2. Chromatography Bovine hemoglobin in an aqueous media is purified using a non-adsorbing size exclusion column (SEC) (30cm in length x 2.5cm in diameter) packed with 75μm diameter porous particles. Below are several experimental parameters. Bed porosity Eb = 0.38 Inclusion porosity 0.498 (the effective stationary phase volume fraction accessible to a specific solute.) Velocity of the fluid v 1cm/min Equilibrium adsorption constant Ke0 (non-adsorbing) kcrot = 4.91 à 10-3cm/min Dispersion coefficient E * 1.80 x 10-5 cm2/min (a) Calculate α ã(1+K2) (b) Calculate the fraction of solute in the moving fluid phase at equilibrium Ï H=2644004) as a function of the fluid velocity v (d) Calculate the number of theoretical chromatographic plates N = LH at v = 1 cm/min. (e) Consider a case where bovine serum albumin is mixed with bovine hemoglobin and loaded to this SEC column with hemoglobin. The inclusion porosity for bovine serum albumin E; 0.600. Calculate the resolution R (f) Using the approximate solution discussed in the lecture, plot the concentration of bovine hemoglobin and bovine serum albumin at the end of the column as a function of time on the same graph. Assume the initial loading was 10mg for both hemoglobin and serum albumin. Are the concentration profiles consistent with R that you calculated in (e)? (g) If we want to achieve R 1 for this mixture, what should be the column length? Plot the concentrations vs time and see if the overlap between two concentration profiles is substantially reduced