Question

what is the principle of gel filtration chromatography?

19 E 3 2 1 AaBbceDdEe ABCDdee AaBbCcDc AaBbCcDdE No Spacing Heading 1 Heading 2 7. A. What is the principle of Gel filtration chromatography? (1 point) B. In gel filtration chromatography, what is the stationary phase and the mobile phase (that we used in our lab experiment)? (1 point) C. In what order would the proteins elute from a size exclusion column (limit 50- 40,000) (1 point) Protein Molecular weight KDa 25,000 KDa 256 K Da

Answer 1

7. A) Gel filtration chromatography is otherwise called as size exclusion or molecular sieve chromatography. In this technique molecules in solutions are separated by their size. Generally an aqueous (mobile phase) solution is used to transport the sample through the column (stationary phase).

This method can be used to calculate the approximate molecular weight of the sample. Based on the elution volume this can be found.

Gel filtration separates molecules purely based on molecular size. Separation is achieved through porous matrix to which molecules have different rate of access i.e. Smaller molecules have greater access to the pores and they get trapped into it and the larger size molecules will get excluded from the pores and they get eluted first in the column.

B) The organic solvent which we use to elute the sample from the column is mobile phase and the porous matrix used to trap the molecules are mobile phase. in general these porous beads are composed of dextran polymers (sephadex), agarose (sepharose) and, poly acrylamide (bio-gel P).

C) The order of elution is first 25000 KDa, then 256 KDa and finally 82 KDa. Larger size molecules get excluded in the matrix and get eluted first.