9.
A student has a mixture of different proteins (molecular weight of Protein A = 30.0 KDa;
Protein B = 35.0 KDa; Protein C = 40.0 KDa; Protein D = 50.0 KDa). The protein
mixture was purified using FPLC, size-exclusion (gel filtration) chromatography. What
size fraction will elute first?
a. Protein A = 30.0 KDa
b. Protein B = 35.0 KDa
c. Protein C = 40.0 KDa
d. Protein D = 50.0 KDa
Protein D (50.0 KDa) elutes first in FPLC size-exclusion chromatography. Larger proteins travel a shorter path through the column, exiting before smaller ones.
Question Analysis
In size-exclusion (gel filtration) chromatography via FPLC, proteins separate based on hydrodynamic size. The column contains porous beads; proteins larger than the pore size cannot enter and elute first in the void volume. Smaller proteins enter pores, increasing their path length and delaying elution.
Option a (Protein A, 30.0 KDa) elutes last among these, as its small size allows full pore access.
Option b (Protein B, 35.0 KDa) elutes after C and D but before A, following decreasing size order.
Option c (Protein C, 40.0 KDa) elutes after D due to partial pore entry.
Option d (Protein D, 50.0 KDa) elutes first, fully excluded from pores.
In FPLC size-exclusion chromatography, protein mixtures separate by molecular weight, with the largest protein eluting first. This gel filtration technique is key for CSIR NET Life Sciences exam questions on protein purification, where Protein D (50.0 kDa) from a 30-50 kDa mix exits the column ahead of smaller ones like Protein A (30.0 kDa).
Core Principle
Size-exclusion chromatography uses porous beads in the FPLC column. Larger proteins (e.g., 50 kDa) bypass pores entirely, traveling the shortest path and eluting first. Smaller proteins (e.g., 30 kDa) enter pores, prolonging retention and later elution. This inverse size-elution relationship ensures clean separation without chemical interactions.
Elution Order Breakdown
| Protein | Molecular Weight (kDa) | Elution Position | Reason |
|---|---|---|---|
| A | 30.0 | Last | Fully enters pores, longest path |
| B | 35.0 | Third | Partial pore access |
| C | 40.0 | Second | Limited pore entry |
| D | 50.0 | First | Excluded from pores |
CSIR NET Exam Relevance
For CSIR NET questions on FPLC gel filtration, remember: elution follows decreasing molecular weight. Practice with 30-50 kDa examples to master protein purification concepts in biochemistry.
