Alan Ibanez

Change in Chlorophyll Through Fall

Principles of Biology 111-101

Abstract
Every year during fall leaves start to change color and eventually fall off the trees. The
pigments in the leaves change color. This is usually an indicator of change. Leaves usually
turn into a yellow tint, then proceed to the iconic orange tone. At Chesapeake College
there are various species of trees located throughout the campus. In this experiment we
will take a look at three different species of trees and analyze the change in color
pigments by taking an in depth look at Chlorophyll A, B, and Carotenoids.

Introduction

The study was conducted to have a better understanding of the fall color of trees.
Looking at different pigments such as chlorophyll A, B, and Carotenoids we can see
exactly how this change takes place. Some trees look beautiful in the fall with their
magnificent leaves. It is amazing that all trees change in a different way. To most, it might
seem the same that all trees go through the same change every fall and spring. Some
trees stay green all year around like conifers, which include pines that we use as
christmas decorations because of their ability to keep the chlorophyll levels almost the
same throughout the year. Whereas, it is obvious that other trees lose their leaves
around the fall. They start by changing their color. Some look yellow, others look reddish,
and others go to a lighter shade of green and fall to the ground. Theres a difference in
every tree as factors affect this process. Location, soil. climate, species, among other
factors play a specific role in the way leaves change.

Materials And Methods

Chromatography solution (Petroleum Ether and Acetone)

Isopropanol (2-propanol)
Chromatography paper
Spectrophotometer
Leaves
Test tubes
Beakers
Pipets
Graduated cylinder
Scissors
Coins

Selected Trees
Weeping Beech
Fagus sylvatica

Leaves are produced between March and April, with flowers in April and may, and nuts in
august and september. It is a large tree with a broad crown and smooth grayish bark. (1)
Usually, the branches touch the ground and cover the trunk of the tree. The leafs are
simple, flat, between 2 and 4. The tree produces a type of acorn that is covered in a
husk.

Figure 1

Downy Serviceberry
Amelanchier arborea
One of the first trees to bloom in the spring. Small tree with smooth bark, narrow and
rounded crown. Grows in most soils. Leaves are simple, flat, with curve veins, and short.
(2)

Figure 2
Sycamore Maple tree
Acer pseudoplatanus

Very common in North America.Very large deciduous tree. Introduced during colonial
ties. Tolerates sandy and clay based soils. It has a smooth and grey bark that becomes
rougher with age as well growing in size. Leaves are flat and V-shaped. (3)

Figure 3
Pigment Separation and Extraction
Male a line with a pencil 1.5 - 2 cm from the bottom edge of the chromatography
paper. Using the capillary tubes apply pigment extract to the pencil line. Roll the edges of
the paper together to form a cylinder with the green line on the outside. The cylinder was
stapled at the bottom and at the top. Then pour 20 mL of the Chromatography solution
into the beaker. The beaker was left undisturbed for up to 15 minutes
Extraction of pigments:
Open the dried cylinder by removing the staples. Three different color bands is
what we look for after removing the staples. Then cut the bands and shed them
separately. Then proceed to fit them into a small beakers with a label on them. Add 5 mL
of isopropyl alcohol to each beaker and seal with small piece of plastic wrap. Allow to
stand until color is completely eluted from the paper and the paper is white. The solution
should have a tint of color when the pigment has been completely eluted.

Figure 4
Directions: Spectrophotometric Analysis
Turn on the spectrophotometer using the 0% T knob on the front left side of the
instrument and all it to warm up for about 15 minutes.

Figure 5
Preparation of samples for spectrophotometric analysis
Fill a spectrophotometer cuvet with isopropyl alcohol. Label "bl" using a marking
pen. This is the blank used to standardize the spectrophotometer. Transfer pigment
solution from the beaker to a second spectrophotometer cuvet. Only one pigment
solution per cuvet. Label cuvets as appropriate. Wipe sides of cuvet with a Kimwipe and
avoid touching surfaces with fingers. Be sure that the label does not interfere with the
path of the light beam.
Measuring absorbance of pigments
Turn wavelength control knob, large knob on top of the machine, to 360 nm. With
the sample chamber lid closed and no cuvet in the chamber set the 0 % transmittance to
0 using the left knob on the front of the machine. Insert the cuvet containing isopropyl
alcohol and close the sample chamber lid. Set transmittance to 100% using the right
knob on the front of the machine. Insert a cuvet containing pigment solution into the

sample chamber and close lid. Record the absorbance of the pigment. Remove cuvet.
If
you are measuring multiple pigments repeat this step for each pigment.
Turn wavelength
control knob the next wavelength listed on the datasheet. If the spectrophotometer you
are using, has a lever on the front, in the bottom left corner, make sure the lever is set to
encompass the wavelength you are measuring.
Calculations
For the calculations you will need the following information:
chlorophyll a = Molecular weight 894 g/mol, Molar extinction coefficient (E) 89 / mM cm
(milimole/L)(cm)
chlorophyll b = Molecular weight 907 g/mol, Molar extinction coefficient (E) 56 / mM cm
carotene = Molecular weight 536 g/mol, Molar extinction coefficient (E)
2500 / mM cm
Then solve for the Beer-Lambert equation (A=
E
lC) for concentration, C, which is
expressed as molarity (M), or moles/L.

Results
The results showed what we inferred. We hypothesized that the Chlorophyll levels would
drop as fall goes on. The levels of Chlorophyll A were the most promising results as they
matched the colors of fall. As the color faded away Chlorophyll levels in the three
samples dropped. The research was not perfect. Chlorophyll B samples were difficult to
obtain during the first week. In the last week however, chlorophyll B was non existent
except for the Sycamore Maple tree. The research of this project allowed me to have a
better of understanding of conducting research. One must be punctual and ready to
conduct the experiment. This was my first real experiment and even though it was
flawed, I managed to create a correlation between the fall color of trees and decline in
Chlorophyll levels throughout fall.

Table 1
Weeping Beech

Chlorophyll B

Chlorophyll A

Carotenoids

Sample 1

6.74210^-4 m/L

.064 um/mL

Sample 2

9.643x10^-4 m/L

.001056 m/L

.022 um/mL

Sample 3

5.281x10^-4 m/L

.376 um/mL

Sycamore Maple Tree

Chlorophyll B

Chlorophyll A

Carotenoids

Sample 1

4.382x10^-4 m/L

.018 um/mL

Table 2

Sample 2

.0015 m/L

.001393 m/L

.0212 um/mL

Sample 3

6.25x10^-4 m/L

.002258 m/L

.0164 um/ml

Downy Serviceberry

Chorophyll B

Chlorophyll A

Carotenoids

Sample 1

3.933x10^-4 m/L

.072 um/ml

Sample 2

5.714x10^-4 m/L

6.74210^-4 m/L

.0164 um/ml

Sample 3

4.607x10^-4 m/l

.112 um/ml

Table 3

Notes
http://eol.org/pages/1143547/details
(1)
http://eol.org/pages/633499/details
(2)
http://eol.org/pages/583073/details
(3)