Question

1. Which type of gel matrix (aka resin) would separate the following proteins using size exclusion chromatography: protein 1 (2,000 Da), protein 2 (7,000 Da), protein 3 (20,000 Da), and protein 4 (90,000 Da)? The answer is C. Please explain the answer!

1. Gel of range 2,500 – 85,000 Da
2. Gel of range 8,000 – 200,000 Da
3. Gel of range 3,000 – 200,000 Da
4. None of the above

Answer 1

Size exclusion chromatography: In this technique of chromatography molecules (proteins) are separated according to their size. The smaller molecules penetrate the pores present on the stationary phase (gel matrix/resin). While the larger molecules unable to enter into the pores and eluted first. So the Larger molecules eluted first and the larger molecules elute later/last (elution is inversely proportional to size of the elutes). The larger molecule eluted with the required volume of solvent which is equal to void volume of column (volume between matrix and beads). The void volume is different for different size of proteins. The protein with larger size (like, 90,000kDa) the void volume is smaller than the protein of smaller size (like; 7,000kDa or 20,000kDa). So the use of smaller volume of solvent first of Protein-4 (90,000 kDa) will be eluted further with increasing the volume of solvent Protein-3 and after that further increasing of solvent volume Protein-2 will be eluted. Lastly Protein-1 will be eluted. So in this type of separation we can get four different peaks. The retention time (Rt); The value of Rt is small for larger protein and Rt is big for smaller protein.