Chapter 3 Tests for Siling LabuyoCapsaicin, a kathamine alkaloid derivative (Phenylalkylamine), does not precipitate like most alkaloids since there’s only a 0.14% yield from the chillii plant. (Arnold Brossi, 1990) N- (4- hydroxyl- 3 methoxybenzyl)- 8- methyl- non- 6- enamide General Test for Alkaloid: General Test Positive Result Mayer’s Cream or pale yellow precipitate Wagner’s Brown or reddish brown precipitate Hager’s Tannic Acid Vitali-Morin Yellow precipitate Amorphous or Crystalline Precipitate Violet precipitate Experimental Result Pale yellow solution Brown or reddish precipitate Yellow solution Amorphous precipitate No reaction brown Procedure for Preparing the Reagents: Reagent Mayer’s (the reagent is available in the laboratory) Wagner’s ( Iodo-potassium iodide) Hager’s (Picric acid) (the reagent is available in the laboratory) Tannic Acid (the reagent is from the extract of the tannin group) Vitali-Morin Procedure Dissolve 1.358g of HgCl2 in 60ml water and pour into a solution of 5g KI in 10ml of water. Add sufficient water to make 100ml. Dissolve 2g of iodine and 6g of KI in 100ml water. Dissolve 1g of picric acid in 100ml of water. Dissolve 10g of tannic acid in 10ml of ethanol and dilute with water to 100ml. See procedure for general test. Procedure for General Testing of Alkaloid: Test Procedure Mayer’s To 1mL of the extract, add 2-3 drops of the Mayer’s reagent. Wagner’s To 1mL of the extract, add 2-3 drops of the Wagner’s reagent. Hager’s To 1mL of the extract, add 2-3 drops of the Hager’s reagent. Tannic Acid To1mL of the extract, add 2-3 drops of the Tannic acid solution reagent. Vitali-Morin To 1mL of the extract, add 5-10 drops nitric acid. Evaporate to dryness. Dissolve the residue in acetone. Add methanolic solution of potassium hydroxide. Principles Behind the Tests: Test Principle Like this. metals like aluminium. the nitro groups are introduced and sulfonic acid groups are displaced. etc. Nitration followed by Complex reaction mechanism (Bronsted-Lowry theory) Complexation reaction mechanism (Bronsted-Lowry theory) Positive for Tropane alkaloid. It should be borne in mind that a very small amount of impurity may conceal or to a great degree. amyl alcohol.Mayer’s Wagner’s Hager’s Tannic Acid Vitali-Morin Halogenation followed by Complex reaction mechanism. strychnine. There is a great difference in the comparative solubility of the alkaloids in many of the organic liquids like chloroform. there must be a base to accept the proton. petroleum ether. and the methods devised for their separation are based upon this. Its weak acidity is due to the numerous phenol groups in the structure which can precipitate many organic and inorganic compounds. in chemistry. Tannic acid complexes with and precipitates alkaloids such as nicotine. and silver. just as ammonia is freed from its salts by the same reagents. In Hager’s test. most of them. Tannic acid is a polyphenol. So the Bronsted-Lowry concept can be defined by the reaction acid + base conjugate base + conjugate acid. Only a few of the alkaloids are liquid. ether. The reaction can proceed in either forward or backward direction. It may also give a false negative result for the impurities of the extracted alkaloid may have interfered with the structure of the capsaicin. Halogenation followed by Complex reaction mechanism. Wagner’s test is similar in principle to the Mayer’s test where the reagent changes the degree or angle of the bonds leading to a complex reaction mechanism. to a lesser extent. (the group’s alkaloid is not a Tropane alkaloid) Discussion: The alkaloids are a class of vegetable compounds which are chemically similar to ammonia. being crystallize-able solids. in each case the acid donates a proton to the base. The separation and identification of the alkaloids is not as simple an operation as that of the metallic compounds. Mayer’s Test gives a negative result for Ephedrine and Purine alkaloids. is defined as the product formed from the neutralization reaction of acids and bases). as the alkaloid is less soluble than its salts. They are decomposed by alkalis. (Arnold Brossi. lead. apomorphine. It follows that if a compound is to behave as an acid. the free alkaloids are of a basic nature and they form salts by uniting with acids without the setting free of hydrogen. the alkaloid being set free. They all have a bitter taste. donating a proton. A salt is formed with the alkaloid since picric acid is an acid and was combined with the alkaloid which is basic. Thus. as well as their salts. mercury. as a rule. they would form a salt (A salt. Specific Test for Alkaloid: . the picric acid slowly sublimes even at room temperature making the test results give a false negative result. The reaction of the picric acid is highly exothermic and over time. and it also precipitates. When the solution is concentrated this process often results in their precipitation. Their salts are. much more easily soluble in water than the alkaloids themselves. the addition of tannic acid in the extracted alkaloid gives an amorphous precipitate since tannic is an acid and alkaloids are basic. 1990) In the Bronsted-Lowry theory defines an acid as any chemical species (molecule or ion) that is able to lose. and the conjugate acid is the species created when the base accepts the proton. modify the reactions. or donate a hydrogen ion (proton) while a base is any species with the ability to gain or accept a hydrogen ion (proton).. as do their salts. benzene. The conjugate base is the ion or molecule remaining after the acid has lost a proton. Shake vigorously. Baeyer’s Addition of distilled water and solid potassium permanganate into the extract.Specific Test 2.4. Jones’ Dissolve 10 mg or 2 drops of the extract in 1 ml of pure acetone in a test tube and add to the solution 1 small drop of Jones reagent (chromic acid in sulfuric acid). Bromine To 1mL of the extract. Add 8 drops of the Nitrating mixture (2mL conc. Sulfuric acid and 2 ml Nitric acid in an Erlenmeyer flask immersed in water bath).4-DNP: The chemistry of the reaction: The overall reaction is given by the equation: .DNP (test for aldehydes and ketones) Baeyer’s (test for active unsaturation) Nitration (test for aromacity) Bromine (test for unsaturation) Jones’ (Chromic Acid test) Positive Result Experimental Result Orange precipitate (positive for Turbid orange solution ketones) Brown solid Brown solution with brown precipitate Yellow oily layer Yellow oily layer Colorless Colorless Further addition turns the solution to reddish color Blue-green solution Blue-green solution Procedure for Specific Testing of the Alkaloid: Test Procedure 2.4.Dinitrophenylhydrazine reagent (available in the laboratory) in a test tube. add 1-2 drops of pure bromine. Principles behind the Tests: Test Principle 2. Then shake the test tube.4. Nitration Place 5 drops of plant extract in a test tube.DNP Addition-Elimination (condensation reaction) Baeyer’s Oxidation with reduction (Redox reaction) Nitration Electrophilic aromatic substitution Bromine Electrophilic addition Jones’ Oxidation reaction Discussion: 2.4-DNP Add 2 drops of the plant extract in 2mL 95% Ethanol to 2. In oxidation with reduction. (Ralph Shriner. When the two solutions react. The reagent is the concentrated nitric acid and the catalyst is the concentrated sulfuric acid. then it is a ketone. thus the purple color disappears. In this case. 2004) Look carefully at what has happened. The KMnO4 (Mn+7) reagent is reduced to MnO2 (Mn+4).4dinitrophenylhydrazine first adds across the carbon-oxygen double bond (the addition stage) to give an intermediate compound which then loses a molecule of water (the elimination stage). the target of which is the double bonds. (Ralph Shriner. MnO2 on the other hand is a muddy brown precipitate which will appear for positive results. To put it simply. If at least one of them is a hydrogen. The target of which is the benzene ring. The positive result is an orange or yellow precipitate from a carbon-oxygen double bond which indicates the presence of an aldehyde or ketone. The reaction is known as a condensation reaction. 2004) Baeyer’s: The Baeyer’s test is similar to that of the Bromine test which tests for active unsaturation. The reagent gets reduced in the process. then the original compound is an aldehyde. it releases NO2+ which is called nitronium ion. Since it is reduced. this is a nucleophilic addition-elimination reaction. The principle involved is the electrophilic aromatic substitution because it substitutes hydrogen with NO2.R and R' can be any combination of hydrogen or hydrocarbon groups (such as alkyl groups). 2004) Potassium permanganate is purple. 2004) You can just use it to test for the presence of the carbon-oxygen double bond. 2004) Nitration: Nitration test is a test for aromacity.4-dinitrophenylhydrazone". the double bonds get broken and the substrate oxidizes and becomes a diol compound. The 2. (Ralph Shriner. A condensation reaction is one in which two molecules join together with the loss of a small molecule in the process. that small molecule is water. this nitronium ion attaches to the benzene ring and to retain the aromatic . (Ralph Shriner. The product is known as a "2. If both are hydrocarbon groups. 2004) In terms of mechanisms. (Ralph Shriner. (Ralph Shriner. it will get electrons from the nucleophile which is Br-. 2004) Jones’: A positive test is marked by the formation of a green color within 5 seconds upon addition of the orangeyellow reagent to a primary or secondary alcohol. Thus. the final product is a dibromo-compound. meaning electron-deficient. the positive result was the disappearance of the red color. A positive test for aldehydes and primary or secondary alcohols consists in the production of an opaque suspension with a green to blue color. But further addition of pure bromine would make the solution saturated. Thus. (Ralph Shriner. The principle involved is electrophilic addition because the double bonds are broken and the electrophile is added to the substrate. will in return release the hydrogen attached to where the NO2 was attached. 2004) . To compensate for it.stability. the red color of the free Br2 disappears. the product is a nitrocompound which is responsible for the yellow oily layer in the specific test result. thus destroying the double bond and making the other side positive. 2004) Bromine: Bromine test is a test for unsaturation and the target is the double bonds. Br2 is red because it was combined with the substrate in the reaction so that there won’t be any free Br2. (Ralph Shriner. It gets the electron. (Ralph Shriner. Thus. The electrophile Br+ will react with the electron-rich portion of the substrate which is the double bond. As a result. Br2 reagent separates to Br+ and Br-.