n To become familiar with techniques separating mixtures
n To learn about chromatography, specifically paper chromatography
The word chromatography means color-writing and the name was chosen at the beginning of this century, when the method was first used, to separate colored components from plant leaves. The name ‘chromatography’ may be misleading, because separation techniques do not depend on color. Many variations of this technique exist today. Chromatography is used as a very reliable and fast technique to separate a variety of natural and synthetic mixtures into their components. Several types of chromatography exist today, among which are paper chromatography, thin-layer chromatography or TLC, liquid-liquid chromatography, gas chromatography, or high performance liquid chromatography or HPLC.
Chromatography is a separation technique used to separate the components of a mixture. Many separation methods rely on physical differences between the components of a mixture. Undoubtedly, you are already familiar with several means chemists use to effect separations based on physical differences. These techniques include: Filtration, where separation may be effected because substances are present in different states (solid vs. liquid); Centrifugation, where separation is effected by differences in density; and Distillation, where separation is effected by taking advantage of differences in boiling temperatures of the various components. In this laboratory exercise, we will effect a separation of a mixture of food dyes using paper chromatography.
In a typical experiment, a mixture of different chemicals is partitioned between a stationary phase and a moving phase (eluent or solvent). The moving phase may be a liquid or a gas; the stationary phase is typically a solid. As the mobile phase flows over the stationary phase, the components in the mixture are carried along. The ability to separate a mixture into its components basically depends on two fundamental concepts: solubility and adsorption. The better soluble a component is in the moving phase the faster it will be transported along the stationary phase. Adsorption refers to the ability of a substance to ‘stick’ (or be adsorbed) to the surface. The better the component is adsorbed to the stationary phase, the slower it will be transported along the stationary phase. As the mixture moves over the stationary phase, the components in the mixture move further and further apart into discrete zones.
Paper chromatography uses ordinary filter paper as the stationary phase, which consists primarily of cellulose. Thin-layer chromatography (abbreviated TLC) uses a thin glass plate coated with either aluminum oxide (alumina) or silica gel as the solid phase. The moving phase is a liquid and is chosen accordingly with respect to the properties of the components in the mixture.
In a typical experiment, a single drop of the mixture is applied at one end of a strip of filter paper. The filter paper is then placed in a shallow layer of solvent or solvent mixture in a jar or beaker. Since the filter paper is permeable to liquids, the solvent begins to rise by capillary action. As the solvent rises to the level at which the spot of mixture was applied, the components of the spot that are completely soluble will be moving along with the solvent upward (refer to Figure 1). The rate at which a component will move along depends on the relative tendency to be dissolved in the solvent and adsorbed on the paper. By this means, the original spot of the mixture is spread out. The net effect is that the components separate from each other and spread out into a series of bands or new spots.
Set up for thin layer chromatography
It is possible to describe the position of spots (so the substances that have separated) in terms of their retention factor, the Rf value (Figure 2). The retention factor is defined as:
Rf = distance traveled by spot
distance traveled by solvent
Because the retention factor for a particular mixture may vary depending on the conditions, a sample of known composition is typically analyzed at the same time on the same sheet of paper or slide. The Rf value is a characteristic property of a given compound in a given solvent in a chromatography experiment. (See page 76 of your text for more information on chromatography)
Figure 2: Example for calculating the Rf value
In today’s experiment, you will analyze (not eat; sorry) the shell coating of M&M candies. Some M & M’s use yellow #5 (tartrazine) as color. This dye causes allergic reactions in some people and must therefore be named as an ingredient in products that contain it. The M&M package states that yellow #5 is one of the colors added. The big question is then: is the dye present in all candies or only in some of the candies of certain colors? The answer to this question is important to the person who is allergic to yellow #5 and wants to know which candies are safe to eat.
In the second part of today’s experiment, you will analyze felt-tip pen inks. Felt-tip pens come in many different colors but most colors are often mixtures of primary color inks. We will use water-soluble pens and further study the importance of the proper solvent or solvent mixture in paper chromatography for optimal separation.
MATERIALS AND EQUIPMENT
felt-tip pens, both water-soluble and permanent
Whatman #1 chromatography paper or filter paper
250 mL beaker
0.1 % NaCl, acetone, ethanol
butanol : water : ethanol : acetic acid mixture (120:40:20:1)
Optional: Bring in some different M&M candies, or any other color coated candies.
More info on M&M chromatography can be found at
Part I (M&M candies)
1. A 10 x 10- cm strip is cut from Whatman #1 chromatography or filter paper. Along one side, 1 cm from the edge, draw a straight pencil line, and make seven marks at about 1 cm intervals. Six of these are identified by code for the different colored candies, and the seventh for the standard. The standard will be the compound tartrazine.
2. To separate the dyes used to coat M&Ms, you must first prepare a solution of the coating dyes. To do this, place four M&M’s of the same color into a small beaker. Add 3 mL of a 50/50 mixture of water and ethanol and swirl the solvent until the candy coating has dissolved. Remove the M&Ms from the solvent before the chocolate center is exposed. The dye solutions are not very concentrated, so you will need to spot these dye mixtures several times to obtain a sufficiently dark spot. Dry each spot between applications of the mixture to maintain as small and concentrated a spot as possible. Try to keep your spots less than 4mm in diameter.
3. Repeat the procedure for all color-coated candies. Gently dry the paper by moving it in the air.
4. Use a 250 mL beaker. Add 0.1% sodium chloride, (solvent or eluting solution) so that the bottom of the beaker is covered. Place the filter paper in the eluting solution, with the sample spots down near the liquid surface, but not in the liquid (Figure 3). Tightly seal the beaker with clear film and let it stand undisturbed. The solvent will gradually rise by capillary action, carrying the components in the spots along.
Figure 3: Arranging the filter paper in a circle
5. The paper is taken from beaker when the solvent front is about 2 cm from the top. Because the paper remains wet for several more minutes and the solvent continues to rise, it is very important to draw the solvent line and circle the colored spots immediately.
6. Measure the distance from the straight line on which you applied the spots to the solvent front. Then measure the distance from the pencil line to the center of each colored spot and calculate the Rf value from the two number you obtained. Repeat the procedure for each colored candy spot that you applied.
7. Compare the Rf values that you calculated to the ones given in the Table to determine which colors are present in each candy that you investigated.
Each organic compound, and many inorganic compounds, has a characteristic Rf value in a certain solvent. The colors of the shells of M & M candies contain food coloring that are organic dyes and therefore display characteristic Rf values.
Table I The composition of the candy shells is shown in the Table below. (J.Chem.Edu, 2002, Vol.79, 847)
1. A 10 x 10- cm strip is cut from Whatman #1 chromatography or filter paper. Along one side, 1 cm from the edge, draw a straight pencil line, and make four marks at 1 cm intervals. Obtain several water-soluble pens from your Instructor.
2. Apply a single small spot of different ink to each of the marks on the filter paper. Allow the spots to dry completely. Gently dry the paper by moving it in the air.
3. Use a 250-mL beaker. Add the butanol : water : ethanol : acetic acid mixture (120:40:20:1), which is the solvent or eluting solution. Make sure that the bottom of the beaker is covered. Place the filter paper in the eluting solution, with the sample spots down near the liquid surface, but not in the liquid (Figure 1). Tightly seal the beaker with clear film and let it stand undisturbed. The solvent will gradually rise by capillary action, carrying the components in the spots along.
4. The paper is taken from the beaker when the solvent front is about 2 cm from the top. Because the paper remains wet for several more minutes and the solvent continues to rise, it is very important to draw the solvent line and circle the colored spots immediately.
5. Measure the distance from the straight line on which you applied the spots to the solvent front. Then measure the distance from the pencil line to the center of each colored spot and calculate the Rf value from the two numbers you obtained.
6. Repeat the procedure with several permanent ink markers using the same solvent.
Dispose of the solvents and the used filter papers in the designated container in the fume hood.