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 or 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 an 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 change is known as the pheophytin,
an olive green color.

It is to say that, in most the plants that survive 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 flown at the bottom of the Whatman
no.1 filter paper.
Objectives

1) To determine the rate of flow of each pigment and determine their solubility using
Whatman no. 1 filter paper.
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
the process of heating.
4) To compare the pH of the 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 and 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 for 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, implies the raw spinach is slightly
alkaline but close to neutral

(B)Extraction of Water-Soluble Plant Pigments

Sample pH value Intensities and shades of

colors
Raw red cabbage 6.38 Purple intensity presented,
raw red cabbage is slightly
acidic but close to neutral
Cooked red cabbage 6.11 (reject) Blue intensity presented,
cooked red cabbage is
slightly alkaline.

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

Sample Color changes of green beans

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

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

 Plant
Band Color
Pigment

Bright yellow Carotene

Pale yellow-green. Xanthophyll

Chlorophyll
Blue-green
a

Chlorophyll
Yellow Green
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 is 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 which is
water-soluble pigment that made the cabbage to form purple color. It is known that the
extracted juice of raw red cabbage is a good natural pH indicator in which it turns the
colors based on the acidity. In very acidic solutions, the color of the indicators will
change into red color due to the presence of number of hydrogen atom. When an alkaline
solution is tested, the color will result in the color change from purple into yellowish due
to the increase in the number of hydroxyl groups. For the neutral solution, it will maintain
the purple color of the solution. 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. In the experiment, the red
cabbage was cooked in the distilled water has resulted in the formation blue color. In
other word, the color had change from purple into blue intensity strongly implies that the
cooked red cabbage is slightly alkaline. This is because that when red cabbage was
cooked, it will not become more acidic in theory. However in the reality, the pH value of
the cooked red cabbage has shown a reading of 6.11 which is more acidic than the raw
red cabbage. This can be explained that the when there is an increase or decrease in
temperature of solution, the pH value from the pH meter will be changed. This is true as
the solution been detected by the pH meter was still in hot condition since we used the
cooked red cabbage in the experiment. Hence, the result we had obtained cannot be
considered as an error but it can be understand that there is a true pH value in a new
temperature.

For the discussion on part C (Effects of Vinegar and Baking Soda on Plant Pigments), we
used distilled water as the constant and green beans were used to contribute the plant
pigments. It is to say that chlorophyll gives the color of beans into light green. However,
when green beans are introduced with vinegar under heating process, acid reacts with the
plant pigment (green beans) will change the color of the green pigment from light green
into olive green which is unfavorable color. This can be explained that green beans
consist of chlorophyll in which they change the color into pheophytin through the
addition of acid by removing the Magnesium atom and it is to say that the magnesium
presents in the chlorophyll now is being replaced by the hydrogen atom through a process
known as acid hydrolysis. On the other hand, baking soda acts as an alkaline solution in
this experiment. The color of green beans change from light green into bright green as it
forbids hydrogen from getting into chlorophyll that will eventually replace the
magnesium atom causing the pigment becomes brighter. Chlorophyllin is said to be
produced under the heating process. In the experiment, frozen green beans were used to
keep the freshness and the quality of the color of the green beans.

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. 1 filter 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.

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 is, 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.

There are some precaution steps have been taken while we conducting the experiment.
Firstly, we made sure that the cap was capped closely when a small amount of acetone
was added in the test tube, as the solvent may evaporated easily (volatiles). Next, gloves
were to be wore while conducting the experiment to avoid undesirable incidents. Lastly,
we made sure that the drop (filtrate) tipped on the filter paper was completely dry before
it was inserted in the test tube containing the acetone.

Conclusion

It can be concluded that anthocyanins contribute purple color pigment to the red cabbage.
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 an
increase in the hydroxyl group, the blue intensity of the foods increases. Besides that, it is
known that when the chlorophyll is mixed with the acidic solution under heating process,
the color will change from its own light green colour of chlorophyll into an olive green
color which is also known as pheophytin. On the other hand, when chlorophyll is mixed
with the alkaline solution, the color will change from light green into the color of bright
green, which is also called as chlorophyllin. Last but not least, in the experiment of Part
D, filter paper has shown to be an accurate method of separating and observing the
various colors of plant pigments in which the pigments are moving upward after
absorbing the acetone. According to the migration distances that have been observed, the
results show that the flow rate of carotene pigment is the highest followed by the
xanthophyll. Chlorophyll b was the less soluble compared to chlorophyll a.
Attachments

Diagram 1 shows the result of the separation of pigments on paper chromatography.

Diagram 2 shows the result of the effect of vinegar and baking soda on plant pigments
whereby the distilled water acts as control at the left, color changes of sodium
bicarbonate (middle) and acetic acid (right) after the heating process.
References

Caloriebee (2015) Red cabbage health benefits, Anthocyanins and indicator fun.
Available at: https://caloriebee.com/nutrition/Red-Cabbage-Health-Benefits-
Anthocyanins-and-Indicator-Fun (Accessed: 20 August 2016).

Jenway (2016) Available at:

http://www.jenhttp://www.jenway.com/faq_pH_Meters.aspway.com/faq_pH_Met
ers.asp (Accessed: 22 August 2016).

Pre AP Lab Reports (no date) Available at:

http://www.biologyjunction.com/chromatography_of_simulated_plan.ht
(Accessed: 20 August 2016).

StasoSphere (2007) Plant pigments. Part 4. Available at:

http://chestofbooks.com/food/science/Experimental-Cookery/Plant-Pigments-
Part-4.html#ixzz4HqM5M5iE (Accessed: 20 August 2016).

Tripod (no date) Pigment Separation Using Chromatography Paper. .

2016 Education (2014) Leaf chromatography. Available at:

http://www.education.com/science-fair/article/find-color-pigments-hidden-green/
(Accessed: 20 August 2016).