COLUMN AND THIN LAYER CHROMATOGRAPHY

Ivy Rose C. Orozco, Arnold V. Paguirigan Jr., Sarrah Mae B. Punzalan,

Jayvee S. Rivera and Rico Maria R. Rivera
Group 8 2E Pharmacy Organic Chemistry Laboratory

ABSTRACT
The pigments present in malunggay leaves and the process of chromatography was the main focus of this study. The
results, both qualitative and quantitative, that was obtained for this experiment was interpreted. The qualitative
results of the experiment were about the colour and dominance of different pigments in malunggay. Both yellow
pigments and green pigments were both seen in both set-ups. Also, the column chromatography set-up was only able
to extract green and yellow pigments with almost equal amount in drops. The quantitative results, in the other hand,
were about the retention factor value of the different pigments especially the green and yellow pigments, and also the
presence of different compounds. The retention factor of the yellow pigment was 0.98 since it travelled 59 cm from
the origin. While the green pigment retention factor was 0.33 since it travelled 20 cm only from the origin. Since
retention factor is inversely proportional to polarity [8], a conclusion that yellow pigment was less polar than the green
pigment was deduced.

INTRODUCTION determining their purity and following the

This experiment is all about the use of progress of a reaction [7].
Chromatography in separation, identification, and
determination of purity of different compounds
present in the sample.
Chromatography is a proven method for
separating complex samples into their constituent
parts, and it is undoubtedly the most important
procedure for isolating and purifying chemicals [4].
Chromatography relies on the differential
solubilities and adsorptive of the components to
be separated with respect to two phases;
stationary phase and mobile phase [1]. Stationary
phase is the part of the chromatographic system
though which the mobile phase flows where Figure 2. Thin Layer Chromatography
distribution of the solutes between the phases
occurs [3]. Mobile phase, on the other hand, is the
The sample we used is the Malunggay
part of the chromatographic system which carries
plant. It is rich in Vitamin A and C, iron, and
the solutes through the stationary phase [2]. The
High-Density Lipoprotein [5]. It is naturally green
two types of chromatography that will be used in
in colour. Its scientific name is Moringa oleifera.
this experiment is Column Chromatography and
The objectives of this experiment are to
Thin Layer Chromatography
(1) separate the coloured components of
Column Chromatography is the most
malunggay leaves using column chromatography,
advantageous over most of the chromatographic
(2) determine the purity of the components using
techniques. It can be used to determine the
thin layer chromatography, and (3) measure the
number of components in a mixture but also to
Rf values of the coloured components in Thin
separate and purify substantial quantities of
Layer Chromatography
those components for further analysis [6].
EXPERIMENTAL
A. Sample Used
Malunggay (Moringa oleifera)

Figure 1. Column Chromatography

Thin Layer Chromatography, on the other

hand, is a very commonly used technique in
synthetic chemistry for identifying compounds,
Figure 3. Malunggay Leaves
viewed under a UV lamp and the Retention Factor
B. PROCEDURE (Rf) values of the components were calculated
1. COLUMN CHROMATOGRAPHY and noted.
First, the malunggay leaves were
Watch Glass
triturated with the use of a mortar and pestle.
Second, 5 mL of hexane: acetone (7:3) was
Thin Layer Plate
poured to the triturated malunggay leaves. After
that, the dropping pipette that will be used for
the set up was plugged by cotton and was Solvent Mixture
uniformly packed with silica gel up to the
indented part of the dropping pipette. 3 mL of
the extract was placed on the top of the column
using a dropping pipette. Then, the pigment
mixture was eluted using 2 mL of hexane:
acetone (7:3), 2 mL of acetone, and 2 Ml of
acetone: methanol (1:1). The solvent systems Figure 5. Thin Layer Chromatography Set-Up
were introduced in portions and the column was
not allowed to run dry. The colourless eluate
gotten from the column was discarded while the Distance traveled by compound
=Retention Factor( R f )
coloured eluates were collected in different test Distancetraveled by solvent front
Figure 6. Formula for Retention Factor (R )
tubes. Data, like the number of drops per eluate f
collected in each test tube, were collected and
noted. RESULTS AND DISCUSSION
Different observations were noted during
the experiment. First of all, the presence of the
yellow pigment and green pigment were seen in
Dried silica gel both set-ups. Yellow pigments and green
pigments dominance was proven by this
Cotton observation. This was also proven by the column
chromatography set-up which only has extracted
Test tube yellow and green pigments. If one focused on the
data of the thin layer chromatography, the yellow
pigments proved to have a higher retention
factor (Rf) value than the green pigments. The
greater polarity of green pigments over yellow
pigments was shown by this because Rf value is
indirectly proportional to polarity.
Figure 4. Column Chromatography Set-Up

Computations:
2. THIN LAYER CHROMATOGRAPHY
First, the malunggay leaves were Yellow pigment:
triturated with the use of a mortar and pestle. 59 cm
Second, 5 mL of hexane: acetone (7:3) was Rf= =0.98
60 cm
poured to the triturated malunggay leaves. After Gray pigment:
that, the eluates were applied by spotting ten 29 cm
times on a 5 cm x 8 cm pre-coated TLC plate. Rf= =0.48
Each spot was allowed to dry before applying the 60 cm
next and each spot was assured to be as small as 20 cm
Green pigment: R f = =0.33
possible. Then, the developing chamber was 60 cm
prepared by placing an approximate amount of 18 cm
solvent system. The inner wall of the chamber Yellow pigment: R f = =0.30Dark green
60 cm
was lined with the filter paper, was covered with 16 cm
a watch glass and was allowed to equilibrate. pigment: R f = =0.27Light yellow pigment:
After that, the plates were carefully placed in the 60 cm
developing chamber. The solvent system was 12 cm
Rf= =0.20Light yellow pigment:
allowed to rice up to 1 cm from the upper end. 60 cm
Then, the plates were removed from the chamber
and the solvent front was immediately marked,
and air-dried. Finally, the components were
 6 cm tutorials/exp3A/columnchrom/
Rf= =0.10Colourless (seen in UV) pigment: 08/24/2010
60 cm
54 cm [7] University of California, Los Angeles. Thin
Rf= =0.90 layer Chromatography.
60 cm http://www.chem.ucla.edu/~bacher/Gene
Table 1. Compound and Solvent system used ral/30BL/tips/TLC1.html 08/24/2010
[8] University of Colorado at Boulder. TLC:
Plant Used Malunggay (Moringa oleifera)
Retention Factor.
Solvent System Used Hexane: acetone (7:3)
http://orgchem.colorado.edu/hndbksuppo
rt/TLC/TLCrf.html 08/24/2010
Table 2. Column Chromatography

Colour of Component Volume of eluate

1 Yellow 31 drops
2 Light green 31 drops
3 Green 65 drops
4 Yellow green 96 drops
5 Light yellow 109 drops
6 Yellow 44 drops

Table 3. Thin Layer Chromatography

Colour of Distance of Retention

Component component Factor (Rf)
from origin (X) values
in centimetres
1 Yellow 59 cm 0.98
2 Gray 29 cm 0.48
3 Green 20 cm 0.33
4 Yellow 18 cm 0.30
5 Dark green 16 cm 0.27
6 Light yellow 12 cm 0.20
7 Light yellow 6 cm 0.10
8 Colourless (UV) 54 cm 0.90

REFERENCES
[1] Bayquen, A.V., Cruz, C.T., de Guia, R.M.,
Lampa, F.F., Peña, G.T., Sarile, A.S.,
Torres, P.C. (2010). Laboratory Manual in
Organic Chemistry. Manila, Philippines. C&
E Publishing Inc.
[2] Chromatography-online.org. Mobile
Phase. http://www.chromatography-
online.org/topics/mobile/phase.html
08/24/2010
[3] Chromatography-online.org. Stationary
Phase. http://www.chromatography-
online.org/topics/stationary/phase.html
08/24/2010
[4] Miller, James (2005). Chromatography:
Concepts and Contrast. New Jersey: John
Wiley & Sons Inc.
[5] Tacio, H.D. Malunggay: The miracle
vegetable.
http://www.sunstar.com.ph/static/dav/20
07/10/13/bus/malunggay.the.miracle.veg
etable.html 08/24/2010

[6] University of British Columbia. Column

Chromatography.
http://www.chem.ubc.ca/courseware/154/