Chromatography
Abstract
This project will teach you the basics of analytical chemistry, which is a must for students who want to
go into chemistry or materials science. The molecules in objects we see all around us are constantly
attracting each other. Different materials can be attracted or repelled depending upon their molecular
structure. We can utilize the different attractive forces between substances to separate a mixture of
compounds. Chromatography is a method to separate mixtures and identify each compound in the
mixture. You will separate ink components found in different marker using a strip of paper, chalk and
different liquids.
Objective
The objective of this project is to compare different chromatography substrates and solvents to see
which combination performs best for separating ink components.
Introduction
Matter makes up everything in the universe. Our body, the stars, computers, and coffee mugs are all
made of matter. There are three different types of matter: solid, liquid, and gas. A solid is something
that is normally hard (your bones, the floor under your feet, etc.), but it can also be powdery, like
sugar or flour. Solids are substances that are rigid and have definite shapes. Liquids flow and assume
the shape of their container; they are also difficult to compress (a powder can take the same shape as
its container, but it is a collection of solids that are very small). Examples of liquids are milk, orange
juice, water, and vegetable oil. Gases are around you all the time, but you may not be able to see
them. The air we breathe is made up of a mixture of gases. The steam from boiling water is water's
gaseous form. Gases can occupy all the parts of a container (they expand to fill their containers), and
they are easily compressed.
Matter is often a mixture of different substances. A heterogeneous mixture is when the mixture is
made up of parts that are dissimilar (sand is a heterogeneous mixture). Homogeneous mixtures (also
called solutions) are uniform in structure (milk is a homogeneous mixture). A sugar cube floating in
water is a heterogeneous mixture, whereas sugar dissolved in water is a homogeneous mixture. You
will determine whether the ink contained in a marker is a heterogeneous or homogeneous mixture, or
just one compound.
In a mixture, the substance dissolved in another substance is called the solute. The substance doing
the dissolving is called the solvent. If you dissolve sugar in water, the sugar is the solute and the
water is the solvent.
For this project, you will be making a small spot with an ink marker onto a strip of paper. The bottom
of this strip will then be placed in a dish of water, and the water will soak up into the paper. The water
(solvent) is the mobile phase of the chromatography system, whereas the paper is the stationary
phase. These two phases are the basic principles of chromatography. Chromatography works by
something called capillary action. The attraction of the water to the paper (adhesion force) is larger
than the attraction of the water to itself (cohesion force), hence the water moves up the paper. The
ink will also be attracted to the paper, to itself, and to the water differently, and thus a different
component will move a different distance depending upon the strength of attraction to each of these
objects. As an analogy, let's pretend you are at a family reunion. You enjoy giving people hugs and
talking with your relatives, but your cousin does not. As you make your way to the door to leave, you
give a hug to every one of your relatives, and your cousin just says "bye."
So, your cousin will make it to the door more quickly than you will. You are more attracted to your
relatives, just as some chemical samples may be more attracted to the paper than the solvent, and
thus will not move up the solid phase as quickly. Your cousin is more attracted to the idea of leaving,
which is like the solvent (the mobile phase).
To measure how far each component travels, we calculate the retention factor (Rf value) of the
sample. The Rf value is the ratio between how far the component travels and the distance the solvent
travels from a common starting point (the origin). If one of the sample components moves 2.5 cm up
the paper and the solvent moves 5.0 cm, then the Rf value is 0.5. You can use Rf values to identify
different components as long as the solvent, temperature, pH, and type of paper remain the same. In
the image below, the light blue shading represents the solvent and the dark blue spot is the chemical
sample.
When measuring the distance the sample traveled, you should measure from the origin (where the
middle of the spot originally was) and then to the center of the spot in its new location.
To calculate the Rf value, we use the equation:
Rf =
distance traveled by the sample component
distance traveled by the solvent
In our example, this would be:
Rf =
2.5 cm
= 0.5
5.0 cm
Note that an Rf value has no units because the units of distance cancel.
Polarity has a huge affect on how attracted a chemical is to other substances. Some molecules have a
positively charged side and a negatively charged side, similar to a magnet. The positive side is
attracted to the negative side of another molecule (opposites attract), and vice versa.
The larger the charge difference, the more polar a molecule is. The reason for the unequal charge is
that electrons (which are negatively charged) are not shared equally by each atom (in water, the
negative electrons are more attracted to the oxygen because of its atomic structure). Some
molecules, like vegetable oil, are neutral and do not have a charge associated with them; they are
called nonpolar molecules. Polarity affects many of a molecule's properties, such as its affinity to
water. Water is a highly polar molecule, so other polar molecules are easily attracted to it. A molecule
is called hydrophilic if it dissolves well in water (hydrophilic essentially means "loves water"). A
nonpolar molecule, such as oil, does not dissolve well in water, and thus it is hydrophobic ("fears
water"). Oil would rather stick to itself than to water, and this is why it forms a layer across water
instead of mixing with it.
Soap can clean oils off of your body because soap has both polar and nonpolar properties. A soap
molecule has a nonpolar, and thus hydrophobic, "tail" made up mostly of carbon and hydrogen atoms,
but it also has a polar (hydrophilic) "head." The nonpolar body of the soap mixes easily with the
nonpolar oils, but not with the water. The polar head is attracted to the water, so the soap/oil mixture
is rinsed off. The negatively charged oxygen of the water is not attracted to any of the "tail's"
hydrogen because the carbon and hydrogen share electrons almost equally, so there is not a major
charge difference (the carbon-hydrogen group is neutral).
The picture below shows water molecules bonding with one another (hydrogen bonding). The
negatively charged oxygen atom (red) on one water molecule is attracted to the positively charged
hydrogen atom (white) on another water molecule.
Below is a picture of a fatty acid (a component of fat molecules) bonding with water. The hydrophobic
tail is not attracted to the water, and thus it stands upright out of the water. The hydrophilic head is
attracted to the water, which bonds to it. Many fatty acid molecules bonded together can form a layer
above the water. Carbon atoms are represented by black circles.
Chromatography is used in many different industries and labs. The police and other investigators use
chromatography to identify clues at a crime scene like blood, ink, or drugs. More accurate
chromatography in combination with expensive equipment is used to make sure a food company's
processes are working correctly and they are creating the right product. This type of chromatography
works the same way as regular chromatography, but a scanner system in conjunction with a computer
can be used to identify the different chemicals and their amounts. Chemists use chromatography in
labs to track the progress of a reaction. By looking at the sample spots on the chromatography plate,
they can easily find out when the products start to form and when the reactants have been used up
(i.e., when the reaction is complete). Chemists and biologists also use chromatography to identify the
compounds present in a sample, such as plants.
Terms—write the definition for each of the following:
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adhesion, cohesion forces
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capillary action
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adsorptive properties (adsorption chromatography)
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polarity of molecules
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hydrophilic, hydrophobic
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miscibility
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stationary phase, mobile phase
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Rf value
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paper chromatography
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adsorptive chromatography
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solvent
Questions
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How does the polarity of a substance affect how far it travels up the chalk/paper?
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Are the polarities of the chalk and the paper different? If yes, how will this affect the spreading
of the different inks?
Bibliography
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Basic chemistry concepts:
http://www.chem4kids.com
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Readily available solvents for chromatography (with polarity information):
Papers and Solvents for Paper Chromatography
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Overview of liquids and capillary action:
http://encarta.msn.com/encnet/refpages/RefArticle.aspx?refid=761571486&sec=18#s18
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Chromatography:
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/chromintro.html
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Capillary action:
http://www.madsci.org/posts/archives/feb98/887637827.Ch.r.html

Polarity:
http://www.biology.arizona.edu/biochemistry/tutorials/chemistry/page3.html
CHROMATOGRAPHY LAB
Materials and Equipment
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rubbing alcohol
water
nail polish remover or turpentine
at least 30 pieces of thin chalk (same size)
at least 30 identically sized strips of Chromatography paper
Note: chromatography paper or laboratory filter paper is preferable, but you can use a paper
ruler
pencils
at least three different types of black markers (including a permanent marker)
a small dish and/or wide-mouth jar for the solvent
Experimental Procedure
Note: To make sure you can compare your results, as many of your materials as possible should
remain constant. This means that the temperature, brand of solvents used, size of paper strips/chalk,
where the ink is placed onto the chalk/paper etc. should remain the same throughout the experiment.
1. Take a piece of chalk and etch a small ring two cm from the end.
2. Take the black marker and thinly trace inside of the etched ring.
3. Place the chalk upright in a small dish of solvent with the ink on the bottom (the ink should be
above the solvent level, it must not be submerged or touching the liquid).
4. Let the solvent rise up the chalk until it is almost at the top.
5. Remove the chalk from the dish and mark how far the solvent rose with a pencil.
6. Analyze the separated ink components:
Measure the distance the solvent and each ink component traveled from the starting position,
then calculate the Rf value for each component. (If there are not different colors in the ink,
you should use a different marker.)
7. Using your knowledge of polarity and the results from your first trial for this marker brand,
predict the order this ink will separate for each solvent and stationary phase.
8. Repeat this experiment for each brand of marker five times.
9. Repeat this experiment for each solvent five times.
Questions
For all of these questions, be sure to consider molecular structures and polarity.
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Which ink components were the most polar for each brand? Least polar? How do you know?
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How did the separation order differ for each solvent system, marker brand, and solid phase?
Why?
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How did the Rf values differ for each solvent system, maker brand and solid phase? Why?
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Which combination of solid phase and solvent separated out each brand of marker the best?
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Were your initial predictions correct?