LAB REPORT

Experiment 06:
THIN LAYER CHROMATOGRAPHY OF VEGETABLES

Course Name: PRACTICE IN ANALYTICAL CHEMISTRY - BT316IU 

Semester 1, Academic Year: 2022-2023
Date of submission: 12/11/2022  
Lecturer: MSc. Bui Xuan Anh Dao

Group 02

Trần Thu Anh BTBTIU20003

Đặng Thị Quỳnh Hương BTBTWE19026

Lại Thuý Na BTBTIU19160

 I. INTRODUCTION

Aim of practical

This labwork equipped students with techniques of thin layer chromatography method,
which can be used widely to analyze chemical mixtures in the real life. Following that,
the procedure of pigment extraction and recognition the pigments was learnt.

What is the method

Chromatography is a qualitative method of separation of components from the mixture

based on characteristics of chemicals. Thin Layer Chromatography is one type of
chromatography, separate components based on different polarity levels.

How does it works

Thin-layer chromatography consists of 2 phases: stationary phase and mobile phase. In

the stationary phase, there is a plate coated with completely silica gel. In mobile phase,
there is a chromatography chamber with a small volume of solvents. The solvent in
mobile phase then will rise up to the plate (stationary phase), to the solvent front. The
silica gel on the surface TLC plate is a polymer linked with many -OH groups, which
make it very polar, thus, it will easily interact with polar components, while interact
poorly with nonpolar compound. After that, the pigments will be visible on the plate at
different distance, otherwise can use UV lamp to visualize it. We then can measure the
distance between different color bands shown, calculate and compare retention factor (Rf
value), then refer Rf values into pigment name.
 Equipment Chemicals
Test tubes x2 Spinach: 4-5 leaves
Test tube rack and holder x1 Shredded carrot prepared
Stone mortar and pestle x1 Hexane
Beaker 250-mL (chromatography chamber) x1 Acetone
Beaker 50-mL x1
Capillary tube x2
Graduated pipette, pump x1
Thin stemmed pipette, pipette bulb x2
Ruler, pencil
TLC silica gel plate x1
(Weighting bottle x1)

Figure 1. Pigment extraction of spinach.

The arrow points the top oil layer that contains pigments
 Figure 2. Pigment extraction of carrot.

The arrow points the oil layer that contains pigments

II. RESULT

Distance solvent moved from the spotting line (origin) Y: 5.80 cm

Observation:
 Before After
Figure 3. TLC sheet before and after placing in the chromatography chamber
Spinach
Distance moved
Color of spot Rf value Identify
(X)
Yellow 1.35 0.23 Xanthophyll
Yellow 1.90 0.33 Xanthophyll
Green 2.10 0.36 Chlorophyll b
Blue- green 2.35 0.41 Chlorophyll a
Grey 3.55 0.61 Pheophytin a
Yellow- orange 5.70 0.98 Carotene

Carrot
Distance moved
Color of spot Rf value Identify
(X)
 Yellow 1.35 0.23 Xanthophyll
Yellow 2.25 0.39 Xanthophyll
Yellow- orange 5.60 0.97 Carotene

Mixture
Distance moved
Color of spot Rf value Identify
(X)
Yellow 1.55 0.27 Xanthophyll
Yellow 2.10 0.36 Xanthophyll
Yellow 2.30 0.40 Xanthophyll
Green 2.55 0.44 Chlorophyll b
Grey 3.75 0.65 Pheophytin a
Yellow- orange 5.70 0.98 Carotene

The pigments in order of the most polar to the least polar/ non- polar:
Xanthophyll > Chlorophyll a > Chlorophyll b > Pheophytin a > Carotene
Based on their affinities for the stationary phase (the polar silica on the thin-layer
chromatography plate) and the mobile phase, the various colors in the vegetables extract
are separated (the solvent – a nonpolar substance). Nonpolar chemicals, or those with a
strong affinity for the solvent, will go farther than those with a high affinity for silica
(i.e., polar compounds). Therefore, xanthophyll moved a shorter distance than carotene,
indicating that xanthophyll is the most polar and carotene is the least polar (non- polar).

III. DISCUSSION 
Based on the result of chromatography:

Spinach: From the result of TLC, there are 6 bands. Depending on the distance and
colors, we can dedicate the pigment in the material, as we can conclude from the result,
spinach has these following pigments: xanthophyll, pheophytin a, chlorophyll a,b and
carotene. In which chlorophyll gives the green color whereas xanthophyll, the dark shade
from pheophytin a and carotene deliver the yellow color, the color of spinach leaf would
be a blend between green and yellow. But the leaf would be more green than yellow since
green color is more dominant based on the color vibration proportional to the
concentration of the spots on TLC. 

Carrot: From the result of TLC, there are 3 bands. The carrot contains carotene,
xanthophyll and no chlorophyll based on the result of TLC; in this manner, the color of
carrot would be a blend of yellow and orange. Despite the concentration of xanthophyll,
which indicate yellow is more than carotene, which indicate yellow, orange could be
more intense by naked eyes than yellow, thus, the carrot’s color would be mainly orange
under normal observation. 
Carrots, however, should have Chlorophyll, this error will be explained further below.

Mixture: From the result of TLC, there are 6 bands. The mixture should have all the
pigments combined by two vegetation, but the result of our group is lack of the
Chlorophyll b The errors of lacking pigment Chlorophyll b in mixture and carrot can be
caused by: 
 The TLC sheet can be contaminated during the procedure due to the lack of gloves
while we perform the experiment, or our technique conducting on the silica gel
might had been scratched during the preparation steps. 
 The oil that we obtain from the test tube may be contaminated with distilled water,
which is still remains after rinsing. Explaining why the spots that we apply on the
TLC plates did not clear enough to see the Chlorophyll since its concentration has
been diluted, and the diameter of the dots was large, thus, gives the inaccurate
result. 
 Chlorophyll might be evaporated before recording our sample. Thus, the
Chlorophyll component might be too opaque to be observed.  
  Our careless in pre checking equipments, leading to the faulty in transferring
chlorophyll via the broken tip of the capillary tubes. 
 The chamber could had been not saturated with solvent since the seal was not
perfectly tight. 
 The unequal data between 3 samples is occurred by our placement into the non-flat
bottom of the beaker. The curve from the bottom can lead to the curve solvent
front and make the 2 sample from the outside (spinach and carrot) run slower in
the chamber than sample in the middle (mixture) 

Identify component of each sample. 

The sample spinach contains xanthophyll, pheophytin a, chlorophyll a,b and carotene
The sample carrot contains xanthophylls and carotene
The mixture sample contains xanthophyll, chlorophyll b and pheophytin a and carotene

Why use the pencil to draw in TLC plate 

The ink contain the chemicals that may also move together with pigments on TLC and
can separate into its own component pigment, affecting the experimental result.

Explain the color of vegetation based on the results of the chromatography. 

Carrot has orange color due to the presence of xanthophyll. As a matter of fact, the
carotene pigment is typically yellow-orange pigments that give carrots their characteristic
color. Nonetheless, chlorophyll do present in carrot, typically carrot top, because
chlorophyll is essential for plant to photosynthesis as it play the role of the main
photoreceptor, it assures that the plant can absorb light to turn it into energy. 
Spinach has green color due to the presence of chlorophyll which has a strong green
color.

Which pigments, if any, were present in both vegetation? Explain 

Due to our result, both vegetation obtain xanthophylls and carotene and carrot lacks of
chlorophyll pigment, which is our errors and we already explain above. Chlorophyll is
essential for plant to photosynthesis as it play the role of the main photoreceptor, it
assures that the plant can absorb light to turn it into energy. While carotene also aids in
the photosynthesis process. 

The functions of Hexane and Acetone in a 70:30 ratio of the chromatography

chamber in the mobile phase 
To produce the eluting solvent in the chromatography chamber in a saturated condition
(70:30) so that the solvent and the analyte is stable in the interaction with the TLC during
stationary phase or the mobile phase. The 70:30 ratio is reasoned as the pigment
extraction are both polar and nonpolar. They also help dissolving pigments and transport
them upward.

Why spot the sample 1cm above the edge of plate? 

If not mark the pigment spot on the original line above 1cm of the plate edge, then the
pigment might be dissolved in the eluting solvent before being able to bind on the
absorbent particles of TLC silica gel via the polaric interaction. 

IV. CONCLUSION 
Thin layer chromatography was used in this lab to analyze the components of the material
by separating them. This approach is also simple and safe to use. We examined the
components of carrot and spinach using this approach. For example, it demonstrates that
spinach has a high concentration of chlorophyll and other pigments such as xanthophyll,
pheophytin, and carotene. In the carrot sample, we could only detect pheophytin b rather
than the expected amount of carotene. This might be explained by the fact that we did not
follow the proper method (in taking extract, adding hexane-acetone and shaking). The
color and Rf are slightly out of sync at times, which might be due to human mistake.
After all, we've established the TLC technique and learned how to compute Rf and
compare it to the color displayed.