Activity pubs.acs.org/jchemeduc
Engaging Students in Real-World Chemistry through Synthesis and Confirmation of Azo Dyes via Thin Layer Chromatography To Determine the Dyes Present in Everyday Foods and Beverages Kristi Tami, Anastasia Popova, and Gloria Proni* Science Department, John Jay College of Criminal Justice, New York, New York 10019, United States S Supporting Information *
ABSTRACT: In order to make science more appealing to students, it is imperative that a real-world approach to the principles of chemistry be taught in the classroom enabling students to see the applicability of chemistry to their everyday lives. In this laboratory activity, students were asked to bring in everyday food items that contain food dyes. The students then synthesized the FD&C dyes yellow 5 and yellow 6. The dyes were then run, along with their food items, on a TLC plate in order to determine what dyes were present in the foods and drinks they consume. KEYWORDS: High School/Introductory Chemistry, Second-Year Undergraduate, Amines/Ammonium Compounds, Chromatography, Aromatic Compounds, Analytical Chemistry, Hands-On Learning/Manipulatives, Applications of Chemistry
■
INTRODUCTION According to a recent Public Agenda poll, 85% of high school students believe that having an understanding of advanced science is important.1 Even though 79% of students think that advanced math and science skills will open up good jobs and career opportunities, they do not see themselves in a career involving science. According to another Public Agenda poll, 41% of the students would be unhappy in a career that required a significant amount of science and math.2 One reason for this might be that some high school students find chemistry courses boring, too abstract, or not relevant to the real world.1 Other studies have found that this is a generalized problem around the world.3 In order to increase interest in science, particularly in chemistry among high school students, new laboratory experiments need to be designed. Since most students do not see how chemistry relates to everyday life, the best way to increase students’ interest in chemistry is to engage them in a lab where the abstract nature of chemistry and the reality of life come together. As many foods today contain some sort of food coloring, laboratories involving food dyes seem to be an appropriate place to involve students in science. Numerous experiments involving FD&C dyes can be found in scientific literature. Most of the experiments involve the synthesis of various dyes or their purification and quantification.4 Others involve paper chromatographic separation of foodextracted dyes.5 This experiment combines the synthesis of two FD&C dyes, yellow 5, 1, and yellow 6, 2, and the verification of the synthesized dyes’ identities by comparison with dyes currently used in coloring foods using thin layer chromatography (Figure 1). Working with familiar materials not only helps spark the students’ curiosity, it also makes the explanation of chemistry concepts more relevant to them.6 Students were shown the molecular structures of the synthesized dyes without emphasizing organic nomenclature or functional groups beyond identifying the azo group, and comparing similarities and © XXXX American Chemical Society and Division of Chemical Education, Inc.
differences in structure. An important note on the structure is the azo group, which is responsible for the carcinogenic properties of some of the azo dyes, since they are known to break down into aromatic amines. While the dyes synthesized in this experiment have not been determined to be carcinogenic, there are several studies that indicate that they can produce very serious health conditions when consumed.7 We believe it is important for students to be aware of what is contained in the foods they eat and drink. This experiment was designed to allow students to work independently, share data, and combine their experiences in order to reach a conclusion. Students learned the basic theory behind organic synthesis and the thin layer chromatography separation technique. Students gained practical experience in using an analytical balance, synthesizing the dyes, working with potentially dangerous solutions, spotting a TLC plate, obtaining results from the TLC plate using UV light, measuring the distance traveled by the spots, and calculating Rf values. Lab time required is 3 h. The procedure is appropriate for high school, and general science classes. This laboratory experience was carried out by a selected group of middle- and highschool students (grades 7−12) participating in the Science and Technology Entry Program (STEP). It can also easily be adapted to an organic chemistry lab with more emphasis on the synthesis, mechanism of the reactions, and structure and nomenclature of the compounds. The organic chemistry students who are currently performing this laboratory experiment at John Jay College as part of their Organic Chemistry II curriculum receive detailed instructions regarding the diazotization reaction during their prelab experience. Any organic chemistry textbook has clear mechanistic details regarding this reaction. Received: May 9, 2016 Revised: January 19, 2017
A
DOI: 10.1021/acs.jchemed.6b00334 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Activity
Figure 1. Reaction scheme of the syntheses of (E)-5-oxo-1-(4-sulfophenyl)-4-((4-sulfophenyl)diazenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (Tartrazine or FD&C yellow 5, 1) and 6-hydroxy-5-((4-sulfophenyl)diazenyl)naphthalene-2-sulfonic acid (Sunset yellow or FD&C yellow 6, 2) from 4-aminobenzenesulfonic acid (sulfanilic acid).
■
EXPERIMENTAL PROCEDURE In the first part of the experiment, two azo dyes were synthesized: FD&C yellow 5 (Tartrazine), 1, and FD&C yellow 6 (Sunset yellow), 2. Aryl diazonium ion was prepared by the reaction between sulfanilic acid (50 mg) and sodium nitrite (50 mg) in the presence of concentrated hydrochloric acid (2 mL) in ice. Since the students may have very limited experience with chemicals, the step involving the use of concentrated hydrochloric acid may be carried out by the lab technician. The students were divided into two groups: every student in one group prepared FD&C yellow 5, 1, and every student in the other group prepared FD&C yellow 6, 2. Students weighed out 50 mg of either 6-hydroxynaphthalene-2-sulfonic acid or 5-oxo1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid, depending on the dye that was assigned to them. They added to each chemical 10 mL of ethanol and 5 mL of 2.5 M sodium hydroxide solution. Then, several drops of the diazonium ion solution were added to the solutions of the activated aromatic compounds. A more stepwise procedure protocol is presented as Supporting Information. Students were able to observe how the reaction between two noncolored solutions produced a colored substance. Furthermore, they got a chance to see how structure relates to function. Both dyes have one part of the molecule in common (indicated in Figure 1 as the 4-sulphophenyl portion), while the other part is different. The structure differences were correlated to the difference in color. In the second part of the experiment, the identities of the synthesized dyes were confirmed by comparison with the dyes present in foods and drinks using thin layer chromatography. Both groups of students spotted separate TLC plates: one for FD&C yellow 5 and one for FD&C yellow 6. Several different foods containing dyes of interest were used for the comparison: powders (Crystal Light, Jell-O, Tang), food coloring (McCormick yellow and green food coloring), and drinks (Mountain Dew, Gatorade, Sunkist). Foods containing FD&C yellow 5 were spotted on a TLC plate with the
synthesized yellow 5 dye, along with a yellow 5 dye standard. Foods containing FD&C yellow 6 were spotted on a TLC plate with the synthesized yellow 6 dye, along with a yellow 6 dye standard. Some samples were spotted on both plates because they contained both dyes. Some foods also contained FD&C red 40 (Allura red, Figure 2), 3. Although the students did not
Figure 2. Structure of disodium 6-hydroxy-5-((2-methoxy-5-methyl-4sulfophenyl)azo)-2-naphthalene-sulfonate (Allura red or FD&C red 40, 3).
synthesize red 40, a standard was used to spot food that contained red 40 on the TLC plates (Figure 3). In order to spot different foods on the TLC plates, samples were prepared: powders (10 mg) were dissolved in methanol (10 mL), while a few drops of the food coloring were diluted in 10 mL of methanol. Drinks were spotted onto the TLC plates right from the bottles (at least 3 or 4 capillary tubes). When each spot dried, the TLC plates were developed in a mixture of isopropanol and concentrated ammonium hydroxide (80:20). The plates were then examined under UV light (254 nm) in the dark. The distance traveled by the spots was then measured, and Rf values were calculated. Dyes were analyzed, and comparisons were made by the students regarding the results.
■
HAZARDS Students should wear safety goggles and gloves at all times. The dyes that are being prepared are harmful if ingested or inhaled. Dyes can also be absorbed through the skin. They can provide allergic reactions, and they are potentially carcinogenic. B
DOI: 10.1021/acs.jchemed.6b00334 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Activity
Figure 3. Plate contains yellow 5 standard in lane 1 (far left), synthesized yellow 5 in lane 2, Gatorade in lane 3, McCormick green dye in lane 4, Mountain Dew in lane 5, McCormick yellow dye in lane 6, Crystal Light in lane 7, and standard red 40 in lane 8 (far right). (The plate is analyzed under white (right) and UV-254 nm (left) lights.)
Table 1. Dyes Present and Rf Values for Each Sample
Students should avoid skin contact with all the remaining chemicals described in this paper. Diazonium salts are explosive when dry. Never let the diazonium salt dry out: use the prepared diazonium solution immediately in the next step. Hydrochloric acid is corrosive. Neutralize any spills immediately with sodium bicarbonate. Should the acid come in contact with skin, wash the affected area thoroughly with copious amounts of cool water. Avoid skin contact with sodium hydroxide since it is caustic. Sodium nitrite is toxic. If powdered mixes of food items are used, avoid inhalation of the dust. Methanol and isopropanol are flammable. Silica gel should not be inhaled. Direct viewing of UV lights is harmful to the eyes and therefore should be avoided. Students should avoid inhaling the solvent mixture used for the TLC development.
■
a
RESULTS AND DISCUSSION Typical students’ results from the TLC separation of the dyes are summarized in Table 1. It was determined that some drinks and powdered mixes are better for this experiment than others. For example, Gatorade and Jell-O dyes are not concentrated enough for the spots to be clearly visualized after the development. The red 40 dye in McCormick yellow and Sunkist was also not visualized (Figures 3 and 4). McCormick food coloring is very concentrated and needs to be diluted (1 drop in 5 mL of methanol) to achieve good resolution. The yellow food coloring contains FD&C red 40, which has an Rf value similar to FD&C yellow 6. On a TLC plate it had a distinctive red color as opposed to the orange color of yellow 6 (Figures 3 and 4). Moreover, in the yellow 5 TLC plate, the synthesized and the reference dye have apparently different Rf values. In order to prove the two molecules are the same, a TLC plate, which includes the two molecules spotted alone and cospotted,
Sample
Dye Present
Rf Value
Synthesized yellow 5 Standard yellow 5 Synthesized yellow 6 Standard yellow 6 Standard red 40 McCormick green McCormick yellow Crystal Light Mountain Dew Gatorade Tang Sunkist Vanilla Jell-O
Y5 Y5 Y6 Y6 R40 Y5/B1a Y5/R40 Y5 Y5 Y5 Y5/Y6 Y6/R40 Y5/Y6
0.074 0.113 0.308 0.323 0.378 0.109/0.313 0.117/NA 0.109 0.117 NA 0.108/NA 0.138/NA NA
FD&C blue 1 standard was not run on the plates.
was prepared and run (Figure 5). The two molecules coelute. However, for unambiguous proof that they are the same molecule, additional techniques may be used (melting point data, and for more advanced students nuclear magnetic resonance spectroscopy) Students were a little disappointed that the TLC plate development took such a long time, but were pleased with the results. They particularly liked looking under UV light at the spots on the plates. Most students were able to determine that the synthesized dyes are indeed the same as dyes used in foods. They were also able to confirm the identity of synthesized dyes by calculation of Rf values.
■
CONCLUSION Students were interested in learning about the synthesis of FD&C dyes used in foods. They were surprised to learn that several dangerous compounds like concentrated hydrochloric acid and sodium nitrite are used to prepare color for drinks they C
DOI: 10.1021/acs.jchemed.6b00334 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Activity
Figure 4. Plate contains yellow 6 standard in lane 1 (far left), red 40 standard in lane 2, synthesized yellow 6 in lane 3, synthesized yellow 5 in lane 4, Jell-O in lane 5, Sunkist in lane 6, Tang in lane 7, and yellow 5 standard in lane 8 standard (far right). (The plate is analyzed under white (right) and UV-254 nm (left) lights.)
Figure 5. Plate contains yellow 5 standard in lane 1 (far left), cospotted dyes in lane 2, and the synthesized one in lane 3 (far right). (The plate is analyzed under white (right) and UV-254 nm (left) lights.)
■
consume. Some students were disappointed to learn that their favorite drinks contain chemicals that are hazardous even to touch. Visualization of the azo group in the structure of the dyes helped students better relate to the laboratory experience. They were not only curious about the effect of the azo group and its degradation pathway, but also the impact on their health. The experiment demonstrated that putting chemistry in context by demonstrating how chemistry is part of everyday life has favorable effects on the students’ interest in the subject.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00334. Instructions for the students and instructors (PDF, DOCX)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. D
DOI: 10.1021/acs.jchemed.6b00334 J. Chem. Educ. XXXX, XXX, XXX−XXX
Journal of Chemical Education
Activity
ORCID
Gloria Proni: 0000-0002-9942-8810 Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Kadlec, A.; Friedman, W.; Ott, A. Important, But Not for Me Parents and Students in Kansas and Missouri talk about Math, Science and Technology Education; Public Agenda: New York, 2007, http:// www.publicagenda.org/media/important-but-not-for-me (accessed Dec 2016). (2) Johnson, J.; Arumi, A. M.; Ott, A.; Remaley, M. H. Issue No. 1: Are Parents and Students Ready for More Math and Science?; Public Agenda: New York, 2006, http://www.publicagenda.org/files/rc0601. pdf (accessed Dec 2016). (3) Understanding Student Participation and Choice in Science and Technology Education; Henriksen, E. K., Dillon, J., Ryder, J., Eds.; Springer: Dordrecht, 2015; DOI: 10.1007/978-94-007-7793-4. (4) Gung, B. W.; Taylor, R. T. Parallel Combinatorial Synthesis of Azo Dyes - A Combinatorial Experiment Suitable for Undergraduate Laboratories. J. Chem. Educ. 2004, 81 (11), 1630−1632. Sigmann, S. B.; Wheeler, D. E. The Quantitative Determination of Food Dyes in Powdered Drink Mixes. J. Chem. Educ. 2004, 81 (11), 1475−1478. Erhardt, W. Instrumental Analysis in The High School Classroom: UV-Vis Spectroscopy. J. Chem. Educ. 2007, 84 (6), 1024−1026. Bird, E. W.; Sturtevant, F. Extraction of FD&C Dyes from Common Food Sources. J. Chem. Educ. 1992, 69 (12), 996−998. (5) Peay, A. Separation of Dyes by Paper Chromatography.www.apsu. edu/sites/apsu.edu/files/chemistry/SP12_1011_Chromatography_ lab.pdf (accessed January 2017). (6) Banks, F.; Barlex, D. Teaching STEM in the Secondary School: Helping Teachers Meet the Challenge; Routledge: Abdington, Oxon, UK, 2014. (7) Rowe, K. S.; Rowe, K. J. Synthetic Food Coloring and Behavior: a Dose Response Effect in a Double-Blind, Placebo-Controlled, Repeated-Measures Study. J. Pediatr. 1994, 125 (5 Pt 1), 691−698.
E
DOI: 10.1021/acs.jchemed.6b00334 J. Chem. Educ. XXXX, XXX, XXX−XXX
