Question

In a Column Chromatography of spinach extract experiment a post lab question is as follows:

Compare the order of elution from the column with the Rf of the observed compounds. Is there a correlation? If yes, what is it?

Answer 1

Spinach extract contains , Chlorophyll a, Chlorophyll b, and beta carotene.

Characteristics of these pigments are : Chlorophyll a -Blue-green, polar.

Chlorophyll b-Green, polar.

beta-carotene-Yellow, non polar.

In column chromotagraphy the solvent used are,1. Hexane, 2. 95% Hexane/5% acetone., 3. 70% Hexane/30% acetone.4. 80% acetone/20%Methanol.

In column, Non-polar components, carotene pigments eluted first. Because polar alumina doesn't absorb much of it.

Polar components, chlorophyll euted after.

Why are column clhlorophyll less mobile on column and why do they have low Rf value than carotene.

Chlorophyll(a and b) both they are consisted with non polar cyclopentenes and polar ester.,ketone and aldehyde.

column is prepared with polar alumina and when chlorophyll run through the column it will interact with polar functional group of chorophyll group molecules. Similararly in TLC, polar parts of Chlorphyll interacts with polar laryer of adsorbent and partially polar solvent in chamber; Therefore, it will ascend slowly.