Chromatography is an important technique in the chemistry lab. This activity uses a popular beverage for a safe, engaging lab experience for students of all ages.
What makes grape-flavored Kool-Aid® purple? Food dye, of course. Color additives are common in the foods on supermarket shelves. From foods and drinks to cosmetics, pharmaceuticals, and toiletries, color additives are used to make products more appealing. When we look at yellow candy, we think lemon. When we see red, we visualize and taste strawberry or cherry. The color orange reminds us of the fruit, and green summons the taste of lime or mint.
An analysis of a mixture requires that the components be separated and studied individually. One of the most powerful techniques used to separate substances is chromatography, a technique used to separate substances by partitioning them between mobile and stationary phases.
Two common methods of chromatography are paper chromatography and thin-layer chromatography, or TLC. In each of these techniques, there is a mobile solvent phase and a stationary solid phase (paper or a silica gel-coated plate). As the solvent migrates up the plate due to capillary action, it comes in contact with an applied dot of a sample to be analyzed. The solvent dissolves the dot into the mobile phase. Equilibrium is established for each chemical in the mixture to be analyzed. Part of the time the particular chemical is moving in the mobile phase, while at other times it is adsorbed onto the stationary phase. The chemicals in the sample separate due to differences in polarity and solubility. Because the silica gel or paper is more polar than the solvent system, nonpolar substances will spend more time dissolved in the less polar mobile phase and will therefore travel farther up the plate. The migration distance is dependent on a number of factors, including the polarity of the stationary phase, the polarity of the mobile phase, the compounds being separated, and the concentration of the sample.
The ratio of the distance a substance moves up the chromatography plate to the distance the mobile phase (the solvent front) travels is called the retardation factor, commonly written Rf.
Rf = distance traveled by substance
distance traveled by solvent front
Figure 1 Chromatography results.
An Rfvalue is characteristic of a substance when the same solvent system and type of chromatography plate are used. It should be noted, however, that Rfvalues are difficult to reproduce accurately. Therefore, Rf values alone are not sufficient to identify a substance. The solvent used to perform chromatography is dependent on the mixture being separated. When performing chromatography, scientists typically test many different solvents and analyze the results. Solvents can even be diluted with other miscible liquids to change the results of the chromatography experiment. Each different solvent produces different Rf values.
Wear appropriate personal protection equipment (PPE) such as gloves, chemical-splash goggles, and lab coats or aprons.
For each student or group
- Chromatography Paper Strip
- Capillary Tube
- Test Tube
- Test Tube Rack or Beaker to Hold Tube
- Disposable Pipette
- Paper Towel
- Straight Pin
For the classroom
- Graduated Cylinders (10 mL and 100 mL)
- Digital Balance (0.1 g)
- Grape Kool-Aid®
- Plastic Wrap
- Medicine Cup
- Table Salt (NaCl)
- Distilled Water
- Prepare a concentrated solution of the grape Kool-Aid® by mixing 2 g of Kool-Aid® powder with 5 mL of water in a medicine cup.
- Prepare 100 mL of a dilute NaCl solution (0.1 g of NaCl in 100 mL water).
- Cut chromatography strips to fit in tubes. Strips should be narrow enough to fit in test tubes but slightly longer than the tubes.
- Measure 1.5 cm from the bottom of the chromatography strip and draw a line across the paper with a pencil. This line will be the starting point or origin for each applied sample.
Figure 2 Draw a line across the paper with a pencil.
- Insert a capillary tube into the concentrated grape Kool-Aid® solution. Place your forefinger of the top end of the tube to retain a small column of sample (3 to 4 mm).
- Place 1 small dot of the sample in the center of the pencil line by touching the paper and briefly releasing your finger from the top of the capillary tube.
- Place a test tube in the rack.
- Transfer about 1 mL of the dilute NaCl solution to the test tube.
- Insert a straight pin near the top of the chromatography strip so that the strip will reach into the solution when suspended in the tube by the pin. See Fig. 3.
Figure 3 Chromatography paper strip suspended in a test tube.
- Adjust the strip’s position as needed so that the strip is in the center of the tube, not touching the walls. The spot should be above the surface of the solvent. See Fig. 4.
Figure 4 Correct location of the dot of a sample on the chromatography plate.
- Allow the solvent front to wick up the paper and through the spot until the front reaches within a centimeter of the top. This should take around 10 to 15 minutes.
- Remove the strip from the test tube, remove the pin, place the strip on a paper towel, and mark the solvent front with a pencil before it dries.
- With a pencil, mark the middle of the colored spots that were deposited above the origin.
- Measure the distance in millimeters that the solvent front traveled. Record this information in a data table.
- Measure the distance in millimeters that each color spot moved, from the origin to the mark in the middle of the spot. Record this distance in your data table.
- Calculate the Rf value for each of the spots, noting their color.
Sample Data Table
Spot Number from Origin
|Spot Color||Distance Solvent Traveled (mm)||Distance Spot Traveled (mm)||
From this experiment students should observe that the grape Kool-Aid® is colored with a mixture of red and blue food dyes. If you have access to FD&C food dyes, students can repeat the procedure to better determine specifically which food dyes are used in this product. Alternatively, students may investigate other foods they suspect of containing food dyes or mixtures of food dyes such as hard candies, other Kool-Aid® flavors, or other color beverages.
- Carolina ChemKits®: Introduction to Thin Layer Chromatography (items #688950 and #688951)
- Carolina ChemKits®: Thin Layer Chromatography of Amino Acids (items #840640 and #840641)
- Carolina Investigations® for AP® Chemistry: Principles of Chromatography Kit (item #840574)
- Thin-Layer Silica Gel Chromatography Plates (item #689456)
- Carolina Chemonstrations®: Rainbow Indicators Kit (item #840333)