Recently as I was mowing my lawn, I noticed that some of the florets on white clover have a slight pink or purple color (Figure 1). Because anthocyanins often impart red, blue, and purple color to plants, fruits, and flowers,1,2 I wondered if these tiny pink florets in white clover contained anthocyanins. Of course, anthocyanins are routinely used as acid-base indicators in science classes, and red cabbage is a widely used source of anthocyanins. I reasoned that if white clover contained anthocyanins, these common flowers could provide yet another easily obtained source of an anthocyanin-based acid-base indicator.
Figure 1: White clover. Slight pink and purple color can be observed in the florets.
I set out to extract anthocyanins from white clover by first collecting about 30 white clover samples in a bowl. Next, I added a quarter cup of tap water, and heated the flowers in a microwave for one minute. When I removed the bowl from the microwave and poured off the water, I saw no indication of pink or purple color – no anthocyanins. Instead, I was quite surprised to see that the water had taken on a deep yellow color (Figure 2, Video 1).
Figure 2: Boiling white clover in water yields a yellow-colored mixture.
On a whim I decided to add some vinegar to the yellow-colored mixture. Upon doing so the yellow color disappeared. Adding baking soda to the colorless mixture caused the yellow color to reappear (Video 1). I found that I could cause the yellow color to appear upon addition of a variety of household bases and disappear upon addition of several household acids. While I was unsuccessful in extracting anthocyanins from white clover, I had nevertheless discovered that the white clover contained an acid-base indicator, after all! But where did this colorless-to-yellow acid-base indicator come from?
A bit of literature searching turned up a likely candidate: anthoxanthins (Figure 3). Anthoxanthins are a class of chemical compounds that are similar in structure to anthocyanins, and they often impart white or yellow color to plants and flowers.3-8 Anthoxanthins, are known to appear yellow colored in the presence of base, but colorless in the presence of acid.3-4
Figure 3: Chemical structure of apigenin, an anthoxanthin
You can see some of my explorations I did with white clover below (Video 1).
Video 1: White Clover Color Chemistry, Tommy Technetium YouTube Channel, July 2, 2021.
In the future I’m hoping to carry out some other investigations with other white-colored plants that might contain anthoxanthins, such as daisies or cauliflower. I’m also hoping to use white clover as a source of an acid-base indicator in my classes from time to time. If you try some similar experiments with white colored plants and flowers and learn anything new, please be sure to let me know in the comments.
Happy experimenting!
References
1. See Kuntzleman, T., Berries are red, berries are blue…I’ve got a berry surprise for you!, ChemEd X, 2020 and references therein.
2. Alkema, J.; Seager, S. L. The Chemical Pigments of Plants, J. Chem. Educ. 1982, 59, 183-186.
3. Asenstorfer, R. E.; Wang, Y.; Mares, D. J. Chemical Structure of flavonoid compounds in wheat (Triticum aestivum L.) flour that contribute to the yellow color of Asian alkaline noodles. Journal of Cereal Science 2006, 43, 108-119.
4. Pate, K. M.; Rogers, M.; Reed, J. W.; van der Munnik, N.; Vance, S. Z.; Moss, M. A. Anthoxanthin polyphenols attenuate Ab oligomer-induced neuronal responses associated with Alzheimer’s disease, CNS Neurosci. Ther. 2017, 23, 135-144.
5. Blank, F. The anthocyanin pigments of plants. The Botanical Review 1947, 13, 241-317.
6. Geissman, T. A. The chemistry of flower color variation. J. Chem. Educ. 1949, 26, 657-655.
7. Lawrence, W. J. C.; Price, J. R. The genetics and chemistry of flower color variation. Biol. Rev. 1940, 15, 35-57.
8. Dutta, S.; Halder, S. A colourful food palette: health benefits and beyond. International Journal of Current Research 2021, 13, 16596-16600.
NGSS
Asking questions and defining problems in grades 9–12 builds from grades K–8 experiences and progresses to formulating, refining, and evaluating empirically testable questions and design problems using models and simulations.
Asking questions and defining problems in grades 9–12 builds from grades K–8 experiences and progresses to formulating, refining, and evaluating empirically testable questions and design problems using models and simulations.
questions that challenge the premise(s) of an argument, the interpretation of a data set, or the suitability of a design.
Scientific questions arise in a variety of ways. They can be driven by curiosity about the world (e.g., Why is the sky blue?). They can be inspired by a model’s or theory’s predictions or by attempts to extend or refine a model or theory (e.g., How does the particle model of matter explain the incompressibility of liquids?). Or they can result from the need to provide better solutions to a problem. For example, the question of why it is impossible to siphon water above a height of 32 feet led Evangelista Torricelli (17th-century inventor of the barometer) to his discoveries about the atmosphere and the identification of a vacuum.
Questions are also important in engineering. Engineers must be able to ask probing questions in order to define an engineering problem. For example, they may ask: What is the need or desire that underlies the problem? What are the criteria (specifications) for a successful solution? What are the constraints? Other questions arise when generating possible solutions: Will this solution meet the design criteria? Can two or more ideas be combined to produce a better solution?
Constructing explanations and designing solutions in 9–12 builds on K–8 experiences and progresses to explanations and designs that are supported by multiple and independent student-generated sources of evidence consistent with scientific ideas, principles, and theories.
Constructing explanations and designing solutions in 9–12 builds on K–8 experiences and progresses to explanations and designs that are supported by multiple and independent student-generated sources of evidence consistent with scientific ideas, principles, and theories. Construct and revise an explanation based on valid and reliable evidence obtained from a variety of sources (including students’ own investigations, models, theories, simulations, peer review) and the assumption that theories and laws that describe the natural world operate today as they did in the past and will continue to do so in the future.
Modeling in 9–12 builds on K–8 and progresses to using, synthesizing, and developing models to predict and show relationships among variables between systems and their components in the natural and designed worlds.
Modeling in 9–12 builds on K–8 and progresses to using, synthesizing, and developing models to predict and show relationships among variables between systems and their components in the natural and designed worlds. Use a model to predict the relationships between systems or between components of a system.
Constructing explanations and designing solutions in 9–12 builds on K–8 experiences and progresses to explanations and designs that are supported by multiple and independent student-generated sources of evidence consistent with scientific ideas, principles, and theories.
Constructing explanations and designing solutions in 9–12 builds on K–8 experiences and progresses to explanations and designs that are supported by multiple and independent student-generated sources of evidence consistent with scientific ideas, principles, and theories. Construct and revise an explanation based on valid and reliable evidence obtained from a variety of sources (including students’ own investigations, models, theories, simulations, peer review) and the assumption that theories and laws that describe the natural world operate today as they did in the past and will continue to do so in the future.
Planning and carrying out investigations in 9-12 builds on K-8 experiences and progresses to include investigations that provide evidence for and test conceptual, mathematical, physical, and empirical models.
Planning and carrying out investigations in 9-12 builds on K-8 experiences and progresses to include investigations that provide evidence for and test conceptual, mathematical, physical, and empirical models. Plan and conduct an investigation individually and collaboratively to produce data to serve as the basis for evidence, and in the design: decide on types, how much, and accuracy of data needed to produce reliable measurements and consider limitations on the precision of the data (e.g., number of trials, cost, risk, time), and refine the design accordingly.
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Comments 4
Clover Extractions
These observations are very interesting! Thank you very much for posting. I have had a try using your procedure, although the clover flowers I could find had a much stronger purple colouration so there are likely some anthocyanins present in my sample.
The top sample picture was taken 10 minutes after extraction using the method described. The bottom picture was taken the next day. (L to R Ferric chloride + extract, HCl + extract, acetic acid + extract, original extract and NaOH + extract).
The red colour of the HCl and acetic acid samples are likely due to anthocyanins from the purple material in the petals. The particularly interesting results are the Ferric chloride test and addition of hydroxide. The dark coloured solution on addition of Fe3+ and the brown solution with hydroxide (aged in air) could also be evidence for the presence of anthoxanthins. I found similar observations reported in 1897 with samples of isolated apigenin (for further detail see work by A. G. Perkin FRSE https://pubs.rsc.org/en/Content/ArticleLanding/CT/1897/CT8977100805#!div...)
I think the clover extraction observations might also explain some of the flower observations we saw last year. in many cases, fruit and vegetable extracts gave blue or violet solutions when iron salts were added, but many flowers gave black/dark green solutions, so perhaps the flowers are a richer source of anthoxanthins? Lots of really interesting chemistry here!
Great extensions!
Wow Iain, thank you for sharing your explorations in some extensions to the clover chemistry experiments. I appreciate you alerting me to the hydroxide test for the presence of anthoxanthins. I always enjoy hearing about your work with various plant pigments. I, too, have noticed a slight pink color in some of the white clover around my home. In fact, I often noticed a slight pink tinge when my clover extracts were acidified. However, the clover you share in your pictures has a LOT of pink in them. Indeed, some anthocyanins are also present in white clover. I always appreciate your input, my friend.
More clover colours
I managed to find some clover that was less purple in colour, the flowers look more like the ones you had. Pleasingly, the results this time align with yours. The yellow colour disappears with acid and forms again when sodium bicarbonate is added. I don't know why our local clover was so purple, I travelled a bit further from the coast when I found these examples. The Fe3+ test works well with these too. Just like your blueberries article last year, this is another great home investigation to try!
clover_pics_2.jpg
Beautiful!
Beautiful work, Iain. Thank you for sharing these explorations with us.