Question

Q6. Given below in Table 2 are the readings obtained from a spectrophotometer, for the above mentioned elution of NADH and Ovalbumin (NADH has a max of 340 nm, whereas Ovalbumin was reacted with Bradford dye to give a blue coloured Protein-dye complex, which absorbs at 595 nm). Draw a chromatogram, make sure to label the axis properly, give a legend to the graph and write a detailed title below the graph. (10 Marks) Table 1: Absorbance readings for NADH at 340 nm and Ovalbumin at 595 nm after the gel filtration chromatographic separation of the two. Elution Volume (ml) Readings at 595 nm Readings at 340 nm 0.01 0.01 2 0.03 0.02 3 0.14 0.01 4 0.56 0.01 5 0.18 0.02 6 0.01 0.01 7 0.02 0.01 8 0.01 0.02 9 0.01 0.11 10 0.02 0.26 11 0.01 0.69 12 0.01 0.29 13 0.02 0.15 14 0.01 0.01 Q7. Analyze the results of the above experiment from the chromatogram. Which molecule eluted first from the size-exclusion column and which eluted later. Why? (10 marks)

Answer 1

Question: 6 Answer: The absorption chromatograph of NADH at 340 nm and Ovalbumin at 595 nm given below. Chromatograph amax calculation 0.8 0.7 0.6 0.5 0.4 Absorption (au) - Reading at 595 nm (Ovalbumin) -Reading at 340 nm (NADH) 0.3 0.2 0.1 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Elute volume (mL)

Question: 7 Answer: The chromatograph recorded at different wavelength shows two peaks with difference in peak intensity. In the chromatograph the first peak assigned for ovelbumin (MW 43000), while second peak assigned for NADH (MW 663.43). This higher peak intensity in case of NADH due to more double bond conjugation compare to ovelbumin will also shift absorption frequency at higher wavelength. Size-exclusion chromatography (SEC) is a chromatographic technique used for separating substances according to their molecular size, or more correctly, hydrodynamic volume. Size exclusion is mainly used for the separation of different bio-polymers, polymers and for the separation of polymers from small molecules. Because molecules ovelbumin (kDa 45) that have a large size compared to the pore size of the stationary phase have very little entrance into the pores, hence ovebum in larger sized molecules elute first from the column. Therefore, smaller in size NADH (kDa 0.7) molecules elute last and larger molecules elute first in Size Exclusion Chromatography. In chromatograph (figure) elution profile of ovelbumin and NADH the majority of ovelbumin was present in fraction 4 (elution volume 4) and that of NADH in fraction 11 (elution volume 11) indicating larger ovelbumin eluted first and smaller NADH eluted latter. Since, fraction 4 was collected before fraction 11. This result confirm that ovelbumin is larger than NADH.