Question

these questions are in reference to gel filtration chromatography

Fost lab questions on lab 8: Analysis of Laboratory Results 1. If the following mix of molecules were parified using sine exclusion chromatography, what would be the 6 WI onder in which the molecules pass chrough the opening in the bottom of the column? Mixture containingR hemoglobin, 65,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000 daltons. rhe tw First molecule to appear Second molecule to appear Third molecule to appear 2. Ifa size exclasion chromatography columm is said to have an exclusion limit of 40,000 daltons, would bemoglobin (60,000 dalhons) be fractionated or excluded from the column? 3. Why do you think the column needed to be "dry" (the absence of buffer on the top of the column bed) when the protein mix was loaded? 4. Why did you need to add more buffer after the protein mixture was loaded onto the column? 5. Examine the 10 fractions that you collected. Which tube contains the "peak" fraction for yellow protea and blue protein? The peak fractions contain the highest concenttration of protein/vitamin and will be the most intense in color. 51 6. Which molecule, yellow or blue, exited the column first? Would this molecule be the larger or smaller of the two? period 2 $7 (n-2)

Answer 1

1.

Gel filtration resin is used in the gel filtration chromatography which is mainly based on the molecular size. It is composed of beads which contain pores of a defined size range. Therefore the large protein molecules cannot enter these pores and pass around the outside of the beads hence the volume of the column appears smaller for the large molecule.

On the contrary the smaller proteins can enter the pores of the beads hence the volume of the column appear larger for small molecule. Overall in the given sample of proteins passing through a gel filtration column and separated on the basis of their molecular size. The he big molecule will elute first and the smallest ones will elute last. Hence the 1^st molecule to appear = Myosin

2^nd molecule to appear = Hemoglobin

3^rd molecule to appear = Myoglobin

2.

The exclusion limit of the gel can be defined as the size above which proteins will elute in the void volume of the column. Since the size of hemoglobin molecules is above than exclusion limit therefore they will fractionated from the column.

3.

When the protein mix was loaded the column needed to be dry as presence of any other molecule can hindered the process of separation.