# Paper Chromatography: Advanced Version 2

 Difficulty Time Required Short (2-5 days) Prerequisites None Material Availability Filter paper can be purchased from the Science Buddies Store. Cost Low ($20 -$50) Safety Alcohol is flammable and toxic

## Abstract

Chromatography is a method used to separate mixtures of compounds and to identify each compound in the mixture. You may have separated the different inks in a black marker by using a strip of paper and water. There are many different types of chromatography: paper, thin-layer, high-performance thin-layer, gas, and more. You will be using paper or thin-layer chromatography to analyze the compounds from different plants. Chromatography is used by analytical chemists, organic chemists, and many other types of scientists since it is so easy and affordable. If you want to get a head start in chemistry, this is a great way to do so.

## Objective

The objective of this project is to use paper chromatography to analyze the leaf pigments found in three different types of plants.

## Credits

Author: Amber Hess
Editor: Andrew Olson, Science Buddies

### MLA Style

Science Buddies Staff. "Paper Chromatography: Advanced Version 2" Science Buddies. Science Buddies, 22 Mar. 2013. Web. 30 Aug. 2014 <http://www.sciencebuddies.org/science-fair-projects/project_ideas/Chem_p010.shtml?From=blog>

### APA Style

Science Buddies Staff. (2013, March 22). Paper Chromatography: Advanced Version 2. Retrieved August 30, 2014 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/Chem_p010.shtml?From=blog

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Last edit date: 2013-03-22

## Introduction

Matter and Mixtures

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.

Chemical Bonding

Matter is made up of small particles called atoms. Each atom is made up of smaller, positively charged particles called protons, neutral particles called neutrons, and even smaller negatively charged particles called electrons. At the center of each atom is the collection of protons and neutrons called the nucleus. Electrons spin around the nucleus in different energy levels called orbitals. Each orbital corresponds to a discrete amount of energy that the electron can have. A really excited electron with a lot of energy is further from the nucleus. In the image below, the electrons are yellow with a negative sign, the protons are green with a positive sign, and the neutrons are red with no sign. Keep in mind that this is a representation of what an atom looks like. Since atoms are so small, no one has seen a full atom before with the electrons orbiting around the nucleus.

Atoms are differentiated from one another by the amount of protons in their nucleus. For example, the carbon atom has six protons, but lithium has three. Each type of atom represents a different element (substances that cannot be broken down any further). The Periodic Table (see the Chemistry Resources page for a link to a printable Periodic Table) shows all of the elements we have discovered so far. The amount of electrons an atom has normally equals the number of protons, unless an electron is added or removed during a chemical reaction.

The objects you see around you are formed when atoms combine together by bonding to each other. Think of a bond between two atoms as a bond between two people. If you like someone, you might give them a hug. In this way, you are joined together. You can also hold hands with your friends and form a long chain of people.

Let's say you are talking to another friend, Mike, about your marble collection. You brought some of your marbles with you, as did Mike. Let's say you have 17 marbles total, and Mike has 11 total. You take your seven favorite marbles out of your bag, and Mike takes out his favorite one. Mike decides he wants to have eight marbles in his hands. Reluctant to give your marbles to him (Mike can be clumsy and easily drops things), you suggest you both hold the marbles. Holding out one of your hands with the seven marbles, Mike does the same with his one. You join hands. Now each of you technically has eight marbles because you are both holding them. Like Mike, a sodium atom (symbol Na) has 11 electrons total, but only one in its outermost orbital. A Chlorine atom (symbol Cl) has 17 electrons total, but seven in its outermost orbital. As a general rule, most atoms need eight atoms in their outermost orbital so they are stable. Both of you become sick of sharing the marbles. Wanting to make some money, Mike asks if you would buy his marble, and you agree. Now you have all eight marbles for yourself! Since you are still best friends though, Mike and you hold each other's hand. You have just formed an ionic bond with Mike! An ionic bond occurs when electrons are more attracted to one atom than another, and thus leave one of the atoms entirely. If you are the chlorine atom, and Mike is the sodium atom, you just formed sodium chloride, which is table salt. The bond between sodium chloride is ionic. The electrons are so attracted to the chlorine, that they leave the sodium atom! Now each atom is not neutral however. The chlorine atom gained one negative electron, so it has a negative charge (-1). The sodium atom lost one electron, so it has a positive charge (+1). Each atom is now stable. You and Mike are both happy; you have one more marble, and Mike has money.

Before Mike sold you his marble, you were sharing all eight marbles. When atoms share electrons with each other to gain eight electrons in their outer orbital, they form a covalent bond. Compounds like sodium chloride do not form covalent bonds because of the large attractive difference by electrons, but other compounds, like water, do. The oxygen atom shares the two electrons from the two hydrogen, and it also shares two of its electrons with them (the lowest orbital—hydrogen's highest—only needs two electrons to be stable). So, each hydrogen has two electrons and the oxygen has eight electrons in its outermost orbital.

The image below shows one way chemists often draw the bonding between atoms, which are called Lewis Dot Structures. For water, the light blue dots represent the oxygen's electrons, and the red dots represent the hydrogen's electrons. The circles show how it is possible to share electrons and still get a full outer orbital. For the sodium chloride molecule, the chlorine takes one electron from sodium (the pink dot), leaving sodium with no electrons in its highest orbital. The circle is fully around the chlorine because it is not sharing the electron, it is taking it to add to its other seven outer electrons (the purple dots).

Sometimes an atom will form a covalent bond called a double or triple bond. Carbon and nitrogen do this a lot. Although there are many different kinds of molecules, each atom can only bond in a few ways depending upon its outer electron configuration. To bond with certain atoms and still obtain a stable outmost orbital, some atoms share two or three pairs of electrons. A carbon atom can double bond with another carbon atom to get a full electron shell (as in ethene, below). A nitrogen atom triple bonds with a carbon atom in the Lewis Dot Structure below. Think of a double bond like holding both hands with Mike, and a triple bond a really tight hug.

All bonds take a lot of energy to break, but double and triple bonds are even more difficult to break. The kinds of bonds compounds have determine their chemical properties. If you've ever looked on the back of a food's package, you might have noticed the amount of saturated fat. Saturated fats are long carbon-hydrogen chains with single bonds. Unsaturated fats are long carbon-hydrogen chains with at least one double bond between two of the carbons. Saturated fats clog your arteries, but unsaturated fats are thought to lower cholesterol. It's amazing how just one double bond can make the difference between being healthy or harmful to your body!

Chromatography

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.

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.

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.

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 very polar molecule, so other polar molecules are attracted to it easily. 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 a water molecule bonding with another water molecule. The negatively charged oxygen atom (red) is attracted to the positively charged hydrogen atom (white) on the other 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.

Plants

Plants need to absorb light in order to create their own food. The chemical chlorophyll absorbs photons, which excite electrons in its central magnesium (Mg) atom. These electrons are channeled away from the chlorophyll to be used as energy to create food. Below is the chemical structure for chlorophyll. Chlorophyll a has one carbon and three hydrogen atoms in place of the "R" group, and Chlorophyll b has a carbon, a hydrogen, and an oxygen atom. The red highlighted part of the image is called the porphyrin ring. Each "point" in the diagram (i.e. each tip of the pentagons, hexagons, and jagged edges) represents a carbon atom. Hydrogen atoms are attached to most of the carbon atoms, but they are not shown in this structure.

When an object absorbs a certain wavelength (color) of visible light, that color does not appear in the object. When an object reflects a certain color (similar to a mirror, the light is not absorbed), you see that color with your eyes. If an object absorbs all colors, it appears to be black. If an object reflects all colors, it appears white to our eyes. The different colors we perceive around us are all produced by reflection and absorption. White light (e.g., sunlight) is actually a combination of all visible wavelengths. Molecules absorb/reflect different wavelengths more efficiently depending upon their structure. For example, the alternations of single and double bonds between the carbons in chlorophyll are what allow it to be so efficient at absorbing light. Chlorophyll absorbs red and blue light from the sun, but it does not absorb green light, it reflects it. This is why tree leaves are normally green. In the fall, the tree shuts down chlorophyll production to save energy, and recycles it for later use. Thus the green-reflecting chlorophyll disappears, and we are left with the orange and red carotenoids (pigments that capture light energy and transfer it to chlorophyll), and so the leaves turn red and orange.

## Terms and Concepts

• capillary action
• polarity of molecules
• miscibility
• stationary phase, mobile phase
• covalent bond, ionic bond, hydrogen bond
• Rf value
• paper chromatography
• thin-layer chromatography
• high-performance thin-layer chromatography
• solvent
• types of plant pigments (chlorophyll A and B, carotenoids, xanthophyll, carotene)
• plant structure (stomata, epidermis, collenchyma, etc.)
• plant cell structure (mitochondria, golgi bodies, chloroplasts, plastids, etc.)

### Questions

• Why do trees leaves turn different colors in the fall?
• What are the different pigments used for in the plant?
• What is the process of photosynthesis?
• What wavelengths of light do plants use?
• What makes a molecule polar or nonpolar? How does its polarity affect its interactions with other molecules?
• How do the polarities of the solid phase and liquid phase affect how far chemicals travel on the paper strip/plate?

## Bibliography

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:
You can start learning more about plants and pigments from these resources:

## Materials and Equipment

• Laboratory filter paper; available for purchase from the Science Buddies Store.
• Note: For this project we recommend using laboratory filter paper and acetone, but for a more detailed discussion about papers and solvents for doing paper chromatography experiments at home see the Paper Chromatography Resources page.
• acetone (nail-polish remover)
• water
• ruler
• pencils
• a small wide-mouth jar for the solvent chamber
• spinach leaves
• iceberg lettuce leaves
• marigold leaves
• small pipette, capillary tube, or eyedropper

## 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 nail-polish remover, size of paper strips, where the ink is placed onto the solid phase, etc., should remain the same throughout the experiment.

1. Grind up roughly equal samples of each of the different plant leaves and distribute them into test tubes. There should be at least three labeled test tubes for each type of plant (if using the Iceberg lettuce, "Iceberg 1," "Iceberg 2," and "Iceberg 3" are good names for the tubes).
2. Add enough acetone (nail-polish remover) to suspend the ground-up leaves.
3. Let the acetone/leaf mixture sit for 24 hours.
4. Take a paper strip use the ruler to draw a horizontal straight line 2 cm above the bottom (this is the origin).
5. Label what sample paper strip will contain (in pencil).
6. Fill the jar to a depth of 1 cm with the acetone (nail-polish remover).
7. Take one of the capillary tubes (or pipette or eyedropper) and fill with one of the samples.
8. Spot the sample in the middle of the origin (see illustration, below). You might want to practice a few times in order to get a nice round spot.

9. Place the strip of paper into the solvent chamber. Place a pencil across the top of the glass and tape the chromatography paper to it if the paper is not firm enough to stand on its own (see illustration, below).

10. Take out the paper strip when the solvent has almost reached the top.
11. Mark how far the solvent soaked up the strip/plate with a pencil.
12. Trace around the newly-moved spots so that if they fade, you can still use them to collect data.
13. Calculate the Rf value for each spot.
14. Repeat this experiment for each of the three samples.
15. Repeat this experiment for each type of plant leaf.

Questions

• What types of pigments do you think are present in each type of leaf? How can you tell? (Consider color, Rf value, etc.)
• How were the Rf values different for each pigment and leaf? Why? (Make sure to consider molecular structure, polarity, etc.)
• How do the different pigments help the plant? Why do different plants have different amounts and different types of these pigments?
An example of the experiment:
http://web.stclair.k12.il.us/splashd/chromexp.htm

## Variations

• If you really want to impress the judges, go even further in depth studying molecular polarity and the different types of molecular bonding (ionic, covalent, hydrogen, single, double, triple). This will help you better understand why different components move different distances depending upon the stationary and mobile phases used. It will also help you understand more how the chemical structure of molecules like chlorophyll enables it to carry out a specific function (like channel away electrons).
• Do plants' pigments vary with more or different types of light? You might try growing a few plants of the same type under different colored lights, and/or more intense light, and then do the chromatography procedure. If you are very precise when taking samples (i.e., measuring how much of the leaf you grind up for each plant), you could even comment how the amount of each pigment changes, if at all.
• You can also analyze the amino acids that are found in orange juice and lemon juice with chromatography.
http://www.baruch.cuny.edu/wsas/departments/natural_science/chemistry/chm_1000/11_chromat.doc
• For a less advanced chromatography project, see:

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