Chromatography of Food Dyes: A Simple Demonstration for Actively Engaging High School Students in the Chemistry of Foods. Grady W. Chism.

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1 INAUGURAL ISSUE Chromatography of Food Dyes: A Simple Demonstration for Actively Engaging High School Students in the Chemistry of Foods Grady W. Chism Abstract: A simple demonstration of the chromatography of food dyes suitable for high school students is presented. Reversed phase mini-columns are utilized to separate dyes in grape and lime drinks. The exercise introduces chromatography and reinforces the concept of polarity. It has been successfully used to actively engage students for periods of 30 to 60 min. Introduction This demonstration was designed to be used with high school students. The purpose of the demonstration was to give an example of how chemistry is used in food science and to get the students actively engaged in learning. This demonstration or variations of it have been used more than 20 times and it always is successful in engaging students. Because of the limited resources needed to conduct the demonstration, it can be easily done off site as well as in a laboratory setting. The demonstration should begin with a brief discussion of safety that includes use of safety glasses, admonishment not to drink any of the samples and a caution about inhaling vapors. The importance of color in the acceptance of foods (Francis 1999; Hutchings 1999) and a brief introduction to chromatography should be presented. Providing examples of foods having poor or strange colors may be an asset to beginning this discussion. Ideally, this should be limited to 5 min or less. Materials Solvents Methanol, ethanol or isopropanol (70% isopropanol can be purchased in drug store and avoids the need to purchase a larger volume of solvent) and water. Food products Grape soda with typical ingredients including carbonated water, high fructose corn syrup, citric acid, sodium benzoate (preservative), modified food starch, artificial flavor, Blue #1, Red #40.) Lime sports drink with typical ingredients including water, sucrose syrup, glucose-fructose syrup, citric acid, natural and artificial flavors, salt, sodium citrate, monopotassium phosphate, ester gum, sunflower oil, Yellow #5, Blue #1. Other drinks using dyes as colorants or mixtures of dyes will work as well. Equipment per student group: 1 10-mL disposable syringe without a needle 1 C 18 minicolumn (Alltech Maxi-clean SPE #20928, , Alltech Associates, Inc., Deerfield, Ill., U.S.A.) 6 Small beakers or cups 1 Small test tube 1 Small graduated cylinder (if students are asked to dilute solvents). Procedure (1) Place the end of the syringe into the beaker containing the grape soda and draw about 2mL of sample into the syringe. (2) Attach the minicolumn to the syringe. (3) Push on firmly. (4) Point the syringe away from you and towards a cup on the bench and then 18 JOURNAL OF FOOD SCIENCE EDUCATION Vol. 1, Institute of Food Technologists

2 Separating food dyes demonstration... slowly push in the plunger until the soda has been forced out of the syringe. (5) Record your observations. (6) Detach the column from the syringe. (7) Draw 2 ml of diluted solvent into the syringe. (8) Reattach the column, push the plunger as before, and collect the fluid in a small test tube. (9) Record your observations. (10) Detach the column and draw 2 ml of 70% isopropyl alcohol into the syringe. (11) Reattach the column and push the fluid through as before. (12) Record your observations. (13) Detach the column and draw 2 mls of air into the syringe, reattach the syringe and remove the remaining solvent from the column. (14) Rinse the column with 2 mls of water and then remove the water. You are now ready to try a different sample. Discussion This activity introduces or reinforces the concept of polarity and provides an introduction to chromatography. It is simple to do and less demanding than the separation of natural pigments (Curtwright and others 1999). This works best with groups of two. It does not work well if the individual groups contain more than three students. If you provide the students with appropriate dilutions of the solvent, the exercise can be completed in less than 30 minutes. Asking the students to make their own dilutions or providing more samples can extend the time to 60 minutes. Most students become actively engaged in the activity for the entire period. With rare exception, the students appear to enjoy the activity and there are always a few that want to take the minicolumn home for further experimentation. Two printable handouts for this exercise are provided. The regular form has been used effectively with a variety of high school students. The advanced one has worked well with honors students and ones who have completed at least a portion of a high school chemistry class. This exercise can easily be extended by collecting the separated dyes and analyzing them spectrophotometrically. Letting the students scan the dyes or providing them with a scan can lead to a discussion absorption and reflection in color vision. References Curtwright R, Emry R, Markwell J Student understanding of chromatography: A hands-on approach. J Chem Educ 76:(2) Francis FJ Colorants. St. Paul, MN: Eagan Press. 144 p. Hutchings JB Food Color and Appearance. 2nd Ed. Gaithersburg, MD: Aspen Publishers. P 1-30, Author is with the Dept. of Food Science and Technology, Ohio State University, 2015 Fyffe Rd., Columbus, OH 43210, ( chism.2@osu.edu) Reproducible worksheets on next three pages Vol. 1, 2002 JOURNAL OF FOOD SCIENCE EDUCATION 19

3 JFSE: Journal of Food Science Education Handout for the short version Chromatography of Food Dyes Put on your safety glasses. Nothing we are using today is very toxic, but we will be using isopropyl alcohol which will cause pain if you squirt it in your eyes. Avoid inhaling the solvent vapors. Do not drink any of the samples we are using today. Chromatography is a means of separating compounds based on differences in the affinity (attraction) to a support. In our case, we will use a column containing silica with a long-chain hydrocarbon (C 18 ) attached to it. The hydrocarbon makes the column have a high affinity for compounds that are soluble in fat. We often refer to this as a non-polar column. If we had a column that had more affinity for water than fat, we would refer to this as a polar column. Since we are using a liquid to move our sample through the column we call this liquid chromatography. Materials: Grape soda Lime sports drink 1 10 ml disposable syringe without a needle 1 C 18 mini-column (Alltech Maxi-clean SPE #20928, , Alltech Associates, Inc., Deerfield, Ill., U.S.A.) 6 Small beakers or cups 1 small test tube Water 70% isopropyl alcohol 14% isopropyl alcohol Procedure Grape soda. (1) Place the end of the syringe into the beaker containing the grape soda. You need to have a couple of milliliters to inject onto the column. What happened when you pulled back the plunger to draw the soda into the syringe? Question for later Why did this happen? What is the principle governing this? (2) Attach the mini-column to the syringe. Push on firmly. Point the syringe away from you and towards a cup on the bench and then slowly push in the plunger until the soda has been forced out of the syringe. What do you observe in the column? What color was the fluid coming out of the syringe? Why? (3) Detach the column from the syringe. (4) Draw 2 ml of 14% isopropyl alcohol into the syringe. (5) Reattach the column and push the plunger as before and collect the fluid in a small test tube. What happens? Comment on both the column and the fluid coming out of the column. Look at the ingredient label to find out which dyes are used in this product. Can you make a conclusion about whether Red #40 or Blue #1 is more fat soluble? (6) Detach the column and draw 2mL of 70% isopropyl alcohol into the syringe. (7) Reattach the column and push the fluid through as before. What do you observe? (8) Detach the column and draw 2mLs of air into the syringe, reattach the syringe and remove the remaining solvent from the column. (9) Rinse with 2mLs of water and then remove the water. You are now ready to try a different sample. Lime sports drink (10) Repeat what you did with grape soda. Compare your observations for the colors in Lime sports drink. What happens? Look at the ingredient label to find out which dyes are used in this product. What conclusions can you make about Yellow #5, Red #40 and Blue#1? 50/50 Lime sports drink /Grape. (11) Repeat what you did with grape soda. What happens? What conclusions can you make about Yellow #5, Red #40, and Blue #1? Questions What does FD&C stand for? Which of the three dyes is the most fat soluble? Which is the least fat soluble? If you had a mini-column that was polar instead of non-polar like the one you used, which of the dyes would you expect to bind most tightly to the column? Look at the ingredient statements for the two drinks. What are the major differences? Why are each of these ingredients added to the drinks? Resources Francis FJ Colorants. St. Paul, MN: Eagan Press. 144 p. Hutchings JB Food Color and Appearance. 2nd Ed. Gaithersburg, MD: Aspen Publishers. 1-30, JOURNAL OF FOOD SCIENCE EDUCATION Vol. 1, 2002

4 Separating food dyes demonstration... Handout for more advanced students. Chromatography of Colors Put on your safety glasses. Nothing we are using today is very toxic, but we will be using alcohols, which will cause pain if you get any in your eyes. Avoid inhaling the solvent vapors. Do not drink any of the samples we are using today. Chromatography is a means of separating compounds based on differences in the affinity (attraction) to a support. In our case, we will use a column containing silica with a long chain hydrocarbon (C 18 ) attached to it. The hydrocarbon makes the column have a high affinity for compounds that are soluble in fat. We often refer to this as a nonpolar column. If we had a column that had more affinity for water than fat, we would refer to this as a polar column. Since we are using a liquid to move our sample through the column we call this liquid chromatography. Color is a very important attribute of foods. While we have chosen to have you work with artificial colors today, the chemistry of the natural pigments like chlorophyll, anthocyanins, and carotenoids is quite interesting and important to foods. The compounds we will be working with today are know as FD&C dyes. This means they are approved for use in food, drugs, and cosmetics. The color of these dyes comes from the large conjugated double bond system which lowers the energy required to excite an electron to the point that light in the visible range has enough energy to make this excitation occur. When this happens, visible light is absorbed and we see color. Ask your chemistry or physics teacher to explain this to you. In addition to the large conjugated system, these dyes have a sulfonic acid group that gives them a negative charge at the ph of most foods. This charge makes these dyes soluble in water. Red #40 Blue #1 Yellow #5 Based on the structures, make a guess as to which of these would be the most polar and which would be the least polar. Hint: Polar groups are OH, SO 3 Na, SO 3 H. Most polar In between Least polar Briefly describe the reason for your choice Now that you have a hypothesis (that is, the relative polarity of the dyes), can you test it using the mini-columns we have provided? Our minicolumn is nonpolar. Which of the dyes do you expect to stick most tightly to the column? Which one would stick the least? Why? Likes attract likes, so our nonpolar minicolumn will have the strongest attraction for the most nonpolar dye. Try this out using the grape soda as the source of the colors. Look at the label to see what colors are present. Materials Grape soda Lime sports drink 1 10-mL disposable syringe without a needle Vol. 1, 2002 JOURNAL OF FOOD SCIENCE EDUCATION 21

5 JFSE: Journal of Food Science Education 1 C,8 minicolumn (Alltech Maxi-clean SPE #20928, , Alltech Associates, Inc., Deerfield, Ill., U.S.A.) 6 Small beakers or cups 1 small test tube Water Methanol or Ethanol or 70% isopropanol Procedure Place the end of the syringe into the beaker containing the grape soda. You need to have a couple of milliliters to inject onto the column. What happened when you pulled back the plunger to draw the soda into the syringe? Question for later. Why did this happen? What is the principle governing this? Attach the minicolumn to the syringe. Push on firmly. Point the syringe away from you and towards the bench and then slowly push in the plunger until the soda has been forced out of the syringe. What do you observe in the column? What color was the fluid coming out of the syringe? Why? Detach the column from the syringe. Draw 2 ml of solvent into the syringe, reattach the syringe and slowly push the plunger so that the solvent flows slowly through the syringe. Observe the colors in the column as well as in the drops falling from the column. What did you observe? Does this prove your hypothesis? Part of it? If all of the color comes out together, the solvent is too strong. This means that it is too nonpolar. You can make the solvent less nonpolar (that is, more polar) by adding water to it. (Suggestion: mix equal volumes of solvent and water. If this is still too strong, dilute the mixture in half again. Keep going until you have good separation.) Rinse the column with 1 ml of water. Remove the syringe, draw 1 ml of air into the syringe, and use this to remove the water from the column. You are now ready to start again. What final concentration worked best? Now that you have figured out Blue #1 and Red #40, it is time to work on Yellow #5 and Blue #1. We will use a lime-flavored sports drink as the sample. First, write your hypothesis for these two colors. Hypothesis: Inject 2 ml of lime drink like you did with the grape soda. What happens? Choose the solvent combination you think will separate the blue from the yellow and try it. If time permits, write a hypothesis for separation of the combination of Blue #1, Red #40 and Yellow #5. Hypothesis: Test it. What combination of solvents works best to separate all three colors? Questions: What does FD&C stand for? Which of the three dyes is the most fat soluble? Which is the least fat soluble? If you had a minicolumn that was polar instead of nonpolar like the one you used, which of the dyes would you expect to bind most tightly to the column? Look at the ingredient statements for the two drinks. What are the major differences? Why are each of these ingredients added to the drinks? Resources Francis FJ Colorants. St. Paul, MN: Eagan Press. 144 p. Hutchings JB Food Color and Appearance. 2nd Ed. Gaithersburg, MD: Aspen Publishers. P 1-30, JOURNAL OF FOOD SCIENCE EDUCATION Vol. 1, 2002

6 Separating food dyes demonstration... Supplementary Material The following supplementary material is available for this article: Spanish version. Cromatografía de Colorantes para Uso en Alimentos: Una simple demostración para alumnos de Preparatoria involucrados activamente con la Química de los Alimentos This material is available as part of the online article from: (This link will take you to the article abstract). Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Vol. 1, 2002 JOURNAL OF FOOD SCIENCE EDUCATION 23