Question

QUESTION 22 The unknown protein sample used in this experiment contained Potassium ferricyanide (329.24 Da), Beta galactosidase (540,000 Da), Pancreatic amylase (51,000 54,000 Dal. Lysozyme (14,300 Da), and Beta-amylase (223,800 Da), and Blue Dextran (2,000,000 Da). State the order in which would you expect them to elute out of the column

Answer 1

Gel filtration technique is one of the important chromatography techniques used for the separation of macromolecules. The principle of seperation is molecular sieving, where the molecules enter into matrix of beads and then elute based on their molecular size/weight. In the beads, the seperating molecules have different access to the pores because of the varieng steric interaction based on their molecular weight. When small molecules enter the pores of the beads and percolates throght the pore at slower rate compared to larger molecules and thus move slowly through the coloumn. However, the large molecules comparably have less access inside the beads, making a comparably faster elution.

Thus in the above sample containing Potassium ferricyanide, Beta galactosidase, Pancreatic amylase, Lysozyme, Beta amylase, Beta amylase, and Blue dextran, the elution will be based on the molecular weight. The molecule with higher molecular weight elutes first followed by the molecules with decreasing size.

The elution will be in the following sequence

1. Blue dextran: 2000 kd
2. Beta galactosidase: 540 kd.
3. Beta amylase: 223.8 kd
4. Pancreatic amylase: 51-54 kd
5. Lysozyme: 14.3 kd
6. Potassium ferrucyanide: 0.329 kd