Photosynthesis AP Lab.
Dr. Lourdes Rueda
Key Concepts I: Plant
Pigment Chromatography
• Paper chromatography is a technique
used to separate a mixture into its
component molecules. The molecules
migrate, or move up the paper, at
different rates because of
differences in solubility, molecular
mass, and hydrogen bonding with the
paper.
• For a simple, beautiful example of
this technique, draw a large circle in
the center of a piece of filter paper
with a black water-soluble, felt-tip
pen. Fold the paper into a cone and
place the tip in a container of water.
In just a few minutes you will have
tie-dyed filter paper!
• The green, blue,
red, and lavender
colors that came
from the black ink
should help you to
understand that
what appears to be
a single color may
in fact be a
material composed
of many different
pigments —and
such is the case
with chloroplasts
Design of the
Experiment I
• In paper chromatography the
pigments are dissolved in a solvent
that carries them up the paper. In
the ink example, the solvent is water.
To separate the pigments of the
chloroplasts, you must use an organic
solvent.
• If you did a number of
chromatographic
separations, each for a
different length of time,
the pigments would
migrate a different
distance on each run.
However, the migration of
each pigment relative to
the migration of the
solvent would not change.
This migration of pigment
relative to migration of
solvent is expressed as a
constant, Rf (Reference
front). It can be calculated
Key
Concepts II
• In the light reactions
of photosynthesis,
light energy excites
electrons in plant
pigments such as
chlorophyll, and
boosts them to a
higher energy level.
These high-energy
electrons reduce
compounds (electron
acceptors) in the
thylakoid membrane,
and the energy is
eventually captured
in the chemical
bonds of NADPH and
ATP.
Using DPIP As
an Electron
Acceptor
• In this activity you will measure
the rate of electron excitation
when light hits chlorophyll. You
will use DPIP, a blue compound,
as an electron acceptor. The
thylakoid membranes of the
chloroplasts are mechanically
disrupted so that the natural
electron transport chain no
longer functions normally.
• When light strikes the
chloroplasts, the DPIP is reduced
by the excited electrons from
chlorophyll, and it changes from
its original blue color to colorless
as it accepts the electrons. You
will use a spectrophotometer to
measure the color change, which
gives an indication of the rate of
the light reactions of
photosynthesis under various
conditions
The Spectrophotometer
• The spectrophotometer is an instrument
that can be adjusted to illuminate a
sample with a specific wavelength of
light. The spectrophotometer then
measures the amount of light energy
that is absorbed or transmitted by the
sample. In this laboratory, as DPIP goes
from blue to colorless, the amount of
light of wavelength 605 nm transmitted
through the sample will increase.
Though you can note this color change
visually, the spectrophotometer
quantifies the change
Design of the
Experiment II
• Pigments absorb in the visible
range of the spectrum (380–760
nm). After illuminating the
reaction tubes as shown below, we
will use the spectrophotometer to
measure the percentage of
transmittance at wavelength 605
nm.
• If DPIP is in an oxidized state, it will be
blue, and the percentage of light
transmitted will be low. If, on the other
hand, chlorophyll's electrons have been
excited and move on to reduce the
DPIP, the sample will become
progressively paler, allowing more light
energy to pass through the sample. We
can measure this change over time
until the sample has been completely
reduced, is almost colorless, and the
percentage of transmittance is very
high.
• For this experiment, one tube (the
blank) will contain all the solutions
used in the reaction except the DPIP.
Since the blank contains chloroplasts, it
will be green; you will use this tube to
calibrate the machine. The other tubes
will be experimental, and will contain
either boiled or unboiled chloroplasts
• Helpful Hints
• 1.The first reading provides a baseline. Be
sure you read the % transmittance
immediately after adding chloroplasts to each
experimental tube.
• 2.Keep the test tube glass clean! Use a
Kimwipe or lens paper to remove your
fingerprints.
• 3.Thoroughly mix each sample before you
measure it.
• 4.Be sure to place all the tubes into the
spectrophotometer in the same orientation
each time so variations in the glass don't mask
your results.
• 5.Maintain a constant distance from the light
source to the sample for all the tubes.