In size-exclusion (gel-filtration) chromatography, which molecules pass the column the fastest?

• A. Large molecules pass quickly around the beads.
• B. Small molecules enter the tiny pores and pass through more slowly.
• C. All molecules move through at the same rate.
• D. Small molecules pass quickly around the beads.

Answer: (A)

In size-exclusion chromatography, separation hinges on size because the beads are porous. Large molecules can’t fit into the tiny pores, so they travel through the column mainly in the spaces between the beads and take a shorter, faster path to the detector. Small molecules can enter the pores and spend more time diffusing inside the bead network, which lengthens their travel time and slows them down. Because of this, the fastest eluting species are the large molecules.