Alkaloidals qualitative & quantitative analysis.

QUALITATIVE TESTS, QUANTITATIVE TESTS, DIFFERENT FORMS OF ALKALOIDS AND THEIR CLASSIFICATION,
OF ALKALOIDS.

1. Assignment no-3 Qualitative and quantitative analysis of alkaloids Presented to: Dr. Inder Pal Singh Presented by: D. Gnanabhaskar 16TMM2364 NIPER, Mohali. SAS NAGAR

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3. 4/17/2017 3 INTRODUCTION Because alkaloids are so heterogeneous chemically and there are so many of them, they cannot be identified in plant extracts using a single chromatographic criterion. In general, because of the wide range of solubility and other properties of the alkaloids, any general screening procedure for alkaloids in plants may fail to detect particular compounds. J. B Harborne , “ Phytochemical Methods” 1978(3)203-213

4. 4/17/2017 4 Forms of alkaloids& their classification Sr.no Alkaloidal form Example 1 Salts with special acids Meconic acid in Opium& Quinic acid in Cinchona 2 Steroidal Glycosidal alkaloid. Solanine in Solanum & chaconine 3 Terpenoidal Alkaloids. Taxol ( diterpinoidal alkaloid) J. B Harborne , “ Phytochemical Methods” 1978(3)203-213

5. Dr. Mazen A . El- Sakka “ Phytochemistry of Alkaloids” 2010(3) Al Azhar University Faculty of Pharmacy Department of Pharmacognos y . 5 Classification According to Hegnauer's classification, which is based on both, the type of nitrogen and the biochemical origin, three main types of alkaloids are distinguished: 1. True alkaloids : that are derived from amino acids and have nitrogen in a heterocyclic ring. 2. Protoalakloids :that are derived from amino acid and do not have nitrogen in heterocyclic ring. 3. Pseudo alkaloids: that are not derived from amino acids but have nitrogen heterocyclic ring. Different systems of classification may be adopted based on: 1. The pharmacological action (biological activity.) 2. The chemical structure (type of nitrogen, heterocyclic or non heterocyclic and type of ring structure.) 3. The biochemical origin (biosynthetic pathway of production in the plant .) 4. The taxonomical origin (plant families rich in alkaloids.)

6. 4/17/2017 6 Volumetric methods: These are based on reaction of alkaloidal bases with acids (Acid-Base titration). They include: Aqueous titration : This is carried by either: 1- Direct titration of the alcoholic solution of the alkaloidal residue with standard acid, or 2- Back titration by dissolving the residue in a known amount of standard acid and back titration of residual acid against standard alkali. Non-aqueous titration : This method is suitable for determination of weak bases e.g. Caffeine. ed. Quantitative Determination of Alkaloids

7. 4/17/2017 7 Gravimetric methods: They can be performed by either: 1- Direct Weighing of the alkaloidal mixtures 2- Precipitation of the total alkaloids and determination of the weight of the precipitate obtain Spectrophotometric Methods.( Spectrophotometric Method for Estimation of Alkaloids Perceptible with Dragendorff’s Reagent in Plant Materials) Polarimetric Method .(The colorimetric determination of various alkaloids Authors F. J. Bandelin ) Fluorimetric Method .(Simplified fluorimetric determination of digitalis alkaloids Datta V. Naik .) Chromatographic Methods.

8. 4/17/2017 8 QUALITATIVE ANALYSIS

9. 4/17/2017 J . B Harborne , “ Phytochemical Methods” 1978(3)203-213 9 Extract dried tissue with 10% acetic acid in ethanol, leaving to stand for at least 4h. Concentrate the extract to one-quarter of the original volume and precipitate of the alkaloid by drop wise addition of conc.NH 4 0H. Collect by centrifugation , washing with 1 % NH 4 OH . Dissolve residue in a few drops of ethanol or chloroform. Chromatograph an aliquot on sodium citrate-buffered paper in n- butanol-aqueous citric acid. Chromatograph another aliquot on silica gel G plates in methanol-conc.NH 4 0H (200: 3). Detect the presence of alkaloids on the paper and plate, first of all by any fluorescence in UV light and then by application of three spray reagents - Dragendorff , iodoplatinate and Marquis. RFs and colours of twelve of the commonest alkaloids are shown in below table. Confirm the presence of a particular alkaloid by measuring the UV spectrum of a sample dissolved in 0.1 M H 2 SO 4, Typical maxima values range from 250 to 303nm Alkaloids with aromatic rings in their structures may also absorb at longer wavelengths, e.g. colchicine"- max 243 and 351 nm. Sample preparation:

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11. 4/17/2017 J. B Harborne , “ Phytochemical Methods” 1978(3)203-213 11  This reagent was invented by the German Pharmacologist, Johann Georg Dragendorff (1836–1898). Dragendroff It is a color reagent to detect alkaloids in a test sample. Alkaloids, if present in the solution of sample, will react with Dragendorff’s reagent and produce an orange or orange red precipitate.  Composition: Dragendroff’s reagent is a solution of potassium bismuth iodide prepared from basic bismuth nitrate(Bi(NO 3 ) 3 ), tartaric acid, and potassium iodide (KI).   Dragendorff’s test VIDEO OF THE TEST DOWNLOAD TO SEE

12. 4/17/2017 J. B Harborne , “ Phytochemical Methods” 1978(3)203-213 12   Wagner's test   . When reagent treated with sample Reddish brown precipitate confirms the presence of alkaloids Wagner’s Reagent (Potassium Triiodide): Iodine = 1.3 g Potassium = 2.0 g Distilled water to make = 100.00 ml

13. 4/17/2017 13 It is freshly prepared by dissolving a mixture of mercuric  chloride (1.36 g) and of  potassium iodide  (5.00 g) in water (100.0 ml). Most alkaloids are precipitated from neutral or slightly acidic solution by Mayer's reagent (potassiomercuric  iodide  solution) to give a cream coloured precipitate Mayer's test

14. 4/17/2017 14 AMMONIA SOLN/VAPORS Murexide test VIDEO OF THE TEST DOWNLOAD TO SEE

15. 4/17/2017 15 Hagers test J. B Harborne , “ Phytochemical Methods” 1978(3)203-213

16. 4/17/2017 J. B Harborne , “ Phytochemical Methods” 1978(3)203-213 16 Solid alkaloid + fuming HNO3 → Evaporate to dryness, dissolve residue in acetone, add methanolic solution of KOH → Violet colour . Vitali-Morin’s test-Tropane alkaloids VIDEO OF THE TEST DOWNLOAD TO SEE

17. 4/17/2017 17 Libermann’s test This test is very useful in distinguishing various alkaloids. It is 10% solution of KMnO 4  in H 2 SO 4 . Whenever a microdrop of this solution is applied to given sample of  alkaloid , the following different colour will show the  alkaloids presence . 1.   Atropine , Hyoscyamine , Hyoscine, Ephedrineq  Orange  colour . 2.   Papaverine , Emetine Black colour . 3.   Cocaine, Physostigmine  Red colour . 4.   Colchicine Yellow colour . 5.   Yohimbine  Blue colour .

18. 4/17/2017 18 1. Dissolve 5-10 mg of the alkaloid in a few drops of hot amyl alcohol or ethanol and allow it cool gradually. The appearance of jelly-like product gives the characteristic test of the solanum alkaloids. 2. When a few mg of the alkaloids is treated with antimony trichloride solution in dry chloroform, it gives rise to a distinct red colouration . The   solanum alkaloids , in general, produces an instant red-violet colour with formaldehyde (HCHO) and sulphuric acid (H 2 SO 4 ). This particular test is so distinct and sensitive that it is used for the quantitative estimation of these alkaloids colorimetrically . Steroidal alkaloids

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20. 4/17/2017 20 J. B Harborne , “Phytochemical Methods” 1978 (3)203-213 William Charles Evans “Trease and Evans' Pharmacognosy ,” (16)301-370 Dr. Mazen A. El- Sakka “ Phytochemistry Alkaloids” 2010 (3) Al Azhar University Faculty of Pharmacy Department of Pharmacognos y . Janet travell , “The potassium mercuric iodide reagent for alkaloids.” From the Laboratory of Pharmacology, University of Illinois, College of Medicine, andassisted by a grant from the American pharmaceutical association July, 1934 , 689-698. Datta v. Naik “Simplified fluorimetric determination of digitalis alkaloids” J Aalytica Chemica Acrta , , 74 ( 1975 ) 29-33. References