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12 Nov 2019
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
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