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?
Size exclusion chromatography (SEC) is a technique that separates molecules based on their size and shape. In SEC, a column is filled with porous beads that have different pore sizes. The sample is injected into the column and eluted with a buffer. As the sample travels through the column, molecules of different sizes interact differently with the pores of the beads.
Elution volume (V_e) is the volume of buffer required to elute a molecule from the column. It depends on the size and shape of the molecule and the pore size of the beads. Larger molecules elute faster than smaller molecules because they cannot enter the pores of the beads and are excluded from a fraction of the column volume. Smaller molecules elute slower because they can enter the pores of the beads and have access to a larger fraction of the column volume.
Void volume (V_o) is the volume of buffer required to elute the largest molecules that are completely excluded from the pores of the beads. It corresponds to the volume outside the beads and is determined by injecting a molecule that is too large to enter any pore, such as blue dextran.
Total volume (V_t) is the volume of buffer required to elute the smallest molecules that can enter all the pores of the beads. It corresponds to the volume inside and outside the beads and is determined by injecting a molecule that is small enough to enter any pore, such as acetone.
The relationship between elution volume, void volume, and total volume can be expressed by a dimensionless parameter called partition coefficient (K_av):
K_av = (V_e - V_o) / (V_t - V_o)
K_av ranges from 0 to 1 and reflects how well a molecule is separated by SEC. A K_av of 0 means that the molecule is completely excluded from the pores and elutes in the void volume. A K_av of 1 means that the molecule can enter all the pores and elutes in the total volume. A K_av between 0 and 1 means that the molecule can enter some but not all of the pores and elutes between the void and total volumes.
By plotting K_av versus the logarithm of molecular weight for a set of known standards, a calibration curve can be obtained for a given SEC column. The molecular weight of an unknown sample can then be estimated by measuring its elution volume and finding its corresponding K_av on the calibration curve.
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