Question

What is the main purpose of running a column chromatography? Similar to TLC. column chromatography is based on analytes being partitioned between a stationary phase (not moving) and a mobile phase (moving). What is most commonly used as stationary phase in a column? Is this material polar or non-polar? In this experiment, you will use different solvents to run column chromatography separation of different pigments extracted from spinach leaves. These solvents are a mixture of acetone and hexanes. (a) What are the composition of these solvents? (b) Rank order the polarity of these solvents. The mobile phase of column chromatography is eluent solvent. The choice of the solvent polarity is extremely important to the success of a column chromatography run. Why are solvents of different polarity chosen as the eluting solvents for different compounds such as carotene and xanthophyll?

Answer 1

Main purpose of running column chromatography: Main purpose of column chromatography is used to separate mixture of organic compounds (either solid or liquid) using stationary phase and mobile phase. Mixture of compounds load into column containing stationary phase and eluted by organic solvent or mixture of organic solvents with different compositions depends on polarity of organic mixtures. Polar compounds bind strongly with stationary phase and elute slowly whereas non-polar compounds elute fastly due to weak binding with stationary phase. This is most prefered technique used by organic chemists in laboratory to purify organic compounds.

Most commonly used stationary phase are silica gel or alumina. These materials are polar due to hydroxyl groups present in it which binds with polar compounds more firmly than non-polar compounds.

Mixture of Acetone and Hexane used to separate the different pigments of spinach leaves. Compositions of these solvents given in below table

Sr. NO.	Composition of Hexane (%)	Composition of Acetone (%)	Polarity
1	100	0	polarity increase from top to bottom as % of acetone increases
2	90	10
3	80	20
4	70	30
5	60	40
6	50	50
7	40	60
8	30	70
9	20	80
10	10	90
11	0	100

Upper composition is non-polar due to % of non-polar solvent (hexane) is higher, used to separate non-polar compounds. The % of polar solvent (Acetone) increases, then polarity of moble phase also increases, hence lower compositions is used to separate polar compounds.

Dipole moment of acetone is higher than hexane hence,

Acetone is - polar solvent

Hexane is - non-polar solvent

Organic solvents of different polarity used to purify / separate organic compounds depends on funtional group present in it. Carotene is highly conjugated hydrocarbons (terpenoids) and is highly nop-polar compound.

Structure of Beta-caroteme

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Hence it can be separated by non-polar solvent hexane or mixture of solvents such as Hexane:Acetone (90:10) mixture.

Where as Xanthophyll contains hydroxyl group hence it is polar compound compare to carotene. Hence it can purified by using polar mobile phase such as mixture of Dichloromethane:Methanol.

Structure of Xanthophyll

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