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Difference in Pigment Levels of the Leaves from Sunny and Shady

Parts of Syringa vulgaris

As my teacher informed me that we were going to conduct an experiment concerning

chromatography of the leaf pigments, I started doing my research. During that process, I found out

that ecologist and emeritus scientist James P. Barnett has determined during his explorations in the

coniferous forests that chlorophyll concentrations in the leaves of individuals of the same species tend

to be higher under lower light levels than under high levels (Barnett). This was a very interesting

disclosure for me and I decided to go deeper into the topic of the impact of the light on the amount of

chlorophyll in the leaves. The above-mentioned study was made on leaves from different trees. This is

why I decided to conduct my experiment on the leaves from the same tree. I identified a tree that

would have both - sunny and shady sides. This tree was Syringa vulgaris. I formulated the following

research question:

“To what extent is there a difference in the chlorophyll concentrations of leaves taken from the sunny

side of Syringa vulgaris versus the leaves from its shady side?”

Theoretical Background

Chloroplasts are the organelles that are responsible for photosynthesis. Pigments in the

chloroplasts are vital for this operation, which is “the process by which green plants and some

other organisms use sunlight to synthesize nutrients from carbon dioxide and water” (Oxford

Dictionaries, “Photosynthesis”). The pigments in the leaves are mainly in the forms of chlorophyll. As

stated by the University of California Museum of Paleontology, “Chlorophyll is a stable ring-shaped

molecule around which electrons are free to migrate (Speer). Because the electrons move freely, the

ring has the potential to gain or lose electrons easily, and thus the potential to provide energized

electrons to other molecules (Speer). This is the fundamental process by which chlorophyll "captures"

the energy of sunlight” (Speer). As the activity of the chlorophylls is highly dependent on the

environment the leaf is in and especially on the amount of sunlight it receives, many leaves have

developed shade tolerance, which “is a function of a species’ ability to efficiently capture and use

limiting light resources and thereby optimize the whole-plant carbon balance in shade” (Khan et al.

172). For instance, “Hawkins and Lister found higher chlorophyll concentrations in needles of 2-year-

old potted Douglas-fir seedlings grown under a 30% light regime than in those grown under open

conditions” (172). Based on these findings, deductively and inductively, a hypothesis was

formulated:

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The leaves from the shady part of the tree are expected to have a higher density of chlorophyll than

the ones from its sunny part.

For determining the density of chlorophyll pigments in the leaves I decided to use a

colorimeter. The Colorimeter determines the concentration of a solution by analyzing its color

intensity (Vernier). It measures the amount of light transmitted through a sample at a user-selectable

wavelength (Vernier). “The ratio of the light energy falling on a body to that transmitted through it is

called transmittance” (“Transmittance,” Oxford Dictionaries). In this experiment, it is assumed that

transmittance is inversely proportional to the concentration of pigments in the leaves.

Identification of the Tree

To conduct this experiment, firstly, a suitable tree must have been identified that had parts

that were almost always in shade and parts that gain a relatively bigger amount of sunlight. I looked

for such a tree for a long time but couldn’t find it. However, after a while of wonderings I identified

that the tree, right in front of our high school campus is the best fit for this experiment, because it is

right in front of the building, that is to say, its back part was almost completely shady and the front

part - entirely exposed to the sun. The tree was Syringa vulgaris.

Factual Background to the fall of sunlight

Due to the absence of appropriate technology and other resources, it was impossible to

identify the exact amount of sunlight that both sides of the tree received. The approximate value of the

independent variable undoubtedly harmed the accuracy of the exploration. To reduce this uncertainty,

the approximate number of sunlight hours that the plant received during a day was observed. This is

also limited, as this figure changes throughout a year; however, there was no other available method

for making estimations concerning this figure, that is why it was decided to use this one. For further

analysis, a secondary information, concerning the fall of sunlight in Armenia was used, so as to

estimate the amount of sunlight that each side of the tree receives during a year.

On average Armenia receives 2700 hours of sunlight a year; 332 days a year (Tourism

Armenia). Full Shade is considered the condition of fewer than 4 hours of direct sun a day. (Sun,

Shade) It was observed that the shady part of the plant examined received no more than 4 hours of

sunshine a day. That equals to approximately 1328 hours a year. Partial Sun or Partial Shade is 4 to 6

hours of direct sun a day (Sun, Shade). Full Sun is 6 or more hours of direct sun a day (Sun, Shade). It

was also observed that the sunny side of the Syringa vulgaris examined received more than 7 hours of

sunlight a day. That equals to more than 2324 hours a year. The differences between the amount of

sunlight that the sunny and shady sides of the tree receive are quite contrasting, thus, the tree is

suitable for this research.

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For determining whether there’s a difference between the two types of the leaves examined,

samples were obtained from the tree. Thirty leaves were harvested from each - sunny and shady sides

of the same tree. The samples were crushed in the mortar and then mixed with isopropyl alcohol,

which is a good solvent because of its chemical properties, to solve chlorophyll of the leaves. The

extracted pigments, solved into the alcohol, were poured into a cuvette and analyzed using a

colorimeter. These results showed whether there is a difference between the concentrations of the

pigments between different samples of leaves.

Variables

Table 1 Independent and Dependent Variables, their Units and Ranges

Variables Units Range

Independent Variable

The amount of sunshine

that leaves receive

depending on their location

Hours

N/A1; Estimation was

used - 1328 hours for

shady and 2324 hours

for sunny leaves

Dependent Variables

Percentage of transmittance

of the light, emitted by

colorimeter through the

solution.

Percent (%) 0%-100%

Table 2 Controlled variables, their possible effects on the accuracy of the research and the methods of

their control

Controlled variables Possible effect(s) on results Method of Control

Location of the Leaf on the

tree

It is impossible to obtain

samples of leaves that have

received exactly the same

amount of sunshine during their

lifetime, thus, it may bring to

inaccuracies.

Harvest leaves from the same parts

of the tree: height – 1.5m – 1.75m;

from the part that is the most exposed

to sun and from the one that is the

least exposed.

Concentration of Isopropanol

Concentration of isopropanol

may affect the rate of the

extraction of chlorophyll from

Use isopropanol of the same

concentration for both ‘sunny’ and

‘shady’ solutions: Isopropyl alcohol

1 Means Not Available unless otherwise noted

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the leaves, thus, in case of

different concentrations of

isopropanol used, the accuracy

of the experiment may be

harmed

with 90% concentration was used in

this experiment.

Health and Size of the leaves.

Leaves may have unhealthy

parts, where the density of

chlorophyll might be harmed.

In addition, leaves have

different sizes, which will

greatly harm the accuracy of

the experiment if will not be

equalized.

Try to harvest leaves that have

similar sizes; cut squares with a set

area (10cm2) from the healthy parts

of each sample and use only these

parts for the experiment.

Duration of the extraction of

pigments from the samples

The crushed samples should

stay in the isopropanol for the

same amount of time, so as to

be comparable to each other

and the data obtained –

accurate.

A time should be set for keeping the

crushed leaves in isopropyl alcohol

(2 hours). The temperature of the

environment in which the samples

are must also be the same (water bath

was used (70˚C)).

The wavelength of light,

emitted by colorimeter

As there are different types of

pigments in the leaves, which

have different color tones, there

is no universal wavelength of

light, emitted by colorimeter,

that will be absorbed by all

pigments.

Choose a wavelength that will be

maximally absorbed by the solution.

It is recommended in the most cases

to focus on the absorption of the

wavelength by the Chlorophyll a, as

this pigment is the most common in

leaves. 430nm was chosen in case of

this experiment, as this was the

closest of the available wavelengths

to the peak of the absorption of

wavelength by Chlorophyll a.

Calibration of Colorimeter

Colorimeter must be calibrated

using a substance, relative to

which it will give a percentage

of a transmittance.

Colorimeter may be calibrated using

a cuvette, filled with distilled water.

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Safety & Environmental Issues

Throughout the experiment, an easily flammable isopropyl alcohol (90%) was used, which,

also has a very strong smell. That is why, latex gloves, safety glasses, and masks are compulsory to be

used throughout the entire process of the laboratory exploration. Also, try to harvest leaves from

different parts of the chosen side of the tree in order not to harm it and its photosynthetic productivity.

Materials

1) 30x leaves from the shady part of the tree

2) 30x leaves from the sunny part of the tree

3) Isopropyl alcohol – 1000ml

4) Distilled water 1000ml

5) 1x Vernier Colorimeter (±0.1%)

6) 30x cuvette 4.0ml (±0.2ml)

7) 10x Mortars and Pestles

8) 10x graduated beakers 40.0ml (±2ml)

9) 10x graduated beakers 100.0ml (±5ml)

10) 10x Syringe 30.0ml, graduated (±1.5ml)

11) 1x Ruler (±0.5mm)

12) 1x Scalpel

13) 1x Tweezers

14) 3x Funnels

15) 1x Scissors

16) 1x A1 sized list of Filter Paper

17) 1x Pair of Latex gloves

18) 1x Mask

19) 1x Safety Glasses

20) 60x A5 Papers

21) 1x Vernier LabQuest Mini

22) 1x Computer; LabQuest Mini Driver

installed

I want to note that due to the absence of sufficient numbers of lab glassware, many of them

were thoroughly cleaned with isopropyl alcohol and then reused. The quantity of the glassware and

other resources described above are what I had in my possession, not what would be ideal for this lab.

Ideally, for every single sample there should have been separate mortar with pestle, beaker and all

other types of glassware, which get into direct contact with the chlorophyll solutions, so as to avoid

the possible interaction of the solutions and their solutes – chlorophylls – which could have been

transmitted from one solution to another, thus, might have harmed the accuracy of the experiment.

Procedure

1. 30 A5 papers were marked “Shiny” and the another 30 – “Shady”

2. 30 Leaves were harvested from both – shiny and shady parts of the tree, from the same

heights - 1.5m-1.75m (Overall 60 leaves)

3. The leaves were washed with distilled water and wrapped into paper envelopes, made of the

marked papers so as to make sure that there are no confusions

4. Squares, equal to 10cm2, were cut from the wide bottom parts of each leaf

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5. The cut parts were cut into smaller pieces

6. The samples were placed in mortars

7. Isopropyl alcohol was poured into 100ml Graduated Beaker, so as to make it easier to use

during the investigation

8. 8.0ml of isopropanol was added to each of the samples (twice as much as is the volume of the

cuvettes, so as to have an extra solution of that particular sample in case it will be lost or

harmed)

9. The samples were crushed with pestles. Make sure to use different pestles, or clean them

thoroughly with isopropyl alcohol, as the one used for another sample will have chlorophylls

on their heads.

10. The solutions were covered, so that the alcohol would not evaporate

11. The mixtures were left in the water bath for 2 hours each. The temperature of the water bath

was maintained stable (70˚C). It is also worth noting that they were in complete darkness, so

as to decrease activity of chloroplasts and chemical reactions of chlorophyll. These measures

were undertaken to maximally extract chlorophylls from the leaves.

12. Round surfaced pieces were cut from the filter papers

13. Filter papers were placed into funnels

14. The funnels were placed into 40.0ml graduated crucibles

15. Extracted solutions of pigments and isopropanol were filtrated through the funnels

16. 4.0ml of the refined solutions were taken from each sample and poured into the colorimeter

cuvettes using syringes

17. Control solution – cuvette (4.0ml) filled with 4.0ml of distilled water – was prepared

18. Computer, LabQuest Mini and the colorimeter were prepared

19. Colorimeter was set on 430nm wavelength intensity, as it was the most absorbed color by the

leaf pigments that the colorimeter featured

20. The colorimeter was calibrated using the cuvette, filled with a distilled water

21. The transmittance (%) of the solutions in the cuvettes, marked ‘Sunny’ were determined2 after

the stabilization of the values

22. The transmittance (%) of the solutions in the cuvettes marked ‘Shady’ were determined3 after

the stabilization of the values

2 You can see the results in the Appendix: Table 5 3 You can see the results in the Appendix: Table 5

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Figure 1 Absorption Spectrum of Chlorophyll a

(University of Michigan)

Figure 1 shows the absorption spectrum

of Chlorophyll a – the percentage of its

transmittance under visible wavelengths (from

400 to 700).

This graph would have been produced

during the experiment if a specialized and

professional colorimeter, that would emit all

these wavelengths, was available and the

absorption percentages of the samples were

determined. Also, this graph shows that the

wavelengths from 430nm to 440nm are the most

absorbed ones by the Chlorophyll a, that is to say, the wavelength of 430 was chosen fairly. Also, this

figure shows which colors are the most absorbed by the Chlorophyll a – Violet-Blue and Red-Pink.

Also, it shows that the green color is the least absorbed one, which is the reason why the leaves seem

green to humans.

Results and Data Analysis

55.0

60.0

65.0

70.0

75.0

80.0

85.0

0 5 10 15 20 25 30

Tran

smit

tan

ce (

%)

Speciemen Number

Transittances of the Sample Solutions

Sunny

Shady

Figure 2 The results of the experiment

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Table 3 Average Transmittances of the "Sunny" and "Shady" Solutions

Number of

Speciemens (n)

Mean

Transmittance

(%, ±0.1%)

Standard

Deviation Variance

Sunny 30 76.4% 3.66 13.4

Shady 30 69.3% 4.0 16.0

Figure 2 depicts the transmittances of all sample – both ‘sunny’ and ‘shady’. From the Table 3 it may

be concluded that there is a significant difference between the two sets of data collected, however, to

verify this statistically and to identify whether this difference is significant, a t-test was carried out.

t-test equation: 𝑡 = │x1 −x2

│

√𝑠1

2

𝑛1−

𝑠22

𝑛2

Where: x = the mean n = sample size s = standard deviation

𝐷𝑒𝑔𝑟𝑒𝑒𝑠 𝑜𝑓 𝐹𝑟𝑒𝑒𝑑𝑜𝑚 = 𝑛1 + 𝑛2 − 2 = 30 + 30 − 2 = 58

Null Hypothesis (N0) was formed which is – there is no significant difference between the

transmittances of 'sunny' and 'shady' solutions.

Alternative Hypothesis (Na) - there is a significant difference between the transmittances of 'sunny'

and 'shady' solutions.

Table 4 Results of the t-Test (Two-Sample Assuming Unequal Variances)

Variable 1 Variable 2

Mean 76.4% 69.3%

Variance 13.4 16

Observations 30 30

Degrees of Freedom 58

t Value 7.15

P Value 0.00000167

t Critical two-tail 2.00

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From the table 1, it can be clearly observed that the t value (7.15) is greater that t Critical

(2.00). Consequently, it can be claimed that the Null hypothesis is rejected and the alternative

supported. There is a significant difference between the transmittances of the 'sunny' and 'shady'

solutions. The outcome is significant at p<0.00001.

In addition, it is worth noting that there was a difference between the qualitative features of the

samples: the ‘shady’ solutions were darker than the ‘sunny’ ones. See this difference in the photo in

Appendix Figure 5. It may be linked to the estimation that the proportion of chlorophyll b, which has

blue-green color, is larger in the leaves from shady areas, which brings to the fact that they have

darker colors (Variations in Green).

Conclusion and Evaluation

The 'sunny' and 'shady' solutions had average transmittances of 76.2% and 68.8%

respectively, that is to say, on average 23.8% of the light, originally emitted by the colorimeter, was

absorbed by the 'sunny' solutions and 31.2% by the 'shady' ones. T-test showed that there is a

significant difference between the two sets of data. These imply that the solutions made of the

chlorophylls from the leaves of the sunny part of the tree had a smaller concentration of pigments than

the ones made of the leaves from the shady part. This is an indicative of the fact that the leaves from

the sunny part of the tree had a smaller amount of pigment than the ones from the shady part. That is

to say, the hypothesis was supported.

“This phenomenon is caused by the adaptation of the leaves to the environment (Variations in

Green). Leaves in the shade have a higher concentration of chlorophyll to improve the light-capturing

capability of the chloroplast (Variations in Green). “Shade leaves, having adaptations for capturing

the low intensities of sunlight, are not designed for optimal photosynthesis when given full exposure

to direct sunlight” (Variations in Green). “They are, in effect, a cheap unit of photosynthetic area,

where performing cellular respiration and producing organic compounds is cheaper – requires less

sunlight - than in a sun leaf” (Variations in Green). Another aspect that fostered the hypothesis to be

supported is the fact that the research was done during autumn and that in the areas, with a climate

like in Armenia, the concentration of chlorophyll decreases during cold sunny autumn days.

“Chlorophyll molecules are destroyed and not replenished when they are exposed to excessive

sunlight and when temperatures are low (Autumn). Cold sunny autumn days make the overall

concentration of chlorophyll to decrease.” (Autumn). The shady part of the tree was exposed to less

sunlight, and the sunny part was exposed to an intensive sunlight. Consequently, more chlorophyll

was decomposed in the leaves from the sunny part than in the ones from the shady part, which

brought to differences between the concentrations of chlorophyll of the two types of the leaves.

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Limitations and the Ways of Improvement

Throughout the experiment, there were some limitations identified. Firstly, the investigation

was carried out in the late autumn, which means that chlorophylls of many leaves started to

decompose, thus, the accuracy of the experiment was harmed. Secondly, there were dust particles in

the refined solutions of alcohol and leaf chlorophyll. Although the amount of these particles was

small, it is undeniable, that they affected the outcome of the tests on the transmittance of the light

through the cuvettes filled with the solutions using a colorimeter. Thirdly, the chosen wavelength

(430nm) for measuring the transmittance percentage of the solutions was generalized. This

wavelength was chosen, as it was the optimal one, not the universal. It was the most absorbed for the

chlorophyll a, which has the biggest proportion in the leaves, nonetheless, it is undeniable that the

accuracy of the experiment was harmed, as the other types of the leaf pigments might have been

missed – they might not have absorbed the light, emitted by the colorimeter and the sensor might not

have identified them. Another point is that “Just like most spectrophotometer sample tubes, individual

plastic cuvettes vary slightly in the amount of light they absorb” (Vernier). These differences are

small, thus, in many cases they are being ignored, nevertheless, it would be unfair not to consider it as

a limitation. Finally, it is worth reclaiming the fact that I did not have sufficient amount of glassware

for this experiment, thus many of the glassware were thoroughly cleaned with isopropyl alcohol and

reused. This had made the interaction of pigments from different samples possible.

The experiment could be further enhanced, if you:

1. Use more specialized and serious colorimeter, which would have a more wavelengths, thus,

would make the experiment more accurate, flexible and multidimensional.

2. Try to keep the equipment used in this lab the way, so that no dust particles will get into the

solutions. “This is done best by plugging with cotton, corking, taping a heavy piece of paper over

the mouth or placing the glassware in a dust-free cabinet.” (Aldrich 2)

3. There are two solutions to the issue connected with the differences between the cuvettes: 1) You

can use the same cuvette, but after washing it carefully, or you can identify the most alike

cuvettes in terms of their properties of light transmission and minimalize their differences.

4. Use separate glassware for every single sample to avoid the possible interaction exchange of the

solutions and their solutes – chlorophylls.

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Bibliography

Aldrich. Suggestions for Cleaning Glassware. n.d. PDF file.

“Autumn.” Netlogo. Northwestern University, n.d. Web. 23 Dec. 2025.

<http://ccl.northwestern.edu/netlogo/models/Autumn>.

Barnett, James. “Shading reduces growth of longleaf and loblolly pine seedlings in containers. Tree

Plant.” Tree Planter’s Notes 40.1 (1989): 23–26. Print.

“Geography and Climate in Armenia.” Tourism Armenia. Tourism Armenia. n.d. Web. 23 Dec,

2015.<http://www.tourismarmenia.net/about-armenia/geography-and-climate-of-

armenia.html>.

Khan, Shafiqur Rehiman, et al. “Effects of shade on morphology, chlorophyll concentration, and

chlorophyll fluorescence of four Pacific Northwest conifer species.” New Forests 19.2 (2000):

172. Web. <http://link.springer.com/article/10.1023%2FA%3A1006645632023>.

“Photosynthesis.” Oxford Dictionaries. Oxford Dictionaries Online, n.d. Web. 30 Nov, 2015.

<http://www.oxforddictionaries.com/definition/english/photosynthesis>.

Speer, Brian S. “Photosynthetic Pigments.” University of California Museum of Paleontology.

University of California in Berkeley. n.d. Web. 30 Nov. 2015.

<http://www.ucmp.berkeley.edu/glossary/gloss3/pigments.html>.

“Sun, Shade or Perhaps Something in Between.” Proven Winners, Proven Winners. n.d. Web 23

Dec, 2015. <https://www.provenwinners.com/learn/sun-shade-or-perhaps-something-

between>.

“Transmittance.” Oxford Dictionaries. Oxford Dictionaries Online, n.d. Web. 27 Dec, 2015.

<http://www.oxforddictionaries.com/definition/english/photosynthesis>.

University of Michigan. Absorption Spectrum of Chlorophyll a. Digital image. Experiment II -

Solution Color, Absorbance, and Beer's Law. University of Michigan, n.d. Web 25 Dec,

2015.

<http://www.umich.edu/~chem125/softchalk/Exp2_Final_2/Exp2_Final_2_print.html>.

“Variations in Green.” Botgard. UCLA. n.d. Web 21 Dec, 2015.

<http://www.botgard.ucla.edu/html/botanytextbooks/generalbotany/shootfeatures/generalstruc

ture/leafcolor/variationsingreen.html>.

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Vernier Software & Technology. Colorimeter. Beaverton. n.d. Print.

Appendix

Table 5 Transmittances of 430nm wavelength by

each sample

Sample Transmittance (%, ±0.1%)

Sunny Shady

1 78.9 69.4

2 73.4 68.3

3 76.7 64.3

4 78.9 66.9

5 74.3 72.2

6 83.5 68.2

7 70.9 71.6

8 79.8 73.6

9 80.1 66.1

10 80.9 70.5

11 75.1 73.5

12 77.6 62.5

13 78.5 67.9

14 78.6 62.8

15 74.4 74.7

16 69.7 70.7

17 71.8 70.2

18 82.5 68.0

19 72.9 66.0

20 77.3 69.7

21 74.2 70.1

22 76.6 76.9

23 77.1 69.6

24 68.3 67.6

25 72.8 76.5

26 75.2 74.2

27 79.7 68.9

28 76.9 71.0

29 79.1 68.1

30 76.0 59.8

Figure 3 Difference of the colors observed between the samples of 'Sunny' and 'Shady' solutions