DEMONSTRATING THE PRESENCE OF CYANIDE IN BITTER SEEDS WHILE

Cyanides are old poisons, with a uniquely long, obscure history, undoubtedly because they grow so abundantly around us. They scent the leaves of the yew tree, the flowers of the cherry laurel, the kernels of apricot and peach, the taste and the aroma of bitter almonds. They are present in exudations of insects (e. g. Harpaphe Haydeniana), they are involved in the poisonous structures of cyanobacteria, they gather in the floating blue-green algae along the edges of the murkiest ponds and lakes and live in plants which populate forests and fields.^{i,ii,iii,iv,v,vi}

At each chemistry education level cyanides are considered in theoretical study because of their historical and commercial employ, for example the Prussian Blue (also called Paris Blue or Milori Blue) or, for German-speaking world, Berliner Blau, the first synthetic coordination compound prepared in the chemical history.^vii

Unfortunately, because of their toxicity, cyanides are practically absent in most laboratory experiments for students. Cyanides of sodium and potassium were widely used in qualitative chemistry for the recognition of Fe, Cu, Cd, Cr, Zn, Co, Ni, Ag, due to the fact that the corresponding complexes could be easily prepared, because of their typical color and corresponding solubility. ^viii,ix,x Today the difficulties connected to the acquiring and preserving of many cyanide salts, along with the danger of their use, discourage many teachers from using them in educational activities.

The main goal of the experiment here proposed is to observe the presence and the reactivity of cyanides in bitter seeds, which are used to prepare and flavour desserts and drinks (such as the Italian liqueur “Amaretto” and “Amaretti macaroons”), prepared with peach or apricot kernels, as well as with bitter almonds. Tart, Macaroons, Marzipan, Christmas Pudding and sugared seeds, similar to the old fashioned comfits, are still a popular sweetmeat prepared with bitter seeds. The use of kernels or seeds (from apricot, peach, almond and apple) in many recipes makes this experiment close to the everyday experience of students. We report also a list of different edible plants and fruits, containing cyanides, which are easy to find and buy.

As previously reported cyanides can be visualized and quantified using ninhydrin^xi,xii and in many other systems.^xiii,xiv Here we report an easy reaction with ninhydrin and copper complex as a qualitative classroom activity.

In the first part of this article a qualitative test for cyanides with ninhydrin in cyanogenic food is proposed. Bitter seeds for cyanide extraction are easy to find and cheap: they can be bought in grocery stores or online. In the second part of this article, different reactions with Copper(II) are proposed in order to investigate the complexation and redox equilibria of cyanide associated with showy color changings of the tested solutions. A spectrochemical series between water, cyanide, ammonia and ninhydrin can be rationalized after the observation of these reaction with copper ions.

ACTIVITY

SAMPLING

80 grams of bitter seeds (apricot, peach, bitter almonds or other cyanogenic vegetables reported in this article) must be crushed and pulverized, then the mixture must be introduced into a flask with a perforated cap (it is very important to buy whole seeds, not already crushed or pulverized ones). Add 250 ml of water, 3 g of Na₂CO₃ and 0.5 g of NaOH. The obtained mixture must be vigorously stirred for 15 minutes. Then introduce a tube for bubbling air in the perforated cap (the tube must get to the bottom of the bottle) and a second pipe for the gas outlet: this outlet tube must be connected to a Pasteur pipette which must be introduced successively into the tubes for colorimetric assays. 70 ml of 1M H₂SO₄ (5 ml at a time) must be slowly added into the bottle in order to release and display the cyanides released (see Figure 1). After each addition, the solution must be stirred again, in order to permit the formation of CO₂ and HCN, which are collected in the basic solutions containing Cu²⁺ complex or ninhydrin detectors. As soon as the solution in the bottle reaches acid pH, all cyanides have been released (for reagents, standard solutions and materials see SI).

Fig. 1 Apparatus for HCN extraction from bitter seeds solution. On the right: testing solutions of ninhydrin (yellow) and [Cu(NH₃)₄]²⁺ (blue).

PREPARATION OF NINHYDRIN REAGENT

The ninhydrin solution has to be freshly prepared by dissolving 50 mg of ninhydrin in 10 mL of 2% Na₂CO₃ and purged with nitrogen for 15 min. All solutions must be prepared using distilled water.

PREPARATION OF COPPER COMPLEX SOLUTION

Copper sulfate is the starting material for the one-step synthesis: when ammonia solution (ammonium hydroxide) is added to a clear, light blue, aqueous solution of Copper(II) sulfate, a powdery, light blue precipitate of copper(II) hydroxide forms.

2Cu²⁺ + (SO₄)²⁻ + 2NH₃ + 2H₂O → Cu(OH)₂-CuSO₄ ↓ +2NH₄⁺ (1)

Further addition of ammonia causes the copper ion to go back into solution as a deep blue ammonia complex.

Cu(OH)₂-CuSO₄ ↓ + 8NH₃ → 2[Cu(NH₃)₄]²⁺ + (SO₄)²⁻ +2OH⁻ (2)

This first syntheses can be done with a magnetic stir plate, standard glassware, and chemicals purchased from hardware stores and supermarkets. Household ammonia (2ml, 10% w/v) must be added dropwise to a solution of 50 mg of CuSO₄*5H₂O (0.0002mol) in 2 mL of distilled water with vigorous stirring. As each drop of NH₃(aq) touches the liquid surface, the characteristic deep blue of the tetraamminecopper(II) complex, [Cu(NH₃)₄]²⁺, can be seen for an instant, then it disappears. The dropwise addition of ammonia solution must be continued until the blue color of the complex remains throughout the reaction mixture. This will require 3-5 mL of NH₃(aq). For “Disposal of wastes and unused solutions” see SI.

QUALITATIVE ANALYSIS OF CYANIDE

WITH NINHYDRIN

Add 5 mL of ninhydrin reagent in a tube and bubble the gas from the sample bottle, observing the color change (see Figure 2). The solution color turns red if HCN is present in the bubbling gas and cyanide ions are trapped in basic solution of ninhydrin. The reaction is very selective for cyanide, because of the ciano-chromophore that forms (see Scheme 2). This experience represent the first strong evidence of the cyanide presence.

Then add some drops of NaOH solution (1M): the color of the solution turns blue. Discussions on chromophore groups and different colors of mono or double-ionized CN−ninhydrin product would be appropriate among undergraduate students.

Fig. 2 Ninhydrin solution reagent (left) and the same solution after 20 sec of bubbling: you can see the red ciano-chromophore product (right).

WITH TETRAMMINECOPPER(II) COMPLEX

Add 3 mL of tetraamminecopper(II) complex reagent in a tube and bubble the gas from the sample bottle, observing the color changing and the consequent precipitation. After 2 minutes the color solution turns green if HCN is present in the bubbly gas; after 10 minutes the solution loses color and dark precipitate forms (see Figure3).

Discussions on chromophore groups and different ligands in complexes equilibrium would be appropriate among undergraduate students.

Fig. 3 Copper-Ammonia solution (left),after 2 min of bubbling (center), and after 10 min of bubbling (right).

REACTION BETWEEN COPPER AND NINHYDRIN

Prepare a solution of copper sulfate in water. Split 1,5 ml of this solution into two tubes. Prepare a solution of tetraamminecopper(II) complex, as previously described, from the first solution. Add 4 ml of basic solution of ninhydrin to both the tubes. Shake vigorously until the dissolution of any possible precipitate (CuCO₃). Then observe the color of the solutions (see Figure 4).

Fig. 4 From the left: CuSO₄ (blue), CuSO₄ and ninhydrin (green complex), ninhydrin (yellow), ninhydrin and [Cu(NH₃)₄]²⁺ (dark yellow), [Cu(NH₃)₄]²⁺ (dark blue).

DISCUSSION

CYANIDE IN BITTER SEEDS

About 2500 species of plants from 80 families, including agriculturally important species, contain cyanogenic compounds (see Figure 5). Some of these plant species include linseeds, sorghum species, white clover, vetch, hydrangea, arrow grass, corn, flax, alfalfa, bamboo, lima beans, soy, millet sprouts, leaves and pits of Prunus species (black cherries, apricots, peaches), pear and apple seeds (see table 1). Cassava is a starchy tuber (like the potato) which, like the plants listed above, contains cyanide. Sudden disruption of growth in the plant, such as frost, drought or cutting can considerably increase the amount of cyanide.^{xv,xvi,xvii,xviii}

For this experience any of the listed vegetables can be used; in each case 150 g of material are enough for the complete activity. In particular bitter seeds from Prunus species contain a compound called amygdalin, which can be transformed into deadly “Prussic Acid” (hydrogen cyanide, HCN) after crushing, chewing, or any other injury to the seed (in the laboratory it is possible to use a blender or mortar).^xix Bitter seeds contain also the emulsin enzyme, which, in presence of water, acts on soluble glucosides, amygdalin, and prunasin, yielding glucose, cyanide and the essential oil, which is nearly pure benzaldehyde. In Table 1 the cyanogenic glycosides content for different species is reported. An apricot kernel contains approximately 0.5 mg of cyanide, bitter almonds may yield from 4–9 mg of hydrogen cyanide per almond.^xx,xxi It is possible to use other vegetables with similar amounts of cyanide for this experience, for example fresh leaves of Laurel Cherry (Prunus Laurocerasus), Cassava root (Manihot Esculenta, 1-4 mg/g), Bamboo shoots (different species, such as Dendrocalamus Giganteus, 1-8 mg/g), Flaxseed (5-6 mg of cyanogenic glycosides in one tablespoon). See “Planning experience with different seeds, kernel and vegetable” in SI. Each reported species contains different cyanogenic glycosides (see Table 1 and Figure 5), usable for the experience. ^{xxii, xxiii, xxiv, xxv, xxvi}

Table 1. Cyanogenic glycosides content for different vegetables:

Fruit Seeds	Cyanogenic glycosides content (mg*g-1)
Apricot	14
Cherry Black	3
Cherry Red	4
Peach	7
Plum Green	18
Plum Black (friar black)	10
Apple (Royal Gala)	3

Fig. 5. Chemical structure for the cyanogenic glycosides. Amygdalin can be found in the kernels of apples, apricots, peaches, almonds, cherries, and plums. Linamarin can be found in cassava, lima beans and flax seeds. Prunasin can be found in cherries and peaches. Dhurrin can be found in sorghum.

In each case bitter seeds or other vegetables contain an enzyme, which, in presence of water, acts on cyanogenic glucosides, yielding glucose and cyanide. When the solution containing the crushed seeds is acidified, HCN is released according to the equation:

H⁺ + CN^- → HCN ↑ (3

HCN flows in the tube, reaching the basic test solutions. In these basic solutions HCN releases CN^-, according to the reverse reaction above reported.

NINHYDRIN REACTION

A chemical reaction between ninhydrin and HCN in presence of Na₂CO₃ and NaOH is showed in Scheme 1. Such a reaction pathway may elucidate the color changes of the absorbing solution, during the addition of the ninhydrin reagent.^{xxvii,xxviii,xxix}

Scheme 1. Ninhydrin Reaction with Hydrogen Cyanide.

The reaction between ninhydrin and cyanides is very selective and specific. We use this reaction to display the cyanides in the used seeds. Ninhydrin is here used as a chromogenic reagent, which forms a red colored product, reacting with cyanide in slightly basic medium. The color turns blue after the addition of some drops of NaOH solution: the blue color is attributed to the divalent anion (pH >12).

COPPER REACTIONS

A coordinate complex is formed when a ligand surrounds the metal atom through coordinate covalent or ionic bonds. Transition metals behave as Lewis acids due to a partially filled d-orbital and are therefore capable of accepting electron pair/s. On the other hand ligands are capable of donating electron pair/s and can thus be referred to as Lewis bases. The stability of a particular metal complex depends on the nature of the metal and ligand used. Ligands with a high pK_b usually form more stable complexes. A higher charge on smaller sized metals and smaller ligands with larger electron density also give stability to the metal complexes. These molecular interactions between the electron donors and acceptors are mostly associated with the formation of colored charge-transfer complexes, which absorb photon in the visible region.^xxx

This very simple demonstration, which requires the preparation of only one solution, can be related to several concepts in general chemistry, including 1) the displacement of equilibria via the concentration changes, and 2) the colors of some complexes of copper ion, which may be observed in qualitative analysis.

One begins with an aqueous solution of copper(II) sulfate, in which the species [Cu(H₂0)₄]²⁺ is responsible for the relatively pale-blue color. The concentration of this solution is not critical, but a greater concentration does not result in a more intense color. Then aqueous ammonia is added, which complexes copper(II) in the deep blue [Cu(NH₃)₄]²⁺. We can assume that the complex ion equilibria in this system can be represented by a single equation in which the Cu²⁺ ion combines with four NH₃ molecules to form the four-coordinate [Cu(NH₃)₄]²⁺ complex.

After HCN flows from the bitter seeds bottle, the Copper(II) in the solution partially oxidizes the cyanide to cyanate, according to the equation:

2Cu²⁺ + CN^- + 2OH^- → 2Cu⁺ + OCN^- + H₂O (4)

Copper(I) is very unstable in solution and tends to disproportionate. The relative stability of Cu(I) and Cu(II) depends strongly on the nature of complexing anions or other ligands present in solution (such as ammonia and cyanide). However, under standard conditions, the formal potentials for the Cu⁺¹|Cu⁺², CN^-|CNO^- pairs are 153 and -970 mV respectively versus the standard hydrogen electrode, SHE. Therefore, the relative stability of Cu(I) and Cu(II) may be altered by the addition of a ligand which forms stable complexes.

In this case a large excess of [Cu(NH₃)₄]²⁺ is present in the solution, so a mixed valence copper amino-cyanide complex, Cu₃(NH₃)₄(CN)₄, forms, as previously reported, according to the equation: ^{xxxi,xxxii,xxxiii,xxxiv}

[Cu(NH₃)₄]²⁺ + 4CN^- + 2Cu⁺ → Cu₃(NH₃)₄(CN)₄ (5)

The cyanide from bitter seeds reacts as a reductive agent and as a ligand with Copper ions, forming a mixed valence complex.

The multicenter-mixed valence complex is green and in Figure 2 it is possible to see the color variation during the oxidation and the complex formation. After the bubbling process, different precipitates can be produced, depending on the kinetic and the presence of O₂ in the solution. As time goes on, more oxygen comes into the system and other copper compounds form and precipitate, such as Cu(OH)₂ and CuO, according to thermodynamic researches.^xxxv

The unstable mixed valence copper ammine-cyanide complex is formed due to the excess of ammonia in the solution.^xxxvi

Otherwise, starting from a pure CuSO₄ solution and adding cyanide, we would obtain:

Cu²⁺ + 2CN⁻ → Cu(CN)₂ ↓ yellow (6)

The precipitate would rapidly decompose into white copper(I) cyanide and cyanogen (highly toxic gas): ^xxxvii

2Cu(CN)₂ ↓ → 2CuCN ↓ + (CN)₂ ↑ (7)

The precipitate would then dissolve in an excess of CN⁻; therefore,colorless tetracyanocuprate(I) complex is formed:

CuCN ↓ + 3CN⁻ → [Cu(CN)₄]³⁻ (8)

This last colorless complex is very stable, but in this experience with bitter seeds, it is impossible to obtain a large excess of cyanides, so the previously reported considerations (such as the color changing and the complexation equilibria) take place.

Starting from [Cu(NH₃)₄]²⁺, this experience also avoids the formation of cyanogen (CN)_2, a very toxic gas.

COPPER AND NINHYDRIN REACTION

A chemical reaction between ninhydrin and Cu²⁺, in presence of Na₂CO₃, is showed in Scheme 1. Such a reaction pathway may elucidate the color changes of the solution.

As one adds the solution of CuSO₄ to the yellow solution of ninhydrin, a green precipitate (CuCO₃) forms. After vigorous stirring, the solution becomes clear and green complex Cu-ninhydrin forms (see Scheme 2).^{xxxviii,xxxix,xl,xli,xlii} Otherwise, using tetraamminecopper(II) complex, the solution of ninhydrin does not change its color; in fact the copper complex solution is not modified by the presence of ninhydrin. In this last case ninhydrin and unreacted [Cu(NH₃)₄]²⁺ remain in the solution whose color is now dark yellow (simply the sum of the yellow ninhydrin and the deep blue of the copper-ammonia complex).

Scheme 2. Reaction of ninhydrin with copper ions in alkaline solution, to form green Cu²⁺ complex.

In the present work ninhydrin is used as chromogenic ligand which forms a green-colored complex with copper(II) ions in slightly basic medium. The copper(II)–ninhydrin complex forms immediately after mixing the reactants, giving green color.

The Formation constant (Kf) describes the formation of a complex from its central ion and the attached ligands. Kf can be defined as follows:

Kf=[M_xL_y]/[M]^x[L]^y (9)

In this equation, M is a metal ion, L is a ligand, and “x” and “y” are coefficients.

The bigger the order of magnitude of Kf value is, the more favorable the products of the complex formation reactions are, as well as the larger the Kf value of a complex, the more stable it is.

In the first part of the experience proposed, two different ligands are present with Cu(I-II) ions: ammonia and cyanide. The relative Kf values are [Cu(NH₃)₄]²⁺=1.1*10¹³, [Cu(CN)₄]^-2=1*10²⁵, [Cu(CN)₄]^-3=2*10³⁰; on the basis of these values, it is possible to understand why cyanides can partially replace ammonia in a tetraamminecopper(II) complex solution. This consideration agrees with the spectrochemical series, a list of ligands ordered on ligand strength, such as:

I^-<Br^-<SCN^-<Cl^-<F^-≤OH^-<H₂O<NCS^-<CH₃CN<NH₃,<CN^-, NO, CO (10)

Generally, complex ions with polydentate ligands have much higher formation constants than those with monodentate ligands. This additional stability is known as “the chelation effect”. In scheme 3 it is possible to see that ninhydrin acts as a bidentate ligand. Nevertheless, in the last part of this experience it is possible to deduce that ninhydrin and copper form a stable complex, but at the same time, ninhydrin is unable to substitute ammonia from tetraamminecopper(II) complex. According to these considerations, we can locate nynhidrine between H₂O and NH₃ in the spectrochemical series. Therefore, at the end of this experience, it is possible to rationalize the spectrochemical series of ligands:

H₂O<ninhydrin<NH₃,<CN^- (11)

HAZARDS

Students must wear safety glasses and laboratory coats at all times. The extraction steps should be performed in a fumehood. The copper(II) salts are harmful if swallowed and by inhalation. Ninhydrin and ammonia too are harmful if swallowed and causes skin, respiratory and eye irritations.

Caution is needed in preparing the solutions. Sodium hydroxide pellets are caustic, so avoid contact with skin, especially near the eyes. To make up a solution, add the weighed pellets to cool water with stirring. When NaOH dissolves in water, heat is evolved. Concentrated sulfuric acid is extremely corrosive and can cause serious burns when not handled properly. This chemical is unique because it not only causes chemical burns, but also secondary thermal burns, as a result of dehydration. This dangerous chemical is capable of corroding skin, paper and metals. If sulfuric acid makes direct contact with the eyes, it can cause permanent blindness. If ingested, this chemical may cause internal burns, irreversible organ damage, and possibly death. Remember to wear safety glasses when preparing or presenting the experiment.

CONCLUSIONS

This demonstration can be easily adapted to a large lecture hall and student laboratory. In addition, it can be particularly interesting for those students who are qualitative analyzing food samples and toxicity. Another interesting feature of this experience is related to the color-changing phenomena, which are always stimulating for students to observe. At a somewhat more advanced level, other issues which might be included in the academic discussion are the structures of the complexes, the splitting of the d-orbitals and the relative field strength of the ligands, as well as the crystal field theory, and the absorption of light.

During the experiment, students can observe the progress of the reaction between cyanide and different reagents (ninhydrin and copper(II)) by the visual examination of these solutions.

Students are often surprised by the results, which indicate a significant presence of cyanide in common food as apricot, peach or bitter almonds (as well as other previously reported vegetables).

We suggest to precede the experiment with an explanation of the theoretical background of several concepts in general chemistry, including: redox potentials, crystal/ligand field theory and spectrochemical series, as well as the colors of some complexes of copper ion, so that the students can properly understand the results.

In conclusion, in this experience they can observe an example of redox processes, complexation-precipitation equilibria and kinetics of cyanide and copper compounds. The experience is also adapted to the time spent by students in laboratory, it is easy to handle and prepare, and the absence of toxic reagents and derivatives render this synthetic approach particularly useful for students in complete safety, without risks, and with low costs and ease to reproduce.

ASSOCIATED CONTENT

Supporting Information

Experimental details, notes for instructor, hazards, question and answer for lab experiences.

AUTHOR INFORMATION

Corresponding Author

Giorgio Volpi E-mail: [email protected]

Present Address

Department of Chemistry, University of Turin, Via P. Giuria 7, I-10125 Turin, Italy.

Notes

Author declare no competing financial interest. The author would like to thank Claudio Garino for the friendship and the scientific support.

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