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Paper Chromatography: Advanced Version 1

Time Required Short (2-5 days)
Prerequisites None
Material Availability Filter paper can be purchased from our partner Amazon.com.
Cost Very Low (under $20)
Safety Alcohol is flammable and toxic


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.


The objective of this project is to compare different chromatography substrates and solvents to see which combination performs best for separating ink components.


Author: Amber Hess
Editor: Andrew Olson, Science Buddies

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MLA Style

Science Buddies Staff. "Paper Chromatography: Advanced Version 1" Science Buddies. Science Buddies, 16 Oct. 2017. Web. 23 Nov. 2017 <https://www.sciencebuddies.org/science-fair-projects/project-ideas/Chem_p009/chemistry/paper-chromatography-advanced-version-1>

APA Style

Science Buddies Staff. (2017, October 16). Paper Chromatography: Advanced Version 1. Retrieved November 23, 2017 from https://www.sciencebuddies.org/science-fair-projects/project-ideas/Chem_p009/chemistry/paper-chromatography-advanced-version-1

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Last edit date: 2017-10-16


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).

In paper chromatography, you can see the components separate out on the filter paper and identify the components based on how far they travel. To do this, we calculate the retention factor (Rf value) of each component. The Rf value is the ratio between how far a component travels and the distance the solvent travels from a common starting point (the origin). For example, if one of the sample components moves 2.5 centimeters (cm) up the paper and the solvent moves 5.0 cm, as shown in Figure 1 below, 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 Figure 1, the light blue shading represents the solvent and the dark blue spot is the colored solution sample.

Rf Example 1
Figure 1. values are how different components are compared to each other in paper chromatography.

Rf values are calculated by looking at the distance each component travels on the filter paper compared to the distance traveled by the solvent front. This ratio will be different for each component due to its unique properties, primarily based on its adhesive and cohesive factors.

When measuring the distance the component 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 then use Equation 1 below.

Equation 1:

In our example, this would be:

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.

Polarity 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.

Nonpolar-Water Polarity

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 and Concepts

  • Adhesion, cohesion forces
  • Capillary action
  • Adsorptive properties (adsorption chromatography)
  • Polarity of molecules
  • Hydrophilic, hydrophobic
  • Miscibility
  • Stationary phase, mobile phase
  • Rf value
  • Paper chromatography
  • Adsorptive chromatography
  • Solvent


  • How does the polarity of a substance affect how far it travels up the chalk/paper?
  • Are the polarities of the chalk and the paper different? If yes, how will this affect the spreading of the different inks?


Here are some basic chemistry and chromatography resources: This resource will give you more information about the history of chromatography, and teach you about the types of chromatography used in research labs today:

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Materials and Equipment

  • 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 filter paper; filter paper can be purchased through our partner Amazon.com.
    • Note: Filter paper is recommended for this project, but for a more in-depth discussion of which types of paper and solvents can be used at home for paper chromatography experiments please see the Paper Chromatography Resources page.
  • 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

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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.
  10. Repeat this experiment with paper instead of chalk. Instead of etching into the paper, use a ruler and pencil to draw a line across it horizontally two cm from the bottom. Place a small dot of ink onto the line. Then tape the paper to a pencil and hang it into the dish of solvent so it is barely touching. See Paper Chromatography: Is Black Ink Really Black?


For all of these questions, be sure to consider molecular structures and polarity.

  • Which ink components were the most polar for each brand? Least polar? How do you know?
  • How did the separation order differ for each solvent system, marker brand, and solid phase? Why?
  • How did the Rf values differ for each solvent system, maker brand and solid phase? Why?
  • Which combination of solid phase and solvent separated out each brand of marker the best?
  • Were your initial predictions correct?
An example of the experiment:

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  • It is very easy to make this project more advanced without doing much additional work. You could test how changing the pH of the water and/or changing the temperature of the solvents affects your results. For changes in temperature, do a few levels of temperature difference: refrigerated, room temperature, and warm (do not let the solvents get so hot they boil). Changing the level of pH is easy, just add a small amount of acid (diluted hydrochloric or sulfuric acid will work). Test the pH of the water with litmus paper, and try a variety of different acidic pHs (maybe a pH of 2, 4, and 7). What is pH? Look here:
  • If you really want to impress the judges, study the different types of molecular bonding (covalent, ionic, double, triple) and study polarity in depth. This will help you better understand why different components move different distances depending upon the stationary and mobile phases used. For information on bonding, see:
    Paper Chromatography: Advanced Version 2
  • Instead of analyzing different inks, you could analyze the compounds found in a plant; see:
    Paper Chromatography: Advanced Version 2
  • For a more basic chromatography project, see:
    Paper Chromatography: Basic Version

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