Experiments: Crucial Reagents

Today, I am going to introduce you three crucial chemical reagents that I will use repeatedly in our up-coming experiments or projects. I've chosen the first two chemical compounds to selectively screen or spot-test certain plant toxins based on a colour changing reactions. That allows us to isolate them even in a kitchen chemistry set up. The last compound is a powerful mutagen that I will be using to induce genetic variations in plants.  

1. Dragendorff's Reagent

Dragendorff's Reagent

The Dragendorff's reagent is perhaps the single most useful substance that we will employ to detect alkaloids. It is invented by the German chemist Johann Georg Dragendorff in the 18th century, but it's still being used by scientists all around the world today. In fact, I used this reagent on a daily basis when I was doing my doctorate study, so it is very dear to me. The Dragendorff's reagent is a chemical mixture comprising of a non-oxidising acid (such as acetic acid), potassium iodide (KI), and bismuth oxynitrate (Bi₅H₉N₄O₂₂). Bismuth is a heavy metal and it reacts with potassium iodide under acidic conditions to produce a complex negative ion called bismuthtetraiodide (BiI₄^–). The Dragendorff's reagent will produce an orange or reddish precipitate when it reacts with most plant alkaloids. The exact nature of this chemical reaction is not well-understood, but it is widely accepted that negative BiI₄^– ion forms a red coloured complex with alkaloids, which are mostly secondary or tertiary amines. It should be noted that all alkaloids have to be protonated (under acidic environment) for the reaction to proceed. The Dragendorff's reagent is quite specific, because it only gives positive result to alkaloids, and not other phytochemicals such as flavonoids, steroids, amino acids, chlorophyll etc. This aspect is particularly favorable because after all, many plant toxins of our interest are alkaloids, from atropine to morphine to strychnine, they all show up on the Dragendorff's test. Thus, we will be using the Dragendorff's reagent to not just detect the presence of alkaloids in plants, but also to guide their isolation attempts. The Drangendorff's reagent is very versatile, stable on storage, and can be used as a spray for alkaloids absorbed on paper or thin layer chromatography (TLC) plates. Don't worry, we will deal with chromatography sooner or later.

Example of various alkaloids separated on a TLC plate, and stained with the Dragendorff's reagent.

 2. Picric acid (2,4,6-Trinitrophenol)

Picric acid crystals (wet). It exists as yellow crystals or in a strong yellowish solution. Note: Picric acid is a relatively hazardous substances, do NOT risk it if you are not experienced.

  

Picric acid is another chemical reagent that I'll be using to selectively detect the presence of cardiac glycosides (cardenolide) and cyanide, two of the most deadly plant toxins. Picric acid is an aromatic compound, which means that it contains a central benzene ring that confers it great stability. Besides, it has a phenol functional group, which is triple nitrated using nitric and sulfuric acids at carbon position 2, 4, and 6, hence it's alternate name 2,4,6-trinitrophenol. The three nitro group sucks electron away from the phenolic hydrogen atom, making it highly acidic. However, as suggested by its name, picric means 'picros' or bitter, so this is an acid that supposedly tastes bitter. Don't asked me if I've tried it because picric acid is actually quite nasty. Picric acid is a structural analogue of the famous military explosive TNT (2,4,6-Trinitrotoluene). When it is dry, or when it in the form of heavy metal salts called picrates, picric acid can explode (detonate) with great violence! It is a dangerously unstable explosive that was responsible for the Halifax explosion (Canada, 1917), which is the most powerful non-nuclear explosion in human history. So please be warned, picric acid is definitely something you do not want to fool around, and I'm using it only for demonstration purposes. The small quantity you see here was freshly prepared, and kept under water, which will render it relatively inert because the trick is to forbid it from drying out or reacting with metals. 

Figure 1: Reaction of picric acid with cardenolide glycoside under alkaline conditions.

Picric acid reacts with most if not all cardiac glycosides containing the cardenolide lactone group. A lactone is a cyclic ester, and cardenolide is a conjugated cyclic ester, which contains a double bond next to the ester functional group (Figure 1). The conjugated cardenolide system can react with a base (alkali) to produce a powerful nucleophile called enolate. This enolate will then attack picric acid at its phenolic carbon 1 to produce a resonance stabilised compound known as the Meisenheimer's complex. It is this complex that gives rise to a colour change, usually from yellow to orange or red, which indicates a positive test for cardenolide glycosides. This reaction is called the nucleophilic aromatic substitution. The Meisenheimer's complex is unstable and it will eventually lose a leaving group (hydroxide or water), while favouring the formation of a substituted cardenolide-picric acid adduct. Hence, the picric acid reagent that's employed to detect cardenolide glycosides is formulated by dissolving saturated picric acid solution into sodium hydroxide, which is a strong alkali, to which test samples are added. The color change (if any) would be instantaneous. This particular reagent is also known as the Beljet's test, and it's still used today in conjunction with more advanced spectroscopic techniques to quantify the concentration of cardiac glycosides from plants. 

 

Example of a positive test with the Beljet's reagent (alkaline picrate). Note: Left over solutions were destroyed using Fenton's reagent. Alkaline picrate may present an explosive hazard when dried.

Apart from cardiac glycosides, picric acid also gives a colour change with free cyanide ions, especially hydrogen cyanide in its gaseous state. To my best knowledge, the precise reaction between picric acid and cyanide is not well-studied, but it is said to give a promising rose-red product, which can be used to quantify cyanide from a given plant sample. Some old literature mentioned that picric acid reacts with hydrogen cyanide under alkaline conditions according to produce isopurpurate, which is the rose-red substance (Figure 2). In that case, the reaction would be more or less similar to nucleophilic aromatic substitution, which was aforementioned apart from cyanide ion being the nucleophile. I must admit that I have not used picric acid to test for cyanide in plants before, but since it is a well-established method that is still used today, I am most excited to give it a try!

 

Figure 2: Reaction of picric acid with hydrogen cyanide under alkaline conditions.

 3. Sodium azide

Crystals of sodium azide.

If you think picric acid is bad enough, sodium azide is probably the most hazardous reagent that we will be using in our future experiment, namely induction of new plant traits by chemical mutation. I'm not here to exaggerate how toxic anything is considering what I like and what I do, but sodium azide is one chemical that you should definitely NOT trifle with without experience. It is extremely toxic, and is rapidly fatal if inhaled, ingested or in contact with the skin. It is also explosive when reacted with metals much like picric acid, if not more sensitive. In the lab, we treat sodium azide as seriously as sodium cyanide, that is with the utmost care. And just like cyanide, sodium azide will react with acid and even water (slowly) to produce a highly toxic and explosive gas called hydrogen azide, which would turn out to be important for us. But guess what? This most unpleasant chemical compound sodium azide is still used today in some car airbags, as an explosive to inflate the live-saving airbags! 

Figure 2: Mechanism of chemical induced DNA mutation in plants. Note: Sodium azide tends to create point mutations (substitution AT – GC).

 

Despite its hazards, sodium azide is one of the most powerful and widely used plant mutagen to improve crop traits. This is because the azide ion can induce point mutations in germinating seeds, which result in different traits when the plants grow up. It is worthy to note that not all mutations are bad, and some may confer 'superhuman' traits to plants, such as improved tolerance to stress and pest, or increased yields or fruits etc. In the ornamental plant industry, chemical mutagens are also used to produce chimera or variegated plants with great success. Here, we aim to produce plants with very high toxin yields (hopefully), or perhaps wilder crazier flowers just for fun. I'll show you an example at the end of this article. Anyways, to achieve mutation of seeds, we will only need very minute quantity of sodium azide (in microMolar range), which is buffered in an acid. This produces hydrogen azide in small quantities, which then permeates the seed coat deep into the plant's DNA. Inside, an enzyme called O-acetylserine sulfahydrase (OASS) will incorporate the azide anion into an amino acid called L-cysteine, leading to the formation of a powerful mutagen called azidoalanine (Figure 3). It is this azidoalanine that gets incorporated into DNA as a faulty base pair, which then promotes point mutations (particularly A-T to G-C) when the DNA replicates during germination (Figure 2 and 3). Many improved or genetically modified crops are induced using sodium azide as a mutagen, and it is a most useful reagent in this aspect. I have personally chosen sodium azide for one reason, because it only mutates plants. There are many other mutagens out there, but others (including gamma rays) tend to mutate human DNA too and I don't ever want to get cancer from them, touch wood. Therefore, I decided on sodium azide because it's very nasty but at least not cancer-causing!

Figure 3: Proposed in-vivo transformation of azide into the potent mutagen azidoalanine.

Example of azide mutated plants with variegated leaves. Ref: Mostafa, G. G. Int. J. Plant Breed. Genet. 2011, 5, 75-85

 

That's it for today, and we will meet these reagents in due course when we decide which experiments we should do. Again, I welcome any constructive comments and suggestions, and we will see what to do. Cheers!