What is size-exclusion chromatography and what determines separation in SEC?

What is size-exclusion chromatography and what determines separation in SEC?

• A. The separation is based on ionic strength of the mobile phase.
• B. The separation is based on boiling point differences.
• C. The separation is based on the mobility of ions in solution.
• D. The separation is by hydrodynamic size; larger molecules elute first as they are excluded from pores; smaller molecules enter pores and elute later; separation depends on pore size distribution and molecular size relative to pore sizes.

Answer: (D)

Size-exclusion chromatography separates molecules by their hydrodynamic size rather than their charge or chemical interactions. The column is packed with porous beads, and large molecules cannot enter the small pores, so they travel mostly through the column’s void space and come out first. Smaller molecules can penetrate the pores and take a longer, more winding path through the pore network, which slows them down and makes them elute later. Therefore, the order of elution reflects how each molecule fits into the pore sizes available. The exact separation depends on the pore size distribution of the stationary phase and the molecule’s size relative to those pores; calibration with standards relates elution position to molecular size. This approach is also known as gel filtration, emphasizing that separation is driven by size rather than by ionic strength, boiling point, or ion mobility.

Size-exclusion chromatography separates molecules by their hydrodynamic size rather than their charge or chemical interactions. The column is packed with porous beads, and large molecules cannot enter the small pores, so they travel mostly through the column’s void space and come out first. Smaller molecules can penetrate the pores and take a longer, more winding path through the pore network, which slows them down and makes them elute later. Therefore, the order of elution reflects how each molecule fits into the pore sizes available. The exact separation depends on the pore size distribution of the stationary phase and the molecule’s size relative to those pores; calibration with standards relates elution position to molecular size. This approach is also known as gel filtration, emphasizing that separation is driven by size rather than by ionic strength, boiling point, or ion mobility.