paper chromatography hypothesis

Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.

They all have a (a solid, or a liquid supported on a solid) and a (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. We'll look at the reasons for this further down the page.

In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.

You probably used paper chromatography as one of the first things you ever did in chemistry to separate out mixtures of coloured dyes - for example, the dyes which make up a particular ink. That's an easy example to take, so let's start from there.

Suppose you have three blue pens and you want to find out which one was used to write a message. Samples of each ink are spotted on to a pencil line drawn on a sheet of chromatography paper. Some of the ink from the message is dissolved in the minimum possible amount of a suitable solvent, and that is also spotted onto the same line. In the diagram, the pens are labelled 1, 2 and 3, and the message ink as M.

The reason for covering the container is to make sure that the atmosphere in the beaker is saturated with solvent vapour. Saturating the atmosphere in the beaker with vapour stops the solvent from evaporating as it rises up the paper.

As the solvent slowly travels up the paper, the different components of the ink mixtures travel at different rates and the mixtures are separated into different coloured spots.

The diagram shows what the plate might look like after the solvent has moved almost to the top.

It is fairly easy to see from the final chromatogram that the pen that wrote the message contained the same dyes as pen 2. You can also see that pen 1 contains a mixture of two different blue dyes - one of which be the same as the single dye in pen 3.

values

Some compounds in a mixture travel almost as far as the solvent does; some stay much closer to the base line. The distance travelled relative to the solvent is a constant for a particular compound as long as you keep everything else constant - the type of paper and the exact composition of the solvent, for example.

The distance travelled relative to the solvent is called the R value. For each compound it can be worked out using the formula:

For example, if one component of a mixture travelled 9.6 cm from the base line while the solvent had travelled 12.0 cm, then the R value for that component is:

In the example we looked at with the various pens, it wasn't necessary to measure R values because you are making a direct comparison just by looking at the chromatogram.

You are making the assumption that if you have two spots in the final chromatogram which are the same colour and have travelled the same distance up the paper, they are most likely the same compound. It isn't necessarily true of course - you could have two similarly coloured compounds with very similar R values. We'll look at how you can get around that problem further down the page.

In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures.

Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.

A small drop of a solution of the mixture is placed on the base line of the paper, and similar small spots of the known amino acids are placed alongside it. The paper is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5.

The position of the solvent front is marked in pencil and the chromatogram is allowed to dry and is then sprayed with a solution of . Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple.

The left-hand diagram shows the paper after the solvent front has almost reached the top. The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin.

There is no need to measure the R values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours.

In this example, the mixture contains the amino acids labelled as 1, 4 and 5.

And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.

Two way paper chromatography gets around the problem of separating out substances which have very similar R values.

I'm going to go back to talking about coloured compounds because it is much easier to see what is happening. You can perfectly well do this with colourless compounds - but you have to use quite a lot of imagination in the explanation of what is going on!

This time a chromatogram is made starting from a single spot of mixture placed towards one end of the base line. It is stood in a solvent as before and left until the solvent front gets close to the top of the paper.

In the diagram, the position of the solvent front is marked in pencil before the paper dries out. This is labelled as SF1 - the solvent front for the first solvent. We shall be using two different solvents.

If you look closely, you may be able to see that the large central spot in the chromatogram is partly blue and partly green. Two dyes in the mixture have almost the same R values. They could equally well, of course, both have been the same colour - in which case you couldn't tell whether there was one or more dye present in that spot.

What you do now is to wait for the paper to dry out completely, and then rotate it through 90°, and develop the chromatogram again in a different solvent.

It is very unlikely that the two confusing spots will have the same R values in the second solvent as well as the first, and so the spots will move by a different amount.

The next diagram shows what might happen to the various spots on the original chromatogram. The position of the second solvent front is also marked.

You wouldn't, of course, see these spots in both their original and final positions - they have moved! The final chromatogram would look like this:

Two way chromatography has completely separated out the mixture into four distinct spots.

If you want to identify the spots in the mixture, you obviously can't do it with comparison substances on the same chromatogram as we looked at earlier with the pens or amino acids examples. You would end up with a meaningless mess of spots.

You can, though, work out the R values for each of the spots in both solvents, and then compare these with values that you have measured for known compounds under exactly the same conditions.

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Paper is made of cellulose fibres, and cellulose is a polymer of the simple sugar, glucose.

The key point about cellulose is that the polymer chains have -OH groups sticking out all around them. To that extent, it presents the same sort of surface as silica gel or alumina in thin layer chromatography.

It would be tempting to try to explain paper chromatography in terms of the way that different compounds are adsorbed to different extents on to the paper surface. In other words, it would be nice to be able to use the same explanation for both thin layer and paper chromatography. Unfortunately, it is more complicated than that!

The complication arises because the cellulose fibres attract water vapour from the atmosphere as well as any water that was present when the paper was made. You can therefore think of paper as being cellulose fibres with a very thin layer of water molecules bound to the surface.

It is the interaction with this water which is the most important effect during paper chromatography.

Suppose you use a non-polar solvent such as hexane to develop your chromatogram.

Non-polar molecules in the mixture that you are trying to separate will have little attraction for the water molecules attached to the cellulose, and so will spend most of their time dissolved in the moving solvent. Molecules like this will therefore travel a long way up the paper carried by the solvent. They will have relatively high R values.

On the other hand, polar molecules have a high attraction for the water molecules and much less for the non-polar solvent. They will therefore tend to dissolve in the thin layer of water around the cellulose fibres much more than in the moving solvent.

Because they spend more time dissolved in the stationary phase and less time in the mobile phase, they aren't going to travel very fast up the paper.

The tendency for a compound to divide its time between two immiscible solvents (solvents such as hexane and water which won't mix) is known as . Paper chromatography using a non-polar solvent is therefore a type of .

A moment's thought will tell you that partition can't be the explanation if you are using water as the solvent for your mixture. If you have water as the mobile phase and the water bound on to the cellulose as the stationary phase, there can't be any meaningful difference between the amount of time a substance spends in solution in either of them. All substances should be equally soluble (or equally insoluble) in both.

And yet the first chromatograms that you made were probably of inks using water as your solvent.

If water works as the mobile phase as well being the stationary phase, there has to be some quite different mechanism at work - and that must be equally true for other polar solvents like the alcohols, for example. Partition only happens between solvents which don't mix with each other. Polar solvents like the small alcohols do mix with water.

In researching this topic, I haven't found any easy explanation for what happens in these cases. Most sources ignore the problem altogether and just quote the partition explanation without making any allowance for the type of solvent you are using. Other sources quote mechanisms which have so many strands to them that they are far too complicated for this introductory level. I'm therefore not taking this any further - you shouldn't need to worry about this at UK A level, or its various equivalents.

Where would you like to go now?

To the chromatography menu . . .

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© Jim Clark 2007 (modified July 2016)

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paper chromatography

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• Open Library Publishing Platform - DRAFT – Organic and Biochemistry Supplement to Enhanced Introductory College Chemistry - Thin Layer (TLC) And Paper Chromatography (PC)
• Chemistry LibreTexts Library - Paper Chromatography - Separation and Identification of Five Metal Cations
• Oregon State University - College of Engineering - Paper chromatography
• Academia - Paper Chromatography
• Journal of Emerging Technologies and Innovative Research - Paper Chromatography: A Review

paper chromatography , in analytical chemistry , technique for separating dissolved chemical substances by taking advantage of their different rates of migration across sheets of paper. It is an inexpensive but powerful analytical tool that requires very small quantities of material.

The method consists of applying the test solution or sample as a spot near one corner of a sheet of filter paper. The paper is initially impregnated with some suitable solvent to create a stationary liquid phase . An edge of the paper close to the test spot is then immersed in another solvent in which the components of the mixture are soluble in varying degrees. The solvent penetrates the paper by capillary action and, in passing over the sample spot, carries along with it the various components of the sample. The components move with the flowing solvent at velocities that are dependent on their solubilities in the stationary and flowing solvents. Separation of the components is brought about if there are differences in their relative solubilities in the two solvents. Before the flowing solvent reaches the farther edge of the paper, both solvents are evaporated , and the location of the separated components is identified, usually by application of reagents that form coloured compounds with the separated substances. The separated components appear as individual spots on the path of the solvent. If the solvent flowing in one direction is not able to separate all the components satisfactorily, the paper may be turned 90° and the process repeated using another solvent.

Paper chromatography has become standard practice for the separation of complex mixtures of amino acids , peptides , carbohydrates , steroids , purines , and a long list of simple organic compounds . Inorganic ions can also readily be separated on paper. Compare thin-layer chromatography .

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Leaf chromatography

In association with Nuffield Foundation

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Try this class practical using paper chromatography to separate and investigate the pigments in a leaf

Most leaves are green due to chlorophyll. This substance is important in photosynthesis (the process by which plants make their food). In this experiment, students investigate the different pigments present in a leaf, from chlorophyll to carotenes, using paper chromatography.

The experiment takes about 30 minutes and can be carried out in groups of two or three students.

• Eye protection
• Pestle and mortar
• Chromatography paper
• Beaker, 100 cm 3
• Small capillary tube (see note 1)
• Cut-up leaves, or leaves and scissors (see note 2)
• Propanone (HIGHLY FLAMMABLE, IRRITANT), supplied in a small bottle fitted with a teat pipette (see note 3)

Equipment notes

• The capillary tubing can be ‘home-made’ from lengths of ordinary glass tubing (diameter: 3–4 mm) using a Bunsen burner fitted with a flame-spreading (‘fish-tail’) jet.
• A variety of leaves can be used. Best results are obtained from trees or bushes with dark green leaves, eg holly.
• Preferably use teat pipettes that do not allow squirting, eg those fitted to dropper bottles of universal indicator.

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Propanone, CH 3 COCH 3 (l), (HIGHLY FLAMMABLE, IRRITANT) – see CLEAPSS Hazcard HC085A .  The vapour of propanone is HIGHLY FLAMMABLE. Do not have any source of ignition nearby.
• Finely cut up some leaves and fill a mortar to about 2 cm depth.
• Add a pinch of sand and about six drops of propanone from the teat pipette.
• Grind the mixture with a pestle for at least three minutes.
• On a strip of chromatography paper, draw a pencil line 3 cm from the bottom.
• Use a fine glass tube to put liquid from the leaf extract onto the centre of the line. Keep the spot as small as possible.
• Allow the spot to dry, then add another spot on top. Add five more drops of solution, letting each one dry before putting on the next. The idea is to build up a very concentrated small spot on the paper.
• Attach the paper to the pencil using sellotape so that when placed in the beaker, the paper is just clear of its base.
• Place no more than about 10 cm 3 of propanone in the beaker and hang the paper so it dips in the propanone. Ensure the propanone level is below the spot.

Source: Royal Society of Chemistry

The equipment required for using paper chromatography to separate the different pigments in leaves

• Avoid moving the beaker in any way once the chromatography has started.
• Leave the experiment until the propanone has soaked near to the top, and then remove the paper from the beaker.
• Mark how high the propanone gets on the paper with a pencil and let the chromatogram dry.

Teaching notes

This experiment works very well providing care is taken over preparing the spot on the chromatography paper. It should be as small and as concentrated as possible. Encourage students to be patient and to wait until each application is dry before adding the next.

At least three spots should be obtained, and one of these should be yellow due to carotenes.

The extent to which any particular component moves up the paper is dependent not only on its solubility in propanone but also on its attraction for the cellulose in the chromatography paper. The yellow carotene spot (with a higher RF value) tends to move up the paper the furthest.

More resources

Add context and inspire your learners with our short career videos showing how chemistry is making a difference .

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry.

Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

• 11-14 years
• 14-16 years
• Practical experiments
• Chromatography

Specification

• 2. Develop and use models to describe the nature of matter; demonstrate how they provide a simple way to to account for the conservation of mass, changes of state, physical change, chemical change, mixtures, and their separation.
• Chromatography as a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase.
• 6 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• Chromatography involves a stationary phase and a mobile phase. Separation depends on the distribution of substances between the phases.
• The ratio of the distance moved by a compound (centre of spot from origin) to the distance moved by the solvent can be expressed as its Rf value: Rf = (distance moved by substance / distance moved by solvent)
• Mixtures can be separated by physical processes such as filtration, crystallisation, simple distillation, fractional distillation and chromatography. These physical processes do not involve chemical reactions and no new substances are made.
• Recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases.
• Interpret chromatograms, including measuring Rf values.
• Suggest chromatographic methods for distinguishing pure from impure substances.
• 12 Investigate how paper chromatography can be used to separate and tell the difference between coloured substances. Students should calculate Rf values.
• 2.11 Investigate the composition of inks using simple distillation and paper chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates…
• C2.1g describe the techniques of paper and thin layer chromatography
• 2.9 Describe paper chromatography as the separation of mixtures of soluble substances by running a solvent (mobile phase) through the mixture on the paper (the paper contains the stationary phase), which causes the substances to move at different rates o…
• C5.1.4 recall that chromatography involves a stationary and a mobile phase and that separation depends on the distribution between the phases
• 3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• C3 Using chromatography to identify mixtures of dyes in a sample of an unknown composition
• 1.9.5 investigate practically how mixtures can be separated using filtration, crystallisation, paper chromatography, simple distillation or fractional distillation (including using fractional distillation in the laboratory to separate miscible liquids…
• 1.9.7 interpret a paper chromatogram including calculating Rf values;
• carry out paper and thin-layer chromatography and measure the Rf values of the components and interpret the chromatograms;

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May 14, 2015

Chromatography: Be a Color Detective

A colorful project from Science Buddies

By Science Buddies

Key concepts Colors Solutions Molecules Chromatography Primary colors

Introduction Do you love to use bright and vibrant colored art supplies such as markers or paints? Do you ever wonder how these colors are made?

The variety of colors comes from colored molecules. These are mixed into the material—whether ink or paint—to make the product. Some colored molecules are synthetic (or man-made), such as "Yellow No. 5" found in some food dyes. Others are extracted from natural sources, such as carotenoid (pronounced kuh-RAH-tuh-noid) molecules. These are molecules that make your carrot orange. They can be extracted from concentrated natural products, such as saffron.

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But there is more to making a color look the way it does in your homemade artwork. You might have learned that many colors, such as orange and green, are made by blending other, "primary" colors. So even though our eyes see a single color, the color of a marker, for instance, might be the result of one type of color molecule or it might be a mix of color molecules responsible. This science activity will help you discover the hidden colors in water-soluble markers.

Background We see objects because they reflect light into our eyes. Some molecules only reflect specific colors; it is this reflected, colored light that reaches our eyes and tells our brains that we are seeing a certain color.

Often the colors that we see are a combination of the light reflected by a mixture of different-color molecules. Even though our brains perceive the result as one color, each of the separate types of color molecules stays true to its own color in the mixture. One way to see this is to find a way to separate out the individual types of color molecules from the mixture—to reveal their unique colors.

Paper chromatography is a method used by chemists to separate the constituents (or parts) of a solution. The components of the solution start out in one place on a strip of special paper. A solvent (such as water, oil or isopropyl alcohol) is allowed to absorb up the paper strip. As it does so, it takes part of the mixture with it. Different molecules run up the paper at different rates. As a result, components of the solution separate and, in this case, become visible as strips of color on the chromatography paper. Will your marker ink show different colors as you put it to the test?

Two white coffee filters

Drawing markers (not permanent): brown, yellow and any other colors you would like to test

At least two pencils (one for each color you will be testing)

At least two tall water glasses (one for each color you will be testing), four inches or taller

Two binder clips or clothespins

Drying rack or at least two additional tall water glasses (one for each color you will be testing)

Pencil or pen and paper for taking notes

Preparation

Carefully cut the coffee filters into strips that are each about one inch wide and at least four inches long. Cut at least two strips, one to test brown and one to test yellow. Cut an extra strip for each additional color you would like to test. How do you expect each of the different colors to behave when you test it with the paper strip?

Draw a pencil line across the width of each paper strip, about one centimeter from the bottom end.

Take the brown marker and a paper strip and draw a short line (about one centimeter) on the middle section of the pencil line. Your marker line should not touch the sides of your strip.

Use a pencil to write the color of the marker you just used on the top end of the strip. Note: Do not use the colored marker or pen to write on the strips, as the color or ink will run during the test.

Repeat the previous three steps with a yellow marker and then all the additional colors you would like to test.

Hold a paper strip next to one of the tall glasses (on the outside of it), aligning the top of the strip with the rim of the glass, then slowly add water to the glass until the level just reaches the bottom end of the paper strip. Repeat with the other glass(es), keeping the strips still on the outside and away from the water. What role do you think the water will play?

Fasten the top of a strip (the side farthest from the marker line) to the pencil with a binder clip or clothespin. Pause for a moment. Do you expect this color to be the result of a mixture of colors or the result of one color molecule? If you like, you can make a note of your prediction now.

Hang the strip in one of the glasses that is partially filled with water by letting the pencil rest on the glass rim. The bottom end of the strip should just touch the water level. If needed, add water to the glass until it is just touching the paper. Note: It is important that the water level stays below the marker line on the strip.

Leave the first strip in its glass as you repeat the previous two steps with the second strip and the second glass. Repeat with any additional colors you are testing.

Watch as the water rises up the strips. What happens to the colored lines on the strips? Does the color run up as well? Do you see any color separation?

When the water level reaches about one centimeter from the top (this may take up to 10 minutes), remove the pencils with the strips attached from the glasses. If you let the strips run too long, the water can reach the top of the strips and distort your results.

Write down your observations. Did the colors run? Did they separate in different colors? Which colors can you detect? Which colors are on the top (meaning they ran quickly) and which are on the bottom (meaning they ran more slowly)?

Hang your strips to dry in the empty glasses or on a drying rack. Note that some colors might keep running after you remove the strips from the water. You might need longer strips to see the full spectrum of these colors. The notes you took in the previous step will help you remember what you could see in case the colors run off the paper strip. Look at your strips. How many color components does each marker color have? Can you identify which colors are the result of a mixture of color components and which ones are the result of one hue of color molecule? Are individual color components brightly colored or dull in color? How many different colors can you detect in total?

Extra: Most watercolor marker inks are colored with synthetic color molecules. Artists often like to work with natural dyes. It is fairly easy to make your own dye from colorful plants such as blueberries, red beets or turmeric. To make your own dye, have an adult help you finely chop the plant material and place it in a saucepan. And add just enough water to cover the plant material. Let the mixture simmer covered on the stove for approximately 10 to 15 minutes. If, at this point, the color of your liquid is too faint, you might want to remove the lid of the saucepan and continue boiling until some liquid has evaporated and a more concentrated color is obtained. Let it cool and strain when needed. Now you have natural dye. (Handle with caution, as it can stain surfaces and materials.) To investigate the color components of this dye, repeat the previous procedure but replace the marker line with a drop of natural dye. A dropper will help create a nice drop. Let the drop of dye dry before running the paper strip. Would the color of your natural dye be the result of a mixture of color molecules or one specific color molecule? Does the marker of the same color as your natural dye run in a similar way as your natural dye does?

Extra: In this activity you used water-soluble markers in combination with water as a solvent. You can test permanent markers using isopropyl rubbing alcohol as a solvent. Do you think similar combinations of color molecules are used to color similar-colored permanent markers?

Extra: You can investigate other art supplies, including paints, pastels or inks in a similar way. Be sure to always choose a solvent that dissolves the material that is being tested to run the chromatography test. Isopropyl rubbing alcohol, vegetable oil and salt water are some examples of solvents used to perform paper chromatography tests for different substances.

[break] Observations and results Did you find that brown is made up of several types of color molecules, whereas yellow only showed a single yellow color band?

Marker companies combine a small subset of color molecules to make a wide range of colors, much like you can mix paints to make different colors. But nature provides an even wider range of color molecules and also mixes them in interesting ways. As an example, natural yellow color in turmeric is the result of several curcuminoid molecules. The brown pigment umber (obtained from a dark brown clay) is caused by the combination of two color molecules: iron oxides (which have a rusty red-brown color) and manganese oxides (which add a darker black-brown color).

In this activity you investigated the color components using coffee filters as chromatography paper. Your color bands might be quite wide and artistic, whereas scientific chromatography paper would yield narrow bands and more-exact results.

Cleanup Throw away the paper strips and wash the glasses.

More to explore Paper Chromatography , from ChemGuide Paper Chromatography: Is Black Ink Really Black? , from Science Buddies Make Your Own Markers , from Science Buddies Candy Chromatography: What Makes Those Colors? , from Science Buddies Find the Hidden Colors of Autumn Leaves , from Scientific American

This activity brought to you in partnership with Science Buddies