CHEM 200L EXPT# 5 - 2DMT - GROUP 2

COLUMN AND THIN LAYER CHROMATOGRAPHY

Balawis, L., Baleta, D., Baluga, R., Baluyot, H., Barbo, J.*, Basa, A.*
Department Medical Technology, Faculty of Pharmacy
University of Santo Tomas
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ABSTRACT
Chromatography is a versatile method of separating mixtures. In the
experiment, the group used two different techniques of chromatography to be able to
attain its objectives.
The first objective of the experiment is to separate the colored components
Capsicum frutescens through the process of column chromatography. DCM - hexane
was used to obtain the extract of the Capsicum frutescens. The extract was then
placed in the silica column, and introduced to three solvent systems in successions.
The first eluate obtained was -carotein with __ drops. The next eluate was
capsorubin with __ drops. The last eluate was capsanthin with __ drops.
The other two objectives of the experiment are to be able to determine the
purity of the eluates and to be able to solve for the rf values of each eluate through
the process of Thin Layer Chromatography. Drops of each eluate were placed on the
TLC plate. It was then placed inside an equilibrated developing chamber. Afterwards,
the chromatogram was subjected to UV light to see the movement of each eluate.
The results showed that only the ___ and the ___ eluates moved away from the
origin. The retention factor was computed to be ___. While the other eluate had __
as an rf value.
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INTRODUCTION
extract obtained was immediately
covered with a watch glass because
METHODOLOGY

the solvent used was highly volatile.

A. Column Chromatography
In column chromatography, the

1ml of the plant extract was

pipetted and placed on top of the silica

group started by preparing the silica

column. Right after this,

column., which is a dropping pipette

systems were introduced into the

plugged with cotton to close the other

column. These solvents are: DCM-

end and then filled with silica gel.

hexane (1:1), DCM, DCM-methanol

While one of the members is doing

(1:1). 3.0ml of each solvent system

this,

the other two members were

was dropped to the column. The clear

preparing the plant extract. The even

eluates obtained were discarded by

group was assigned the plant siling

the group while the colored eluates are

labuyo, so the members of the group

covered with parafilm for checking and

sliced 3 pieces of siling labuyo into

to be used for the next experiment,

smaller pieces. It was placed in the

the Thin Layer Chromatography.

the solvent

pestle and 5ml of DCM-hexane was

poured into the Pestle to be used as

B. Thin Layer Chromatography (TLC)

Then it was

While the first process was

pounded with the mortar to maximize

being done, one of the members also

the extract that can be obtained. The

drew the original line and the end line

the extracting solvent.

CHEM 200L EXPT# 5 - 2DMT - GROUP 2

line on the TLC plate. For the Thin

labuyo extract, was applied on the

Layer

group

TLC plate by spotting each eluate and

prepared their developing chamber

the plant extract 5 times on the line

ahead of their TLC plate. The chamber

drawn on the TLC plate.

was prepared by placing an amount of

solvent reached the top of the filter

the solvent system DCM-hexane in a

paper, the TLC plate was placed inside

beaker. The chamber was first needed

the Developing chamber and then

to be equilibrated. The group used

covered with a watch glass. The group

filter paper for this process. They

now waited for the solvent to reach the

placed a piece of filter paper inside the

end line drawn on the TLC plate. After

beaker and waited for the filter paper

the solvent reached the said line, it

to be fully submerged in the solvent

was dried and was subjected to UV

before placing the TLC plate inside.

light. The group went to the Satellite

While

Laboratory for the UV light.

Chromatography,

waiting

for

the

the

developing

After the

chamber to equilibrate, the members

of

the

group

assigned

to

this

experiment prepared the TLC plate.

The eluates, together with the siling

RESULTS AND DISCUSSION