Assignment # 04 Course Code 2597

[Year]

SHAHZAD ASLAM
BN436891
BS CHEMISTRY
UNIT #04
Column
Chromatography

Submitted to
Dr IZMA YOUNAS
Q-1 Explain the basic Principle of Column Chromatography.
Column Chromatography
In chemistry, Column chromatography is a technique which is used to
separate a single chemical compound from a mixture dissolved in a
fluid. It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move through the column
at different rates which allow them to get separated in fractions
Principle:
When the mobile phase along with the mixture that needs to be
separated is introduced from the top of the column, the movement of the
individual components of the mixture is at different rates. The
components with lower adsorption and affinity to stationary phase travel
faster when compared to the greater adsorption and affinity with the
stationary phase. The components that move fast are removed first
whereas the components that move slow are eluted out last.
The adsorption of solute molecules to the column occurs in a reversible
manner. The rate of the movement of the components is expressed as:
Rf = the distance travelled by solute/ the distance travelled by solvent
Rf is the retardation factor.
Q-2 Name at least four materials used as adsorbents in Column
Chromatography.
Adsorbents used in Column Chromatography:
The usual adsorbents employed in column chromatography are
• silica,
• alumina,
• calcium carbonate,
• calcium phosphate,
• magnesia,
• starch
Q-3 How many components were present in carrot extract?
Carrots are a multi-nutritional food source. They are an important
root vegetable, rich in natural bioactive compounds, which are
recognised for their nutraceutical effects and health benefits.
Composition of carrot extract:
Major component of orange carrot extract is B-carotene.
Pigments:
Lesser amounts of α-carotene, γ-carotene, lutein, and zeaxanthin.
Other components:
Phenolics , Polyacetylene, Ascorbic acid and tocopherol.
Caffeic acid is the predominant phenolic acid in carrots. Thiamin,
riboflavin, niacin, folic acid and vitamin C are present.

Q-4 At which number carotenes were eluted from the column?

By using non-polar eluent. Yellow band of carotene eluted first from the
column

Q-5 Write down the composition of eluent (solvent system) used to

elute the carotenes from the column.

Composition of eluent use in column chromatography of

carotene:
Β-Carotene is a non-polar compound, so it is separated with a non-
polar solvent such as hexane.
A solution of 10% ethyl acetate in hexanes is use as an eluent in
column chromatography.

Q-6 How do you know that you got pure carotene as a result of
column chromatography?
Purity of carotene:
Purity of carotene can be determined by TLC.
Procedure:
• Draw 1cm line on TLC plate and mark three points at regular
internal.
• Concentrate the carotene extract by evaporating solvent.
• Apply spot of three solutions on TLC plate. One of pure carotene
use as standard, second of extracted carotene and third of crude
extract.
• Place TLC plate in solvent chamber containing 30% acid + 70%
hexane. Never allow the bulk solvent to rise above the line drew.
• Allow capillary action to draw the solvent up the plate until it is
approximately 1 cm from the end.
• Never allow the solvent to migrate all the way to the end of the
plate. Remove the plate
and immediately draw a pencil line across the solvent front.
• Carotene is a colored compound so it can easily be visualized.
Results:
If extracted yellow carotene appears as only one spot on TLC plate
same as that of standard carotene solution than it will pure
otherwise not. If more than one spot appears for extract it will not
pure.