Chromatography
Have you ever mixed different colors of markers or colored pencils? If so,
you probably noticed that they formed an entirely new color. What you may not
know is that many of the regular colors of markers and pens that you use are
already mixed. For example, a black marker might be made up of purple, yellow,
and red inks mixed together.
Today you will be using a method called chromatography to separate
pigments. In chromatography, a mixture is moved over a stationary substance.
Some parts of the mixture go faster and farther than others, so they end up in
separate places. This method is often used by chemists and biologists to identify
parts of a mixture. You will be using it to separate pigments in markers, pen ink,
and spinach leaves.
To carry out the separations, you will make a mark with ink or spinach juice
on a coffee filter. When you put the filter in contact with a liquid, the liquid will
travel through it and carry along any pigment that can dissolve in it. How far the
pigment is carried depends on how big the pigment molecules are and how much
they stick to the paper. For the marker, you will use water as the liquid. For pen
ink and spinach juices, which do not dissolve in water, you will use isopropanol.
A quick note about plant pigments: Plants use pigments in their leaves to
capture light from the sun, which they need to make food. The most important
pigment for this is chlorophyll a, which is what makes plants look green. Leaves
also contain “helper” pigments such as chlorophyll b (green), xanthophylls
(yellow), and carotenoids (orange). Hopefully you will be able to see some of
these different pigments in your separation.
Separation 1: Plant Pigments
The different pigments in plants take a long time to separate, so you will need to set this up first
and let it run while you are doing to other two experiments.
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Cut a strip of coffee filter. Draw a horizontal line with a pencil (not pen) about half an inch
from the bottom. Place a spinach leaf on the line and roll a penny over it so that you get a
line of green pigment on the filter. Using a different part of the leaf, roll the penny again
over the same line. Repeat this process until the line is fairly dark.
Put about an inch of isopropanol in a plastic cup. Tape the top of the coffee filter strip to a
pencil and balance the pencil across the top of the cup. See the image below for the set-up.
*It is very important that the bottom of the filter strip is in the isopropanol, but the green spot
is not in the liquid. If the isopropanol touches the spot directly, the pigment will just dissolve
away.*
Set the cup aside. The isopropanol will move up the filter paper slowly and deposit the
pigment components along the way.
Separation 2: Pen Ink
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Draw a horizontal line with a pen on the bottom of a coffee filter strip.
Set up the strip in a cup of isopropanol just as you did for the plant pigment.
This separation might also take some time, so you can set it aside as well while you do the
third one.
Separation 3: Markers
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This separation will be set up differently than the others. Cut a half-inch strip from around
the edge of the coffee filter. This will be your “wick.” Also cut a small hole in the center of
the coffee filter.
 Draw with a marker or markers in a circle around the hole. Black often separates very well,
but you should try other colors as well. Keep in mind that the water will be moving from the
middle, causing the pigments to expand outward (see picture on the first page).
 Loosely twist up your wick and insert it so that the end goes through the hole in the center of
the coffee filter.
 Put a couple of inches of water in a cup, and set up your filter so that the wick is in the water
and the round filter is resting on the top of the cup.
 You should be able to see the water moving up the wick. When it gets to the marks you
made, it will spread them out and separate any that have more than one pigment.
 Which colors separated the best? Were you surprised by this?
Questions
1. Chromatography separates parts of a mixture by moving them over a stationary
substance. What was the stationary substance in this experiment?
2. For the first two separations, you had to use isopropanol so that the ink and
plant pigments could dissolve in it. Why is it important that the pigments be able
to dissolve in the liquid used for the separation?