How does size-exclusion chromatography differentiate molecules, and what is meant by the void volume?

How does size-exclusion chromatography differentiate molecules, and what is meant by the void volume?

• A. Separation by hydrodynamic size/shape; large molecules elute first while small molecules penetrate pores and elute later; void volume is the pore-excluded fraction where non-penetrating solutes elute.
• B. Separation by polarity; large molecules elute first; void volume is the volume inside the detector.
• C. Separation by charge; large molecules elute first; void volume is the volume of mobile phase outside column.
• D. Separation by chemical reactivity; large molecules elute first; void volume is the entire column volume.

Answer: (D)

Size-exclusion chromatography separates molecules primarily by their ability to access the pores in the stationary phase. Large molecules can’t fit into the pores, so they travel through the column mainly through the spaces between beads and come out first. Smaller molecules can diffuse into the pores, which makes their path longer and leads to later elution. Void volume refers to the portion of the column’s volume that is outside the pores of the stationary phase—the mobile phase space that cannot be accessed by solutes that would have to enter pores. Molecules that are too large to enter the pores elute at this void volume. In contrast, molecules that can enter the pores elute later, after traversing the internal pore volume. So, SEC uses size as the sorting principle, not polarity or charge, and the void volume marks the earliest possible elution point for non-penetrating species.

Size-exclusion chromatography separates molecules primarily by their ability to access the pores in the stationary phase. Large molecules can’t fit into the pores, so they travel through the column mainly through the spaces between beads and come out first. Smaller molecules can diffuse into the pores, which makes their path longer and leads to later elution.

Void volume refers to the portion of the column’s volume that is outside the pores of the stationary phase—the mobile phase space that cannot be accessed by solutes that would have to enter pores. Molecules that are too large to enter the pores elute at this void volume. In contrast, molecules that can enter the pores elute later, after traversing the internal pore volume. So, SEC uses size as the sorting principle, not polarity or charge, and the void volume marks the earliest possible elution point for non-penetrating species.