elution volume
View Answer
Explanation: Void volume is the free space inside a column, whereas the elution volume is the total buffer volume required for the elution of a particular protein. Elution volume / Void volume is constant for a particular type of column. It doesn’t depend on the column size.
14. If the total volume of a size exclusion chromatography is 205cc and the void volume for that column is 100cc, then what will be the volume of beads?
a) 105cc
b) 305 cc
c) 75cc
d) 150cc
View Answer
Explanation: Volume occupied by the bead = total volume of the column – void volume. Therefore the bead volume = 205-100 = 105cc.
15. To perform a gel filtration chromatography, you require 77cc buffer to completely fill the column. If the total column volume 156 cc. What is the bead volume present inside the column?
a) 233cc
b) 105cc
c) 79cc
d) 25cc
View Answer
Explanation: The total bead volume of a column = total volume of the column – the void volume. Here 77cc is the void volume. So the bead volume present inside the column is = 156-77 = 79cc.
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