bio003_Fluorescence of Chlorophyll

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					University of Pittsburgh at Bradford                 Science In Motion                       Biology Lab 003

                                       Fluorescence of Chlorophyll
Safety Notes:
Safety glasses should be worn during this lab.

         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. 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.

    1. The student will be able to distinguish between the light and dark reactions.
    2. The student will be able to understand the energy levels in studying atomic theory.
    3. The student will better understand the photosynthetic process.

        Green leaves (spinach works well)               flashlight                  graduated cylinder
        mortar                                          test tube                   stirring rod
        pestle                                          filter paper                funnel

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.

Juniata College                                                                                            1
5. Observe the fluorescence at a 90 degree angle to the flashlight.

                               Fluorescence of Chlorophyll
                                          Student Evaluation

What color does the chlorophyll fluoresce? __________________

1. Discuss the usual return of the excited electrons in chlorophyll (they will return to the ground state and
   the energy released will contribute to the photochemical series of reactions in the LIGHT reaction of

2. Discuss the effect that the destruction of the cell membrane has on the energy dissipated as the
   electron returns from the "excited to the ground" state.

3. Why do we see the color of fluorescence that we see?


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