NAME: ELIZABETH NANA ADWUBI AFRIFA

INDEX NUMBER: 01230228B

COURSE: PRACTICAL FOOD BIOCHEMISTRY

THIN LAYER CHROMATOGRAPHIC METHOD FOR PLANT PIGMENT

CHARACTERIZATION AND QUALITATIVE TEST FOR ANTIOXIDANTS
ABSTRACT
The experiment aimed to separate and analyze plant pigments using paper chromatography. The
photosynthetic pigments in leaves, chlorophyll b, and carotenoid, were identified, and their
separation behavior was observed, the principles of capillarity and solubility were crucial in
conducting. The stationary phase is the paper and silica gel. The mobile phase is the solvent
which moves along the stationary phase to allow separation. Retentions factors were calculated
and the got color pigment of orange, green and yellow with values of 1cm, o.62cm, 0.14cm
respectively.

INTRODUCTION
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.
Chromatography was discovered by M. Tswett in 1906.Thin layer chromatography is performed
on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminum oxide, or cellulose (blotter paper). This layer of adsorbent
is known as the stationary phase. After the sample has been applied on the plate, a solvent or
solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because
different analytes ascend the TLC plate at different rates, separation is achieved. Thin layer
chromatography can be used to: Monitor the progress of a reaction, identify compounds present
in a given substance, determine the purity of a substance. Separation of compounds is based on
the competition of the solute and the mobile phase for binding places on the stationary phase. For
instance, if normal phase silica gel is used as the stationary phase it can be considered polar.
Given two compounds which differ in polarity, the more polar compound has a stronger
interaction with the silica and is therefore more capable to dispel the mobile phase from the
binding places. Consequently, the less polar compound moves higher up the plate (resulting in a
higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it
is more capable of dispelling solutes from the silica binding places and all compounds on the
TLC plate will move higher up the plate. Practically this means that if you use a mixture of ethyl
acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for
all compounds on the TLC plate. Changing the polarity of the mobile phase will normally not
result in reversed order of running of the compounds on the TLC plate. Carotenoids are a class of
natural fat-soluble pigments found principally in plants, algae, and photosynthetic bacteria,
where they play a critical role in the photosynthetic process. They also occur in some non-
photosynthetic bacteria, yeasts, and molds, where they may carry out a protective function
against damage by light and oxygen. Although animals appear to be incapable of synthesizing
carotenoids, many animals incorporate carotenoids from their diet. Within animals, carotenoids
serve as antioxidants, and can be a source for vitamin A activity (Ong and Tee 1992; Britton et
al. 1995) .Carotenoids are responsible for many of the red, orange, and yellow colors of plant
leaves, fruits, and flowers, as well as the colors of some birds, insects, fish, and crustaceans.
Some familiar examples of carotenoid coloration are the oranges of carrots and citrus fruits, the
reds of peppers and tomatoes, and the pinks of flamingoes and salmon (Pfander 1992). Some 600
different carotenoids are known to occur naturally (Ong and Tee 1992), and new carotenoids
continue to be identified (Mercadante 1999).
AIM
To characterize plant pigment using Thin Layer Chromatography
MATERIALS AND EQUIPMENT
TLC paper, TLC chamber with lid, pencil, ruler, sample mixture (plant pigment), solvent mixture
(Petroleum ether: Acetone: Water, ratio 3:1:1)
METHOD
Test for leave pigment
A leave was placed in a mortar and mashed using a pistil with absolute ethanol. A thin line of
2cm was drawn on the TLC paper with a pencil. The sample mixture was spotted on the line
drawn. The TLC chamber was developed by filling the chamber with the mobile phase up to
1.5cm and covered with a lid. The chamber with lid was left undisturbed for the mobile phase to
evaporate and saturated for about 5minites. The paper was placed in the chamber and covered.
There was a clear separation after some time and it was marked and circled before the separation
disappears.
RESULTS
(Petroleum ether: Acetone: Water, ratio 3:1:1)
Petroleum
3÷5 ×20
=12ml
Acetone
1÷5 ×20
=4ml
Water
1÷5 ×20
=4ml

Figure 1: Thin layer chromatography setup with distance

Rf calculation
Distance travelled by the substance ÷Distance travelled by the solvent
Solvent Front =5cm
Yellow - 0.7
0.7 ÷5 =0.14cm
Green- 3.1
3.1 ÷5= 0.62cm
Orange- 5cm
5÷5=1cm
Component on TLC Distance travlled Retention factor
paper /cm
Orange 5 1
Green 3.1 0.62
Yellow 0.7 0.14
Test for Antioxidants
A ml of the sample and distilled water was pipetted into their respective clean test tubes. Two
drops of 5% ferric chloride was added to the contents in each tube. Blue-black or dark green
coloration indicated the presence of phenolic compounds
RESULTS

Figure 2: Negative control and test sample

Figure 3: Antioxidant test in leaf sample

DISCUSSION
As the solvent rises by capillary action up through the TLC paper, the components of the
pigment mixture are partitioned between the mobile phase (solvent) and the stationary phase
(silica gel) due to their different adsorption and solubility strength. The more strongly a given
component is adsorbed to the stationary phase, the less easily it is removed by mobile phase. The
weakly a component is adsorbed, the faster it will migrate up the TLC paper. On the other hand,
the running distance depends on the solubility of the pigment in the solvent. Since the
experiment employs a high non-polar solvent (petroleum ether), the pigments that are least polar
(carotenes) will be best solved in the non-polar solvent and will thus have the largest running
distance. The pigment with an Rf value of 0.14 indicated that it moves slowly on the TLC paper
which suggest strong interaction with the stationary phase. The pigment with Rf values shows
moderate migration which implies balance interactions with the stationary and the mobile phase
leading to moderate separation. The pigment with an Rf of 1 migrates at the same rate as the
solvent front, indicating no interaction with stationary phase and complete affinity for the mobile
phase which represents very polar compounds.
SOURCES OF ERROR
Too much sample applied overwhelmed the TLC plate, causing smearing and poor band
resolution.
SAFETY PRECAUTIONS
Consistent and uniform spotting of samples on the TLC paper was ensured. Uniform
development conditions, such as solvent saturation levels, chamber saturation was maintained.
Samples were applied in small, concentrated spots to prevent overloading and maintain band
resolution

CONCLUSION
Carotene is identified as having the lowest molecular weight by its yellow to orange tint near the
top of the paper. In the pigment separation of chlorophyll, chlorophyll was distinguished by its
blue or dark green hue. When chlorophyll pigments are separated, the colour yellow-light green
identifies chlorophyll B. In the chromatography solvent, xanthophyll is more soluble since it has
gone up the paper. This describes the conclusion of paper chromatography. The pigments are
light-absorbing molecules and are separated by using paper chromatography techniques The
pigments move on the paper based on their solubility in the solvent. Along with the alcohol,
pigments also migrate along the strips of paper. Some pigments in the leaf travel more quickly
than others because of their properties.

REFERENCE
Shioi, Y., Brotosudarmo, T. H. P., & Limantara, L. (2015). Separation of photosynthetic pigments by high-
performance liquid chromatography: comparison of column performance, mobile phase, and
temperature. Procedia Chemistry, 14, 202-210.

Sherma, J., & Zweig, G. (1967). Separation of chloroplast leaf pigments by thin-layer chromatographer on
cellulose sheets. Journal of Chromatography A, 31, 439-445.

Britton, G., S. Liaaen-Jensen, and H. Pfander. (1995). Carotenoids today and challenges for the future.
In: Briton, G., S. Liaaen-Jensen, and H. Pander [eds], Carotenoids vol. 1A: Isolation and Analysis. Basel:
Birkhuuser.