Introduction

Anthocyanins are one of the derivatives in Benzopyran. They are water soluble formed in
vacuole fluid. Besides that, they are generally discovered fundamentally in natural
products additionally show in a few vegetables such as black currant, asparagus, plums
red cabbage, grapes and so on. Because of the pigment of anthocyanins, it causes the
berries and red cabbage to have a color of blue and red. The brighter the appearance of
red and blue colors, they are considered having higher level of antioxidants.
Anthocyanins exist as a plant compound that play an important role as antioxidant in our
body. The color of anthocyanins is depended on the pH changes and the basic structure. It
appears to be red when there is a substitution of methoxy group. When there is a increase
in the hydroxyl group, the blue intensity of the foods increases.

Chlorophyll is one of the tetrapyrrole compounds that appears to be in green color and
presents in the chloroplast of plant cells. It is built up with the centre of Magnesium
surrounded by 4 pyrrole groups and a phytol tail. It is known that chlorophyll a and
chlorophyll b will change its colors from green into olive green when they are immersed
in an acid condition under high temperature. This is because the reason that when an acid
is added into the chlorophyll, the Magnesium will be removed and hydrogen atom will be
added into the chlorophyll at the same time. The color changes is known as the
pheophytin, an olive green color.

It is to say that, in most the plants that live in land consist of carotenes, xanthophylls and
chlorophyll a as well as b. In the experiment of the chromatography, we used the acetone
as the solvent to separate the each of the pigment resulted in the different rate of flowing.
The pigment which is not so soluble will be at the bottom of theWhatman no.1 filter
paper.

Objectives

1) To determine the rate of flow of each pigment and determine their solubilities.
2) To compare the intensities and the colors of pigments between the raw and cooked
samples.
3) To determine the color changes when the pigment immersed in an acid solution under
high temperature.
4) To compare the pH of pigments between the raw and cooked samples.

Apparatus and Equipment

Balance, blender, Whatman no. 1 filter paper, test tube rack, pH meter, beakers, 600ml,
hot plates, rubbery stopper, scissors, forceps, micro capillary tubes, chopping board, knife,
spatula, tong, measuring cylinder 100ml, filter funnel, weighing tray, hollow glass tube.

Reagents and Materials

Raw spinach, raw and cooked red cabbage, acetone-phosphate buffer, pH 8 (80:20) (v/v),
acetone (98%), acetic acid (0.1N), sodium bicarbonate (0.1), frozen green beans, distilled
water.

Procedures

(A) Extraction of Lipid-Soluble Plant Pigments

1. Raw spinach was weighed to 25g.
2. It was transferred to a blender.
3. 50ml of acetone-phosphate buffer was added and blended for 2 minutes.
4. The blended spinach was filtered through Whatman no. 1 paper and the filtrate
was saved.
5. The pH of the filtrate was measured and recorded.
(B) Extraction of Water-Soluble Plant Pigments
1. 100g of raw red cabbage was weighed.
2. It was transferred to a blender. 200ml of distilled water was added and blended
for 2 minutes.
3. The blended red cabbage was filtered through Whatman no. 1 paper and the
filtrate was saved.
4. The pH of the filtrate was measured and recorded.
 5. Steps 1-3 were repeated using cooked red cabbage.
6. The intensities and shades of colors were compared between raw and cooked
samples.
(C) Effects of Vinegar and Baking Soda on Plant Pigments
1) 100ml of 0.1 N acetic acid, 100 ml of 0.1 N sodium bicarbonate and 100ml of
distilled water added into separated 600ml beakers.
2) It was brought to boil on the hotplate.
3) 50g of frozen green beans to each beaker. The test tube was brought back to
boiling and boiled an additionally 5 minute.
4) The colors of the green beans were observed after treatments.

(D) Separation of pigments by filter paper

1) A piece of Whatman no. 1 filter paper was cut one end into a V shaped tip.
2) A small amount of acetone was added into test tube and it was capped.
3) The filtrate (raw spinach) was obtained from section A and it was tipped using a
hollow glass tube on a strip of filter paper with V-shaped.
4) The line pigment (filtrate) was left to dry. The process was repeated until a dark
green line of pigments is evident.
5) The Whatman no. 1 filter paper was placed in the test tube so that the pointed
end is barely immersed in the solvent.
6) The cap was put on the test tube.
7) When the solvent was about 1cm from the top of the Whatman no. 1 filter paper,
the Whatman no. 1 filter paper was removed from the test tube. The pigment was
avoided to be in the solvent.
8) The Whatman no. 1 filter paper was left for 2 minutes to evaporate
9) It was examined for the presence of different bands of color. Each color band
was a different pigment.
a. Chlorophyll a appears blue-green,
b. Chlorophyll b appears yellow-green,
c. Carotene appears bright yellow, and
d. Xanthophyll appears pale yellow-green.
Results

(A) Extraction of Lipid-Soluble Plant Pigments

Sample pH value
Raw spinach 7.57

(B)Extraction of Water-Soluble Plant Pigments

Sample pH value
Raw red cabbage 6.38
Cooked red cabbage 6.11

(C)Effects of Vinegar and Baking Soda on Plant Pigments

Sample Color changes of green beans

Before After
Acetic acid Light green Bright green
Sodium bicarbonate Light green Olive green
Distilled water Light green Light green

(D) Separation of pigments by Whatman no. 1 filter paper

Band Color Plant Pigment

Bright yellow Carotene

Pale yellow-green. Xanthophyll

Blue-green Chlorophyll a

Yellow Green Chlorophyll b

Discussions

For the discussion on part A (Extraction of Lipid-Soluble Plant Pigments), we performed

the experiment by blending the raw spinach leaves and adding acetone-phosphate buffer to
extract its lipids from the leaves. Filtration of the spinach was carried out during the experiment
to prevent any of the small pieces of spinach from getting into the solution. It is known that
green liquid after blending consist mixture of the pigments such as Carotene, Xanthophyll,
Chlorophyll a and Chlorophyll b in which they are used in capturing the light during
photosynthesis. The pH of the raw spinach is 7.57, this shows as that the raw spinach s slightly
alkaline but close to neutral.

For the discussion on part B (Extraction of Water-Soluble Plant Pigments), the

experiment for the extraction of water soluble plant pigments is aimed to compare the
intensities and shades of colors between raw and cooked samples. Filtration of the cabbage
was carried out during the experiment to prevent any of the small pieces of cabbage from
getting into the solution. The samples we used in this experiment were red cabbage. It is
known that red cabbage consists of the pigment of anthocyanins that made the cabbage to
be in reddish-purple color. The raw red cabbage shows a pH of 6.38 while for the cooked
red cabbage shows a pH of 6.11 on the pH meter. It explains that the raw red cabbage has
a higher pH value compared to the cooked red cabbage.

https://caloriebee.com/nutrition/Red-Cabbage-Health-Benefits-Anthocyanins-and-
Indicator-Fun

For the discussion on part C (Effects of Vinegar and Baking Soda on Plant Pigments),

For the part D discussion (Separation of pigments by Whatman no. 1 filter paper), we
used Whatman no. 1 filter paper instead of paper chromatography in this experiment as
both of the filter papers will give us the same result. In this experiment, Whatman no.
1filter paper plays an important role in separating the mixtures and compounds since it
has the same function to measure the flow rate of solvent. In this case, Whatman no.
1filter paper was placed stationary in the test tube and it was tipped with the filtrate (raw
spinach) obtained from section A using a hollow glass tube. Acetone was used while
conducting the experiment, this is because the reason that plant pigments are hydrophobic,
they will not soluble in water but they are able to soluble themselves in organic solvent
(acetone). The result that we obtained in part D shows three separated layers instead of
four layers that presented in different flow rate. It might due to the reason that the green
line pigment that had been tipped was not so dark and obvious that resulted in the missing
of a pigment. The movement of solvent up to the paper strip is due to the capillary action.
The capillary action can be defined as the force of attraction of the solvent to the solid
surface http://gb96.tripod.com/pigment.html.

From the result, the higher rate flow among the pigments that we obtained is the Carotene
(bright yellow), while for the pigment of Chlorophyll b, it shows the slowest rate of
flowing presented on the filter paper as it moved a little distance away from the line of
filtrate. It is known that the pigment of Chlorophyll a (blue-green) has a flow rate faster
than the pigment of Chlorophyll b (yellow green) but slower than the Xanthophyll
(pale yellow-green). Since out of four layers we only get three layers of the pigments,
hence it is predicted that Xanthophyll was the missing layer on the filter paper. From the
source of internet, we know that Xanthophyll (pale yellow-green) shows the second
fastest pigment. The distinction in the developments of every pigment was because of
the dissolvability of a specific pigment and capacity of that specific pigment to adhere to
the cellulose strands of the paper. It is to say that the dissolvability of a pigment decide
the rate, and the separation it will travel. The higher the solubility of a pigment, the
faster the rate of flow. Thereby, we can conclude that Chlorophyll b (yellow green) is
less soluble towards the acetone hence it moved the slowest, Carotene (bright yellow)
moves the fastest as it is very soluble towards the acetone. Chlorophyll a is slightly soluble
than the Chlorophyll b but it falls lower than the position of xanthophyll.
Conclusion

It can be concluded that the

References