Download Fluorescence of Chlorophyll

Fluorescence of Chlorophyll
Name:
Safety Notes:
Safety glasses should be worn during this lab.
Introduction:
What happens when chlorophyll and other pigments absorb photons? The colors
corresponding to the absorbed wavelengths disappear from the spectrum of the transmitted and
reflected light, but energy cannot disappear. When a molecule absorbs a photon, one of the
molecule's electrons is elevated to an orbital where it has more potential energy. When the
electron is in its normal orbital, the pigment molecule is said to be in its ground state. After
absorption of a photon boosts an electron to an orbital of higher energy, the pigment molecule is
said to be in an excited state. The only photons absorbed are those whose energy is exactly equal
to the energy difference between the ground state and an excited state, and this energy difference
varies from one kind of atom or molecule to another. Thus, a particular compound absorbs only
photons corresponding to specific wavelengths, which is why each pigment has a unique
absorption spectrum
The energy of an absorbed photon is converted to the potential energy of an electron
raised from the ground state to an excited state. But the electron cannot remain there long; the
excited state, like all high-energy states, is unstable. The chlorophyll electrons become excited
by the light energy, but have no electron transport chain to flow along because the chloroplast
thylakoid membranes have been dissolved away. Therefore, the chlorophyll electrons give up
their excited energy state by releasing energy in the form of a reddish glow. This is essentially
the same phenomenon as a neon light, except the electrons of neon gas molecules in the glass
tube become excited and then release their energy as a white glow.
Generally, when pigments absorb light, their excited electrons drop back down to the
ground-state orbital in a billionth of a second, releasing their excess energy as heat. Some
pigments, including chlorophyll, emit light as well as heat after absorbing photons. The electron
jumps to a state, of greater energy, and as it falls back to ground state, a photon is given off.
This afterglow is called fluorescence. The fluorescence has a longer wavelength, and hence less
energy, than the light that excited the pigment.
If a solution of chlorophyll isolated from chloroplasts is illuminated, it will fluoresce in
the _____?_____ part of the spectrum and also give off heat.
Materials:
Green leaves (spinach works well)
mortar
pestle
acetone
flashlight
test tube
filter paper
graduated cylinder
stirring rod
funnel
Procedure:
1. Grind green leaves using a mortar and pestle.
2. Add acetone to the grinded green leaves. Use enough acetone and green leaves to get about
10-15 ml of extract.
3. Next, filter the extract through to a test tube
4. Shine a flashlight through the test tube.
5. Observe the fluorescence at a 90 degree angle to the flashlight.
Student Evaluation
1. What color does the chlorophyll fluoresce? __________________
2. Why do we see the color of fluorescence that we see?
Discussion
Chlorophyll is a natural pigment found in green plants. It is the primary pigment that absorbs
light energy from the sun for photosynthesis. This energy is then used by the plant to synthesize
glucose from carbon dioxide and water. The structure of the chlorophyll molecule consists of
several conjugated nitrogen-containing rings surrounding a magnesium ion by coordinate
covalent bonds. Molecules such as this with a metal ion coordinated to an organic compound are
called coordination compounds. Coordination compounds are found elsewhere in nature, and
generally have distinct spectral characteristics that account for their energy-transfer function in
metabolic reactions. This chemical behavior is also evident in the photoresponse of these
compounds. For example, hemoglobin changes from a deep purplish red to a bright red upon the
binding of oxygen.
In the case of chlorophyll, a spectral analysis shows the wavelengths of sunlight absorbed, which
is actually the combined absorption of two different chlorophylls, a and b. The maximum
absorbance of chlorophyll a is at 420 and 660 nm and the maximum absorbance of chlorophyll b
is at 435 and 643 nm. In leaves, chlorophyll is bound to thylakoid membranes in the chloroplasts,
and absorbed wavelengths of light are converted to chemical energy. When chlorophyll is
extracted from leaves, light energy cannot be transferred to the chloroplasts. Instead, the light is
re-emitted and/or absorbed as heat. The emission of light is known as fluorescence and occurs
between 675 and 685 nm (in the red region of visible light). In the laser analysis, the
fluorescence is distinguished from scattered red light by the use of a cut-off filter that allows
only this range of wavelengths to pass through. This longer wavelength light has a lower energy,
-34
as predicted by the expression: E = hc/
J s), c is
8
the speed of light (3 x 10

can be calculated by subtracting the energy emitted from the energy absorbed as ultraviolet light
(E4.00 - E6.75 = Eheat).
Calculations:
1. Energy at 4.00 x 10-9 m __________J
2. Energy at 6.75 x 10-9 m __________J
3. Heat absorbed (1-2) __________J