Ratio of chlorophyll a to b in various plants increases due to color of leaf

Olha S. Lee
BIO 1306 B5 12/10/2013

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Table of contents

Table of contents..1 Abstract...2 Introduction.3-4 Materials and Methods5-7 Table 18 Results9 Table 1a..9 Graph 110 Discussion .11-12 Literature cited..13

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Abstract:
The objective of this research was to discover the ratio proportions of chlorophyll a to b depending on the shade of green the leaf was by using the Rfactor formula. Several methods were tested in order to find the best results, final method used to collect the data was called the chromatography technique. Each leaf sample was pressed onto the bilbous chromatography paper in a straight line, using the edge of a quarter, 4cm from the tip of the paper strip. This technique was repeated three times for each different leaf sample to ensure the same data. The tip of each strip was placed into a graduated cylinder filled with 10mL of Ethanol solvent attached by tape and a toothpick until the pigments stopped separating. The data collected confirms the hypothesis which stated that deeper green leaves

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Introduction:
Chlorophyll a, chlorophyll b and carotenoids are types of pigments present in plants in the thylakoids of chloroplast. The function of chlorophyll is to absorb light necessary for photosynthesis in plants and turn it into energy it needs to function properly. Through the process of photosynthesis, carbon dioxide and sunlight are absorbed by the plant in order to produce oxygen and glucose the plant uses for food. The purpose of having four different pigments instead of just one is to increase the plants absorption spectrum so it can absorb a wider range of light more efficiently. Out of all the pigment types, chlorophyll a and b are the most abundant photoreceptors and are responsible for the green coloration of most plants. The two chlorophylls differ from each other only in chemical composition of one sidechain. Chlorophyll a has a CH3 sidechain while chlorophyll b has a CHO sidechain. This slight variation in the sidechains causes the two chlorophylls to absorb different wavelengths of light thus working together to absorb the maximum light. On the light spectrum, chlorophyll a best absorbs light at 665 Nm and 425 Nm, chlorophyll b best absorbs light at 470 Nm and 630 Nm, and carotenoids absorb light best at the orange end of the spectrum. On the bilbous chromatography paper, chlorophyll a will appear to be green-blue while chlorophyll b will appear more green-brown.

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Retention value is a simple ratio calculation used to compare the distance traveled from the origin by the solute divided by the distance traveled by the solvent.

Solvent Front Separated Pigments

R(f)Center=
Application Line/ Origin

Center of Separated Pigment Solvent Front

This calculation uses distances to compare amounts of different compounds that are separated on the chromatography paper due to the parting of different pigments. The purpose of this experiment is to observe the chlorophyll levels present in different types of plants and compare the proportions of chlorophyll a to chlorophyll b. Because different species of leaves have different methods of using their two types of chlorophyll (a and b) for absorbing light during photosynthesis, chlorophyll a and b levels can differ. I predict that the deeper green colored leaves will have a higher ration/concentration of chlorophyll a rather than b.

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Materials and Methods:

Levels of chlorophyll a and b were observed in Mrs. Restivos laboratory at Waxahachie Global High School during tutoring between the hours of 3pm and 4:30pm. All leaf samples of the Crape Myrtle- Lagerstroemia indica, Bradford Pear- Pyrus calleryana, Fan Tex Ash- Fraxinus velutina, Choctaw Pecan- Carya illinoensis, Chinese Pistache- Pistacia chinensis, Red OakQuercus rubra, Saucer Magnolia- Magnolia x soulangiana, Oakleaf Hydrangea- Hydrangea quecifolia, Red Tip Photinia -Photinia fraseri, Dwarf Burford Holly-Ilex cornuta, LorapetalumLoropetalum chinense were collected from Rowlands Nursery in the city of Waxahachie. The Purple Wandering Jew- Tradescantia pallida came from Mrs. Restivos backyard and the Carolina Buckthorn- Frangula caroliniana was picked from a local neighborhood.

Method 1: Leaf paste on Chromatography paper Fifty Carolina Buckthorn leaves (Frangula caroliniana) were collected between 8-9pm. The leaves were placed in a blender for approximately 3 minutes to be mixed into a paste. The Blender was stopped occasionally and a total of 30mL of water was added to the mixture. The paste was placed in a zip-lock bag and stored in the refrigerator overnight. The next day, three samples with a mass of 1g each were placed in three different cups. 3mL of water was added to each cup and was gently blended in with the leaf paste. The leaf paste was then spread on three strips of chromatography paper (2.5cm by 25.5cm with a 3cm point) 2.5cm thick. The paste was left to dry for approximately 10 minutes. Then, the dried up paste was scraped off of all three strips and placed into a graduated cylinder with the tip of the chromatography paper slightly into 10mL of chromatography solvent. A toothpick was taped to the end of all the strips

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to keep the chromatography paper in place and from completely sliding into the graduated cylinder.

Method 2: Leaf paste on coffee filter paper 3g of the Carolina Buckthorn leaves (Frangula caroliniana) paste from the previous method was mixed with 3g of mater. Then, the mixture was placed on a piece of coffee filter paper and the results were observed.

Method 3: Leaf paste on bilbous paper 5 leaves of the Purple Wandering Jew- Tradescantia pallida and the roots were place in a cup of water. The leaves were placed in a blender for approximately 3 minutes and 3mL of water was added. The leaves were then placed in a ziploc-bag overnight. The next day, 3g of the paste were gently mixed with 3mL of water and placed in the center of the bilbous paper.

Method 4: Using a quarter to extract pigment One leaf from Crape Myrtle- Lagerstroemia indica, Bradford Pear- Pyrus calleryana, Fan Tex Ash- Fraxinus velutina, Choctaw Pecan- Carya illinoensis, Chinese Pistache- Pistacia chinensis, Red Oak- Quercus rubra, Purple Japanese Honeysuckle- Lonicera japonica, Saucer MagnoliaMagnolia x soulangiana, Gardenia-Gardenia augusta, Oakleaf Hydrangea- Hydrangea quecifolia, Red Tip Photinia -Photinia fraseri, Dwarf Burford Holly-Ilex cornuta, LorapetalumLoropetalum chinense and was collected from Rolands Nursery. Each leaf was placed 4cm

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from the tip of the chromatography paper and a quarters edge was used to press down on the leaf. A line about the size of the thickness of the quarter was made on the paper strip. Then, each paper tip was dipped into 10mL of Ethanol solvent inside a graduated cylinder. Then, the distance traveled by the pigments was observed.

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Table 1. Leaf Name

Pistacia chinensis

Leaf appearance

Carya illinoensis

Magnolia x soulangiana

Quercus rubra

Lagerstroemia indica

Pyrus calleryana

Fraxinus velutina

Hydrangea quecifolia

Loropetalum chinense

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Results:
Table 1a. Each plant sample was ranked 1-9 based on shades of green. 1 is the darkest/deepest shade of green and 9 is the lightest shade of green. Trial 1 Trial 2 Trial 3 Rfactor Chlorophyll a (1)Chinese PastichePistacia chinensis (2)Choctaw Pecan - Carya illinoensis (3)Saucer MagMagnolia x soulangiana (4)Red OakQuercus rubra (5)Crape MyrtleLagerstroemi a indica (6)Bradford Pear- Pyrus calleryana (7)FanTex Ash- Fraxinus velutina (8)Oak Leaf HydrangeaHydrangea quecifolia (9)Lorapetalu mLoropetalum chinense
(9.5cm/9.6cm)

Chlorophyll b
(9.3cm/9.6cm)

Chlorophyll a
(9.6cm/9.7cm)

Chlorophyll b
(9.5cm/9.7cm)

Chlorophyll a
(9.5cm/9.6cm)

Chlorophyll b
(9.4cm/9.6cm)

Average a=0.991 b=0.976

a=0.991

b=0.969

a=0.991

b=0.980

a=0.991

b=0.980

(9.9cm/10cm)

(9.8cm/10cm)

(9.8cm/9.9cm)

a=0.990
(10.1cm/10.2c m)

b=0.980
(9.9cm/10.2cm )

a=0.991
(9.2cm/9.3cm)

(99.7cm/9.9cm )

(9.4cm/9.5cm)

(9.2cm/9.5cm)

b=0.981
(9.1cm/9.3cm) (9.8cm/9.9cm) (9.6cm/9.9cm)

a=0.990

b=0.970

a=0.990 b=0.977 a=0.987 b=0.970

a=0.990
(9.9cm/10cm)

b=0.91
(9.7cm/10cm)

a=0.989

b=0.978

a=0.990

b=0.970

(9.9cm/10cm)

(9.7cm/10cm)

(9.7cm/9.8cm)

(9.5cm/9.8cm)

a=0.990
(9.4cm/9.5cm)

b=0.97
(9.1cm/9.5cm)

a=0.990
(9.4cm/9.5cm)

b=0.970
(9.3cm/9.5cm)

a =0.989
(9cm/9.1cm)

b =0.969
(8.8cm/9.1cm)

a=0.990 b=0.973 a=.990 b=0.971

a=0.990

b=0.961

a=0.990

b=0.981

a=0.990

b=0.970

(9.2cm/9.3cm)

(9cm/9.3cm)

(9.3cm/9.4cm)

(9.1cm/9.4cm)

(9.2cm/9.3cm)

(9cm/9.3cm)

a=0.990
(9.9cm/10cm)

b=0.971
(9.7cm/10cm)

a=0.990
(9.8cm/9.9cm)

b=0.970
(9.6cm/9.9cm)

a=0.990
(9.5cm/9.6cm)

a=0.991
(9.3cm/9.6cm)

a=0.900 b=0.971 a=0.991 b=0.971 a=0.990 b=0.970

a=0.990
(10.1cm/10.3c m)

b=0.970
(10cm/10.3cm)

a=0.991
(9.8cm/9.9cm)

b=0.971
(9.6cm/9.9cm)

a=0.991
(9.4cm/9.5cm)

b=0.971
(9.2cm/9.5cm)

a=0.981
(9.6cm/9.9cm) a = 0.960

b=0.971

a=0.990

b=0.970

a=0.989

b=0.968

(9.8cm/9.9cm) b =0.980

(9.8cm/10cm) a= 0.980

(9.9cm/10cm) b = 0.990

a=0.970 b=0.985

Table 1. The Lorapetalum leaves had a purple appearance and were tested to see if leaves other than the color green would differ in proportion of chlorophyll a to b. The (*) is in place because a third sample could not be collected due to the season and the death of the plant. Chinese Pistache - Pistacia chinensis was the control group of this experiment because it had the deepest green colored leaf. The data collected demonstrates that in most green leaves, the chlorophyll a ratio will be greater than that of chlorophyll b.

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Chlorophyll a vs. Chlorophyll b

0.99

Retention Factor

0.985

0.98 chlorophyll a 0.975 chlrophyll b

0.97

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Discussion:
Several different samples were collected at different times of each leaf and their chlorophyll a and b levels were measured. The leaves were ranked from darkest green to lightest green based on the exterior appearance of the leaf sample. The data collected proves the hypothesis; Ratio of chlorophyll a to b in various plants increases due to color of leaf, true. The control leaf, Chinese Pastiche- Pistacia chinensis, had the deepest green exterior coloration out of all the samples and the data collected confirms it has the highest chlorophy a levels. The lightest leaf, Oak Leaf Hydrangea- Hydrangea quecifolia, did not have the lowest Rfactor value of all the samples which concludes that chlorophyll a levels cannot be predicted based on the color of the leaf. Magnolia x soulangiana, even though not with the lightest exterior coloration, had approximately the same R(f) factor of chlorophyll b as the hydrogea quecifolia. Its interesting to note that even though the chlorophyll b levels were very similar, the chlorophyll a levels were drastically different. The season in which the experimentation occurred may have skewed or altered the retention factors of the data collected. The leaves were collected in the months of January which is the time in which the amounts of sunlight decrease, resulting in the death of leaves. Therefore, the leaves collected may not have been alive or to their maximum potential. The leaves were losing their coloration which also made difficult to accurately judge the coloration of the exterior pigmentation which may have altered the results. During experimentation, several factors may have altered the results including the procedure of the main method. When a strip of chromatography paper was lowered into the

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ethanol solution and a few mL of solvent was absorbed, some strips became too short to reach the remaining solvent. This could have resulted in the improper absorption of the solvent thus altering the maximum distance reached on the strip. Also, after the ethanol was absorbed it was difficult to analyze and differentiate between the two chlorophylls because they are so alike in their coloration. Over all, the experiment produced results with minimal error which provides evidence that proves the hypothesis. The data concludes that most leaves have higher levels of chlorophyll a than b.

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Literature Cited:
Journal Article Zablackis, Earl, Jing Huang, Bernd Muller, et al. "Characterization of the Cells-Wall polysaccharides of Arabidopsis thaliana Leaves." Plant Physiology. (1995): n. page. Web. 4 Dec. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC157245/>. Online Book Fried, George, and George J. Hademenos. Schaum's Outlines Biology. New York: McGraw-Hill, 2009. eBook Collection (EBSCOhost). Web. 21 Oct. 2013. Walker, Sharon, and David McMahon. Biochemistry Demystified. New York: McGraw-Hill, 2008. eBook Collection (EBSCOhost). Web. 21 Oct. 2013. Internet May, Paul. "Chlorophyll." http://www.chm.bris.ac.uk/. School of Chemistry, University of Bristol. Web. 21 Oct 2013. <http://www.chm.bris.ac.uk/motm/chlorophyll/chlorophyll_h.htm>. Savonen, Carol. "Colored leaves have chlorophyll too."Oregon State University Extension Service. N.p., 19 02 2003. Web. 6 Nov 2013. <http://extension.oregonstate.edu/gardening/colored-leaves-havechlorophyll-too>.