Question

**Answer the questions below based on Figure 3 (next page) *** 3. What is the elution volume of the blue (11) and the orange (42) molecule? 4. Which one is the larger? The blue (41) or the orange (42) molecule? 5. What is the void volume? (assume A is sufficiently larger than &/or Az is sufficiently smaller than the average pore size for the resin selected) LLL Abs. A Figure 3. Idealized separation through Size Exclusion Chromatography and detection at multiple wavelengths. 0 2 4 6 Volume 8 10 Samples can be analyzed by measuring absorption at various wavelength. HEWA Real samples usually have various overlaps that can complicates analysis. + + + + Abs. 12 smenisine 2 nur non Your sample will contain 3 different samples instead 2. 0 2 8 10 4 6 Volume lesbo

Answer 1

Elution volume is the volume of eluent(solvent) requires for the elution of sample/compound.

3. From chromatogram (Fig.3) the elution volume of the blue molecule is ($\lambda$1) = 6

elution volume of the orange molecule is ($\lambda$2)= 8

4. Generally the stationary phase consists of small inert particles with pores of controlled size. When the molecules of different sizes pass through the column with the help of solvent, the molecules larger than pores size can not enter inside the pores of gel beads, so the large molecules will come out with solvent earlier. But the small size molecules than pore size will enter into the pores, so that they will be delayed in elution inside the column bed.

Here the blue molecule($\lambda$1) is eluting first, so its size is larger than orange molecule ($\lambda$2)

5. The elution volume for larger molecules is considered as void volume (V₀)

It is given that blue molecules ($\lambda$1) ares larger than orange molecules($\lambda$2), so the elution volume for blue molecules ($\lambda$1) is void volume. So the Void volume= 6