Please answer these questions thoroughly: 4. What could you do if you wanted to

Question

Please answer these questions thoroughly:

4. What could you do if you wanted to also elute the chlorophyll, which remains at the top of your column but keep it separate from the beta-carotene?

5. A student using dichloromethane as a solvent runs a chromatography column and elutes with a 10:1 ratio of hexane/ethyl acetate. The student found that all the mixture came off the column at nearly the same time. What mistake did the student make in running the column?

6. Use the Beer-Lambert Law to calculate concentration given the following information. The absorbance of a sample is 0.16. The molar absorptivity is 120L/(mol cm) and the path length is 10mm.

Explanation / Answer

4. Beta carotene being a hydrocarbon is highly non polar. It can be eluted by hexane. As it is yellow in colour the elution can be monitered. Once all the beta carotene has eluted chlorophyll can then be eluted with a more polar solvent eg acetone. A solution of hexane in acetone of suitable polarity can be made and used to elute the chlorophyll.

5.Dichloromethane is a polar solvent. Chlorophyll being polar elutes in a polar solvent, The princliple behind separatation of chloophyll is to make use of the polarity difference between beta carotene and chlorophyll ie. to elute beta carotene in a non polar solvent and then chlorophyll with a polar solvent .H owever, As Dichloromethane is used as a solvent in this experiment , it would elute chlorophyll along with the non polar beta carotene immediately. Therefore we get a mixture of products.

6. A=eCl

a=absorptivity= 0.16

e=molar absorptivity=120L/mol cm

C concentration

l=path length =10mm=1cm

C=A/el

c=0.16/120

c=0.0013 mol/L

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