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Extraction Method Reflux What is heating under a reflux?

? During extraction, heating under a reflux is heating of a mixture of compounds and the material for extraction in a flask with a condenser on top. This is done for its principle of heating without the loss of any solvents and compound. 1. Grind plant leaves to make 50g of powdered plant leaves 2. Fifty grams of the powdered plant leaves will be extracted by heating under reflux for 60 minutes with 750 mL 50% ethanol, accompanied by the addition of 250 mL 10% lead (II) acetate solution. Heating under reflux enables a mixture containing volatile liquids to be heated for a long time without the loss of the solvent. 3. The mixture is cooled and filtered; the filtrate is collected and is further extracted by shaking with three 375 mL quantities of dichloromethane/ isopropanol (3:2). The shaking should be gentle to avoid formation of emulsion. 4. The combined lower phases are filtered over anhydrous sodium sulfate and evaporated to remove the excess water. 5. The residue is collected and dissolved in 25 mL dichloromethane/ isopropanol (3:2). The solution obtained can then be ready for analysis for the presence of cardiac glycoside in saluyot leaves.

In heating under reflux, the water should enter from the bottom of the condenser. Also, there must not be a stopper at the top of the condenser, as the whole system cannot be sealed or else it may burst. The mixture of 50% ethanol, powdered saluyot leaf extract and 10% lead (II) acetate solution will be placed in the flask at the bottom.

General tests With FeCl3 A. Reagents 1. 4mL of glacial acetic acid 2. One drop of ferric chloride solution 3. 2mL of concentrated H2SO4 B. Materials and Apparatus 1. Serological pipette 2. Test tube 3. Dropping pipette 4. Beaker C. Procedure 1. Dissolve 1ml of extract in 4ml of glacial acetic acid containing one drop of ferric chloride solution. 2. Under lay the solution with 2ml of concentrated H2SO4. 3. A brown ring obtained at the interface indicates the presence of glycosides. D.Positive Result A brown ring obtained at the interface indicates the presence of glycosides. E. Principle The brown ring obtained is due to the presence of glycone sugar.

Foam test A. Reagents

1. 20mL distilled water. 2. 3gtts of olive oil B. Materials and Apparatus 1. Serological pipette 2. Graduated cylinder 3. Beaker 4. Filter paper 5. Hot paper C. Procedure 1. Boil about 2ml of sample in 20 ml of distilled water in a water bath and filter. 2. Mix 10ml of the filtrate with 5 ml of distilled water and shake vigorously for a stable persistent froth. 3. Mix the frothing with 3 drops of olive oil and shake vigorously, then observe for the formation of emulsion. D.Positive Result Formation of Emulsion E. Principle The formation of an emulsion is due to the presence of amphiphilic compound.

Specific tests Keller-Killiani A. Reagents 1.Glacial acetic acid 2.Ferric chloride solution 3.Concentrated sulphuric acid B. Materials and Apparatus

1.Dropping pipette 2.Test tubes C. Procedure 1.Treat five ml of each extracts with 2ml of glacial acetic acid containing one drop of ferric chloride solution. 2.Underlay the mixture with 1 ml of concentrated sulphuric acid. 3.A brown ring of the interface indicates a deoxysugar characteristic of cardenolides. 4.A violet ring may appear below the brown ring, while in the acetic acid layer, a greenish ring may form just gradually throughout thin layer D. Positive Result Presence of brown ring on the interface and greenish ring gradually on the upper phase. E. Principle Involved The brown ring indicates the presence of 2-deoxysugar in the glycone of the cardiac glycoside.

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Salkowski A. Reagents 1. Chloroform 2. Sulfuric acid B. Materials and Apparatus 1. Dropper 2. Test tubes C. Procedure 1. Add 1ml of the extract to 2ml of chloroform. 2. Carefully add H2SO4. A reddish brown color at the interface indicates the presence of aglycone portion of cardiac glycoside. D. Positive Result Reddish-brown color at the interface E. Principle Involved

In the Salkowski reaction based method, cholesterol is oxidized in the presence of an excess amount of phosphoric acid and ferric ions to give a reddish brown derivative. The presence of the aglycone portion of the cardiac glycoside structure is detected. The aglycone part is the portion of the structure where there is the absence of sugar.

Baljet A. Reagents 1. Picric Acid 2. Ethanol 3. Sodium Hydroxide B. Materials and Apparatus 1.Dropper 2.Test tubes C. Procedure 1. To prepare Solution I for Baljet Test, place 1g of picric acid in of EtOH. 2. To prepare Solution II for Baljet Test, add 10g NaOH in 100mL 3. Combine the two solutions. 4. Add 2-3 drops of the combined solution to 2-3mg of sample; a reaction is indicated by orange to deep red color. D. Positive Result Orange to deep red coloration E. Principle Involved Reactions due to (-CH2-) group of the lactone ring. cardenolide + Baljet's reagent (picric acid +NaOH) orange or red

100mL water. positive

Barfoed A. Reagent Barfoeds reagent (mixture of acetic acid and copper (II) acetate B. Materials 1. Serological pipette 2. Aspirator 3. Parafilm 4. Test tube 5. Hot plate 6. Beaker C. Procedure 1. In a test tube, add 1mL of Barfoeds reagent to 5gtts of the extract. 2. Seal the test tube with parafilm so as to prevent volatile substances from evaporating. 3. Place the solution in a boiling water bath. 4. When the solution yielded a reaction, remove from the water bath. 5. Observe a brick red precipitate indicating the presence of monosaccharide. D. Positive result Brick red precipitate E. Principle Involved Cardiac glycosides contain a sugar portion called glycone and a non sugar portion called the aglycone. If a reducing sugar is present, a brick red precipitate of Copper (II) oxide is form. The reaction will be negative in the presence of disaccharide sugar as they are weaker reducing agents. One can distinguish monosaccharide from disaccharide based on how fast the brick red precipitate is form. Monosaccharide reacts within 2-3 minutes, whereas disaccharide takes longer. The reaction involved is oxidation in acidic medium.

Seliwanoff A. Reagents Seliwanoffs reagent (dissolving 50 mg of resorcinol in 100 ml of dilute HCl or resorcinol in 6M HCl) B. Materials 1. Dropper 2. Breaker 3. Hot plate 4. Test tube C. Procedure 1. Place 0.5ml of a sample solution is in a test tube. 2. Add two ml of Seliwanoff's reagent (a solution of resorcinol and 3. Heat the solution in a boiling water bath for two minutes. D. Positive result Cherry red solution E. Principle Involved Cherry red solution indicates the presence of ketohexoses. The test reagent dehydrates ketohexoses to form 5-hydroxymethylfurfural. 5hydroxymethylfurfural further reacts with resorcinol present in the test reagent to produce a red product within two minutes. Aldohexoses react to form the same product, but do so more slowly.

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