Why Does P60 Elute Faster Than P40 in a Sephadex G-100 Column?

Question

A researcher separates P40 (40 kDa) from a mixture containing P60 (60 kDa) using a Sephadex G-100 gel filtration column.

Which protein will come out of the column first?

A. P40 will run faster
B. P60 will run faster
C. A 50% mixture of P40 and P60 will always come out
D. P40 and P60 will come out in a ratio of 3:2

Correct Answer: B. P60 will run faster

Principle of Gel Filtration Chromatography (Size Exclusion Chromatography)

Sephadex G-100 separates proteins based on their molecular size.

• Large molecules cannot enter the pores of the Sephadex beads.
• Therefore, they travel mainly through the spaces between the beads and reach the bottom of the column quickly.
• Small molecules enter the pores of the beads, take a longer path, and hence move more slowly.

Why Does P60 Elute Before P40?

P60 (60 kDa)

• Larger protein
• Enters fewer pores
• Travels a shorter path
• Elutes first (runs faster)

P40 (40 kDa)

• Smaller protein
• Enters more pores
• Takes a longer path
• Elutes later

Therefore, P60 comes out of the Sephadex G-100 column before P40.

Easy Trick to Remember

“Big proteins bypass the beads and come out first.”

Or

“In gel filtration chromatography, Bigger = Faster.”

Summary Table

Final Answer

✅ B. P60 will run faster

Because in Sephadex G-100 size exclusion chromatography, larger proteins are excluded from the pores and therefore elute earlier than smaller proteins.