Preliminary Test for the Determination of Plant Constituent It was done to conduct a photochemical screening of a particular plant and he identification of plant constituent present in its extract. Preparation of the Plant Extract Place 1 Go of dried ground plant sample or egg of fresh sample in an Erlenmeyer flask covered with a funnel. To this, add ml of 80% ethyl alcohol. Place the flask over a hot water bath and reflux for an hour. If exhaustive extraction is desired, boil the sample for ten minutes only, macerate in an oleander and then continue alcohol extraction for an hour.
Pour the mixture while hot through a Bummer funnel line with filter paper and fitted to a suction flask. Rinse the flask and plant material with fresh portions of 80% ethanol. Include rinsing with the extract. Concentrate the extract to about 1 ml and keep in a tightly stopper container. Concentrated extracts should be stored with a trace of toluene or chloroform to prevent fungal growth. A. Test for Alkaloids a. Preliminary Alkaloid Test Take an equivalent of ml of plant sample. Evaporate to a syrupy consistency.
Add ml of MM HOC to the extract, stir while heating for 5 minutes and then cool. Add about 0. G powdered NCAA while heating for 2 minutes. Cool and filter the mixture. Add enough fresh MM HCI to wash the residue and then bring the volume of the filtrate to ml. Place Mil each of the filtrate to 2 test pubes as follows: 1. To Mil of the filtrate, add a few drops of Mayor’s Reagent. Result: Light brown solution with precipitate Remarks: There is a formation of precipitate, therefore positive in Alkaloids 2. On Mil of the filtrate, add a few drops of Wager’s Reagent.
Result: Light brown solution with precipitate Remarks: There is a formation Of precipitate, therefore positive in Alkaloids b. Confirmatory test To the remaining ml of the extract, add drowses enough 28% NH to render the solution alkaline. Extract the alkaline solution 3 times with 1 Mol of CHECK (Chloroform). Evaporate the combined CHECK extract over steam bath. The alkaline layer is reserved for the test for quarterly and/ or amine oxide bases). Take up the residue with ml of MM HCI stirring over a steam bath for about 2 minutes. Filter and divide into 2 equal portions and test as follows: 1.
With Mayor’s Reagent Result: Clear solution with clear precipitate With Wagner-‘s Reagent Result: Clear solution with precipitate Remarks: There is a formation of precipitate, therefore positive in Alkaloids c. Test for Quaternary and/or Amine Oxide Bases Add drop wise with stirring with sufficient MM HCI to the alkaline layer until the solution is acidic. Filter and divide into two portions and test. A positive result may be taken as indicative for the presence of quaternary and/or amine oxide bases. 1. With Mayor’s Reagent With Wager’s Reagent B.
Test for Proteins Evaporate the plant extracts equivalent to ml of the plant sample and take up the residue with 1 Mol of water. A. Centerpiece test To Mil Of the aqueous, add 1 Mol drops of HON. and observe for the formation of white precipitate Result: Yellowish solution with red precipitate Remarks: There is no formation of white precipitate, therefore negative in protein. B. Million’s test To Mil of the aqueous extract, add 1 Mol drops of Million’s reagent. Place in oiling water bath. Observe the color. Result: Yellow to light brown solution Remarks: There is no formation of light yellow to pinkish color, therefore negative in protein.
C. Test for Fixed Oil a. Stain Test Boil mi of the plant sample in Mimi of petroleum ether. Take a place of white paper and place on its surface 2 drops of the petroleum ether extracts. Result: No stain produced Remarks: There is no formation of stain, therefore negative in Fixed oil D. Test for Tannins Take the ethanol extract equivalent to ml of plant materials. Evaporate to incipient dryness over a water bath and cool. Add 5 drops of NCAA elution to salt out understate constituent. Filter. Divide the filtrate into 3 test tubes labeled A, B, C. Reserve the contents of test tube A as blank. A.
Gelatin Test To test tube 8, add 3 drops of gelatin salt reagent. Observe any formation of a precipitate. Precipitate of the gelatin indicates the presence of Tannins. Result: No precipitate was formed Remarks: There is no formation of precipitate, therefore negative in Tannins b. Ferric Chloride test To the test tube C, add 3 drops of ferric chloride solution. Observe any color change. A black color may indicate the presence of hydrothermal tannins, if he gelatin test is positive. Result: It produced dark green solution Remarks: There is no formation of blue black color, therefore negative in Tannins.
E. Test for Carbohydrates Evaporate the 80% alcohol extract equivalent to ml of plant sample. Take up the residue with 1 Mol of distilled water. Divide into 2 equal parts. A. Feelings test Boil ml of freshly prepared feelings solution in a test tube, then add an equal quantity of plant extract and boil again. Result: Light red solution with brick red precipitate Remarks: There is no formation Of brick red precipitate, therefore positive for Carbohydrates b. Moor’s test Boil mi of the plant extract with an equivalent of caustic soda(Noah) solution.
Result: Formation of brown solution Remarks: Brown solution indicates the presence of Carbohydrates F. Test for Anthracnose a. Brontosaur test Evaporate the equivalent of 1 ml of the plant extract to incipient dryness using water bath. Take up the residue in Mimi of distilled water and filter. Extract the filtrate with mm’ of benzene, јice. Divide the combined benzene extracts into 2 portions. Reserve one portion as blank. To the second portion, add ml of ammonia solution. Shake. Observe the alkaline layer for color changes. The formation Of red color is a positive test for Anthracnose compounds.
Results:Test tube A produced orange solution,Test tube B produced orange solution Remarks: There is no formation of red color, therefore negative in anthracnose compounds. B. Modified Brontosaur test a water bath. The residue is taken up with ml of 0. MM KOOK and Mil of diluted hydrogen peroxide. Heat on a water bath for 10 minutes. Cool and filter. Add glacial acetic acid drowses until acetic to litmus paper. Extract with 5 ml of benzene, twice. Divide the combined benzene extract into 2 portions reserved one portion as blank. Alkalinity the second portions with 2-ml of ammonia solution.
Observe the alkaline layer for color changes. A red or pink color is indicative of the presence of Antihistamines. Result: The upper portion produced clear solution while the lower portion produced yellow solution. Remarks: There is no formation of pink or red color, therefore negative in Anthracnose compounds. G. Test for the Cardinalities and Faddishness’s a. Keller-killing test Take alcohol extract equivalent to 1 Mol Of plant material. Evaporate to incipient dryness over water bath, defeat extract by titration with hexane to remove the residual hexane solvent. Add ml of Feces reagent.
Stir and transfer to a test tube. Hold the test tube in an inclined position and carefully add Mil of concentrated HOSES, allowing the acid to run along the inside wall of the test tube. Allow the liquid mixture stand undisturbed. Result: Light brown solution with brown precipitate Remarks: There is no formation of golden ring, therefore negative in Cardinalities and Faddishness’s. H. Test for Aspirin Glycoside a. Froth test Take a volume of the alcohol extract equivalent to mi of plant material. For control, use ml of 80% go extract in a separate tube. Add Mimi of distilled water to each test tube.
Stopper and shake the test tube vigorously for 30 seconds, allow to stand and observe over a period of 30 minutes. Result: The test tube with go extract produced bubbles, Test tube with plant extract produced bubbles. B. Lieberman-Buchwald test Evaporate the equivalent of 1 mm’ of plant extract to dryness using a water bath. Add about 1 Mol of hexane or petroleum ether, to the cool residue. Stir for a few minutes, allow to settle and decant off the supernatant. Repeat until all the color has been removed. To the residue and about 10 ml of chloroform and stir for 5 minutes. Decant into a test tube containing about MGM of anhydrous Nassau.
Shake and pass through dry filter paper. Divide the filtrate into 2 clean dry test tubes. TO one position, add 3 drops acetic anhydride. Mix gently. Then add one drop of concentrated sulfuric acid and mix gently. Observe color changes immediately over period of an hour. Use the other test tube as reference. Result: Red brown color was produced Remarks: There is no formation of red brown color, therefore positive in Aspirin Glycoside. Extraction and Purification About log of the plant sample macerated with 1 ml of chloroform for 3 days. And addition of activated charcoal if undesirable color appear.