Please explain the concepts of elution volume and void volume as they pertain to size exclusion chromatography?

Question:  Please explain the concepts of elution volume and void volume as they pertain to size exclusion chromatography?

In size exclusion chromatography (SEC), also known as gel filtration chromatography, molecules are separated based on their size as they pass through a column packed with porous beads. Here are the key concepts:

Elution Volume (Ve)

- Definition: The elution volume is the volume of the mobile phase (buffer) required to elute a particular molecule from the column.

- Significance: It indicates when a specific molecule exits the column. Larger molecules elute earlier (with a smaller elution volume) because they cannot enter the pores of the beads and thus travel through the column more quickly. Smaller molecules elute later (with a larger elution volume) because they can enter the pores and take a longer, more convoluted path through the column¹².

Void Volume (Vo)

- Definition: The void volume is the volume of the mobile phase in the column that is outside the beads. It represents the volume at which molecules that are too large to enter the pores elute.

- Significance: It is the first peak observed in the chromatogram and corresponds to the elution of very large molecules that are excluded from the pores entirely. These molecules travel through the column without entering the beads and thus elute quickly.

Relationship Between Elution Volume and Void Volume

- The elution volume of a molecule is always greater than or equal to the void volume. Molecules that are too large to enter any pores will elute at the void volume, while smaller molecules will elute at volumes greater than the void volume, depending on their size and the extent to which they can penetrate the pores.