Paper Chromatography: Is Black Ink Really Black?
AbstractHave you ever looked at sunlight through a prism? If so, you know that the prism can separate the sunlight into many different colors of light — a rainbow. Like sunlight, chemical mixtures can also be broken into their component parts. One way of doing this is a simple technique called paper chromatography. What do you think you will see if you use paper chromatography to look at the components of black ink? Is black ink just black? Find out for yourself!
Editors: Andrew Olson, PhD and Sandra Slutz, PhD, Science Buddies
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The objective of this project is to use paper chromatography to analyze ink components in black markers/pens.
What color is black ink? Sounds like a trick question doesn't it! But sometimes things are not just what we think they are. Often things can be broken down into component parts. For example, milk looks like one thing but it is actually made up of several components including water, fat, and protein. Which brings us back to black ink — it looks like one thing, but is it actually made up of more than one component? One way to find out it to use a common chemistry technique called chromatography.
Chromatography is a technique used to separate the various components in a complex mixture or solution. There are two parts: a stationary phase and a mobile phase. The stationary phase does not move. It is the platform on which you put the mixture you want to analyze on. The mobile phase does exactly what you would expect given the name — it moves. It sweeps the components in the mixture along the stationary phase separating them by how much they "stick" to each other. It is also referred to as the solvent.
In this science project you will use paper chromatography to see if black ink can be separated into components. The ink will be spotted onto strips of chromatography paper and put in a beaker containing a solution of alcohol and water. The paper (or more precisely the water that is adsorbed to the paper molecules) is the stationary phase and the alcohol and water solution is the solvent (mobile phase). The solvent will move by capillary action. The attraction of the solvent to the water embedded in the paper (adhesion force) is larger than the attraction of the solvent to itself (cohesion force), hence the solvent moves up the paper. The ink will also be attracted to the adsorbed water inside the paper, to itself, and to the solvent differently, and thus a different component will move a different distance depending upon the strength of attraction to each of these objects, as shown in Figure 1. 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 interact more with your relatives, just as some chemical samples may interact more with the stationary phase than the solvent, and thus will not move up the stationary phase as quickly. Your cousin is more attracted to the idea of leaving, which is like the solvent (the mobile phase).
A homemade paper chromatography testing box is made from a tall box with a lid. A dowel spanning the width of the box is placed near the top with a binder clip to hold a paper strip that has been marked by colored pigments. The paper strip is long enough to reach the bottom of the box where there is a small pool of solvent. As the solvent is absorbed by the paper and moves upward it brings some of the colored pigment markings with it.
Figure 1. Paper chromatography. Molecules are separated from each other, depending on how fast they migrate with the solvent up the chromatography paper. (Wikipedia, 2008.)
In paper chromatography, you can see the components separate out on the chromatography 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 (mobile phase) 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 2 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 2, the light blue shading represents the solvent and the dark blue spot is the colored solution sample.
Diagram of a paper chromatography strip shows how marker ink travels up the length of a paper strip when the strip absorbs a liquid solvent. An origin line marks the original position of the ink and the solvent is colored blue so the distance the solvent travels up the paper strip can be measured. In the diagram, solvent traveled 5 centimeters, while the sample traveled 2.5 centimeters from the origin line.
Figure 2. For each compound, an Rf value is calculated based on how far it traveled along the stationary phase. In paper chromatography, Rf values are used to compare different components to each other.
Rf values are calculated by looking at the distance each component travels on the chromatography paper compared to the distance traveled by the solvent front. This ratio will be different for each component due to its unique chemical properties.
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.
In our example, this would be:
Note that an Rf value has no units because the units of distance cancel.
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.
If you want to find out more about paper chromatography, you can watch the video below. The video gives an overview of what paper chromatography is, shows how it is done, explains the separation processes involved, and also provides tips and tricks for troubleshooting your experiment.
In this science project, you can use a simple paper chromatography setup to see if black ink is just one component or a mixture of several components. Will the answer be the same for all types of black ink?
Terms and Concepts
- Stationary phase
- Mobile phase
- Capillary action
- Adhesion force
- Cohesion force
- Why do different compounds travel different distances on the piece of paper?
- How is an Rf value useful?
- What is chromatography used for?
- Andrew Rader Studios. (2012). Chem4Kids. Retrieved November 29, 2012.
- Clark, Jim. (2007). Paper Chromatography. Retrieved August 20, 2013.
- United States Geological Survey Staff. (2012, October 31). Capillary Action. Retrieved November 29, 2012.
- Waters Corporation Staff. (2012). High Performance Liquid Chromatography. Retrieved November 29, 2012.
- Science Buddies. (n.d.). Paper Chromatography Resources. Retrieved January 14, 2018.
These specialty items can be purchased from our partner Home Science Tools:
- Candy Chromatography Science Kit (1). You will need these items from the kit:
- Chromatography paper strips; at least 10. The kit comes with 20 strips; additional chromatography paper can be purchased separately from our partner Home Science Tools.
- 100 mL beaker
- 500 mL beaker
- 90% isopropyl alcohol
- Mini binder clips (2)
- Wooden splints
- Different black pens and markers (3):
- Permanent marker
- Washable black marker
- Black ballpoint pen
- Note: This kit contains additional items to do other chromatography science projects. See the kit instructions page for details. Downloadable materials safety data sheets (MSDS) are available for all kit chemicals if required by your science fair.
You will also need to gather these items:
- Ruler; metric
- Plastic wrap
- Lab notebook
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To make sure you can compare your results, as many of your materials as possible should remain constant. This means that the temperature, type of water used, size of paper strips, where the ink is placed onto the paper etc. should remain the same throughout the experiment.
- Cut the chromatography paper into strips approximately 2 centimeters (cm) wide by 6.5 cm long. Prepare a total of 15 chromatography strips this way.
- Science Buddies Kit: The kit comes with 20 long strips of chromatography paper; two 6.5 cm strips can be cut from each long strip.
- Take one of the chromatography strips and use a ruler and pencil to draw a line across it horizontally 1 cm from the bottom. This is the origin line or baseline, see Figure 3 below for details. Repeat this step for all 15 of the chromatography strips.
Figure 3. Each chromatography strip will have an origin line (baseline). The pen/marker ink to be tested will be spotted in the middle of the origin line.
- Using one of the pens/markers, place a small dot of ink at the center of the origin line of a chromatography strip. This is your spotted sample as shown in Figure 4 below.
- Use a pencil to label which pen/marker you spotted on the chromatography strip. Do not use a pen labeling the strips: the ink will run when the solvent passes through the strips.
- Repeat this step until you have spotted ink on 5 chromatography strips for each pen/marker.
Figure 4. A marker or pen should be used to put a single spot of black ink in the middle of the origin line on the chromatography strip.
- Make a 45% isopropyl alcohol solution to use as your chromatography solvent.
- Pour 20 milliliters (mL) of 90% isopropyl alcohol into the 100 mL beaker. Add 20 mL of water to the beaker so that the final volume is 40 mL. Stir thoroughly with the wooden splint.
- Pour the 40 mL of approximately 45% isopropyl alcohol solution into the 500 mL beaker. Cover the beaker with plastic wrap, so that the solution does not evaporate. This is your solvent, or mobile phase.
- Pour about 8 mL of the solvent back into the 100 mL beaker and run two prepared chromatography strips in the beaker.
- Clip two of the prepared chromatography strips to a wooden splint. Make sure the two strips do not touch each other and the bottoms align. Rest the splint on top of the beaker so that the strips hang into the beaker and do not touch the sides of the beaker.
- If necessary, add more solvent to the small beaker. The goal is to have the end of each chromatography strip just touching the surface of the solvent solution as shown in Figure 5 below. Add solvent as needed to achieve this goal.
- Cover the top of the beaker with plastic wrap.
- Set aside the remainder of the unused solvent (covered with a lid or plastic wrap) for additional runs.
Figure 5. The edge of the chromatography strips should just barely touch the solvent. Remember to cover the top with plastic wrap so that the solvent does not evaporate.
- Let the solvent rise up the strip (by capillary action) until it is about 0.5 cm from the top. Depending on the chromatography paper, this can take anywhere from 30 minutes to several hours. Once the solvent has almost reached the top of the strip, remove the strip from the solvent. Regularly check on your chromatography strip and the solvent front — if you let it run too long the dye may run off the paper and become distorted.
- Be patient and do not take the paper strip out of the solvent early. The longer the paper strip stays in the solvent, the better the separation will be!
- Use a pencil to mark how far the solvent rose.
- Allow the chromatography strip to dry, then measure (in centimeters) and calculate the Rf value for each pen/marker dye component. Record your results in your lab notebook.
- Tip: The equation for calculating the Rf value is given in the Introduction.
- Repeat steps 5 - 8 until you have run all of the chromatography strips.
- Each time you run the experiment make sure there is enough solvent in the beaker. The chromatography strips should be just touching the surface of the solvent. Add more solvent (45% alcohol solution) as needed.
- Using the five repeated strips for each pen/marker, calculate the average Rf for each dye component.
Analyzing Your Results
- Create a data table like Table 1 for each marker or pen that you tested in your lab notebook.
|Type of Marker or Pen:|
|Component Color||Component Rf value|
|Total number of components:|
- Record all your results for one marker/pen in a different data table.
- Make a bar graph that shows the Rf values for each marker/pen. The x-axis of the bar chart should have a separate bar for each color component (label each bar appropriately), and the height of the bar on the y-axis should correspond to the Rf value for each color component.
- Did the inks from the different pens/markers separate differently? By looking at the Rf values, can you tell if any of the ink components from the different pens/markers are the same?
- If the ink components separated differently for each marker, why did this happen? Hint: Think about the strength of the attractions.
For troubleshooting tips, please read our FAQ: Paper Chromatography: Is Black Ink Really Black?.
Ask an Expert
- You could try using other solvents than 45% isopropyl alcohol (or mixture of solvents) and see if the inks are separated differently (rubbing alcohol, vinegar, nail polish remover, and turpentine would be good to try). Which solvent separates the ink the best? Why? (Remember to account for attractions between chemicals.)
- After buying a black light, you could try to see if any more components are visible on the paper. Some chemicals are invisible under white light, but can be seen with a black light. If you decide to do this variation, you should analyze the paper strips the same day or the next day, otherwise the spots will fade! Why are some chemicals visible under one type of light but not another? Read more here at Light Waves and Color.
- For more advanced chromatography projects, see:
Explore How Chromatography Can Unmix Mixtures
Discover Chlorophyll Variety in Different Plants Using Paper Chromatography
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