Colour Changes with Caffeine - Part 2

Co-Authored by Iain A. Smellie*, Alexandra G. Armitage*, Iain L. J. Patterson*, Renald Schaub*

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

In our most recent article, Colour Changes with Caffeine - Part 2,¹ we investigated the effect on the colour of anthocyanin-containing solutions when they were mixed with caffeine. The experiments showed that addition of caffeine to anthocyanin solutions resulted in a shift of the absorption maximum to longer wavelengths (to the human eye, they became shifted from red to violet in colour). These observations were likely due to a “copigmentation” effect, where caffeine and anthocyanin molecules associate in solution by non-covalent interactions.^2-5 Copigmentation was discussed in the previous article,¹ however the key points are highlighted in figure 1. Binding of anthocyanins to metal ions (figure 1, interaction A) or non-covalent π-π stacking assemblies of anthocyanins (figure 1, interactions B and C) can change the colour of anthocyanin solutions.^2-5 We have discussed the effects of metal ions⁶ and caffeine¹ in previous reports; however, we had not examined the effect of adding metal ions and caffeine at the same time. In this article, we highlight some simple experiments where anthocyanin-containing fruit extracts are mixed with aqueous solutions of caffeine in the presence of Al³⁺ or Sn²⁺ ions. The objective of this work was to investigate whether aqueous anthocyanin solutions from fresh fruit extracts would give different colours in the presence of caffeine and metal ions.

Figure 1. Selected copigmentation interactions of anthocyanins (R = sugar units)

 

Inspiration from blue flowers

Copigmentation effects play an important role in the colours of flowers, especially species that exhibit blue colouration.^7,8 Hydrangea (Hydrangea macrophylla), Asiatic dayflower (Commelina communis) and cornflower (Centaurea cyanus) all have beautiful, blue-coloured varieties. The blue colours arise from anthocyanin-containing compounds; however, the chromophores have more complex structures than a simple anthocyanin core.^7,8 Complexation of aluminum ions with delphinidin is believed to be responsible for the colour changes observed in hydrangea flowers.⁸ The blue colouration of cornflowers and Asiatic dayflowers is ascribed to even more complex supramolecular assemblies of anthocyanin containing structures.^2,7 These large molecular assemblies involve simultaneous binding of metal ions, and association with flavones, to a molecule containing a metal-complexed anthocyanin core.^2,7 The metal complex isolated from blue cornflowers is called protocyanin and serves as a useful example of a naturally occurring supramolecular assembly that includes an anthocyanin component. The key constituent molecules of protocyanin are succinylcyanin, which is a derivative of dephinidin, and malonyl flavone (Figure 2).⁷  

Figure 2. Key component molecules of protocyanin.

 

Succinylcyanin can bind to Mg²⁺ or Fe³⁺ ions, the complexed anthocyanin is also able to engage in non-covalent π-π stacking interactions, where the aromatic regions of anthocyanins arrange parallel to each other (or with malonylflavone).^7,9

Figure 3. Metal complexes derived from succinylcyanin.

 

Figure 4. Selected copigmentation interactions present in protocyanin (R = sugar units)

 

Experiments with raspberries, wild blackberries “brambles” and elderberries

Experiments were conducted with commercially sourced raspberries (Rubus idaeus), foraged wild blackberries (Rubus fruticosus known in the UK as “brambles”) and foraged elderberries (Sambucus nigra). Frozen berries were soaked in hot water and mashed to release as much juice as possible. Samples of the resulting fruit extract were then filtered to remove the fruit pulp. The extracts were filtered before use, the filtered solutions were then stored in a domestic freezer and thawed when required.  

 

Results from addition of potassium alum and caffeine solutions to berry extracts

As expected, the initial berry extracts were pale red/pink in colour, this is because the fruit acids, present in the berries, make the solutions acidic (pH 3-4). We were aware from previous work¹ that addition 2% caffeine solution would lead to the extracts becoming violet/pink in colour. We also expected that addition of 10% potassium alum solution to the extract would lead to violet/purple-coloured solutions (these result from formation of aluminum complexes of anthocyanins).⁵ For best results with Al³⁺ ions, we found that it was best to adjust the pH of the berry extracts to pH 6-7 by adding a few drops of saturated sodium bicarbonate. Once the extract pH had been adjusted, 10% potassium alum solution was added dropwise until there was a colour change. Addition of 2% caffeine solution to berry extracts containing Al³⁺ resulted in very pleasing violet/blue coloured solutions in all cases, the raspberry and bramble examples were particularly impressive! (see figures 5, 6 and 7 for pictures. Videos 1 - 3 show the preparation of raspberry and bramble). We found that reversing the order of addition of caffeine and potassium alum solutions to the extracts also gave violet/blue solutions (see video 3). This result is perhaps unsurprising, since our most recent investigations¹ showed that caffeine is basic enough to raise the berry extract pH to ~6, so within an acceptable range for aluminium complexes of anthocyanins to form.    

Figure 5. Experiments with raspberry extracts. A = raspberry extract, B = raspberry extract mixed in 2% caffeine solution, C = raspberry extract containing 10% potassium alum solution, D = raspberry extract mixed with potassium alum and caffeine solutions.

 

Video 1. Raspberry extract, Alum and Caffeine. (Raspberry extract mixed with saturated alum solution, then 2% caffeine.) Gardenindicators YouTube Channel (accessed March 10, 2024)

 

Figure 6. Experiments with bramble extracts. A = bramble extract, B = bramble extract mixed in 2% caffeine solution, C = bramble extract containing 10% potassium alum solution, D = bramble extract mixed with potassium alum and caffeine solutions.

 

Video 2. Bramble extract, Alum and Caffeine. (Bramble extract mixed saturated alum solution then 2% caffeine.) Gardenindicators YouTube Channel (accessed March 10, 2024)

 

Video 3. Bramble extract, Alum and Caffeine. (Bramble extract mixed with 2% caffeine then saturated alum solution.) Gardenindicators YouTube Channel (accessed March 10, 2024)

 

Figure 7. Experiments with elderberry extracts. A = elderberry extract, B = elderberry extract mixed in 2% caffeine solution, C = elderberry extract containing 10% potassium alum solution, D = elderberry extract mixed with potassium alum and caffeine solutions.

 

Results from addition of tin(II) chloride and caffeine solutions to raspberry extracts

In earlier studies, we have examined the effect of exposing anthocyanin-containing solutions to sources of Sn²⁺,⁶ in many cases these solutions became violet in colour. In this study we examined the effect of adding SnCl₂ in combination with caffeine to see if there was a similar effect to adding potassium alum (see figure 8 for pictures). The pH of raspberry extract was adjusted to pH 6-7 by adding a few drops of saturated sodium bicarbonate. Once the extract pH had been adjusted, 2.5% SnCl₂ in glycerol was added dropwise until there was a colour change. Addition of 2% caffeine solution to berry extracts containing Sn²⁺ resulted in a deeper violet coloured solution than was observed from addition of Sn²⁺ in the absence of caffeine. The solution containing tin ions was not violet/blue, as had been observed when potassium alum and caffeine were added to raspberry extract (UV-vis spectra from these experiments are provided in the supporting information).

Figure 8. Experiments with raspberry extracts. A = raspberry extract, B = raspberry extract mixed in 2% caffeine solution, C = raspberry extract containing 2.5% SnCl₂ in glycerol, D = raspberry extract mixed with SnCl₂ and caffeine solutions.

 

Possible explanations for the berry extract experiment results

In our previous study,¹ it was concluded that the colour changes observed in berry extracts resulted from a copigmentation effect from the addition of caffeine. UV-visible spectra of anthocyanin-containing berry extracts showed a shift of the absorption maximum to longer wavelengths when dissolved caffeine was present. It is known from studies of blue cornflower and Asiatic dayflower pigments that the absorption maxima can be in the range of 600-680 nm,⁷ so they appear blue to the human eye. The pigments (protocyanin and commelinin) require anthocyanin π-π stacking interactions and metal ion binding in order to have the right properties to absorb light in the correct range (varying the metal ion often results in a change to the wavelength of light absorbed).⁷ In the experiments reported here, the absorption maxima of solutions containing a metal ion and caffeine were found to be in the 550-580 nm range (UV-vis spectra from these experiments are provided in the supporting information). The shift in absorption maximum is significant, since anthocyanins present in berry extracts, in the absence of caffeine and Al³⁺ or Sn²⁺ ions, show absorption maxima in the range of 510-520 nm. The shifts in absorption when Al³⁺ or Sn²⁺ ions are added to anthocyanin solutions in the presence of caffeine are larger than adding the metal ions in the absence of caffeine.

Spectroscopic and computational investigations of the copigmentation interaction of rutin (a flavone) with caffeine have been reported in recent years.¹⁰ These experiments may suggest that analogous π-π interactions between caffeine with the anthocyanins in berry extracts can take place. It is also possible that metal-bound anthocyanins can interact in a similar way (see figure 9), however, there is some uncertainty whether Sn²⁺ ions are oxidised in-situ to Sn⁴⁺ under the conditions used in our experiments.    

Figure 9. Possible π-π stacking arrangements of metal bound anthocyanin structures with caffeine.

 

 

Summary and further work

We have further investigated the effect on the colour of anthocyanin-containing solutions when mixed with caffeine. The latest experiments we have conducted show that addition of caffeine and Al³⁺ or Sn²⁺ ions to anthocyanin solutions results in a distinct colour change from red to violet/blue. The observations from this study could provide further scope for investigation using other anthocyanin-containing plant extracts. A diverse range of anthocyanins are available from other plant sources, so there is potential for a series of open-ended experiments for educators in classes or science clubs to investigate.

 

 

Supporting Information

Experimental procedures (and safety information) are provided for experiments described. In addition, UV-visible spectra of the relevant extracts have been provided. (Log into your ChemEd X account to access. Don't have an account? Register here for free!) 

 

References

 

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3. Cruz, L.; Baslio, N.; Mateus, N.; de Freitas, V.; Pina, F. “Natural and synthetic flavylium-based dyes: The chemistry behind the color”. Chem. Rev., 2022, 122, 1416-1481.
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5. Kajiya, D. Demonstrating purple color development to students by showing the highly visual effects of aluminum ions and pH on aqueous anthocyanin solutions. J. Chem. Educ., 2020, 97, 4084-4090.
6. Smellie, I.A., Patterson, I.J., “Colourful chemistry of canning”, ChemEd X, July 2022. (Accessed 4th March 2024).
7. Yoshida, K.; Mori, M.; Kondo, T. “Blue flower color development by anthocyanins: from chemical structure to cell physiology”. Nat. Prod. Rep., 2009, 26, 884-915.
8. Ito, T.; Aoki, D.; Fukushima, K.; Yoshida, K. Direct mapping of hydrangea blue-complex in sepal tissues of Hydrangea macrophylla. Sci. Rep., 2019, 9, 5450.
9. Kondo, T.; Ueda, M.; Isobe, M.; Goto, T. “A new molecular mechanism of blue color development with protocyanin, a supramolecular pigment from cornflower, Centaurea cyanus ” Tetrahedron Lett., 1998, 39, 8307-8310.
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