ABSTRACT

Chromatography is a technique that separate particle based on differential partitioning

between mobile and stationary phase. The aims of this experiment are to practice
chromatography separation technique using size exclusion chromatography (SEC) to separate
molecules in mixture and to learn the properties of chromatography technique. The slurry
(silica gel) was entered into the pipette. The top layer of column should be covered with layer
of sea sand. The sample included when mobile phase has surpassed the sand layer. The
sample was allowed to flow pass the sand layer level. The mobile phase was added and
samples were separated by different colors. The colour of solution collected was indicated.
However, the colour of solution obtained was varied with the reference obtained. In this
experiment, carotenes layer with golden in colour was adsorbed at the bottom, showing that
the carotenes compound had largest size and followed by pheophytin, chlorophyll a,
chlorophyll b and lutein. Xanthophyll compounds with light yellow layer were observed to be
formed at the top, showing that xanthophyll has smallest size of compound and highest ability
to be adsorbed by stationary phase.

INTRODUCTION
One of the technique to separate mixture is by chromatography. Chromatography is a
technique that separate the particle based on differential partitioning between the mobile and
stationary phase. Mobile phase is the fluid that dissolved with the mixture which carries it
through a structure holding another material called the stationary phase. The various size of
particles in the mixture will travel at different speed. This chromatography was first employed
by Russian scientist, Mikhail Tsvet in 1900. In the first decade of 20th century, he continued
to work with chromatography for separation of plant pigments such as chlorophyll, carotenes
and xanthophylls. Various types of chromatography techniques were developed during 1930s
and 1940s mainly used for many separation processes. Chromatography can be used for
preparative or analytical purposes. For the preparative, it used to separate the components of a
mixture for further process. For the analytical purpose, it usually used with smaller amounts
of material mainly for measuruing the relative proportions of analytes in a mixture. One of the
chromatography techniques to separate component is size exclusion chromatography (SEC).
SEC separates molecules on the based on the partitioning of molecules between the bulk fluid
between the beads of chromatographic media and the aqueous space with the pore of these

beads. The smaller the molecule the larger fraction of the total volume it sees and the slower
it moves through the column. So, biggest molecules will elute first and smallest molecules
will elute last.The advantages of SEC are it has good separation of large molecules from the
small molecules with a minimal volume of eluate and that various solutions can be applied
without interfering with the filtration process and also it can preserce the biological activity of
the particles to be separated. This elute results from this method can be purified more by using
other purification techniques (Stephanie et. Al., 2007).

OBJECTIVES
1) To practice a chromatography separation technique by using size exclusion
chromatography (SEC) to separate the molecules in the mixture.
2) To learn the properties of chromatography technique.

MATERIALS AND METHODS

Material
1. Silica gel 60 (0.063 0.200mm)
2. Petroleum ether
3. Acetone
4. Sodium Cloride (NaCl)
5. Sodium Carbonate (CaCO3)
6. Sodium Sulphate (Na2SO4)
7. Fresh Mengkudu Leaves
8. Sea sand
9. Distilled water

Apparatus
1. Glass Column with porous membrane (glass pipette 10 ml)
2. Separatory Funnel 100 ml
3. Dry blender / motar & pestle
4. Glass funnel
5. Filter paper
6. Beaker 100ml
7. Measuring cylinder (100ml)
8. Pipette 10 ml with pipette filler
9. Silicon Tubing (small size)
10. Hoffman clip
11. Retort stand with clamp
12. Glass wool
13. Forcep
14. Scissor
15. Test tube
16. Test stand
17. Reagent bottle (250ml)
18. Goggles
19. Rubber Hand Glove
20. Long wire (0.5m)
21. Pasteur pipette & Rubber teats
22. Wash bottle

Methods to make leave sample

First, several leaves was needed to be obtained. The leaves must covered all types of groups
that were old, middle and young. Then, the leaf was finely cut using scissors and this leaf
samples did not need to be washed. The leaves were put into a dry blender container, and was
ensured that it was not too dense. The leaves were blended until they were evenly mashed.
(Besides blending, the sample leaves could also be crushed using mortar & pastle. However,
22 ml Acetone, 3 ml of petroleum ether and a spatular CaCO3 need to be added into the
punch)

Method to make leave extract

3 ml of ether and 22 ml petroleum acetone and a spatula CaCO3 were added (depending on
the total sample) into the sample leaves that had been blended with suitable amount. Sample
leaf was filtered using filter paper. Then, leave samples were added into separatory funnel
(100ml) and were added with 20 ml of petroleum ether and 20 ml (10% NaCl). The flask was
shaken from the top to bottom so that the solution could be mixed. After the flask was shaken,
the flask was left at the vertical condition so that the separation between the solvent solvent
could occur. The solution in the bottom was discarded and waste was discharged into the sink.

Method to wash leave sample

Leaves sample were washed with 5 ml distilled water and repeated for 3-4 times. The flask
was shaken from the top to bottom so that the solution could be mixed. After the flask was
shaken, the flask was left at the vertical condition so that the separation between the solvent
solvent could occur. The solution in the bottom was discarded and waste was discharged into
the sink. The result leaves the sample extracts were cleaned and it was ready to be use.

Method to make column

Any glass tube was tapered at its end (glass pipette) and was connected to the silicon tube.
Some glass wool was added to prevent the resin off from the slurry. A fine wire was used to
push the glass wool to the bottom. Then, Hoffman clip was used (any clip tubing) to prevent
mobile phase from flowing to the exit.

Method to fill column with slurry(resin)

Slurry (silica gel), mobile phase (eluting solvent), mixture of petroleum ether and acetone
(7:3) was mxed together. Mixing was done using a Pasteur pipette. Slurry (silica gel) was
entered into the pipette. The process should be done little by little so that the silica gel could
be filled compactly. Goggle was used as a safety measure. The column was ensured for not
too dry. The top layer of the column should be covered with a layer of sea sand. The sample
was included when the mobile phase had surpassed the sand layerThe sample was allowed to
flow pass the sand layer level. The mobile phase was added and the samples were separated
by different colors.

Substances

Color

0. Mobile phase

Clear

1. Carotenes

Golden

2. Pheophytin

Olive green

3. Chlorophyll a

Blue green

4. Chlorophyll b

Yellow green

5. Lutein

Yellow

6. Xanthophylls

Yellow ( Light)

RESULTS

Figure 1: Colour reference of the solution.

Table 1: Colour reference and compound of the solution.
Test tube

Compound

Colour

Mobile phase

Colourless

Carotenes

Golden

Pheophytin

Olive green

Chlorophyll a

Blue green

Chlorophyll b

Yellow green

Lutein

Yellow

Xantophyll

Light yellow

Light yellow

yellow

colourles
s

Blue green
Olive green

golden

Yellow green

Figure 2: The colour indicated for the solution flowed out from the column.

The solution was collected and separated according the different in colour after the sample
had ran through the chromatograph. The Figure 1 showed the reference colour that ran by the
demonstrator. The colour different for each compound was recorded as shown in Table 1.
Next, the result obtained was collected and showed in Figure 2. The colour of the solution
collected was indicated. However, the colour of the solution obtained was varied with the
reference obtained as shown in Figure 1. However, the colour were similar for both of the
result in Figure 1 and 2. The variation would be discussion in the discussion.

DISCUSSIONS
Column chromatography was generally used as a purification technique to isolates
desired compounds from a mixture. The principle of column chromatography is based on
differential adsorption of substance by the stationary phase so called solid adsorbent. The rate
at which the components of a mixture which is the sample are separated depends on the
activity of the stationary phase and polarity of the mobile phase so called solvent (University
of Colorado at Boulder, Department of Chemistry and Biochemistry, 2013). The mobile phase
that we used in this experiment was petroleum ether and acetone, while the stationary phase
was silica gel.
In column chromatography of this experiment, the stationary phase, silica gel was
made into slurry with the mobile phase and was placed in a vertical column. Noni leaves
extract which is the sample then was placed at the top of the column. Then, the mobile phase
was added to the top of the column to allow the sample to moves down and passed through
the column by gravity force (University of Colorado at Boulder, Department of Chemistry
and Biochemistry, 2013). An equilibrium was established between the sample on the silica gel
and the mobile phase that flowing down through the column.
The compounds found in noni leaves extract are carotenes, pheophytin, chlorophyll a,
chlorophyll b, lutein and xanthophyll. The presence of different types of compounds leads to
formation of layers with different colours as the leaves extract flows through the column. This
is because different compounds will have different interactions with the mobile and stationary
phases (silica gel) in which the compounds are adsorbed at different regions depending on
their ability for adsorption (Virtual Amrita Laboratories Universalizing Education, 2013). The
separation of the layers is depends on the size of the compounds. The compounds with

smaller in size have greater adsorption power and will be adsorbed at the top. While the
compounds, that is much bigger in size will be adsorbed at the bottom.
In this experiment, carotenes layer with golden in colour was adsorbed at the bottom.
This shows that, the carotenes compound do have a largest size and followed by pheophytin,
chlorophyll a, chlorophyll b and lutein. Xanthophyll compounds with light yellow layer were
observed to be formed at the top. This shows that the xanthophyll has smallest size of
compound and highest ability to be adsorbed by the stationary phase.
In addition, the xanthophyll layer was obviously more polar than the other layers as it
does not travel through the column very quickly. This shows that the smaller size of
xanthophyll make it able to formed a strong hydrogen bonding to the silica gel and adsorbed
more strongly than the others compounds. It can be said that, the smaller the size of
compounds, the stronger the hydrogen bond and the binding of compound to the silica gel and
therefore the longer it takes to travel through the column. The polarity of the compounds is
decreased from lutein followed by chlorophyll b, chlorophyll a, pheophytin, and carotenes
which are depend to the size of compounds. These less polar compounds will spends more
time in the mobile phase, thus were washed much faster from the column (Jim Clark, 2007).
More mobile phase was added at the top to dissolving out the different compounds
from the silica gel. This process is known as elution in which it allows the different
compounds to desorbed and collected separately. The most weakly adsorbed component like
carotenes in this experiment will be eluted more rapidly than the other. The individual layer
was collected as the mobile phase drips from the bottom of the column (Virtual Amrita
Laboratories Universalizing Education, 2013).
The colours of layers that obtained from the experiment were little bit different from
the reference shown by demonstrator. This happened probably because we unable to
recognize well some of the colour different between the two layers once the layer were drips
from the bottom. Thus, the mixing of the different layers of colours resulting change in
obtaining colours which are much different from the demonstrators results.
Some precautions need to be highlighted when do this experiment. We must careful
when prepare the column to prevent the column from crack. This is because, once it is crack,
the separation of the sample into layers will be effected. The mobile phase needs to be added
slowly to the top of the column so that it will not disturb the layers that had been formed (Jim

Clark, 2007). We also need to make sure that the mobile phase continually added to the top so
that the column never dries out.

CONCLUSION
Based on the experiment, it could be concluded that column chromatography was
generally used as a purification technique to isolates desired compounds from a mixture
where the principle was based on differential adsorption of substance by the stationary phase
so called solid adsorbent. In column chromatography of this experiment, the stationary phase,
silica gel was made into slurry with the mobile phase and was placed in a vertical column.
The sample was placed at the top of the column, then, mobile phase was added to allow
sample to move down and passed through column by gravity force. Carotenes layer with
golden colour was adsorbed at the bottom indicate that carotenes compound got largest size,
followed by pheophytin, chlorophyll a, chlorophyll b and lutein. Xanthophyll compounds
with light yellow layer were formed at the top, showing that xanthophyll has smallest size of
compound and highest ability to be adsorbed by stationary phase. The colours of layers
obtained from experiment were a bit different from the reference which probably because of
inability to recognize well some of the colour different between the two layers once the layer
were drips from the bottom. Mixing of different colour layers resulting change in obtaining
colours which are quite different from the reference one.

REFERENCES
University of Colorado at Boulder, Department of Chemistry and Biochemistry. (2013).
Column Chromatography. Retrieved from
http://orgchem.colorado.edu/Technique/Procedures/Columnchrom/Columnchrom.html

Clark. J. (2007). Column Chromatography. Retrieved from

http://www.chemguide.co.uk/analysis/chromatography/column.html

Virtual Amrita Laboratories Universalizing Education. (2013). Separation of Compounds

Using Column Chromatography. Retrieved from
http://amrita.vlab.co.in/?sub=2&brch=191&sim=341&cnt=1

Stephanie P.D, Fatma K., Trevor J. M., Marcos M., Alan A. H. and Rafael K., (2007).
Probing Size Exclusion Mechanisms of Complex Hydrocarbon Mixtures: The Effect
of Altering Eluent Compositions, Energy Fuel, American Chemical Society.

BIOSEPARATION & PURIFICATION (BTC 3302) SEMESTER 1

(2013/2014)

LAB REPORT
PRACTICAL 5
Chromatographic Techniques: Ion Exchange Chromatography and Gel
Filtration

NAME:
1) MOHAMAD ZUL IKHWAN NURHAKIM

(164814)

2) NUR HIDAYAH BINTI KAMARUZZAMAN

(164749)

3) NURUL MAISARAH BINTI ABDULLAH

(163709)

4) NOOR EZZAH BINTI MEOR ARIS

(162294)

5) WAN SUET YING

(161407)

LECTURERS NAME : PROF. DR. ARBAKARIYA BIN ARIFF.

: DR. MURNI BINTI HALIM.

DATE OF SUBMITTED: 9th DECEMBER 2013.