In this lab activity, we're going to be looking
at photosynthetic pigments.
The goals for this lab: we want to discuss
the purpose and function of photosynthetic
pigments, we want to separate pigments from
each other using paper chromatography, we're
going to discern some of the chemical properties
of these pigments based on their behavior
during the paper chromatography, and we are
also going to calculate the Rf value for these
pigments.
So, what is a pigment?
A pigment is a molecule that absorbs and reflects
light.
White light is made up of many different wavelengths,
in fact, all possible colors of visible light.
Pigments are molecules which absorb certain
wavelengths of light and reflect others.
The reflected light is the light that we see,
so the reflected light is the light that was
not absorbed.
Photosynthetic pigments use the energy of
the absorbed light to power the conversion
of carbon dioxide and water into glucose and
oxygen gas.
Most plants have multiple photosynthetic pigments,
and having multiple pigments allows plants
to get energy from a wider range of wavelengths
of light.
Reflected wavelengths of light are of no use
to the pigments reflecting those colors.
When we look at a leaf and see the green color,
that should tell us that those pigments are
absorbing all of the colors except for green.
Green light is not used in photosynthesis,
but the other colors are.
Paper chromatography is a technique that we
can use to separate molecules from each other
based on their chemical properties.
So, the pigments in plants may have slightly
different chemical properties in addition
to reflecting different wavelengths of light.
One way to separate different chemicals is
through paper chromatography.
A chromatography solvent flows through a sample,
and the components of that sample move at
different rates depending on their solubility
in the solvent and their attraction to the
cellulose of the filter paper.
So for this activity, for this protocol, we're
going to start with a strip of filter paper.
Now, we want to measure to make sure that
strip of filter paper is no longer than nine
centimeters, or ninety millimeters, and that's
because the chromatography chambers that we'll
be using only support strips of filter paper
about that long.
Once you have your strip of filter paper,
you want to measure one and a half centimeters,
or 15 millimeters, from one end, and then
draw a line using a graphite pencil.
This will be called the starting line, and
our future measurements will all take place
from this starting line.
Our next step is to taper the filter paper.
We're going to cut the end of the filter paper
into a point where the widest part of that
point ends up being where that starting line
is, up through the rest of the length of the
filter paper.
So you're going to trim the two corners of
that strip of filter paper so we have a pointed
piece of filter paper in which the widest
part of the filter paper is where our starting
line is present.
We are then going to take a spinach leaf and
a ridged coin or some smashing tool, and we're
going to crush that spinach leaf along the
pencil line.
This is going to be the pigment sample that
we will be separating.
Now clearly that spinach sample is going to
be a bit wider than a pencil line, but we
want to make sure that our pencil line is
within the area of smashed pigments.
We are then going to take one of our chromatography
chambers, which has a chromatography solution
within it, and we're going to take our strip
of filter paper, we're going to remove the
lid and gently put our filter paper within
the chromatography chamber, quickly sealing
the lid after we are finished.
The chromatography solution within the chamber
is very volatile, meaning it will evaporate
very quickly (you might even notice it as
a strong smell) and that's why we want to
reduce the amount of time that the cap is
off that chromatography chamber.
This process is going to take about 7 to 10
minutes, but here we'll see an animation of
what we would expect.
As the chromatography solution moves up the
strip of filter paper, we're going to notice
pigments that had been all together down at
the bottom will start to separate from each
other.
This is our paper chromatography experiment,
and these pigments have separated from each
other because of their chemical properties.
Now, as soon as the solvent front, or the
liquid, gets to about one centimeter from
the end of the strip of filter paper, we want
to stop this reaction and pull the strip out
of the chromatography chamber.
We quickly want to use either a pencil or
a pen and mark how far the solvent front had
traveled.
That distance between the starting line and
how far the solvent front traveled will be
very important, and we'll use it in calculating
a variety of other values.
For each of the pigments, and we see four
pigments that separated from that green stain
down at the bottom—those four pigments:
the two yellow pigments, a brighter green
pigment, and a dark green pigment—we want
to calculate the Rf value for each of them.
The Rf value is the distance that the pigment
traveled, divided by the distance that the
solvent traveled, and these measurements will
all be taking place from that starting line
down at the bottom.
The Rf value will always be between one and
zero because the pigment cannot travel farther
than the solvent did, but the pigment will
travel some distance from their initial starting
point.
I would recommend measuring from the middle
of each of the pigment bands from the starting
line, and so the distance that the farthest
yellow pigment traveled would be the center
of its band, whereas the distance the yellow
pigment traveled (or the second yellow pigment
traveled) would be about in the middle.
The bright green pigment traveled so far,
and the dark green pigment traveled a certain
distance.
For each of these, the distance the solvent
traveled is the same, but the distance that
the pigment traveled is different, and so
we'll have a different Rf value for each.
You're going to be using a ruler to measure
the Rf value.
Now a few words about the Rf value.
It turns out that the Rf value is a measurement
of the chemical properties of that pigment.
It actually has nothing to do with the pigment’s
color or which wavelengths it absorbs.
Instead, it has to do with the pigment’s
solubility in our chromatography solution
and also its attraction to the cellulose of
the filter paper.
As long as the same solvent and same type
of filter paper are used, the Rf value is
a constant.
Even if the filter paper was twice as long,
if we allow this to run twice as long as we
did here, the Rf value would still be the
same because that ratio between how far the
pigment traveled versus how far the solvent
traveled is a constant.
For this lab activity, when you make your
measurements, please make sure you are measuring
using millimeters and recall there are 10
millimeters in one centimeter.
That's all for this activity.
If there are any questions, please do not
hesitate to ask.
