CHEM 200L - Expt # 5 - 2DMT Group 4

Column Chromatography and Thin Layer Chromatography

Cayetano, J. , Chumacera D., Cruzada A., Detras A., Dionio R., Dionisio J.*
Department of Biochemistry , Faculty of Pharmacy
University of Santo Tomas
ABSTRACT
Chromatography is the most modern and sophisticated of separating mixtures using the
difference in solubility. There are different types of chromatography, In this experiment we made
use of column chromatography and thin layer chromatography. Pigments of siling labuyo were
used as samples for the experiment. They were extracted with the use of DCM-Hexane. DCM,
DCM, and DCM methanol was then introduced to the column chromatography to obtain the
eluate and thin layer chromatography was applied to attain the purity and the Rf values of the
components.
INTRODUCTION

components of the mixture with it. Different

Chromatography can be defined as a

components travel at different rates. In the

laboratory technique of separating mixtures

experiment chromatography was used in

of

the separation of the colored components of

substances

Chromatography

into
is

their
a

components.

commonly

used

siling labuyo. There are many variants of

method for determining the purity of the

chromatography namely solid-liquid, liquid-

components and separating mixtures of

liquid, gas liquid with each being used

liquids into their individual components by

depending on which of the two phases are

using the differential solubilities of the

used. The experiment focuses on the solid-

components to be separated with respect to

liquid chromatography which are the thin

two phases, one of which is stationary and

layer chromatography and the column

the other, mobile. A compound that weakly

chromatography. Column chromatography

interacts with the stationary phase will

was

spend most of its time in the mobile phase,

components of red siling labuyo extract

it will move rapidly in the chromatographic

while thin layer chromatography was then

system. On the other hand, compounds that

used

interacts strongly will move slowly in the

components.

system. The mobile phase flows through the

stationary

phase

and

carries

the

used

to

to

separate

determine

the

the

purity

colored

of

the

The solid used in the experiment is

silica gel since the solid should be any solid

CHEM 200L - Expt # 5 - 2DMT Group 4

that is cannot be dissolved in the liquid

2
plate develops, the colors would not be

phase. The aim of the experiment were to

disarray. The Developing Chamber was

1.) separate the colored components of the

prepared

siling labuyo, 2.) determine the purity of the

amount of DCM hexane. The inner wall of

components, 3.) find the RF value.

the chamber was lined with filter paper to

allow

by

the

placing

TLC

the

plate

approximate

to

stand.

The

developing chamber was covered with

METHODOLOGY

watch glass and was allowed to equilibrate.

Three pieces of siling labuyo were

The TLC plate was carefully introduced in

chopped to pieces, and the seeds were

the

taken by the group. DCM hexane (1:1) was

system was allowed to rise up until it

added to the chopped siling labuyo to

reaches just 1cm from the upper end. The

extract the pigments. Silica Gel Column was

TLC plate was then removed carefully from

prepared by plugging the column with cotton

the

followed by the silica gel which was

immediately marked and the plate was

uniformly packed

the

allowed to dry. The components were

indented part of the Pasteur pipette. 0.5 ml

visualized with the use of a UV lamp. The Rf

of the extract was placed on top of the

values

column using a Pasteur pipette. 0.3 ml of

chromatoplates were documented.

until it

reached

developing

chamber.

were

chamber.

The

The

solvent

measured

solvent

front

and

was

the

DCM hexane, DCM, and DCM methanol

were then introduced in succession to

RESULTS AND DISCUSSION

obtain the colored eluates. The column was

Four components were obtained

not allowed to run dry and the colorless

from the column chromatography. The 4

eluates were disposed. The colored eluates

colors obtained are Peach orange, Orange

were collected to separate vials. After

yellow, Pale yellow, and Red orange. The

collecting the eluates from the column, Thin

volumes of each follow 18 drops, 19 drops,

Layer Chromatography was performed. The

50 drops, and 20 drops, respectively.These

eluates were applied on the 5cm X 8cm pre-

colors were obtained because each color

coated TLC plate by equidistantly spotting

has

each spot 10 times. The spot was allowed

represents capsorbin, and yellow represents

to dry first before applying the succeeding

beta-carotene. Other colors were obtained

spots. It was ensured that the spots made

due to human error.

were small as possible so that when the

different

polarities.

Red

orange

CHEM 200L - Expt # 5 - 2DMT Group 4

After measuring the distance
travelled for each spot, The Rf value was

3
Color of
component

Distance of
component

Rf Value

Peach orange

6.7 cm

0.8375

Orange yellow

5.1 cm

0.6375

Pale yellow

0.4 cm

0.05

Red orange

2.8 cm

0.35

computed. The Rf(Retardation factor) value

is the ratio of time spent in the stationary
phase relative to the time spent in the
mobile phase.The solvent rises up through
capillary action. The distance of the solvent
is 8.0 cm.
The distance travelled by each of the
components as follows: the Peach orange
travelled 6.7 cm from the origin, The Orange
yellow travelled 5.1 cm, The Pale yellow
travelled 0.4 cm, And lastly the Red orange
travelled 2.8 cm.
The color of the developed plate was
not visible to the naked eye, UV light was
used to view the results.

Table

1.

Column

Chromatography

Results
Color of
component

Volume of eluates

Peach orange

18 drops

Orange yellow

19 drops

Pale yellow

50 drops

Red orange

20 drops

Table 2. Thin Layer Chromatography

Results

Figure 1. Column chromatography

CHEM 200L - Expt # 5 - 2DMT Group 4

4
have been too big which have caused the
disarray in color. In addition another source
of error might be not covering properly the
developing

chamber

during

the

development of the TLC plate.

Reference
Figure 2. Thin layer chromatography
Calculations:
RF value= Distance travelled by compound/
Distance travelled by solvent
Peach Orange= 6.71cm/8cm =0.8375

By doing this repeatedly, you can identify

which of your samples collected at the
bottom of the column contain the desired
product, and only the desired product.
(n.d.). Column chromatography. Retrieved
October
16,
2016,
from
http://www.chemguide.co.uk/analysis/chrom
atography/column.html

Orange yellow = 5.1cm/8cm =0.6375

Pale yellow

= 0.4cm/8cm =0.05

Red orange

= 2.8cm/8cm =0.35

Conclusion
We were

able

to

achieve

Thin
Layer
Chromatography.
(n.d.).
Retrieved October 16, 2016, from
http://www.chem.ucla.edu/~bacher/General/
30BL/tips/TLC1.html
our

objectives in the experiment. The devloped

plate was not able to show complete
separation of colors. The possible sources
of error might be from the spotting of the
TLC plate. When the extracted pigments
were spotted on the plate, the spots might

(n.d.). Retrieved October 16, 2016, from

https://www.sciencedaily.com/terms/solubilit
y.htm