Colourful Chemistry of Canning – Part 5

Co-Authored by Iain A. Smellie*, Iain L. J. Patterson*

*University of St Andrews, School of Chemistry, North Haugh, St Andrews KY16 9ST, United Kingdom

In previous articles in this series, we have examined reactions of the tinplate interior of discarded food cans with a variety of substances. We have found that tin cans react with anthocyanins to form colourful tin complexes,¹ we have prepared SnI₄ by reacting tinplate with iodine,² and we have used tin cans to prepare colloidal gold.³ In this article, we examine the ability of tinplated food cans to reduce aromatic azo compounds to amines (scheme 1). We show that the tinplate interior of a food can is able to quickly reduce and decolourise an acidic solution of commonly encountered azo dyes (methyl orange and methyl red).

Scheme 1: Reduction of an azo compound to form two amines.

 

 

Background

Azo compounds will be familiar to high school and undergraduate students due to the use of these colourful substances as dyes in various household products (such as textiles and foodstuffs).^4,5 Furthermore, methyl orange and methyl red are commonly encountered as indicators in acid/base titrations (scheme 2).

Scheme 2: Acid/base equilibria for methyl orange and methyl red

 

Video 1: The reduction of ago compound in a tinplated food can

 

Azo compounds were very important in the early development of effective antimicrobial drugs. Prior to the introduction of penicillin in the 1940s,^6,7 sulphonamide drugs were used to treat bacterial infections, “Prontosil” is one of the best-known examples from this period. Prontosil was also known as “prontosil rubrum” due to its distinctive red colour, however, a colourless product (sometimes called “prontosil album”) followed. Latterly, it was found that azo compounds were readily metabolised in the body, one of the key steps in this process being cleavage of the N=N bond to form aromatic amine products.^6-9 This observation led to the realisation that prontosil was in fact a prodrug for sulphanilamide as the active (and less colourful) antimicrobial compound (scheme 3).⁶

Scheme 3: Enzymatic reduction of prontosil to form sulphanilamide and benzene-1,2,4-triamine.

 

The ability of bacteria and other organisms to reduce azo compounds has been extensively studied, this type of reaction is now known to be catalysed by a large and diverse class of enzymes called azoreductases.^8,9 In the case of prontosil, one of the amine products was a useful antimicrobial compound, however this type of reduction can also have negative consequences for human health. Certain azo dyes are now considered to be carcinogenic, this is due to the formation of amines that have long been known to be potent carcinogens.⁴ As a result, the use of certain azo dyes in household products and textiles are now subject to strict legal controls in many countries. A particularly well studied example is the azo dye known as “congo red”, this compound forms benzidine when metabolised (scheme 4), this product is a health hazard since benzidine is a known carcinogen.¹⁰

Scheme 4: Formation of carcinogenic benzidine (1,1'-biphenyl-4,4'-diamine) by reduction of congo red.

 

More recently, attention has been directed toward the safety of tattoos, some of the inks used in tattoos are aromatic azo compounds (see figure 1 for examples¹¹). There are concerns that enzyme induced reduction of azo dyes in tattoo inks may be related to certain skin conditions and other health problems.

Figure 1: Example azo dyes used in tattoo inks.

 

We became aware that the treatment of aromatic azo dyes with tin(II) chloride in hydrochloric acid can result in reduction of the starting material to the corresponding amines.⁵ We reasoned that this reaction could be visible to the human eye if the azo compound was coloured, since the reaction mixture would likely become colourless once all the azo compound had been consumed. From our previous studies, we anticipated that tinplated food cans would facilitate this reaction and that this would serve as a simple and inexpensive method to demonstrate the reduction of azo compounds.

 

Demonstrations

Early in the testing phase, we limited our attention to aromatic azo compounds that had already been cleared for use in laboratory classes. We also aimed to use very small quantities of the azo compounds to minimise the amount of material needed and the amount of waste produced. Our initial tests showed the most suitable azo dyes were dilute alcohol/water mixes of methyl orange or methyl red (these formulations are often used as titration indicators). Before attempting reactions in tin cans, some control experiments were performed with granules of metallic tin and iron. The tinplate layer inside food cans is very thin, so we wanted to see if iron (present in the external structure of a can) in the presence of HCl would reduce and decolourise a dilute solution of methyl orange. 5 Drops of 0.04% methyl orange in aqueous ethanol were added to 20 mL aliquots of 5 M HCl. The solutions were mixed and granules of tin metal (figure 2, flask 1) or iron metal (figure 2, flask 2) were then added. After swirling the mixtures for 5 minutes, the indicator in the flask containing tin had decolourised, this showed that the azo compound had been reduced. In contrast, the indicator in the flask containing iron did not decolourise, this remained the case even after 30 minutes of mixing. After 90 minutes, the indicator in the flask containing iron began to fade, and after 180 minutes, the red colour of the azo dye was no longer visible.

Figure 2: Reduction of methyl orange (in 5 M HCl) in the presence of metallic tin (flask 1) and metallic iron (flask 2). Panel A shows the initial solution of methyl orange in aqueous HCl. Panel B shows the methyl orange solutions after a 5 minute mixing period. Panel C shows shows the methyl orange solutions after 180 minutes.

 

 

The control experiments worked well, however, 5 M HCl was undesirable for classroom use, so we selected more dilute HCl solutions for the reaction in tin cans. Before adding methyl orange or methyl red, 20 mL portions of 1 M or 2 M HCl were swirled inside a discarded food can for 1-2 minutes. We found that best results were obtained when the acid solution was allowed to contact a large area of the tinplate interior. We assume that the initial treatment with acid allows some SnCl₂ to form, this can then react with the azo compounds that are added subsequently. Once the can had been treated with HCl, 5-6 drops of methyl orange or methyl red indicator solutions were added, and the contents swirled until the solutions turned from red to colourless (figure 3). In most cases the colour change was observed to be complete within 1-2 minutes (see video 2 and 3). We also noted that the cans treated with 2 M HCl reacted slightly faster than those treated with 1 M HCl.

Figure 3: Reactions of a tinplate plate can with methyl orange and methyl red in aqueous HCl. Panel A shows the initial mixture containing methyl orange, Panel B shows the reaction mixture after swirling the can contents for a few minutes. Panel C shows the initial mixture containing methyl red, Panel D shows the reaction mixture after swirling the can contents for a few minutes.

 

Video 2: Reduction of methyl orange in a tin can

 

 

 

Video 3: Reduction of methyl red in a tin can

 

In all the experiments described above, the solutions of methyl red and methyl orange are red in colour prior to reduction. This observation is due to the acidic conditions favouring the hydrazo, rather than azo forms (schemes 1 and 5). A similar situation is believed to exist as part of the mechanism of in vivo reduction of the azo compounds by azoreductases. Recent work suggests that the mechanism of N=N reduction by azoreductase enzymes involves binding of the azo compound to the enzyme, followed by isomerisation to the hydrazo form to facilitate the reduction step.^8,9

Scheme 5: Reduction of methyl orange and methyl red

 

Summary

We have devised a set of very simple, inexpensive, and colourful demonstrations that involve the reduction of aromatic azo compounds. The processes described show that tin metal granules or the tinplate interior of a discarded food can are both capable of reducing familiar acid/base indicators to colourless products. The reduction of azo compounds to form amines is relevant to health-related topics, household dyes and pigments, this process can also serve as a useful prompt for discussions about enzyme function. In addition to photographs of the demonstrations described, we have also provided a videos of azo dyes being reduced in a tin can.

 

References

1. Smellie, Patterson, “Colourful Chemistry of Canning” (Accessed 19th Augus 2023).
2. Smellie, Patterson, Chalmers, “Colourful Chemistry of Canning – Part 3” (Accessed 19th August 2023).
3. Smellie, Patterson, “Colourful Chemistry of Canning – Part 4” (19th August 2023)
4. Ahlström, L. H.; Eskilsson, C. S.; Björklund, E. “Determination of Banned Azo Dyes in Consumer Goods”, TrAC, Trends Anal. Chem., 2005, 24, 49-56.
5. Voyksner, R. D.; Straub, R.; Keever, J. T.; Freeman, H. S.; Hsu, W. H. “Determination of Aromatic Amines Originating from Azo Dyes by Chemical Reduction Combined with Liquid Chromatography/Mass Spectrometry”. Environ. Sci. Technol., 1993, 27, 1665-1672.
6. Wainwright, M.; Kristiansen, J. E. “On the 75th Anniversary of Prontosil”, Dyes Pigments, 2011, 88, 231-234.
7. Altowyan, M. S.; Soliman, S. M.; Ismail, M. M. F.; Haukka, M.; Barakat, A.; Ayoup M. S. “New Bioprecursor Prodrugs of Sulfadiazine: Synthesis, X-ray Structure and Hirshfeld Analysis”, Crystals, 2022, 12, 1016.
8. Ryan, A.  "Azoreductases in Drug Metabolism", Br. J. Pharmacol, 2017, 174, 2161-2173.
9. Misal, S. A.; Gawai, K. R.  "Azoreductase: A Key Player of Xenobiotic Metabolism", Bioresour. Bioprocess., 2018, 5, 17.
10. Oladoye, P. O.; Bamigboye, M. O.; Ogunbiyi O. D.; Akano, M. T.  "Toxicity and Decontamination Strategies of Congo Red Dye", Groundw. Sustain. Dev., 2022, 18, 100844.
11. Gaudron, S.; Ferrier-Le-Bouëdec, M. C.; Franck; F.; D’Incan, M.  "Azo Pigments and Quinacridones Induce Delayed Hypersensitivity in Red Tattoos", Contact Derm., 2014, 72, 97-105.
12. Giulbudagian, M.; Schreiver, I.; Singh, A. V.; Laux, P.; Luch, A. "Safety of Tattoos and Permanent make-up: A Regulatory Review", Arch. Toxicol., 2020, 94, 357-369.

Acknowledgements – We would like to express our sincere thanks to Isobel Everest for very helpful discussions during the course of this study.

Supporting Information: Experimental procedures (and safety information) are provided for experiments conducted in discarded food cans. Videos of methyl orange and methyl red reacting with the tinplate surfaces of food cans has been provided. (Log into your ChemEd X account to access. Don't have an account? Register here for free!)