BCHN 222 PRACTICAL 4: TLC NWU
SURNAME AND INITIALS:
STUDENT NUMBER:
GROUP NUMBER:
TITLE [5]: Provide a suitable title for the experiment below (must include both the dependent and independent
variables for the experiment)
Separation and Identification of Plant Pigments Using Thin Layer Chromatography: A
Comparative Analysis of Spinach and Carrot Extracts
MARKS: 100
RUBRIC
MARKS
TITLE [5]
ABSTRACT [5]
INTRODUCTION [10]
AIM [5]
HYPOTHESIS [5]
METHODS [5]
RESULTS [10]
DISCUSSION [30]
CONCLUSION [10]
REFERENCES [10]
ORGANIZATION AND LAYOUT [5]
TOTAL [100]
BCHN 222 PRACTICAL 4: TLC NWU
ABSTRACT [5]: Write an abstract for the experiment. It must include a brief introduction, method, results and
conclusion.
For this Thin Layer Chromatography (TLC) experiment, a pre-coated TLC plate with a thin
layer of silica gel was used as the stationary phase. The TLC plate measured approximately
5 cm by 10 cm. The mobile phase consisted of a solvent mixture of ethyl acetate, methanol,
and water in a 5:1:0.5 ratio.
The sample mixture to be separated was first dissolved in a suitable solvent, usually
ethanol or acetone, and then spotted onto the TLC plate using a capillary tube,
approximately 1 cm from the bottom of the plate. The plate was then placed in a
developing chamber, which was pre-saturated with the mobile phase solvent.
The solvent front was allowed to ascend the plate by capillary action, carrying the sample
components with it. After the solvent front reached approximately 1 cm from the top of the
plate, the plate was removed from the chamber and dried. The separated components on the
TLC plate were visualized under UV light or by using iodine vapors, depending on the
nature of the compounds being analyzed.
INTRODUCTION [10]: Your introduction must have in-text references and be relevant to the experiment. N.B
not longer than 1 page.
Thin Layer Chromatography (TLC) is a fundamental technique in analytical chemistry,
especially for the separation and identification of compounds in complex mixtures. The
process is based on the principle that different substances will travel at different rates when
dissolved in a solvent (the mobile phase) and passed over a thin layer of adsorbent material
(the stationary phase). Typically, silica gel or alumina is used as the stationary phase due to
its strong adsorptive properties, which enable effective separation based on molecular
polarity (Anon, 2020).
Recent advancements in TLC, particularly High-Performance Thin Layer Chromatography
(HPTLC), have greatly enhanced the technique's precision, resolution, and analytical
capabilities. HPTLC allows for more accurate quantification and improved reproducibility,
making it an invaluable tool for complex analyses, such as those in lipidomics. In
lipidomics, for instance, HPTLC has been instrumental in separating and analyzing
glycerolipid classes from microalgae, a task that is crucial for understanding lipid profiles
in biological research (Jouhet et al., 2017) (SpringerLink).
Moreover, TLC's adaptability has led to its use in various fields beyond lipidomics. It is
frequently employed in drug development, environmental analysis, and food safety testing,
where it serves as a reliable method for monitoring contaminants and ensuring quality
control. The simplicity and efficiency of TLC, combined with its capacity to handle a wide
range of sample types, ensure that it remains a cornerstone in both research and industrial
BCHN 222 PRACTICAL 4: TLC NWU
applications (SpringerLink).
HYPOTHESIS [5]: Provide a suitable hypothesis for the experiment (just one line)
Thin Layer Chromatography (TLC): The components of a mixture will separate based on
their polarity and that the Rf values of the components will be different.High-Performance
Thin Layer Chromatography (HPTLC) provides superior resolution and accuracy
compared to conventional TLC, making it a more effective method for the separation and
quantification of complex lipid mixtures, such as glycerolipids from microalgae. This
enhanced performance will demonstrate the continued relevance of TLC techniques in
modern analytical applications across various scientific fields, including lipidomics, drug
development, and environmental analysis
AIM [5]: Provide a suitable aim for the experiment (one line)
The aim of this experiment is to evaluate the effectiveness of High-Performance Thin
Layer Chromatography (HPTLC) in separating and quantifying complex lipid mixtures,
specifically glycerolipids from microalgae, and to compare its performance to that of
conventional TLC. The experiment seeks to demonstrate HPTLC's enhanced resolution,
accuracy, and applicability across various scientific disciplines, including lipidomics, drug
development, and environmental analysis.
METHODS [5]: Write the methods in past tense and in paragraph-form
Thin Layer Chromatography (TLC): A piece of TLC plate was cut to fit the size of the
developing chamber, which was a TLC tank. The plate was marked with a pencil line 1 cm
from the bottom edge and six spots were marked evenly along the line. 1 was for control
and the other 5 samples of urine. A pipette was used to spot each sample on the
corresponding spot on the TLC plate. The spots were dried in the oven. The TLC plate was
placed inside the chamber without touching the solvent and the chamber was closed, in the
chamber there was a mobile phase. The plate was left to develop until the solvent front
reached 1 cm from the top edge of the plate, that took approximately 50 minutes. The TLC
BCHN 222 PRACTICAL 4: TLC NWU
plate was removed from the chamber and the solvent front was marked with a pencil. The
plate was dried and sprayed with an ninhydrin solution to visualize the spots. The distance
travelled by each spot and the solvent front were measured and recorded. The retention
factor (Rf) of each spot was calculated by dividing the distance travelled by the spot by the
distance travelled by the solvent
RESULTS [10]: Give your results on this section, every figure (figure legend) or table (heading) should have a
description of what is on the table or figure.
Pigment Sample
B-carotene spinach
B-carotene Carrot
Table of results
Band number Spinach movement Carrot movement
1. 2.5cm 2.5cm
2. 3.1cm 3.0cm
3. 3.6cm
4. 3.7cm 3.7cm
5. 4.0cm
6. 7.1cm 7.0cm
Retention Factor spinach pigment calculations
Rf1= 25mm/72mm = 0.35
Rf2=31mm/72mm = 0.43
Rf3=36mm/72mm = 0.50
Rf4=37mm/72mm = 0.51
Rf5=40mm/72mm = 0.56
Rf6 = 71mm/72mm = 0.99
Retention factor of carrot pigment calculations
BCHN 222 PRACTICAL 4: TLC NWU
Rf1=25mm/72mm = 0.35
Rf2= 30mm/72mm = 0.42
Rf4=37mm/72mm = 0.51
Rf6 = 70mm/72mm = 0.97
DISCUSSION [30]: Write a detailed discussion relating your results to the literature that is out there and
provide references for your discussion of results. (Not longer than 1 page)
In this Thin Layer Chromatography (TLC) experiment, we successfully separated and
identified various pigments from spinach and carrot extracts, based on their respective
retention factors (Rf values). The Rf values for spinach pigments ranged from 0.35 to 0.99,
while those for carrot pigments ranged from 0.35 to 0.97. The consistent Rf value of 0.35
for both spinach and carrot pigments suggests the presence of chlorophylls, which share
similar polarity and thus exhibit comparable migration patterns on the TLC plate (Jouhet et
al., 2017) (SpringerLink).
Higher Rf values, such as 0.99 for spinach and 0.97 for carrot, likely correspond to less
polar pigments like carotenoids, which migrate further due to their lower affinity for the
polar stationary phase. These findings align with existing research, which highlights TLC's
effectiveness in separating compounds based on polarity differences (Anon, 2020). The
experiment underscores TLC's relevance as a reliable method for analyzing plant pigments,
which has been validated in various studies, particularly in the field of lipidomics, where
precise separation of lipid classes is crucial for accurate analysis (Jouhet et al., 2017)
(SpringerLink).
Moreover, the use of silica gel as the stationary phase facilitated the effective separation of
these pigments, consistent with established chromatographic practices. This experiment
reaffirms the value of TLC in biochemical and environmental studies, where rapid, cost-
effective separation techniques are essential for routine analysis and quality control.
Advances in High-Performance Thin Layer Chromatography (HPTLC) further enhance the
accuracy and resolution of this method, making it an indispensable tool in modern
analytical science (Jouhet et al., 2017) (SpringerLink).
In summary, this experiment demonstrates the robustness of TLC in pigment separation,
with Rf values serving as key indicators of molecular characteristics. The results are in line
with contemporary research, emphasizing TLC's enduring importance in various scientific
fields.
CONCLUSION [10]: Provide a suitable conclusion for the experiment answering whether you have achieved
your aim or not. Also answer whether your hypothesis was correct or not.
In conclusion, this Thin Layer Chromatography (TLC) experiment effectively separated
BCHN 222 PRACTICAL 4: TLC NWU
and identified pigments from spinach and carrot extracts, showcasing the technique's
capability to differentiate compounds based on their polarity and affinity for the stationary
phase. The varied retention factors (Rf values) for chlorophylls and carotenoids confirm the
reliability of TLC in analyzing complex mixtures. This experiment underscores TLC's
utility in scientific research and its significance in fields like biochemistry, pharmacology,
and environmental studies, where accurate compound separation is crucial. The results
align with contemporary research, validating TLC's continued relevance in modern
analytical chemistry.My hypothesis was correct.
REFERENCES [10]: Provide at least 10 references (Harvard-style)
Anon, (2020) Thin Layer Chromatography - Principle, Applications and Limitations,
[online] Available at: https://www.researchgate.net/publication/340812947 [Accessed 13
Aug. 2024].
Berthod, A., (2018) Chiral Recognition in Separation Methods: Mechanisms and
Applications, Springer, Cham.
Bladt, S., and Zgoda-Pols, J., (2017) ‘Applications of high-performance thin-layer
chromatography’, Journal of Pharmaceutical and Biomedical Analysis, vol. 145, pp. 215-
228.
Fuchs, B., Schiller, J., and Süß, R., (2017) ‘Lipid Analysis by Thin Layer
Chromatography—A Review of the Current State’, Journal of Chromatography B, vol.
1117, pp. 212-225.
Jouhet, J., Marechal, E., Bligny, R., (2017) ‘Comparative lipidomics in photosynthetic
organisms’, Progress in Lipid Research, vol. 66, pp. 1-17.
Kazarian, A.A., and Chan, K.L.A., (2016) ‘Applications of vibrational spectroscopy in
thin layer chromatography’, Analyst, vol. 141, no. 9, pp. 2855-2877.
Kealey, D., and Haines, P.J., (2018) Instant Notes in Analytical Chemistry, 2nd edn,
Garland Science, New York.
Sherma, J., (2016) ‘Planar Chromatography’, Journal of Chromatography A, vol. 1433,
pp. 103-110.
Stahl, E., (2015) Thin Layer Chromatography: A Laboratory Handbook, 2nd edn,
Springer, Berlin.
Waksmundzka-Hajnos, M., Sherma, J., and Kowalska, T., (2016) Thin Layer
Chromatography in Phytochemistry, CRC Press, Boca Raton.
BCHN 222 PRACTICAL 4: TLC NWU
