International University, Vietnam National University - HCMC

 Organic Chemistry Laboratory

 LAB REPORT
EXPERIMENT 4      
 COLUMN CHROMATOGRAPHY
 

 Instructor: Dr. Hoang Le Son

Group: 2                              
Group members: 
Nguyễn Huỳnh Thanh Thảo         BTBCIU16084
Lê Phương Dung                           BTBCIU17054
Lê Ngọc Hà My                             BTBCIU17029

Trần Nguyễn Hồng Phúc              BTBCIU18052             

Date: October 16 , 2019 th

Score:____________
I. Abstract
The purpose of the experiment is to separate the compounds in spinach leaves of with high level
of purity through column chromatography technique. This method exploits some of the
molecular characteristics of a substance such as polarity and molecular weight to obtain different
portions of the sample. The material used in this experiment was fresh compressed spinach leaf
juice and the eluent (mobile phase) was ethanol, silica as stationary phase. After column
chromatography performance, four individual vials were collected according to fractions’s color.
TLC test for the obtained products showed pretty high degree of purity. Finally, the TLC was
observed under UV light

II. Introduction
Column chromatography is an important method that enables the separation, identification, and
purification of the components of a mixture for qualitative and quantitative analysis. Similar to
TLC, the technique operates based on the difference in polarity of substances, which leads to
their dissimilar affinity for two phases: stationary phase (silica gel) and mobile phase (usually a
solvent or mixture of solvents). For that reason, eluent can be chosen by pior performance of
TLC. The column is set up with silica gel inside and the sample (whose layer is above silica’s) is
allowed to go downward with the facilitation of gravity. The mobile phase on the top of the
column phase is continuously passed to cause separation throughout stationary one. Separation
of molecules occurs due to their differences in interaction with the mobile and stationary phase
as well as molecular weight. When the separation is completed, the samples are usually collected
as fractions.  

The advantage of this technique over TLC is that it can separate a large amount of sample, which
can be then used for further analysis or synthesis.

In this experiment, the material used was spinach leaves. This type of veggie contains folic acid,
some flavonoid compounds, chlorophyll (a green pigment), some minerals like iron, manganese,
magnesium, etc. 

III. Materials and method

Materials

Equipment assigned included 5 vials, capillaries, a 50ml cylinder, a spatula, a pipette, 2 beakers
of 250ml, cotton, aluminium foil, a juicer, a syringe and a silica plate for TLC. Chemicals
utilized were silica, ethanol and D.I water. Spinach leaves were used to make sample.

Method

1. Prepare spinach leaf sample

 The juicer was utilized to make spinach juice. Then, the juice was poured into falcon and be
centrifuged to separate the liquid and the ground. Two falcons put in centrifuge machine had
to be equal in weight.

2. Prepare the column

The plunger was taken apart from the syringe, which was then used as the column for
chromatography. A thin layer of cotton was put at the bottom of the syringe. Silica was
mixed with ethanol (eluent) to be the next layer of column. The slurry was expected to be
neither too wet nor dry and occupied about 4cm of the column. To ensure the silica layer was
flat, when pouring into syringe, the mixture was required to be stir well and quickly poured.
It was not ideal if the layer had bubbles. A toothpick might be used to flatten the layer and
omit bubbles. The eluent was used to remove silica stuck to the wall. The layer had to be wet;
hence, solvent had to be added to be 1cm above the layer while the eluent was allowed to
drip out. It took a while for the silica to settle.

3. Run the column and identify the fractions

When solvent was drained. The mixture of spinach extract and silica was carefully
transferred to the column to ensure the even plane. The texture of mixture was expected to be
as that of silica slurry. The solvent’s level had to be 1cm above the third layer. As the green
eluent was about to drip out, vials were used to collect the eluent. Different vials were
expected to contain different color liquid. These liquids were used to run TLC with
developing solvent of 80:20 ethanol:water. The silica plates were observed under room light
auv light.

IV. Result
Part 1: Prepare spinach leaf sample for column chromatography

Figure 1:The spinach leaf after extracting by Figure 2: The measurement of 2 falcons
the juicer before were put in the centrifuge
 Figure 3: The centrifuge (5000rs/m for 7 Figure 4: The spinach solution after
minutes) when extracting the spinach extracted in the centrifuge

Before 2 falcons were been in the centrifuge, they were weighted equally to balance out the
machine and prevent it from shaking.

Part 2: Run the column

Figure 5: Washing around syringe walls Figure 6: The silica gel in syringe

The thickness of the cotton layer was thin enough to prevent silica from flowing out. The silica
had been mixed well, and was poured into the syringe quickly.

The mixture of silica gel and ethanol was thick enough, so the remain solution (ethanol solution)
flowed down easily.
Part 3: Run the column and identify the fractions

Figure 7: The mixture of the spinach leaf Figure 8: The syringe containing silica gel
liquid and silica gel. layer, spinach layer and ethanol layer.

The mixture of spinach solution and silica gel was thick enough, so the remain solution (ethanol
solution) was flown down easily.

A B C D E

Figure 9: 5 samples for TLC analysis

Sample A was the pure spinach solution after extract in the centrifuge. It took half an hour to
have 4 samples B, C, D, E according to fractions in column. When the drop of solution from the
syringe was turned pale, vial B was removed and replaced by vial C and so on until the color of
drop was colorless again.
 A B C D E

A B C D E

Figure 11: the plate in a UV chamber

Figure 10: the plate after the solvent reached
the solvent front in TLC chamber

V. Discussion
At the beginning of the experiment, the centrifuge machine was used to remove the remained
ground out of the spinach solution. Denser portions of the sample with grounds settled to the
bottom of the tube, while low-density substances rose to the top. For preparing the experiment, a
very thin piece of cotton was added into the syringe with the aim of avoiding very small particles
(silica or sand) to appear in the collected sample. The syringe was rinsed with ethanol to help
settle down the cotton shred and to prevent the silica gel to stick to the wall when being added
later. Mixture of silica and ethanol was used as stationary phase. Meanwhile, ethanol was the
mobile phase. Silica was polar, thus attracted polar compound to attach. The more polar the
compound, the stronger the attraction; as a result, the longer it stayed in silica layer while the
least polar compound was eluted first. Solvent (mobile phase) was chosen according to
absorption of compounds tested. In this case, ethanol, which has strong polarity was used to
enhance the movement of compounds through layer of polar silica. The silica’s layer surface was
flattened to ensure separation of the mixture into equal fractions later. The solvent should always
be kept about 1cm above the surface of the silica gel so that it would not go dry and crack, which
could prevent the separation of the sample.

After column run, 4 vials were obtained with color from pale yellow (B) to green (C’s was
darker than D’s) and colorless (D). Vial A was the sample solution. The first layer collected was
solution B and it was pale yellow. Solution B had the fastest rate, which implied that B contains
the least polar substance and high molecular weight. Solution C was the second liquid layer
collected and it had dark green color, which means C contained a lot chlorophyll pigment and it
is more polar than B. D was collected last and its color was a little bit brighter than C so the
substance was the most polar and contained less chlorophyll pigment than C. D’s was the
colorless; as solvent was added to keep the surface of silica not to be dry, E might be the solvent.
One thing had to be considered that as the spinach-silica layer was not evenly distributed, the
fractions’ movement was not equal, which might affect the accuracy via collecting to vials.
 For the TLC test, it was observed that spot A separated into five different visual spots in its trace
(can be seen under both room and UV light) because A was the original spinach leaves solution.
Each spot was a particular compound in spinach leaves with different polarity. The fact that A
separation included five spots but only three colored liquid components were collected induces
that there should be substances that share almost the same polarity and molecular weight in the
obtained samples. This could be proved by observation of spot C and D as both of them
separated into two spots. Moreover, it can be seen that in the separation of spot A, two couples of
spots were very close to each other. Spot B, C, D were invisible under room light but UV light.
Spot D were invisible for both type of light as it was just solvent solution.

The choice of solvent depended on what type of target components. Nonpolar solvents are for
weakly absorbed compounds. The more polar the compounds, the more polar solvents should be.
In the experiment, quite strong solvent such as ethanol was used to test polar components in
spinach extract. Alcohol is less polar than water because the alkyl groups have positive inductive
effect, so the density of the electron around the oxygen atom is higher compared to that in water.
The O-H bond is less polar and hydrogen bonds between the molecules are weaker than in water.
Nonpolar molecules are better solvated by nonpolar solvents, so the larger the alkyl group, the
better the solvent, in this case isopropyl is used and it is larger in size and nonpolar compared to
other alcohol. 

There are some key points that must be memorized when conducting Column chromatography.
First, appropriate solvent is chosen with regard to its polarity. Solvent should be polar enough to
dissolve the target compounds and also bring them downward the silica layer. However, strongly
polar solvent may dissolve the stationary phase. Solvent can be tested by TLC method. Second,
silica must be mixed with adequate liquid so that the layer is firm, neither too wet nor dry. Third,
the layer in column must be evenly flat to ensure equal fractions collected. Last but not least,
solvent must be always 1cm above the layer to prevent cracks.

VI. Conclusion
Column chromatography is a crucial technique used to separate components in a mixture with
large amount and high accuracy. In this experiment, spinach was used as the sample. After
conducting column chromatography, 4 vials were collected and, together with original sample,
were tested to identify the components via TLC. Via color, the components could be predicted
and their polarity were revealed.

VII. References
https://studylib.net/doc/8291232/experiment-1.-thin-layer-chro-column-chromatography.-in-l...

https://studylib.net/doc/7955695/8.-column-chromatography

http://www.columbia.edu/cu/chemistry/ugrad/hssp/EXP_8.html