North Mississippi GK-12 Project
Project #132
Chromatography
Activity Summary:
Students will learn the principles of chromatography by separating the components of black
ink. Before performing this lab, students should be familiar with the concepts of polar and
nonpolar molecules.
Introduction:
Chromatography is a method for analyzing complex mixtures (such as ink) by separating
them into the chemicals from which they are made. Because molecules in ink and other
mixtures have different characteristics (such as size and solubility), they travel at different
speeds when pulled along a piece of paper by a solvent (in this case, water). For example,
black ink contains several colors. When the water flows through a word written in black, the
molecules of each one of the colors behave differently, resulting in a “rainbow” effect. Many
common inks are water soluble and spread apart into the component dyes using water as a
solvent.
Paper chromatography is a method chemists use to separate compounds from one another,
but not change them. In this section we will explore how this separation is made, using
different inks as mixtures and dyes as different compounds. Molecules with similar
arrangements of their atoms or molecular structures are attracted to each other. Water
molecules have the structure shown below in which the two hydrogen atoms form a 104o
angle with the oxygen at the vertex.
Because of this structure the oxygen end of the molecule has a small negative electrical
charge and the hydrogen end has a small positive charge. Liquid water is held together by
the attraction between the charges on different molecules. This is shown below for a small
cluster of water molecules.
A molecule with these charged regions is called a polar molecule. Methanol (CH3OH) has a
similar structure (see below), and the methanol molecules are very soluble in water because
of the mutual attraction between the two polar molecules.
A more complex, yet still similar molecule is cellulose, a molecule that is the basic
component of paper. It is a very long molecule (a polymer) in which thousands of rings of six
atoms each are linked together like beads. A portion of a cellulose molecule is shown below.
The polar -OH regions of these molecules are attracted to OH groups on adjacent cellulose
chains helping to hold the fibers together in paper. Not surprisingly, water molecules, being
polar, are also attracted to these regions. Paper loses its strength when wet because the water
molecules get between the cellulose chains and weaken the attraction between them.
When the end of a piece of paper is dipped into water the water molecules keep finding new
places (polar regions) to stick to, and so the water molecules climb up the paper being
replaced by new water molecules below. Other molecules which might be dissolved in the
water will also be carried along up the paper. This is applied to the separation of dyes in a
technique known as paper chromatography. The paper is called the stationary phase,
because it does not move; the solvent is known as the mobile phase because it does move,
traveling through the mobile phase as the experiment progresses.
A spot of dye is placed on the paper above the level of the water. As the water moves up, the
dye molecules will move with it if they are more strongly attracted to the water molecules
than to the paper molecules. If the dye molecules are more strongly attracted to the paper
than to the water, they will move more slowly than the water or even not at all. What if the
dye is a mixture? If two or more dyes have been mixed to form an ink, then they may move
at different rates as the water moves up the paper. If this happens, they will separate and we
can identify them. This is depicted in the sketches below.
After running the chromatogram, each separated "spot" can be assigned a Retention Factor
(RF) which is characteristic of the specific dye(s) associated with it. The RF is a ratio of the
distance the "spot" travels relative to the distance the solvent (water in this case) travels. The
RF is calculated by dividing the "spot" distance by the solvent distance. This ratio should be a
constant that is characteristic of the dye(s) in a particular spot under a particular set of
chromatographic conditions (i.e. paper chromatogram, water solvent, etc.)
Source: http://wwwchem.csustan.edu/chem1002/mrsketch.htm
Lab Activity
Time required:
5 minutes teacher preparation
30 minutes lab time
Materials needed for each lab group:
2 Large filter paper discs: one unmarked, one previously marked with unknown
1 Smaller filter paper disc
Scissors
Round object for tracing, like a quarter
2 Petri dishes
5 different brands of black felt-tip non-permanent pens
water
Preparation:
Assign each brand of pen a number, and label each pen. On a large filter paper disc,
use a pencil to trace a circle onto the center using a quarter as your guide. Choose one brand
of pen to use as the “unknown.” Using this pen, make a small dot on the penciled circle.
Repeat this procedure so that each lab group will have a large filter paper marked with the
unknown. Distribute the above materials on each lab table.
Procedure:
1. Use a pencil (do not use a pen!) to trace a round object, like a quarter, onto the center
of the large filter paper disc. Use your pencil to poke a small hole into each of the
large filter paper discs.
2. On the inside of the circle you just drew, number from 1-5, spacing the numbers
evenly. Using the 5 labeled pens provided at your table, make a small dot on the
penciled circle above each number, using the appropriate pen for each number. For
example, above the number 1, you would make a small dot using Pen #1.
3. Cut two small right triangles approx. ¾ ” on the bottom and 2” high out of the smaller
filter paper disc:
2”
Roll up this way
¾”
Roll up each triangle lengthwise. These will serve as wicks to draw the water into the
filter paper.
4. Fill each petri dish with about 1 cm of water. Insert each wick into the hole you
previously punched in the middle of each large filter paper disc. Then place each
5.
6.
7.
8.
filter paper into a petri dish; they should balance in the center of the dishes on their
wicks.
Wait about 15 minutes as the wick draws water from the petri dish and into the filter
paper. Each pen should produce a different spectrum of colors. Identify which pen
was the unknown by comparing the unknown’s color pattern to the patterns made by
Pens 1-5.
Record the colors that were produced from each ink spot, beginning with the ones
closest to the center and ending with the ones that traveled farthest from the center.
Write your group’s name on each filter paper, and set aside to let dry. You will then
staple these filter papers to your lab questions.
Answer these questions:
 Identify the mobile phase and the stationary phase. Is the mobile phase polar
or nonpolar? Which molecules will be carried by the mobile phase: nonpolar
or polar species?
 Which pigment travels farthest on the paper? Why? Answer in terms of
solubility of the ink in the water.
 Some pens are “permanent” ink; if they were used in this experiment, they
would not travel across the paper. Why? What solvent could be used as the
mobile phase in order to separate the components of permanent ink?
Students are expected to make the following observations:


marks made from the same pen always produce the same separation
pattern, i.e., the different colors are in the same order after the separation.
The size of the original dot on the paper has no effect on the separation
pattern, although the separation is better defined if the dot is smaller.
different brands of pen produce different separation patterns.
Reinforcements:


Students should be shown that different pens from the same batch (same brand
and same model) produce the same separation pattern.
The separation patterns produced by different brands of pen are governed by
several factors:
1. The composition of the ink. Different companies use different dyes to make
their ink. Some are doing it to produce special physical or visual effects, some
are doing it so they cannot be accused of copying other people’s product.
2. The solubility of each component dye in the carrying liquid. If the dye is not
soluble in the carrying liquid, it cannot be carried up the paper, i.e., if the dye
is nonpolar, it will not dissolve in the polar water (use the “water and oil don’t
mix” analogy). This is the case if a permanent ink pen is used with water as
the carrying liquid. Since the permanent ink is not soluble in water (hence the
term, "permanent"), the mark stays in the starting place. Since most permanent
inks are soluble somewhat in organic solvents, one can get it to produce a
pattern using rubbing alcohol as the carrying liquid. Typically, the most
soluble dye will move up the paper the most and the least soluble dye will
move up the least. If a dye is very soluble in the carrying liquid, it will follow
the top of the water level up the paper closely.
3. The extent to which the ink clings to the paper. A dye that is strongly attached
to the paper will not move up the paper much.
4. The length of the paper. The separation of the different dyes along the paper
increases if the carrying liquid is allowed to carry the ink up a longer distance.
At the beginning of the experiment, the colors are still bundled together. As
the carrying liquid moves across the paper, the separation becomes more and
more complete. Note, however, that the order of the colors does not change
with the distance traveled.
Internet Links
http://library.thinkquest.org/50070/intro/TQCHROM.html
This site gives information on teaching chromatography, as well as additional
investigative activities that students may perform in order to apply their skills of paper
chromatography.
http://www.buildingrainbows.com/CA/lesson/lessonid/1013034045
This gives another example of a paper chromatography experiment that is a little
more advanced than the one described in this project. Instead of ink pens, staining solutions
are used. Different solvents are also used as the mobile phase.