International University – Vietnam National University HCMC

School of Biotechnology
Deparment of Applied Chemistry
Biochemistry Program

REPORT
ORGANIC CHEMISTRY LABORATORY
SEMESTER II (2023-2024)

EXPERIMENT 3
THIN LAYER CHROMATOGRAPHY
Group 4:

1. Nguyễn Thị Cẩm Tiên BTBCIU20070

2. Nguyễn Hoàng Khánh Ngân BTBCIU20086
3. Đậu Ngọc Anh BTBCIU21042
4. Bùi Ngọc Mai BTBCIU21076
5. Trần Cao Bảo Ngọc BTBCIU21081
6. Đinh Thị Thanh Vân BTBCIU21016

Instructor: Dr. Le Quang Phong

Teaching assistant: Nguyen Thanh Phong

Section: Wednesday Afternoon

Date of submission: April 10th, 2024

I. ABSTRACT
The primary aim of the experiment was to showcase the methodology and applications of Thin thin-
layer chromatography (TLC). This technique is commonly utilized for discerning substance quantity
and identity, monitoring reactions, and differentiating unknown substances based on compound
polarity. The experiment comprised three distinct phases: the preparation of spinach leaf extract, the
analytical chromatography of the extract, and the observation and detection of plant components.
Notably, five spotting solutions were derived from spinach leaf extracts namely methanol extract
(A), concentrated methanol extract (B), hexane extract (C), concentrated hexane extract (D), and
co-spot of B and D (E). A TLC chamber was prepared with a solvent featuring a fixed and sealed
70:30 ratio of hexane and acetone. The TLC plate underwent scrutiny using various techniques,
including UV light exposure and staining with iodine, as well as a solution comprising 3% boric
acid and 10% oxalic acid. The TLC plate's visual interpretation could be inadequate due to many
factors, including sample concentration and the range detected by the solvent system. While more
spots were observed under iodine staining compared to UV light, these remained unclear.
Additionally, the inclusion of a solution containing 3% boric acid and 10% oxalic acid did not
significantly enhance the visibility of yellow-brown dots on the TLC plate. Due to their low Rf
values, spots 1, 2, 3, 5, 6, 7, 9, 10, 11, 12, 15, 16, 17, and 18 were determined to have higher polarity
than spots 13 and 19. Notably, spot numbers 4, 8, 14, and 20 exhibited an Rf value of 1, indicating
the least polar compound that the utilized solvent system could detect.

II. INTRODUCTION

Thin-layer chromatography (TLC) is a fundamental method to qualitatively analyze sample

compositions, predict whether the two substances are identical, and monitor reactions, based on
molecular interactions with the stationary and mobile phases. TLC is considered solid-liquid
chromatography with the stationary phase as a solid plate coated by a thin layer of absorbent. In
the case of separating organic compounds with low levels of polarity, silica gel as a polar absorbent
is most commonly used while nonpolar cellulose absorbent is utilized to separate highly polar
compounds. The mobile phase, often referred to as the eluent, is a solvent or solvent mixture that
is used to elute the sample once it has been placed on the plate. The solvent travels up the plate,
and the chemicals in the mixture have different affinities for the mobile and stationary phases. As
a result, whereas less polar molecules spend more time in the mobile phase and travel up the plate
more quickly, more polar molecules interact more with the stationary phase and move up the plate
at slower rates[1].

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Since many compounds can absorb light in the visible range and have color, the spots' locations
and movements may be seen under the normal condition of room light both during and after the
plate's development[1]. However, since the wide range of organic compounds is colorless under
visible light, they cannot be observed directly on the TLC plate, so conducting the additional
visualization method is necessary. There are two types of visualization techniques: destructive (the
compound's molecular structure is destroyed and changed) and non-destructive (the compound
stays untouched). For TLC plates, UV light is the most used non-destructive visualization
technique. UV light with a wavelength of 254 nm or 365 nm may be directed onto a TLC plate
using a UV lamp. When examining with short-wavelength UV light, the majority of commercially
purchased TLC plates include fluorescent materials, which turn the background green. When a
substance absorbs UV light at 254 nm, it appears dark. Especially for aromatic molecules and
highly conjugated systems, this approach is frequently the first to be explored[2]. Additionally, an
old approach for examining chemical molecules on TLC plates is iodine staining. It is a chemical
process that includes binding iodine vapors to analytes on the plate, creating dark brown spots on
a lighter brown background. Since complexation is reversible and the iodine will ultimately
evaporate, leaving the original compound behind, this process is regarded as "semi-destructive".
This substance may have evaporated by then, and another visualization method can be applied as
the hue starts to fade[2]. Using the destructive visualization approach, a plate is dipped in a stain
solution that interacts with a chemical on a TLC plate to produce a visible, colored compound.
Heating the plate slightly can speed up this process. Stains are made to act with certain functional
groups; therefore, not all compounds can be seen with every reagent. The specific stain selected is
determined by the chemicals' assumed structures[2].

The mobility of compounds along the TLC plate is measured using the retention factor (Rf). Rf is
defined as the distance a specific molecule travels relative to the solvent front (the leading edge of
the mobile phase). This distinguishing feature of a compound in a certain solvent system enables
the identification of unidentified compounds by comparison with known standards[1].

Many sectors, including pharmaceuticals, clinical diagnostics, environmental toxicity, food, water,
pesticide analysis, and cosmetics, employ TLC extensively as a chromatographic method. It may
be used for chemical identification, purity analysis, reaction monitoring, and medicinal plant
identification. TLC provides quicker, more selective separation, is easy to use, and is affordably
priced. However, because of the short stationary phase, it has a restricted separation length, cannot
distinguish between enantiomers and isomers, and requires Rf values for compound identification.
Overall, TLC offers advantages and limitations in various fields[3].

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The experiment's goal was to remove samples from spinach leaves and dissolve them in methanol,
condensed methanol, hexane, and condensed hexane solvents. After that, TLC procedures were
performed by adding a sample to a plate to create a small concentrated spot, allowing the plate to
develop in the beaker, and finally, visualization. The composition of the compounds in the leaves
was seen with the naked eye, a UV lamp, iodine staining, and acid staining. The outcomes were then
compared using various solvents.

III. MATERIAL & METHODS

1. MATERIALS

In these experiments, a variety of types of equipment were utilized namely a capillary, forceps,
mortar and pestle, alcohol lamp, aluminum foil, UV chamber, silica plate for TLC, 3 vials, 1 spatula,
2 glass pipets, 4 beaker 100 mL and a 5mL cylinder. Other chemicals required for these procedures
include methanol, hexane, chloroform, petroleum ether, acetone, boric acid, oxalic acid, and iodine.

2. PROCEDURE

First of all, the spinach extract was prepared by grinding 5 g of spinach leaves and stems with 10 ml
of methanol until the extracted solution became dark green. The liquid must be then poured into a
glass vial while avoiding the ground leaves and stems. The vial containing methanol extract was
labeled as A sample. The majority of A was put in a beaker and gently heated by a hot plate to
condense the extract to 1/5 of its original volume. The small amount of condensed liquid was
transferred into vial B as the concentrated methanol extract sample. After heating, 5 ml of hexane
was added to the beaker containing condensed methanol extract and mixed thoroughly. The upper
layer of hexane extract was taken and transferred to a new vial (labeled C). The process was repeated
until the supernatant became colorless. Then, a half amount of C was placed into another vial
(labeled D) and gently heated by the hot plate to condense the extract to 1/5 of its initial volume.

For TLC plate preparation, a faint pencil line was drawn about 1 cm from the bottom border of the
TLC plate, and the line was divided into 5 markers labeled A-E. A capillary with two open ends,
one of which is thinner, was utilized to load small spots of the extract samples at the respective
marked positions on the TLC plate. The spots should be the same size and not too huge to avoid
smears and dispersed streaks. The patches were thoroughly air-dried. Plates were then developed in
a TLC chamber containing the solvent system of hexane-acetone (70:30) until the solvent reached
the marked front line. Notably, the TLC chamber should be carefully covered by aluminum foil to
ensure the saturation between the atmosphere and solvent vapor.

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After developing, the phytocomponents[1] of the spinach extract were observed and analyzed by
visualizing the result of the TLC plate with various staining methods. The four visualization methods
were performed, including observation under visible and UV light, iodine stain, and acid stain.
Firstly, the plates were examined in room light and in a UV chamber. Secondly, the plate was stained
in the chamber containing iodine. Thirdly, the TLC plate was sprayed with a reagent of 15 ml of 3%
boric acid solution and 5 ml of 10% oxalic acid. The weight of oxalic acid required for the 5 ml of
10% oxalic acid in this experiment, was computed using the formula:

% Solid = g/(100ml solvent) = 10% = 10g/100ml = 0.1g/ml

=> moxalic acid = 0.1g/ml x 5ml =0.5g

Besides that, the weight of boric acid was applied to determine the 15 ml of 3% boric acid solution,
which was calculated as follows:

% Solid = g/(100ml solvent) = 3% = 3g/100ml = 0.03g/ml

=> mboric acid = 0.03g/ml x 15ml = 0.45g

After that, the TLC plate was heated on a hot plate. The last section adopted an alternative method
for seeing and developing TLC plates. The plate was put in an iodine-containing chamber. Finally,
the observation of each section was recorded.

IV. RESULTS
Part 1. TLC analysis of spinach leaf extract

Figure 1. Spinach leaf extract in methanol and hexane. Vial A contained methanol-containing
spinach extract, while vial B contained concentrated methanol extract. Vial C contained hexane-

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based spinach extract, Vial D contained concentrated hexane extract, and vial E contained both
concentrated methanol and hexane extract.

Figure 2. The TLC plate result was observed under room light. A - loaded sample of methanol
extract, B - loaded sample of concentrated methanol extract, C - loaded sample of hexane extract,
D - loaded sample of concentrated hexane extract, E - co-spot of sample B and sample D, number
1-4 - spots of sample A, number 5-8 - spots of sample B, number 10-14 - spots of sample D, number
16-20 - spots of sample E

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Figure 3. The TLC plate result that observed under UV light. A - loaded sample of methanol
extract, B - loaded sample of concentrated methanol extract, C - loaded sample of hexane extract,
D - loaded sample of concentrated hexane extract, E - co-spot of sample B and sample D, number
2, 3 - spots of sample A, number 6, 7 - spots of sample B, number 11, 12, 13, 21 - spots of sample
D, number 17, 18, 19, 22 - spots of sample E

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Figure 4. The TLC plate with Iodine stain. A - loaded sample of methanol extract, B - loaded
sample of concentrated methanol extract, C - loaded sample of hexane extract, D - loaded sample
of concentrated hexane extract, E - co-spot of sample B and sample D, number 1-4 - spots of
sample A, number 5-8 - spots of sample B, number 9-14 - spots of sample D, number 15-20 - spots
of sample E

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Figure 5. The TLC plate result after staining with a mixture of 3% boric acid and 10% oxalic
acid solution. A - loaded sample of methanol extract, B - loaded sample of concentrated methanol
extract, C - loaded sample of hexane extract, D - loaded sample of concentrated hexane extract, E
- co-spot of sample B and sample D, number 1-4 - spots of sample A, number 5-8 - spots of sample
B, number 9-14 - spots of sample D, number 15-20 - spots of sample E

A standard ruler measured the distance traveled by each spot on the silica plate. The distance
traveled by the solvent front was recorded at 6.7cm. Their retention factor values calculated using
the formula (*) are shown in Table 1.

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 Table 1. Distance traveled and Rf values of the spots located on the silica plate

Spot no. Traveled distance Rf values

1 2.7 0.40

2 2.9 0.43

3 3.1 0.46

4,8,14,20 6.7 1

5 2.7 0.40

6 2.8 0.42

7 3 0.45

9 0.5 0.07

10 2.6 0.39

11 2.8 0.42

12 3 0.45

13 4.5 0.67

15 0.6 0.08

16 2.5 0.37

17 2.8 0.42

18 3 0.45

19 4.4 0.66

21 5.5 0.82

22 5.5 0.82

V. DISCUSSION
Since the sample of spinach’s stem and leaf predictably contained many components with different
properties, it was extracted using two different types of solvent: methanol and n-hexane. The
methanol extract was meticulously prepared, involving the grinding of the sample to break down the
rigid cell wall and dissolve the polar chemicals. Thereafter, as the spinach consisted of various
compounds with different polarities, the hexane extract could effectively collect the other

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phytochemicals with low polarities. The different compositions between the two extracted samples
could be qualitatively observed by thin-layer chromatography using silica gel as the polar absorbent.

To monitor the changes in sample compositions throughout the process, the four samples of the
methanol extract, the concentrated methanol extract, the hexane extract, and the concentrated hexane
extract were loaded on a silica gel plate in the same amount of 10 dots and denoted as sample A, B,
C, and D respectively. It was crucial to note that the pen must not be used for labeling since the
chemicals in the ink could contaminate the sample and affect TLC performance. Instead, all labels
on the TLC plate should be slightly drawn by an unsharpened pencil to avoid damaging the silica
gel layer. The fifth spot labeled by E on the TLC plate was a co-spot of samples B and D in equal
ratio; the spot comprised five dots of concentrated methanol extract and five dots of concentrated
hexane extract. The comparison among the spot observations of samples B, D, and E was to predict
the composition changes among the two extractions.

Sample B was made by vaporizing sample A until the solution was ⅕ of the initial volume so that
the components in B were predicted to be the same as sample A but in higher concentration unless
there were volatile compounds lost in the condensing step. This prediction was similar to sample D,
which was condensed from the previous sample C. However, despite a high possibility that there
were the same compositions between samples A and B and between samples C and D, the presented
spots on the TLC plate could possess differences since there were changes in concentration. The
higher the concentration of a compound in the sample, the bolder the respective spot appeared on
the TLC plate. In particular, if the concentration of a component was too low, the presence of the
compound could not be detected on the TLC plate. Therefore, since the colored spots could fade
quickly over time and the TLC result was not marked immediately after developing, combined with
the very light color of sample C’s spots because of its low concentration, the composition of sample
C could not be predicted on the TLC result of any visualization method.

After loading the five samples at marked positions on the baseline of the TLC plate, the paper was
then developed by the solvent system of 70:30 Hexane: Ethyl Acetate to separate components of the
mixture into different bands. These different bands appeared in distinct spots on the TLC plate that
could be visualized by various staining methods. Since similar molecular structures show similar
interactions with the stationary and mobile phases of thin-layer chromatography, the Rf value of the
same compound could be approximately constant if the chromatography is performed under the
constant conditions of stationary phase, mobile phase, and temperature. Besides, the Rf value is
affected by other uncontrollable factors such as sample condition, quality of spot, and
chromatography developing technique so the Rf value could be uncertain for identification[5].

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However, the Rf value can still be used to have a preliminary prediction about the number of
components and the relative identities of those in the unknown mixture.

Following the calculation in Table 1, it was observed that certain spots on the TLC plate share
similarities in their retention factor. Specifically, spots 9 and 15 had a relative Rf of 0.08; spots 1, 5,
10, and 16 had a relative Rf of 0.4; spots 2, 6, 11, and 17 had a relative Rf of 0.42; spots 3, 7, 12,
and 18 had a relative Rf of 0.45, spots 13 and 19 had a relative Rf of 0.67, spots 21 and 22 had a
relative Rf of 0.82, and spots 4, 8, 14, and 20 had a relative Rf of 1. The spots with similar Rf values
could represent the same compounds, resulting in the possibility of the presence of the same
components among different extracted samples of A, B, C, and D. In addition, because the greater
Rf value indicates the higher migration up the silica gel plate which results in the lower polarity of
the compound, the relative polarity among components could be preliminarily predicted based on
the order of Rf values.

As shown in Figure 2, when the TLC plate was observed under the room light after the development,
four components were detected in the methanol extracts A and B, while one additional spot appeared
in sample D of concentrated hexane extract. It was evident that the Rf values of spots 1, 5, 10, and
16 in Table 1 were approximately the same at 0.4; these spots could express the same compound in
both four samples A, B, D, and E. Similarly, the four compounds of the spot 1, 2, 3, and 4 of sample
A were remained and appeared in all extract samples since their respective Rf values were relatively
determined at the same number. Especially in sample D and sample E, which was the co-spot of
samples B and D, spot 13 was shown with a different relative position to other spots of methanol
extract samples, resulting in this new compound being collected by hexane extraction and being
predicted as a low-polar compound due to its relative high Rf value at 0.67. Notably, TLC could not
clearly detect any components of sample C since the sample was highly diluted after the hexane
extraction. Moreover, as the colored spots could fade quickly over time, combined with the very
light color of sample C’s spots because of its low concentration, the composition of sample C could
not be predicted on the TLC plate result of any visualization method.

For the following visualization method, the TLC plate was observed under UV light to examine
further if any component did not absorb visible light and was not detected when observing under the
room light. As shown in Figure B, while spots 1, 5, 10, and 16 disappeared on TLC observation, two
new spots, 21 and 22, were detected by UV light. These spots illustrated that one compound in the
hexane extract did not absorb light in the visible range but in the UV range. The two spots, 21 and
22, were not even observed on the TLC plate result after being stained by iodine and the reagent
made of acid boric and oxalic acid. However, by performing these two oxidation staining methods,
two new spots of 9 and 15 were detected in the line of samples D and E, meaning that another new

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compound was detected in the hexane extract. To sum up, four compounds were collected from the
spinach methanol extract, while a total of 7 compounds, including the three additional compounds,
were detected after performing the hexane extraction.

Nevertheless, the total number of spinach extract components detected could be insufficient due to
several factors related to the extract and the chromatography technique. The number of samples
loaded on the TLC plate should be increased for the diluted mixture, such as the spinach extract, to
prevent a blurred and undetected appearance. Additionally, some compounds with similar
interactions with chosen stationary and mobile phases could overlap and appear on the TLC plate as
one spot. The separation could be improved by optimizing the solvent system to interact differently
with the components. Besides, two-dimensional thin-layer chromatography is also useful in more
accurately qualifying the number of components in the mixture by providing better separation than
the one-dimensional TLC performance[5].

Furthermore, the accuracy of identification using the TLC technique could be enhanced by using
authentic compounds as the standard. By co-spotting the unknown sample with the standard
compound, the conditions of the two samples were the same at the spot position, and the
identification of the unknown could be determined without depending on the retention factor[6]. In
addition, many advanced spectroscopy techniques, such as NMR and FITR spectroscopy, have been
utilized to identify molecular structures with high accuracy based on radiation-matter interaction[7].

VI. CONCLUSION
The thin-layer chromatography (TLC) experiment on spinach extracts aimed to separate and detect
the presence of the various components in the sample. The experiment performed 4 different
visualization methods of visual light and UV light observation, iodine stain, and acid stain to fully
obtain the presence of the components. The similarity in Rf values among spots in samples A, B,
D, and E indicated the presence of the same compounds with 0.08 of spot 9 and 15; 0.4 of spot 1,
5, 10, and 16; 0.42 of spot 2, 6, 11, and 17; 0.45 of spot 3, 7, 12, 18; 0.67 of spot of 13 and 19;
0.82 of spot 20 and 21; and 1 of spot 4, 8, 14, 20. In total, four compounds were collected from
the methanol extract of the spinach, while performing the hexane extraction led to the detection of
seven compounds, including three additional ones. However, it is crucial to acknowledge that the
total number of detected components might be insufficient due to factors related to the extract and
the chromatography technique. For future improvement, increasing the number of loaded samples
on the TLC plate and optimizing the solvent system are recommended to enhance separation and
detection. Two-dimensional thin-layer chromatography can also provide better separation and
more accurate identification of components in the mixture[5]. Additionally, the identity and purity
of a compound sample have recently been identified by using spectroscopic techniques[7].

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VII. REFERENCE

[1] Son, H. L. (2022). Thin Layer Chromatography. Organic Chemistry Lab Manual (pp. 12-15).
Ho Chi Minh City: International University, HCMC.

[2] Libretexts. (2022, April 7). 2.3F: Visualizing TLC Plates. Chemistry LibreTexts.
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistry_Lab_Technique
s_%28Nichols%29/02:_Chromatography/2.03:_Thin_Layer_Chromatography_%28TLC%29/2.3
F:_Visualizing_TLC_Plates

[3] Aryal, S., & ChurchillAnthony. (2023, September 11). Thin layer chromatography:
Principle, parts, steps, uses. Microbe Notes. https://microbenotes.com/thin-layer-
chromatography/

[4] Hais, I. M. (1968). Factors which influence paper chromatographic RF values. Journal of
Chromatography A, 33, 25–27. https://doi.org/10.1016/s0021-9673(00)98611-4

[5] Rabel, F., & Sherma, J. (2016). A review of advances in two-dimensional thin-layer
chromatography. Journal of Liquid Chromatography & Related Technologies, 39(14), 627–
639. https://doi.org/10.1080/10826076.2016.1214844

[6] Meyers, C. L. F., & Meyers, D. J. (2008). Thin‐layer chromatography. Current Protocols in
Nucleic Acid Chemistry, 34(1). https://doi.org/10.1002/0471142700.nca03ds34

[7] Penner, M. H. (2017). Basic principles of spectroscopy. Food Science Text Series, 79–88.
https://doi.org/10.1007/978-3-319-45776-5_6

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VIII. LAB NOTE

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