Flower Color Change Demonstration as a Visualization of Potential

Demonstration Cite This: J. Chem. Educ. XXXX, XXX, XXX−XXX

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Flower Color Change Demonstration as a Visualization of Potential Harmful Effects Associated with Ammonia Gas on Living Organisms Andrzej Sienkiewicz,*,† Iwona Rusinek,‡ Anna Siatecka,§ and Sonia Losada-Barreiro∥

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Department of Adsorption, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska square 3, Lublin 20-031, Poland ‡ Department of General and Coordination Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska square 2, Lublin 20-031, Poland § Department of Environmental Chemistry, Faculty of Chemistry, Maria Curie-Skłodowska University, Maria Curie-Skłodowska square 2, Lublin 20-031, Poland ∥ Facultad de Quimicas, Departamento de Química Física, Universidad de Vigo, 36-200 Vigo-Pontevedra, Spain S Supporting Information *

ABSTRACT: This manuscript describes a simple yet meaningful demonstration that can be easily adopted by highschool teachers to emphasize some potential dangers associated with gas inhalation. The demonstration aims to illustrate the undesirable effects of some gases on living organisms by employing freshly cut, colored flowers and ammonia gas. When in direct contact, the ammonia gas causes abrupt, easily observable changes in the appearances of the flowers, leading to their wilting, discoloration, or color fading. The experiment can be carried out at any time within an academic course because flowers with colorful petals can be easily obtained from any local store throughout the year. The proposed demonstration may also be used, if necessary, to introduce students into important biological and chemical concepts, including simple chemical calculations, the cell structure of flowers, and the effects of harmful gas molecules on living cells. KEYWORDS: High School/Introductory Chemistry, Demonstrations, Acids/Bases, Biological Cells, Heterocycles, pH, Plant Chemistry

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INTRODUCTION The proposed demonstration1−3 is aimed at highlighting the potentially harmful effects of some gases on living organisms such as flowers by presenting the changes in the appearance of a flower exposed to ammonia gas. Particularly, we are concerned with the abuse of inhalants by the youth, which leads to serious, long-lasting health problems, including “sudden sniffing death syndrome”.4−8 Although the described demonstration is based on ammonia action upon flower petals, it can be used as a trigger activity to discuss with students the impacts of numerous chemicals in the gaseous phase on living organisms and their consequences; such chemicals most typically include volatile aliphatic, aromatic, and halogenated hydrocarbons originating from paint thinners, glues, aerosol propellants, gasoline fumes, and others. The present demonstration is based on a demonstration organized as a part of an open lecture given by Peter Wothers in 2008 in the Chemistry Department of the University of Cambridge.9 The adopted demonstration takes 10−20 min and was successfully implemented in 2017, during the “chemistry show” delivered in the Faculty of Chemistry of Maria Curie-Skłodowska University (UMCS) in Lublin, Poland, as a part of the activities carried out under the patronage of the © XXXX American Chemical Society and Division of Chemical Education, Inc.

Polish Chemical Society. Since then, the demonstration has been repeated more than 30 times to an overall audience of more than 5000 students.

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RUNNING THE DEMONSTRATION

Apparatus and Chemicals

To run the demonstration, the following materials and chemicals are required: • Fresh flowers of the same or different colors that can be collected from a garden (whenever possible) or purchased from any florist or local store. • A 1 L glass jar with a lid that sits on the rim of the jar (see Figure 1 for an example) or a desiccator. The lid’s edges are covered with desiccator grease to ensure a tight connection between the lid and the jar. This setup also enables the release of gas if too much ammonia is produced during the demonstration. Received: January 2, 2019 Revised: May 24, 2019

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DOI: 10.1021/acs.jchemed.9b00001 J. Chem. Educ. XXXX, XXX, XXX−XXX

Journal of Chemical Education

Demonstration

Figure 1. Experimental setup before addition of water to the small container. Upon addition of a few milliliters of water, NH3(g) is produced immediately.

• Ammonium chloride (NH4Cl), sodium hydroxide (NaOH), and a few milliliters of tap water in a separate beaker. • Pasteur pipettes, plastic gloves, safety glasses, and a lab coat. Procedure Figure 2. Photographs showing the observed changes in the appearances of flowers (a,c,e) before and (b,d,f) after their exposure to the produced ammonia gas. (a,b) Rose, (c,d) aster, and (e,f) carnation.

In a vessel, mix approximately 2.5 g of NH4Cl and 2 g of NaOH. At room temperature (T = 20−22 °C) and atmospheric pressure, these quantities will produce approximately 1 L of NH3(g). Both NH4Cl and NaOH are hygroscopic and, upon mixing, the instructor may notice the faint but characteristic smell of ammonia that is being produced in small amounts. Place the fresh flower in the glass jar and add a few milliliters of tap water to the small container. Swiftly close the lid (see Figure 1). Ammonia gas is produced rapidly and some bubbling occurs in the small container. Almost immediately, the color of the petals darkens and becomes more bluish than it was prior to its exposure to ammonia gas. Some flowers need more time for the change to occur; however, usually after 5 min the color changes (see Figure 2). If the flower remains in the container longer than 2−3 min, it becomes black (e.g., red roses), yellowish, or colorless. Longer exposures cause flowers to wilt (i.e., the rigidity of the plant’s stem and petals is highly affected, as can be seen in Figure 3). Disposal

The flowers employed in the demonstration can be directly thrown into a garbage can. The chemicals in the small container should be neutralized with weak acid (e.g., diluted acetic acid) or watered down with tap water and subsequently disposed of by being flushed down the drain in a well-ventilated place.

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Figure 3. Visual appearance of (a) rose and (b) aster after exposure to ammonia gas for more than 10 min. In comparison with the plants in Figure 2a,c (the same flowers), the burning, blackening, and death effects are evident.

HAZARDS AND SAFETY The relevant material safety data sheets should be consulted before use, and institutional risk-assessment procedures and safety regulations should be followed for any demonstration. Students should not be allowed to experiment with these chemicals on their own. The instructor and students nearby ought to use protective material: gloves, eye protection, and laboratory coats are highly recommended. The demonstration needs to be done in a well-ventilated place because, during its course, ammonia gas is produced.

• Ammonium chloride (NH4Cl): hazardous in the case of eye contact (irritant), slightly hazardous in the case of skin contact (irritant, sensitizer). • Sodium hydroxide (NaOH): corrosive. Extra precautions need to be taken because skin contact can produce inflammation and blistering; the degree of damage B



Demonstration

depends on the elapsed time of contact. When in solution, eye contact can result in corneal damage or blindness. • Ammonia gas (NH3): flammable gas with a characteristic noxious smell. Exposure for a long time can cause severe skin burns and eye damage; it is toxic if inhaled and may cause respiratory irritation. The recommended amounts of NH4Cl and NaOH are given in the Procedure section, above.

Scheme 1. Chemical Structure of the Flavylium Cation (2Phenyl-1-benzopyrilium Cation or 2-Phenylchromenylium Cation)a

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CHEMICAL BACKGROUND The presented method of ammonia gas production is similar to that reported by Thomas.10 It is simple and cost-effective, does not require heating, and can be considered as highly affordable by most schools. Upon addition of water, both NH4Cl and NaOH dissolve and dissociate readily and completely to produce ions. The NH4+ ions behave as an acid, and they react with OH− ions to yield NH3(g). The overall reaction is given in eq 1: H 2O NH4Cl + NaOH ⎯⎯⎯⎯⎯→ Na + + Cl− + H 2O + NH3↑

a

The A and B rings are indicated, and the atoms are numbered in red.

(1)

There are two main factors affecting the colors of the anthocyanidins: the nature and position of functional groups substituting the H atoms and the pH of the solutions.13−16 Because of the hydroxyl substituents, anthocyanin has bluish color; a higher degree of substitution results in a bluer color. Conversely, methoxy groups change the colors of the compounds to more reddish hues. The colors of anthocyanidins are very sensitive to the acidity of the solution. In aqueous acid solutions, anthocyanidins exist predominantly as flavylium cations, and their color is red. Upon increasing the pH, the color shifts from red to purple, blue, green, and yellow and finally to colorless, depending on the chemical species present in the solution (see Scheme 3). In aqueous acid solutions, the flavylium cation can undergo two parallel reactions: the first one is deprotonation to form the corresponding quinoidal base (a very fast process); the second one is hydration of the flavylium cation (a very slow process), which leads to the formation of hemiketal and subsequently to the production of colorless chalcone (via the tautomerization process).13 However, upon further increasing the concentration of hydroxide ions, the quinoidal base is deprotonated, and it gains a negative charge, leading to the subsequent formation of chalcone in an anionic form. The resulting color changes of the anthocyanidins depend on the relative concentrations of the different species that may coexist in solution. The ammonia molecule, which is similar in size to the water molecule, penetrates readily the flower petals, spreading through all the compartments and components of the cells (i.e., the cytosol, plastids, mitochondria, vacuoles, nitrogen-fixing symbiosomes, etc.).19,20 NH3(g) (Brønsted base, pKa ∼ 9.3), when in contact with water in the vacuoles, forms ammonium and hydroxide ions, causing an increase in the vacuole pH. Because the colors of anthocyanins are pH-dependent, the color of the flower petals changes. The transformation of anthocyanins is a reversible process; however, the presence of ammonia and ammonium ions inhibits oxidation of the diphosphopyridine nucleotide by blocking electron transfer from oxidized substrates to oxygen, thus inhibiting cell respiration21 and causing destructive oxidative stress to plant cell components.22,23 Moreover, basic environments disrupt hydrogen bonding between DNA bases, leading to their denaturation.24 All the above-mentioned processes are

The density of ammonia gas is lower than that of air, and so the produced NH3(g) flows to the top of the container, where the flower is located. Flower color is one of the most important features of plants. It attracts pollinators and protects floral organs, as most chemical compounds responsible for flower color can act as antioxidants. When petals are exposed to sunlight, the light penetrates the epidermal pigment layer and is partially absorbed by certain chemical components of the petals. The remaining light is reflected and is perceived by animals and humans as color. Since ancient times, people have extracted pigments from colorful flowers to study their composition and employed them in the preparation of new products. The color of a flower is mainly related to the type and amount of pigments in the petal and, to a minor extent, to the tissue structure. A flower’s color comes mainly from the presence of watersoluble hydroxylated and methoxylated derivatives of 2-phenyl1-benzopyrilium chloride. It usually gathers in the hydrophilic components of epidermal petal cells, where it dissociates to the flavylium cation or 2-phenylchromenylium cation. Its chemical structure is given in Scheme 1. The color of a 2-phenyl-1-benzopyrilium cation derivative aqueous solution originates mainly from the presence of eight double bonds in the cation structure. This conjugated electron system absorbs a part of the incoming energy in the form of visible light, producing very strong colors. Anthocyanidins are obtained when one or more of the (3, 5, 6, 7, 3′, 4′, 5′) hydrogen atoms are substituted by hydroxyl (−OH) or methoxy (−OCH3) groups. Anthocyanins are obtained when sugar molecules (e.g., glucose) are linked through ether bonds at the 3 or 5 carbon atoms.11 The presence of sugar molecules in the anthocyanins does not affect the maximum absorption wavelength and thus does not affect their color. The six most abundant anthocyanidins found in plants are called cyanidin (2(3,4-dihydroxyphenyl)-chromenylium-3,5,7-triol), delphinidin (2-(3,4,5-trihydroxyphenyl)-chromenylium-3,5,7-triol), pelargonidin (2-(4-hydroxyphenyl)-chromenylium-3,5,7-triol), malvidin (2-(4-hydroxy-3,5-dimethoxyphenyl)-chromenylium3,5,7-triol), peonidin (2-(4-hydroxy-3-methoxyphenyl)-chromenylium-3,5,7-triol), and petunidin (2-(3,4-dihydroxy-5-methoxyphenyl)-chromenylium-3,5,7-triol),12 and their chemical structures are displayed in Scheme 2. C



Demonstration

Scheme 2. Chemical Structures of Six Most Abundant Anthocyanidins as Cationsa

The corresponding counter ions, typically Cl−, are not shown for the sake of clarity.

a

Scheme 3. Chemical Structures of Pelargonidin That May Coexist in Solution as a Function of pHa

a

The scheme is oversimplified for the sake of clarity. A detailed description of anthocyanidin reactivity in aqueous solutions can be found elsewhere.15,17,18

accompanied by osmotic swelling of the cells in which NH3 was dissolved, leading to cell wall breakage.25 Thus, the plant tissue is no longer able to carry out its regular functioning. As a result, even if the flower is removed from the ammonia-rich atmosphere, the changes do not recede. The flower changes its color and wilts (Figure 3). Ammonia is an essential chemical compound for plant, animal, and human life. It originates from both natural and artificial sources. It is produced during the spontaneous breakdown of manure and the decomposition of dead plants and animals; it is also synthesized from nitrogen and hydrogen and used in the production of fertilizers and in a variety of household products, including cleaning products. However,

ammonia, in both gaseous and liquid states, can be irritating to the eyes, respiratory tract, and skin because of its alkaline nature. Its biological effects are dose-related (i.e., they depend on their environmental concentration, the amount taken up by the body, and the duration of exposure). A comprehensive report on ammonia’s impact on human health provided by the Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, indicates that exposure to NH3(g) might result in irritation of the respiratory tract.26 For example, most ammonia-based household cleaning products contain 5− 10% aqueous ammonia, and if some concentrated ammonia is spilled on the floor, a strong ammonia odor appears, and the eyes might water. However, if a person is exposed to very high levels D



Demonstration

(5) Vural, M.; Ogel, K. Dilated cardiomyopathy associated with toluene abuse. Cardiology 2006, 105 (3), 158−161. (6) Marjot, R.; Mcleod, A. A. Chronic Non-Neurological Toxicity from Volatile Substance Abuse. Hum. Toxicol. 1989, 8 (4), 301−306. (7) Ventura, F.; Barranco, R.; Landolfa, M. C.; Gallo, M.; Castiglione, A. G.; Orcioni, G. F.; De Stefano, F. Fatal poisoning by butane sniffing: A forensic analysis and immunohistochemical detection of myocardial hypoxic damage. J. Forensic Leg Med. 2017, 51, 57−62. (8) Hanson, G. R.; Venturelli, P. J.; Fleckenstein, A. E. Drugs and Society, 13th ed.; Jones & Bartlett Learning, 2018. (9) Wothers, P.; Royal Society Of Chemistry. Free Range Chemistry Rose in Ammonia, 2013. YouTube. https://www.youtube.com/ watch?v=yVLTmAWuFAw (accessed April 5, 2019). (10) Thomas, N. C. A Convenient Method to Prepare Ammonia and Hydrogen-Chloride Gases. J. Chem. Educ. 1990, 67 (5), 431−431. (11) Delgado-Vargas, F.; Jiménez, A. R.; Paredes-López, O. Natural Pigments: Carotenoids, Anthocyanins, and Betalains  Characteristics, Biosynthesis, Processing, and Stability. Crit. Rev. Food Sci. Nutr. 2000, 40 (3), 173−289. (12) Castañeda-Ovando, A.; Pacheco-Hernández, M. d. L.; PáezHernández, M. E.; Rodríguez, J. A.; Galán-Vidal, C. A. Chemical studies of anthocyanins: A review. Food Chem. 2009, 113 (4), 859−871. (13) Brouillard, R.; Iacobucci, G. A.; Sweeny, J. G. Chemistry of Anthocyanin Pigments 0.9. Uv Visible Spectrophotometric Determination of the Acidity Constants of Apigeninidin and 3 Related 3Deoxyflavylium Salts. J. Am. Chem. Soc. 1982, 104 (26), 7585−7590. (14) Fossen, T.; Cabrita, L.; Andersen, O. M. Colour and stability of pure anthocyanins influenced by pH including the alkaline region. Food Chem. 1998, 63 (4), 435−440. (15) Fleschhut, J.; Kratzer, F.; Rechkemmer, G.; Kulling, S. E. Stability and biotransformation of various dietary anthocyanins in vitro. Eur. J. Nutr. 2006, 45 (1), 7−18. (16) Torskangerpoll, K.; Andersen, Ø. M. Colour stability of anthocyanins in aqueous solutions at various pH values. Food Chem. 2005, 89 (3), 427−440. (17) Trouillas, P.; Sancho-Garcia, J. C.; De Freitas, V.; Gierschner, J.; Otyepka, M.; Dangles, O. Stabilizing and Modulating Color by Copigmentation: Insights from Review Theory and Experiment. Chem. Rev. 2016, 116 (9), 4937−4982. (18) Pina, F.; Melo, M. J.; Laia, C. A. T.; Parola, A. J.; Lima, J. C. Chemistry and applications of flavylium compounds: a handful of colours. Chem. Soc. Rev. 2012, 41 (2), 869−908. (19) Howitt, S. M.; Udvardi, M. K. Structure, function and regulation of ammonium transporters in plants. Biochim. Biophys. Acta, Biomembr. 2000, 1465 (1−2), 152−170. (20) Solubility of Gases in Water, 2008. Engineering ToolBox. https:// www.engineeringtoolbox.com/gases-solubility-water-d_1148.html (accessed April 5, 2019). (21) Vines, H. M.; Wedding, R. T. Some Effects of Ammonia on Plant Metabolism and a Possible Mechanism for Ammonia Toxicity. Plant Physiol. 1960, 35 (6), 820−825. (22) Shengqi, S.; Zhou, Y.; Qin, J. G.; Wang, W.; Yao, W.; Song, L. Physiological responses of Egeriadensa to high ammonium concentration and nitrogen deficiency. Chemosphere 2012, 86 (5), 538−545. (23) Kovácǐ k, J.; Hedbavny, J. Ammonium ions affect metal toxicity in chamomile plants. S. Afr. J. Bot. 2014, 94, 204−209. (24) Bimboim, H. C.; Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979, 7 (6), 1513−1523. (25) Chernyshev, A. V.; Tarasov, P. A.; Semianov, K. A.; Nekrasov, V. M.; Hoekstra, A. G.; Maltsev, V. P. Erythrocyte lysis in isotonic solution of ammonium chloride: Theoretical modeling and experimental verification. J. Theor. Biol. 2008, 251 (1), 93−107. (26) Agency for Toxic Substances and Disease Registry (ATSDR). Toxicological Profile for Ammonia; U.S. Department of Health and Human Services, Public Health Service: Atlanta, GA, 2004. (27) Cruz, S. L.; Rivera-García, M. T.; Woodward, J. J. Review of toluene action: clinical evidence, animal studies and molecular targets. Journal of drug and alcohol research 2014, 3, 235840.

of ammonia or if the skin comes into contact with concentrated ammonia, their skin, eyes, throat, or lungs may be severely burned. These burns might be serious enough to cause permanent blindness, lung disease, or even death. Numerous aliphatic, aromatic, and halogenated hydrocarbons that originate from paint thinners, glues, aerosol propellants, and gasoline fumes are volatile and may be present in the environment in the form of vapors. These products should be used with caution, because the acute health effects of hydrocarbon mixtures are generally associated with exposure concentrations as low as thousands of parts per million. Their effects are also dose-dependent, but adverse effects caused by them are much more serious, and warnings about their harmful effects should be given to people who sometimes use them to achieve altered mental states. Breathing toxic hydrocarbons may cause asphyxia; when volatile organic molecules displace oxygen, they impair normal breathing, which can induce unconsciousness and efficiently prevent an individual from self-rescue during the incident. Because of their chemical natures, these vapors (e.g., toluene27) can easily penetrate cells and replace naturally occurring substances within cell metabolic pathways, permanently impairing their functions and leading to cell death and, consequently, to the degradation of tissues, which may result in overall death of the organism,28 just like how NH3 interrupts important cell mechanisms and speeds up the wilting process of flowers.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.9b00001. Notes for instructors (PDF, DOCX)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Andrzej Sienkiewicz: 0000-0001-8670-5420 Sonia Losada-Barreiro: 0000-0002-9447-5626 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank Anna Deryło-Marczewska and Jolanta Narkiewicz-Michałek (the current president of Lublin’s division of the Polish Chemical Society) for their support and fruitful cooperation. The authors also want to thank Carlos Bravo-Diaz (University of Vigo, Spain) for his encouragement and scientific discussions.

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REFERENCES

(1) Maxwell, G. Chemistry Demonstrations For High-School Teachers; Cornerstone Science Publishing: New York, 2008. (2) Shakhashiri, B. Z. Chemical Demonstrations: A Handbook for Teachers of Chemistry; University of Wisconsin Press: Madison, WI, 2011; Vol. 5. (3) Kumar, K. S.; Krishna, K. R.; Bhaskara, D. Methods of teaching chemistry; Covery Publishing House: New Dheli, 2004. (4) Brouette, T.; Anton, R. Clinical review of inhalants. Am. J. Addiction 2001, 10 (1), 79−94. E



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(28) Bowen, S. E.; Batis, J. C.; Paez-Martinez, N.; Cruz, S. L. The last decade of solvent research in animal models of abuse: Mechanistic and behavioral studies. Neurotoxicol. Teratol. 2006, 28 (6), 636−647.

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Flower Color Change Demonstration as a Visualization of Potential

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