Question

1. Did the column separate NADH and BSA? Explain based on your graph

2. What assumptions have you made in determining the absorbance of the mixture at two different wavelengths?

3. Is the reaction catalysed by GPT a reversible reaction? Give evidence from your experimental results in Table 3 to support your answer. (I DONT UNDERSTAND THIS QUESTION PLEASE HELP)

Separation of BSA and NADH using Gel filtration chromatography 0.060 0.040 0.020 0.000 0.000 4.000 3.000 5.000 6.000 2.000 -0.020 0.040 Elution volume (ml) 280 nm340 nm Table 3: Results obtained from Chromatogram A Distance Distance Deduced Rfvalue identity for each of each Standard(s) or Number travelled by travelled by reaction no. of spots substancesolvent spot (cm) (cm) spot Alanine 5.2 0.48 2.5 a-keto Pyruvate 0.33 5.2 Glutamate 5.2 0.5 2.6 Alanine + a-ketoglutarate 2.7 5.2 0.52 Pyruvate + glutamate aianine t 0.5 alnha 2 5.2 2.6 0.31 yruvate 0.51 2.65 5.2 and 0.33 0.51 glutamate yruvate 0.51 2 5.2 2.6 and 0.33 0.51 glutamate Table 2: Reaction reversibility of liver GPT Vol. of a Vol. of keto- Tissue Vol. of Vol. of Tube keto- Pyruvate (l) GlutamateVolume no. (u) (A) glutarate Pyruvate (Hl) (A) (ul) 800 ul 11 10) 2 100 800 μ1 1000 μι 100 3

Answer 1

1. The absorbance of NADH and BSA are different and both the graph are present hence the elution has seperated BSA and NADH

2.I have made the assumption that the two absorbance a at different wavelengths belong to two different elements

And also that the absorbance of the mixture is not more than 340nm

3.The reaction is a reversible reaction by GPT

Because

The amount of pyruvate and alpha ketoglutarate in tube 2 and 3 are the same