International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
ORGANIC CHEMISTRY LABORATORY
REPORT 3: THIN LAYER CHROMATOGRAPHY
INSTRUCTOR: PhD. Hoàng Lê Sơn
TEACHING ASSISTANT: Nguyễn Thanh Phong
DATE OF SUBMISSION: 08-07-2019
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:
Thin layer chromatography is a simple and effective type of chromatography mainly
used to separate and study individual components of any mixture. Separation in TLC
involve distributing a mixture of two or more substances between a stationary phase
and a mobile phase. Silica-gel plate was used as stationary phase. The technique based
on the polarity of solvent by using various ratio polar solvent in mobile phase. The aim
of the experiment was to emphasize about chromatography technique, apply in
separating the mixture of compound in spinach. The retention factor (R f) of sample in
Petroleum Ether: Acetone with the ratio of 5:5 was 0.42, which the identify of sample
was Chlorophylls and Rf was 0.99, which the identify of sample was Carotene. Hence,
preparation of TLC chamber between 100% chloroform and 5:5 Petroleum Ether:
Acetone, the sample would give the good result in 5:5 Petroleum Ether: Acetone.
II. INTRODUCTION:
Thin layer chromatography (TLC) is a chromatographic technique used to separate
the components of a mixture using a thin stationary phase supported by an inert
backing. It may be performed on the analytical scale as a means of monitoring the
progress of a reaction, or on the preparative scale to purify small amounts of a
compound. Stationary phase is a thin layer of adsorbent (silica gel or alumina) coated on
a plate. Mobile phase is a developing liquid travels up the stationary phase, carrying the
samples with it. Components of the samples will separate on the stationary phase
according to how much they adsorb on the stationary phase versus how much they
dissolve. Polarity is important factor in TLC. A more polar (or more strongly eluting
solvent) will move all of the analytes to a greater extent, than a less polar, weakly
elution solvent.
A small amount of sample was spotted near the bottom of the plate, and the plate
was placed in the mobile phase. This solvent was drawn up by capillary action.
Separation occurred as each component, being different in chemical and physical
composition, interacted with the stationary and mobile phases (100% chloroform or 5:5
Petroleum Ether: Acetone) to a different degree, creating the individual bands on the
plate.
distance traveled by substance
Retention factor: R f =
distance traveled by solvent front
Rf can be used to aid in the identification of a substance by comparison to
standards. Rf value is not a physical constant, and comparison should be made only
between spots on the same plate, run at the same time in mobile phase.
Organic Chemistry Laboratory 2
International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
Application of thin layer chromatography were to identify of components of a
mixture, analyze fractions collected during purification, and analyze the purity of a
compound.
Spinach would use for in the experiment. The pigments in vegetables, flowers
and leaves can be separated and identified by using thin-layer chromatography. Green
pigments, known as chlorophylls, serve as the main photoreceptor molecules of plants.
Carotenoids, yellow pigments, aid the plant in the photosynthesis process.
Table 1: Retention factor value of some pigment
Spinach is an extremely nutrient-rich vegetable. It packs high amounts of
carotenoids, vitamin C, vitamin K, folic acid, iron, and calcium. Spinach has low in
carbohydrate but high in insoluble fiber. This type of fiber may benefit your digestion.
Spinach is extremely healthy and linked to numerous health benefits. It may reduce
oxidative stress, promote eye health, fight cancer, and regulate blood pressure.
Table 2: The nutrition facts for spinach, according to the U.S. Food and Drug
Administration.
III. METERIALS AND METHODS:
Organic Chemistry Laboratory 3
International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
1. Materials:
In this experiment, the material is used plant crude extract with five solvent
hexane, methanol, chloroform, petroleum ether and acetone. In addition, we need TLC
plate which is silica gel (stationary phase) . The mortar and pestle are used to grinding
the spinach leaf and using the glass pipets to transfer it into the vial. The cylinder 5ml is
used to adding 5ml hexane in sample until hexane is colorless. The mobile phase of TLC
is collected in 2 beakers with different solution (100% chloroform and petroleum ether:
acetone with the ratio of 5:5). Using the alcohol lamp to create the injection to extract
the spot on the TLC.
2. Methods:
Preparation of plant extract: firstly, weigh spinach and add 10 ml methanol into a
mortar. Grinding the mixture slowly until the solvent is dark green. Transfer the liquid
into the vial and heat it in hot plate until the extract to 1/5 of the initial volume. Adding
more 5 ml of hexane to this vial and shake it well. After shaking, hexane is taken out and
continue to do it until the hexane which put into the complex has colorless. The hexane
which took out in several time put in one vial. Taking this vial to hot-plate and heat it
until the extract to 1/5 of initial volume.
Analytical Chromatography of plant extract: Two eluents were prepared (1) a 5
mL of 100% chloroform (2) a 5 mL of 5:5 petroleum ether/ acetone eluent mixture. The
TLC plate was placed in a small amount of two prepared eluents in separate beaker
along with filtered paper with covered by aluminum foil and observed/analyzed . For
each chamber we prepared one separated TLC plates. In the silica gel plates, draw a
faint pencil line around 1cm from the bottom edge (original line). Using a capillary
loaded with the extraction in the vial. Each plate should be even in size and not too large
to prevent smears and disturbed streaks. Drying them with the air. Waiting to the
solvent reach the edge (about 1cm) of the top of TLC plates. Remove the plate carefully.
Observation and detection of phytocomponents: observe the plates in room light. We
took the picture of thin layer and marked the spot on the thin layer. After that, the data
will be used to calculate and determine what pigments in this sample.
IV. RESULTS:
Organic Chemistry Laboratory 4
International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
Figure 1: TLC development by using Figure 2: TLC development by using 5:5
100% chloroform as developing solvent petroleum ether: acetone as developing
solvent
Note: Before adding hexane denoted by A and After adding hexane denoted by B
The results should be shown following the developing solvent and also before/ after
adding hexane
a. 100% chloroform:
Distance solvent moved from the spotting line (origin): 5.30
* Before adding hexane:
Color of spot Distance moved (cm) Rf value Identify
Light Green 0.11 0.021 Unknown
Green 0.45 0.085 Unknown
Light grey 0.89 0.17 Unknown
Yellow 5.27 0.99 Carotene
* After adding hexane:
Color of spot Distance moved (cm) Rf value Identify
Organic Chemistry Laboratory 5
International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
Yellow 0.13 0.025 Unknown
Yellow 0.31 0.058 Unknown
Green 0.62 0.12 Unknown
Yellow 5.22 0.98 Carotene
b. Petroleum Ether: Acetone= 5:5
Distance solvent moved from the spotting line (origin): 5.40 cm
* Before adding hexane:
Color of spot Distance moved (cm) Rf value Identify
Yellow- green 2.25 0.42 Chlorophylls
Yellow 2.92 0.54 Unknown
Blue- green 3.12 0.58 Unknown
Yellow 5.34 0.99 Carotene
* After adding hexane:
Color of spot Distance moved (cm) Rf value Identify
Yellow 1.68 0.31 Xanthophylls
Yellow 2.22 0.41 Xanthophylls
Yellow- green 2.30 0.43 Chlorophylls
Yellow- green 2.72 0.50 Unknown
Blue- green 2.89 0.54 Unknown
Yellow 5.37 0.99 Carotene
V. DISCUSSION:
Organic Chemistry Laboratory 6
International University, Vietnam National University - HCMC
School of Biotechnology
Department of Biochemistry
According to the result from TLC, there are Chlorophyll, Xanthophyll, and Carotene
found in spinach, the color of Chlorophyll serves a function of reflect the green color of
spinach, and yellow color of xanthophyll, orange color of Carotene are masked by green,
so we only observe the green color from spinach’s leaf. These pigments play the role of
antioxidant and capturing light energy for photosynthesis in spinach.
Between Chloroform and Petroleum ether: Acetone = 5:5, the sample with
Chloroform as mobile phase was slower and did not separate as well as the mixture.
This is because of the polarity difference between stationary phase and mobile phase.
Stationary used in this experiment was plastic plate cover with silica gel, polar
molecular, and Chloroform is medium polar solvent, generally more polar compare to
the mixture mobile phase, which included petroleum ether, non-polar molecular.
Therefore, the less polar mobile phase, petroleum ether: Acetone=5:5 would allow the
speed at which molecular move up the plate higher compare to Chloroform.
Between before adding hexane and after adding hexane sample, the sample after
adding hexane was separated better. Chlorophylls and carotenes do not dissolve very
well in water, but they dissolve well in hexane. Therefore, hexane was used to extract
the green chlorophylls and yellow carotenes that hide under the water at the bottom. At
the sample after adding hexane, chlorophylls and carotenes are able to characterize by
thin layer chromatography, showed the result clearer with three types of pigments and
better separate dots.
VI. CONCLUSION:
In this experiment, we use thin layer chromatography to determine the compound
in plant crude extraction. Its mechanism based on polarity of solvent is used and
samples. Different solvents give different in result. When using stationary phase is
silica-gel plate (polar compound), the lower polarity of solvent is used, the higher
effective of experiment. With some compound are colored, we can easy determine by
visual. However, other compounds are colorless, it is necessary to use TLC staining
technique to detect them.
VII. REFERENCES:
Lecture 7: Introduction to chromatography: TLC by dr. Nguyen Thao Trang
Spinach 101: Nutrition Facts and Health Benefits Written by Kris Gunnars, BScon May
14, 2019
Retention value of spinach by Dr. Nguyen Thao Trang
Organic Chemistry Laboratory 7
