Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. To prepare the individual dye solutions, add each 0.5 g of solid dye to separate beakers with 100 mL of distilled or deionized water each and mix thoroughly.
2. Dissolve 1 g of solid sodium chloride in 1000 mL of distilled or deionized water and mix thoroughly.
3. See the Lab Hints section for suggestions for unknown mixtures of dyes.

Safety Precautions

The FD&C dyes are slightly hazardous by ingestion, inhalation and eye or skin contact. Red No. 40 may be absorbed through skin and Yellow No. 5 may be a skin contact sensitizer. All are irritating to skin and eyes. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Excess dye solutions and sodium chloride solution may be disposed of down the drain with plenty of excess water according to Flinn Suggested Disposal Method #26b.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. Both parts of this laboratory activity can reasonably be completed in one 50-minute class period. The Prelaboratory Assignment may be completed before coming to lab, and the data analysis and Post-Lab Questions may be completed the day after the lab.
• Suggestions for unknown mixtures: a) Red No. 3, Blue No. 2 and Yellow No. 5; b) Red No. 40, Yellow No. 6 and Blue No. 1; c) Red No. 3, Green No. 3 and Blue No. 1; d) Red No. 3, Red No. 40 and Blue No. 2. Mix equal volumes of each dye desired to create a mixture.
• In this activity, it is extremely important that the initial sample spots are as small as possible. If the spots are too large or if there is too much material (dye) on the initial spot, the students may only see a streak of color up the entire chromatogram.
• Allowing enough time for the development of the chromatography paper is critical. The chromatography paper must be left in the chromatography chamber long enough for the solvent to be drawn up near the top of the strip. Do not stop the development until the solvent front nears the top of the strip. Underdevelopment will lead to incomplete separation. Do not allow the solvent front to move off the paper, however.
• Solvent used for development can be reused. Leftover chromatography solvent can be saved and used by another class.
• Other size beakers or mason jars will also work for chromatography chambers—however, the taller the better. Covering alternatives for the chromatography chambers include plastic wrap, Petri dishes and other suitable inert coverings such as Parafilm M^®. If changing chromatography chamber components, reconfigure the chromatography paper to fit the alternative chromatography chamber. If needed, other filter paper sizes and shapes can be used, such as radial circular paper. Confirm all modified materials are acceptable by conducting a lab trial before introducing the procedure to students.
• Extra dyes are provided for chromatography and spectroscopy lab activities. Other FD&C food dye kits available from Flinn Scientific include Exploring Chemical Reactions with Food Dyes, Catalog No. AP7418, and Quantitative Determination of Food Dyes, Catalog No. AP7428.

Teacher Tips

• The structures of the food dyes are shown in the Supplementary Information on the Teacher PDF.
• Connect with the history of food dyes and have students find a substance now considered toxic that was once purposely added to food.
• Encourage students to try other solvent mixtures to achieve different or better separations.
• Students can construct a design of experiment (DOE) to identify the dyes in for colored food product,s such as M&Ms or Skittles. Candy may be placed in 5–6 drops of water. Stir the candy until the color dissolves. Repeat with two more candies. This is the color extract sample.
• Expand the students chromatographic repertoire by using SepPak C₁₈ Cartridges, such as the Flinn Scientific Kit AP9093.

Further Extensions

See Teacher PDF.

Answers to Prelab Questions

1. What is the primary factor determining the distance a sample will move along the chromatography paper?

   The attraction between the sample and the chromatography paper determines how much time the sample spends bonded to the paper and how much time it remains in solution. These factors determine the distance/time a sample will move.

2. Check for food labels at home or in a grocery store and find items with food dyes in them. List the food(s) and the FD&C dyes included in the ingredient list.

   Student answers will vary. Much of the processed food today has dyes in it.

   {12073_PreLabAnswers_Figure_6_Sample unknown mixtures results}

Sample Data

Answers to Questions

1. Calculate the R_f value for each dye in both the pure solutions and unknown mixtures. Record the results in the data table.

   See data table for sample data.

2. Identify the dyes present in the unknown mixtures. Include supporting data and reasoning for your conclusions.

   Answers will vary depending on the unknown mixtures. Students should cite references to visual color and rate of flow—R_f.

3. Compare the R_f values of the pure dyes. Which pairs of dyes appeared to have very similar properties, based on their R_f values, despite their different colors?

   Red No. 40 and Blue No. 2 had similar R_f values as did Blue No. 1 and Green No. 3.

4. Which food dye(s) had the greatest interaction with or affinity for the paper versus the solvent? Explain.

   A high interaction or affinity for the paper would take quite a bit of time and the sample would not move very much resulting in a low distance traveled. Red No. 3 has a very low distance traveled, showing it has more affinity for the paper than the solvent.

5. You are asked to mix an additional experimental unknown and want to make sure the mixture is a challenging one. Using observations and data from the completed experiment, develop a new three-dye component mixture that may be difficult to analyze. Explain why you chose this mixture.

   Several student answers may be correct. Examples may include: A mixture containing Blue No. 1 and Green No. 3 might be hard to separate due to rate-of-flow values. A mixture containing Yellow No. 5 and Yellow No. 6 is visually hard to separate.

Teacher Handouts

12073_Teacher1.pdf

References

Markow, P. G. The Ideal Solvent for Paper Chromatography of Food Dyes. J. Chem. Ed. 1988, 65, 10, pp 899–900.

Introduction

Food dyes have been used extensively for more than 100 years. Would you eat maraschino cherries if they were their natural color of beige instead of red or green? Explore the properties of the seven certified artificial food dyes with this chromatography activity.

Concepts

• Chromatography
• Polarity
• Food chemistry

Background

The use of color additives increased dramatically in the United States in the second half of the the nineteenth century. As the economy became more industrial, demographics shifted, fewer people lived on farms, and city populations grew. People were becoming more dependent on mass produced foods.

Color additives were initially used to make food more visually appealing to the consumer and, in some cases, to mask poor-quality, inferior or imitation foods. For example, meat was colored to appear fresh long after it would have naturally turned brown. Jams and jellies were colored to give the impression of higher fruit content than they actually contained. Some food was colored to look like something else—imitation crab meat, for example. Many of the food colorings and additives were later discovered to be harmful or toxic.

In 1883, Dr. Harvey Wiley began leading the United States Department of Agriculture (USDA) Bureau of Chemistry agency. His mission was to protect people by regulating the food industry and thus ensuring a safe food supply. Food coloring regulation is just one example of his efforts.

Food colorants were being added to food with little or no health testing. To propagate the food safety effort, in 1906 the USDA hired a consultant, Dr. Bernance Hess, to determine colorants that would be safe to consume in food. In 1907, the number of synthetic food dyes approved for use in the United States was reduced from 695 to just seven. As additional data was collected through consumer reports and laboratory testing, more dyes were eliminated or restricted. Only two of the original dyes from 1907 are still accepted for use today. Five others have been added between 1907 and 1971. In total, only seven dyes color all U.S. food today. All of the FD&C approved food dyes are charged, water-soluble organic compounds that bind to natural ionic and polar sites in large food molecules, including proteins and carbohydrates.

The seven food dyes can be separated and identified by paper chromatography. The word chromatography is derived from two Greek words meaning color (chroma) and writing (graphein)—“color writing.” The term was coined by the Russian chemist Michael Tswet (1872–1919) in 1903 to describe a new technique he had invented to separate the pigments in green plant leaves. Since Tswet’s discovery, many different types of chromatography have been developed for separating the components in a mixture. Paper chromatography is an example of a more general type of chromatography called adsorption chromatography. The paper acts as an adsorbent, a solid which is capable of attracting and binding the components in a mixture (see Figure 1). The mixture to be separated is “spotted” onto the surface of the paper and a solvent is then allowed to seep or flow through the paper by capillary action. If one of the components in the mixture is more strongly adsorbed onto the paper than another, it will spend a smaller fraction of time free in solution and will move up the paper more slowly than the solvent. Components that are not strongly adsorbed onto the paper will spend a greater fraction of time free in solution and will move up the paper at a faster rate. This “partitioning” of the components of a mixture between the paper and the solvent separates the components and gives rise to different bands or spots. If the components of the mixture are colored, like the food dyes or pigments in an ink, the bands are easily distinguished.

Different samples will spend varying amounts of time interacting with the paper and the solvent. Through these different interactions, the samples will move different distances along the chromatography paper. In general, food dye molecules that are more highly charged, that is, have more ionic binding sites, and are more polar will be attracted to the paper more strongly and will thus have lower R_f values. The distance a sample moves along the chromatography paper is compared to the overall distance the solvent travels—this ratio is called the R_f or rate of flow.

Experiment Overview

The purpose of this experiment is to use paper chromatography to separate the components of the seven Food, Drug and Cosmetic (FD&C) food dyes. The R_f value of each substance will be calculated and compared to determine the composition of food dyes in an unknown mixture.

Materials

Prelab Questions

1. What is the primary factor influencing the distance a sample will move along the chromatography paper compared to the solvent?
2. Check for food labels at home or in a grocery store and find items with food dyes in them. List the food(s) and the FD&C dyes included in the ingredient list.

Safety Precautions

The FD&C dyes are slightly hazardous by ingestion, inhalation, eye and skin contact. Red No. 40 may be absorbed through skin and Yellow No. 5 may be a skin contact sensitizer. All are irritating to skin and eyes. Avoid contact with eyes, skin and clothing. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Use proper exhaust ventilation to keep airborne concentrations low. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

1. Cut 2 cm off of one side of the chromatography paper making the new paper dimensions 20 cm x 18 cm (see Figure 2). Note: Handle the paper by the edges so the analysis area is not accidently compacted or contaminated.
   {12073_Procedure_Figure_2}
2. Orientate the chromatography paper so that it is 20 cm wide and 18 cm tall.
3. Using a ruler and a pencil, draw a faint line 1.5 cm from the bottom of the paper across the entire width of the paper (see Figure 3).
   {12073_Procedure_Figure_3}
4. Using the same ruler and pencil, draw nine small dots. Measure 2 cm from the edge for the first dot on the line drawn in step 3 and then add a dot every 2 cm across the line. Label the first dot 1 (see Figure 3).
5. Obtain the seven 0.5% individual dye solutions and two “unknown” dye mixtures.
6. List on the data table the location where each dye will be placed. Note: Dye samples may be spotted in any order but location must be recorded.
7. Label the top of the chromatography paper in pencil, dot 1 = 1, dot 2 = 2.
8. Using a clean toothpick for each individual dye sample, spot the chromatography paper by putting the toothpick into the dye sample solution and then touch the tip of the toothpick gently onto a self-designated pencil dot. Repeat the procedure as necessary to increase the concentration of the sample but do not increase the size of the dot.
9. After spotting all nine samples, wait 1–2 minutes for the samples on the chromatography paper to completely dry.
10. While the sample is drying obtain the 1000-mL tall-form beaker and watch glass that is sized to fit the tall-form beaker. (Note: See teacher list for other options if tall-form beakers are not available.)
11. Pour 50 mL of 0.1% NaCl solution into the tall-form beaker and cover the top of the beaker with a watch glass. This is the chromatography chamber. The 0.1% NaCl is the developing solvent.
12. Once the sample is dry, wrap the chromatography paper into a cylinder, and slightly overlap the blank ends. Staple, being careful not to disrupt the samples (see Figure 4).
    {12073_Procedure_Figure_4}
13. Remove the watch glass from the tall-form beaker and carefully place the cylinder-shaped chromatography paper into the prepared chromatography chamber with the sample end down (as shown in Figure 5). Do not get any solvent on the upper portion of the chromatography paper. The sample spots must remain above the level of the solvent. If the solvent level is too high, the samples will dilute into the solvent!
    {12073_Procedure_Figure_5}
14. Place the watch glass back on the tall-form beaker. Allow the chromatography paper to develop. Note: This should take 15–25 minutes.
15. When the developing solvent is within 1–2 cm of the top of the chromatography paper, stop the run by removing the rolled chromatography paper from the beaker.
16. With a pencil, lightly draw a line to mark the distance the solvent traveled to the top of the chromatography paper. This is called the solvent front.
17. Gently remove the staples and lay the chromatography paper flat.
18. Measure the distance from the pencil line at the bottom of the chromatography paper to the solvent front. Record this distance in cm on the worksheet.
19. In pencil, trace the shape of each dye band or spot to mark the location of each separated band on the chromatography paper. This should be done immediately because the color and brightness of some spots may fade over time.
20. Measure the distance traveled in cm by each dye in each pure solution or mixture. Measure from the line at the bottom of the paper to the center of each band. Record the results in cm on the worksheet.

Student Worksheet PDF

12073_Student1.pdf