PHYSIOLOGICAL PROCESS: PIGMENTS AND PHOTOSYNTHESIS

Exercise no. 13

Pecon , Margel
Department of Biological Sciences, College of Arts and Sciences, Visayas State University, Visca, Baybay City Leyte,
Philippines 6521-A

Bachelor of Science in Biology

INTRODUCTION
Plants contain pigment that produce many the different colors we see. Flowers and fruits
contain many organic molecules that apparently absorb light. Leaves, stems and roots also
contain various pigments. Such pigment molecules include anthocyanins, flavonoids, flavin,
quinones and cytochromes However, none of these should be considered photosynthetic
pigments Photosynthetic pigments are the only pigments that absorb energy from sunlight and
are available in the photosynthetic apparatus

Pigments are chemical compounds which reflect only certain wavelengths of visible light. This
makes them appear "colorful". Flowers, corals, and even animal skin contain pigments which
give them their colors. More important than their reflection of light is the ability of pigments to
absorb certain wavelengths.

Another technique for separating mixtures is chromatography. Chromatography is a term for

color writing. When the technique was first employed to isolate colored components from plant
leaves around 1900, the term was chosen. The most essential technique for the chemical
examination of mixtures is chromatography, in all its forms. Simple methods for separating
mixtures into their constituent parts include paper and thin-layer chromatography. The
methods' workings and underlying ideas are remarkably similar. The main distinction is the
medium employed.

METHODOLOGY

A1. Solubility of Pigments

The four different pigments that each group will do are carotenoids, betalains , anthocyanins,
and chlorophyll. There will be two setups for carotenoids, one for tomato and the other for
carrots. For the last group, one will use Basella alba for chlorophyll, Pigweed (Portulaca
oleraceae) for betalains and Purple Heart (Tradescantia pallida) for anthocyanin. However, for
this experiment, we used Tradescantia pallida for the anthocyanin and Basella alba for the
chlorophyll. The plant materials should be chopped and sliced into small bits before being
placed in a blender, blended, and then homogenized in 100 mL of an acetone-water solution.
Notably, both hydrophilic and hydrophobic pigments are soluble in the polar organic solvent
acetone. By utilizing two layers of cheesecloth with various pore sizes, the leftover tissue should
be filtered out, If the filtrate is less than 50 mL, add enough acetone-water to bring the volume
back to 100 mL. To remove the remaining tissues, centrifuge the filtrate (ask the laboratory
instructor in operating the centrifuge). The colored homogenate will now be freed from any
residual tissues, decant and measure 50 mL of the sample and place in a separatory funnel
added with 50 mL of petroleum ether. Shake carefully the funnel with the point-finger holding
the cap of the funnel. Leave the set-up for an hour or overnight that depends on the rate of
separation of the pigments. The funnel will have two phases in it: a hydrophobic phase
(petroleum ether) and a hydrophilic phase (acetone-water). The pigments in the sample will
move into the phase that best suit their properties, hydrophobic pigments into the petroleum
ether and hydrophilic pigments into the acetone-water phase. Since petroleum ether is less
dense than the acetone-water solution, the hydrophobic phase will be the top phase. The
sample's pigments will transition into the phase that best matches their characteristics:
hydrophilic pigments will transition into the acetone-water phase, while hydrophobic pigments
will move into the petroleum ether. The hydrophobic phase will be on top because petroleum
ether is less dense than the acetone-water solution

A2. Pigmentation Separation by Paper Chromatography

Every group will be given a specific plant sample to work on. The plant samples will be
Codiaeum variegatum, Basella alba, and Hibiscus rosa-sinensis. Whatman No. 1 filter paper
that has been pre-cut into square sheets that fit neatly within the chromatography jar or any
other specimen tube will be made available to the class. Remind yourself to touch the paper as
little as possible, and only on the edge, because your fingers' proteins and oils might interfere
with how the chromatogram develops. Draw a line one-half inch above the bottom of each sheet.
For each of the three plant samples, each group will obtain three filter paper strips. Separate
plant samples are cut into thin strips, placed in a clean mortar, and ground into pulp using a
pestle. Leaf pulp is mixed well with 50 cc of precooled acetone before being filtered through fine
linen fabric. To wet the entire pencil line, dip a toothpick in the leaf extract and run it across it.
Be careful not to wet the line too much, as this could cause the extract to bleed off the line. Until
there is enough dried leaf extract on the paper, let the leaf extract dry before applying it again.
Usually, the solvent dries within 15 seconds. When dried, apply the plant extract again along the
same line using a toothpick; 5-8 applications are sufficient to achieve a solid green tint in the
paper. Dry it out. Add enough solvent into the Erlenmeyer flask that will serve as the
chromatography jar, fill it to approximately an inch from the bottom, using the solvent mixture
of petroleum ether and acetone in a 100:12 ratio. Place the slide or piece of paper that has the
stain vertically, right above the solvent level. Give the development process one to two hours
and cover the flask tightly with the lid. The solvents should flow up the paper, and once they
reach the top, the chromatogram should be removed and let to dry. Examine the paper or plate
when it is still fresh because the pigments' colors fade over time. From top to bottom, the colors
must be Carotenes (orange-yellow), Xanthophylls (one or more yellow bands), and Chlorophyll
a(blue-green)and Chlorophyll b: yellow green
1. Determine the Rf values by following formula:

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 (𝑝𝑖𝑔𝑚𝑒𝑛𝑡)

𝑅𝑓 𝑉𝑎𝑙𝑢𝑒 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡 𝑓𝑟𝑜𝑚 𝑜𝑟𝑖𝑔𝑖𝑛

B2. Evolution of Oxygen during Photosynthesis

Mark three sizable test tubes as Replicate 1, Replicate 2, and Replicate 3, and secure them.
Then after, insert one wholesome Hydrilla sprig inside each tube. Ten milliliters of tap water
and.2% sodium bicarbonate should be added to each tube. Carbon dioxide will come from
sodium bicarbonate. Next, set the tubes in artificial lighting and tally the number of bubbles
that each tube created. The oxygen-containing bubbles are a by-product of photosynthesis' light
response stage.

RESULTS
The experimentation aims to ascertain the results of pigment solubility to establish whether the
material is soluble or not. This also sought to compare the differences between of Rf values of
Hibiscus rosa-sinensis and Codiaeum variegatum. Additionally, the outcomes of this experiment
will demonstrate how oxygen evolves during photosynthesis. Last but not least, additional
research is being done to identify the pigments that are present and in charge of each sample of
a plant as well as to see if photosynthesis actually happens in the presence of light.

Table 1. Results for solubility of pigment

Plant Sample Color Pigment Phases
Basella alba Green Chlorophyll Hydrophobic
Tradescantia pallida Purple Anthocyanin Hydrophobic
 A. Codiaeum variegatum
0.7
0.68
0.66
0.64
Rf Value

0.62
0.6
0.58
0.56
0.54
Chlorophyll B Chlorophyll A Xanthopyll Carotenoid

Pigments

B. Hibiscus rosa-sinensis
0.8
0.7
0.6
0.5
Rf Value

0.4
0.3
0.2
0.1
0
Chlorophyll B Chlorophyll A Xanthopyll Carotenoid

Pigments

Figure 1. (A) Rf value of Codiaeum variegatum. (B) Rf value of Hibiscus rosa-sinensis.

Table 2. Results for oxygen evolution during photosynthesis.

 Oxygen Released (no. of bubbles)
TIME
Replicate 1 Replicate 2 Replicate 3
1 minutes 1 2 2
3 minutes 4 1 4
5 minutes 3 2 4
8 minutes 3 2 6
10 minutes 2 2 6
12 minutes 3 4 6

DISCUSSION

Using Table 1, Results for pigment solubility show that the chlorophyll-containing Basella alba
enters the hydrophobic phase. Since the chlorophyll pigment in Basella alba has a porphyrin
ring with a magnesium ion (Mg2+) at its center and is non-polar, when it is combined with the
polar molecule acetone, the result is typically a lengthy hydrophobic chain (Morançais and
Dumay, 2018). Additionally, the anthocyanin-pigmented Tradescantia pallida forms a
hydrophilic phase. Anthocyanins are soluble in water and the majority of organic solvents, or if
they are polar molecules, they result in a hydrophilic face when mixed with a polar molecule
like acetone, according to Khoo and colleagues (2017).
Figuring 1. Carotenoid has the highest Rf value of 0.68 and chlorophyll B has the lowest Rf
value of 0.6, according to the Rf value of Codiaeum variegatum (A). Carotene, which was the
most soluble pigment in the solvent, traveled the farthest. Because chlorophyll moves far more
quickly than carotenoids do, its Rf value is lower than that of carotene. As it was the least
soluble in the solvent, chlorophyll B traveled the least. This illustrates that although carotene is
a less polar molecule than chlorophyll. Xanthophyll has the highest Rf value of 0.76 and
chlorophyll B has the lowest Rf value of 0.6 in Hibiscus rosa-sinensis (B). According to the
findings, xanthophyll is more valuable since it is soluble in the solvent to a greater extent than
chlorophyll B, which is less soluble. Because they are bigger and/or have a stronger affinity for
the hydrophilic paper (they have more polar groups) than molecules with a larger Rf, which are
more likely to be soluble, the moving molecules with a small Rf are not highly soluble in the
hydrophobic (non-polar) solve
In Table 2, Results for oxygen evolution during photosynthesis demonstrate that each replicate
has a different variety of bubbles. The replicas are around 1.5 inches away from the light
source. For replication 1, the largest number of bubbles released was 4 during the first three
minutes, followed by 4 bubbles for 12 minutes in replicate 2 and 6 bubbles for 12 minutes in
replicate 3. This indicates that, out of the three replications, replication 3 produces. A faster
rate of photosynthesis results from increased oxygen generation caused by increased
temperature and light intensity oxygen than the other two. Replica 3 has a better rate of
photosynthesis since it has more oxygen. A product of photosynthesis is oxygen gas. A faster
rate of photosynthesis is required to produce more oxygen.
CONCLUSION

Tradescantia pallida is hydrophilic in terms of pigment solubility, but Basella alba is

hydrophobic, according to the results of the experiment. This indicates that Tradescantia
pallida is soluble in the solvent as opposed to Basella alba, which is not. Additionally, it was
concluded from the paper chromatography tests conducted on the plant samples Codiaeum
variegatum and Hibiscus rosa-sinensis that xanthophyll and carotenoid had higher Rf values
than other pigments, while chlorophyll B has the lowest value. This indicates that when
compared to photosynthetic pigments, non-photosynthetic pigments are more soluble.
Additionally, it was deduced that replicate 3 has the maximum number of bubbles present,
which indicates that it has the highest oxygen content and the highest rate of photosynthesis

REFERECES
https://www.khanacademy.org/science/class-11-chemistry-india/xfbb6cb8fc2bd00c8:in-in-
organic-chemistry-some-basic-principles-and-techniques/xfbb6cb8fc2bd00c8:in-in-methods-of-
purification-of-organic-compounds/a/principles-of-
chromatography#:~:text='Chromatography'%20is%20an%20analytical
%20technique,components%20can%20be%20thoroughly%20analyzed.
https://letstalkscience.ca/educational-resources/backgrounders/plant-pigments#:~:text=There
%20are%20three%20major%20pigments,chlorophylls%2C%20carotenoids%2C%20and
%20flavonoids.
https://harvardforest.fas.harvard.edu/leaves/pigment#:~:text=The%20chlorophylls%2C%20a
%20and%20b,phytol%20tail%20in%20the%20molecule.