International University, Vietnam National University - HCMC

School of Biotechnology
Department of Biochemistry

ORGANIC CHEMISTRY LABORATORY

REPORT 4: COLUMN CHROMATOGRAPHY
INSTRUCTOR: PhD. Hoàng Lê Sơn

TEACHING ASSISTANT: Nguyễn Thanh Phong

DATE OF SUBMISSION: 15-07-2019

Group member:

1. Trương Thị Ngọc Hằng - BTBCIU16041

2. Võ Minh Hạnh - BTBCIU16083

3. Nguyễn Hồng Anh Phương - BTBCIU16015

4. Nguyễn Tấn Phú - BTBCIU16057

Organic Chemistry Laboratory 1

 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

I. ABSTRACT:
The purpose of this experiment was to utilize the technique of
columchromatography (liquid chromatography) in order to separate various compounds
from each other. This particular experiment was performed in order to isolate the large
amounts of the individual components in pure form from the mixture of spinach and silica
gel.The mixture solvent is separated into three layer for collecting. After the column
chromatography was completed, thin layer chromatography, or TLC, was used in order to
assess the purity of each compound that had been separated, and to verify whether or not
the compounds were pure. As the result of this experiment , base on the Rf value, the pure
carotene is separated in all three trials ( Rf =0.92- 0.9).

II. INTRODUCTION:
In this experiment there was no real reaction that took place. Instead, all that occurred
was the simple separation of compounds from each other and test of their purities. The
main objective of this experiment was to separate the compounds from each other, and to
utilize the differences in their polarities, as well as their affinities for the stationary phase
(silica gel), in order separate the two compounds from each other.

While thin layer chromatography was used in one of the past experiments in order to
separate compounds from each other, this experiment used column chromatography.
Column chromatography has certain advantages over TLC, for example, when using column
chromatography gram-quantities of compounds can be separated from each other instead
of just micro- or milli-quantities. In this experiment, the compound silica gel (SiO 2.nH2O) was
used as the stationary phase, and because it is a polar compound it was useful for
separating polar compounds from non-polar compounds due to the fact that like attracts
like

III. MATERIALS AND METHODS:

 Materials:

In the experiment, the chemical reagents were used for column chromatagraphy was
that Silica powder, 2 mL of Spinach solution, Disiled Water, and Ethanol. For the equipement
included five of Vials, one of Cylinder 100 mL and 5 mL, with a beaker,a glass pipet, a
weighing bottle, a cotton, a silica plate for TLC was used for column chromatography, a
spatula, and some capillaries were used for spot techniqued.

Organic Chemistry Laboratory 2

 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

 Methods:

The chemical solution should be prepared before experiment. Spinach was used to
experiment, which were ground into solution. Moreover, the solvent solution also prepared
by mixing of 10 mL mixture corresponding to ratio 80:20 of ethanol and distiled water.
The preparation of the column was required in the next step. Using enough cotton put
into the bottom of the column. The sufficient silica powder was taken out the chemical
bottle, and put into the beaker. Mixing the silica powder with prepared solvent of ethanol
and distiled water frequently, until the mixture turned into a slurry. Pouring the silica slurry
into the column. The next step, 2 mL solution of spinach was continuously mixed with silica
powder, the stirred until it was converted green slurry. Pouring the sample of spinach into
the column. Additionally, The solvent which included of methanol and water (80:20) should
add into column, It’s been important to notice that the solvent did not run dry, meaning the
solvent should always be 1 cm higher than the surface of silica layer.Adding solvent early,
that was, when there was still solvent in the column, would also help to minimize
disturbance to the silica surface and the resultant disruption of any bands still present near
the top of the column.
Finally, Collecting the fraction of different color of the column was put in five of vials and
Bring the different kinds of solution of sample after column chromatography to perform
under TLC analysis with mobile was ethanol and water (80:20) and stationary was silica plate
for TLC.

IV. RESULTS:

Figure 1: 5 different vials Figure 2: 3 vials selected to

collected from column perform TLC

Organic Chemistry Laboratory 3

 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

Figure 3 : TLC development

 Note: 1: sample in vial 1; 2: sample in vial 2; 3: sample in vial 4.
 Distance solvent moved from the spotting line (origin): 5.30

Table 1 : Rf value and identity from TLC development.

Vial Color of spot Distance moved (cm) Rf value Identify
1 Yellow 2.20 0.92 Carotene
2 Yellow 2.20 0.93 Carotene
3 Yellow 2.25 0.94 Carotene
4 Yellow 2.05 0.86 Unknown
5 Green 1.70 0.71 Unknown

Organic Chemistry Laboratory 4

 International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry

V. DISCUSSION:
According to the result obtained from column chromatography, 3 fractions of
different colors were collected, particularly light yellow, yellow and green, which
represented for 3 specific compounds.
According to the theory, Rf value is based on polarity and how far it travels on the
TLC plate. The less polar the molecule is, the further the distance moved is. From the result
obtained after performing TLC, the yellow spot developed from vial 1 at Rf value of 0.92 and
vial 2 at Rf value of 0.93, which represented for Carotene or particularly β-Carotene pigment
in spinach. While the spot introduced from vial 3, there were four spots developed in TLC
plate. There was a yellow spot at Rf value of 0.94, which pointed out that there was the
presence of Carotene. Additionally, based on the green color of spot, Chlorophyll might be
expected to be defined. However, only one spot at Rf of 0.45 represented for Chlorophyll
and the other which was at Rf value of 0.71, was not.
The only problem in this experiment was in spots development from vial 3.
According to principle of column chromatography, after performing, only single chemical
compound from a mixture was collected based on differential adsorption of compounds to
the adsorbent. Therefore, it was expected that spot introduced from vial 3 should be
separated into one component. However, the result showed that there were both Carotene
and Chlorophyll. This could be explained by several reasons. First, the intersectional area of
two layers was also collected, affected the purity of fraction of vial 3. Second, during
preparation of column, slurry of silica still remained bubbles or was not uniformly filled with
the stationary phase. These can interfere with the separation of the column if they are not
adequately removed and can crack the adsorbent material in the column. The other could
be because the surface of each layer poured into the column. If the surface was not flat
or/and sharp, the efficiency of column chromatography would be reduced.

VI. CONCLUSION:
Column chromatography is a commonly used purification technique in labs
across the world. It also use in diagnostic labs for determine amount of drug present in
blood, urine sample, separations and preparation of biological macromolecules.

VII. REFERENCES:
1. Tips and Tricks for the Lab: Column Troubleshooting and Alternatives by Sarah Millar
2. Lecture 7: Introduction to chromatography by dr. Nguyen Thao Trang
3. Lab manual Organic Chemistry laboratory by dr.Hoang Le Son sem 2016-2017

Organic Chemistry Laboratory 5