Results and discussion

Green tea constituent analysis was done using two-dimensional thin layer chromatography.
There are different visualization techniques done namely analysis under UV light, exposure to
iodine vapors and using vanillin-sulfuric acid spray reagent.
There are two solvents used in the isolation of green tea constituents via 2D-TLC. The role of
these solvents is to show how the differences in polarities affect how various compounds were
isolated. Solvent A, 9:1 dichloromethane and acetone, is more non-polar than solvent B,
35:45:20 hexane-ethyl acetate-glacial acetic acid. Solvent A separated all the nonpolar
compounds into different non polar mini-mixtures, spots consisting of different compounds, then
later on this mini mixtures are separated further by solvent B.
The chromatogram viewed under 254 nm and 366 nm of UV light is shown in figures 1 and 2
respectively. The spots in the 254 nm which appeared as black spots and did not fluoresce are
said to be the constituents while the spots that fluoresce in 366nm are the plant constituents.

Figure 1. Chromatogram of green tea constituents separated by 2D-TLC viewed under short UV,
254 nm.

Figure 2. Chromatogram of green tea constituents separated by 2D-TLC viewed under long UV,
366 nm.

Exposing the TLC plate to iodine vapors reveals the constituents in a way that the spots appear
as brown as if it was stained by iodine, refer to figure 3. This vapor consists of iodine which
chemically reacts with double bonds in the said constituents. This, in turn, is considered a semi
destructive method as the color it made to reveal the spots is a reversible process, heating the
plate fades the brown color and then another indicator can be used.

Figure 3. Chromatogram of green tea constituents separated by 2D-TLC and visualized by

exposure to iodine vapors.

Vanillin-sulfuric acid spray reagent was made to spray and reveal the spots. It was done the last
as it was deemed to be a destructive technique, an irreversible process. The chromatogram
produced using vanillin-sulfuric as a spraying agent is shown in figure 4. Vanillin sulfuric acid
spray reagent produced spots of different colors. Sulfuric acid a potent acid when reacted with
organic compounds turns to char. The charred spots were said to be organic compounds.
Steroids and terpenes are the common constituents revealed by vanillin. Other compounds like
essential oils, catechins, carotenoids, phenols and flavonoids can be detected using vanillinsulfuric acid spray (Spangenberg, Weins & Poole, 2011). These can be attributed to the spots
which turned blue or brown. The spot that fluoresce in 366nm then later on stained by vanillin is
prospected to be a steroid. The red spot is prospected to be a catechin.

Figure 4. Chromatogram of green tea constituents separated by 2D-TLC and visualized using
the 1% Vanillin-Sulfuric Acid spray.

There are spots which are revealed by selected visualization techniques, for example, spot A
only appeared after the plate is sprayed with vanillin-sulfuric acid. These are all summarized in
figure 5. RF values are shown in table 1 in reference to figure 5.

Figure 5. Diagram of all the spots revealed by different visualization techniques

Table 1.
Retardation factor values of Different Spots found in the TLC of Green Tea that had undergone
2D-TLC
SPOT

SOLVENT A

SOLVENT B

0.02

0.72

0.02

0.72

0.29

0.64

0.31

0.71

0.33

0.79

0.32

0.84

0.42

0.45

0.89

0.94

Conclusion
The 2D-TLC techniques success in the separation of green tea constituents cannot be
assessed. It can be deemed successful as it separated different constituents but it can also be
inconclusive as we dont have pre-TLC data of constituents which can be compared with the
separated spots or compounds.
A qualitative analysis of components of the sample should be done to know the nature of the
constituents present. Aqueous extracts consists mainly of carbohydrates, phenolic compounds,
tannins, proteins and others. Now depending on their presence, determined by the
recommended qualitative chemical tests, you can design the TLC experiment. You can choose
the perfect solvent combinations depending on the solubility of the phytochemical constituents
present. Use of other indicators to show other constituents like alkaloid reagents, Mayers and
others, to reveal spots in the TLC plate is also recommended given the premise that we know
what constituents are present.
It is also recommended to use or develop a more effective way to isolate constituents and also
to experiment different solvents to explore and exploit the different polarities of this compounds.
One can use more accurate instruments such as HPLC to isolate constituents desired.

References
Spangenberg, B., Poole, C. & Weins, C. (2011) Quantitative Thin-Layer Chromatography: A
Practical Survey. Berlin: Springer Science & Business Media