Exercise 20 Aim: To detect the presence of carbohydrates like ghicose, sucrose and starch, Principle: Carbohydrates with the free aldehyde or ketone groups (ie., simple sugars) reduce copper sulphate of Benedict and Fehling's reagent to cuprous oxide forming 2 yellow or brownish-ted coloured precipitate depending on the concentration of sugat. No change of blue colour ‘Absence of reducing sugar Blue changes to green precipitate 0.1.0.5 % of reducing sugar Blue changes to yellow precipitate to 05 to 1.0% of reducing sugar Blue changes to orange-red precipitate | to 1.0-2.0 % of reducing sugar Blue changes to brick-red precipitate Over 2.0 % of reducing sugar Requirements: Glasswares - test tubes, funnel, beaker; Chemicals - Benedict's solution, Fehling's solutions A and B; concentrated HCl, saturated solution of NaOH, dilute iodine solution; Equipments - water bath; Miscellatcous - test tube holder, test tube stand, spirit lamp. Sample for test: Fruit juice of apples/banana/leaves Of onion/sugar cane extract, milk, ete. Preparation of reagents (Benedict's reagent: Mix 173g of Sodium citrate and 100g of anhydrous sodium carbonate in 600mL. of water in a beaker and warm gently (solution A). Dissolve 17.3g of hydrated CuSO, in 100mL of distilled water (solution B). Add solution B to solution A with constant stirring, Cool and transfer to a One litre flask and make the volume upto the mark with water (ii) Fehling's reagent A: Dissolve 6.93g of copper sulphate in 100ml. of distilled water. (ii) Fehling’s reagent B; 20g of KOH and 34.6g of sodium-potassium tartarate (Rochelle’s salt) dissolved in 100 ml. of distilled water. A. Test for glucose Procedure : | ‘Take small pieces of banana, apple and grapes. Crush them separately and strain their juices in different (est tubes. Take milk in another test tube. CutLaboratory Manual: Biology fresh leaves of onion bulb into small pieces and boil for 2 to 3 minutes ina test tube and filter it, (a) Benedict's test + Take 2 ml of juice (fruit extract) / milk / onion leaf juice in a test, tube, + Add 2 mL of Benedict's reagent te it and boil. Direct heating of test tube should not be done. It should be carried out in a water bath at the boiling point of water. + The colour changes from blue to green and finally to orange or brick red indicating the presence of simple sugar (glucose). Benedict's Test 1. Apple juice 2. Banana extract 3. Grapes juice 4, Onion juice 5. Milk (b) Fehting'’s test + Mix equal volume/ofFehling's sclution A and B (1-2mL} in a test tube. + add equal volume of the fruit juice or onion leaf juice or milk and place it in water -bath at the boiling of water. + The initial blue colour turns green to yellow and finally a brick-red precipitates formed. Febling’s Test 1, Apple juice 2. Banana extract 3, Grapes juice 4, Onion juice 5. Milk. 23.04.2018Exercise 20 B. Test for Sucrose Procedure Collect sugarcane juice or cut sugarbeet into small pieces. Crush them and strain their juice, (a) Benedict's and Fehling's test + Perform Benedict's and Fehling’s test with cane sugar or sugar beet, Juice as described above. * No change of colour indicates the absence of simple sugars (Le., monosachharides). (b) Hydrolysis test + Take 10 mL of sample juice in a beaker, add few drops of concentrated HCl and boil * After cooling, add 4 mL of saturated solution of NaOH or Na,CO, to neutralize the solution, Now divide the solution into two parts. Transfer one part in test tube ‘A’ and the other in test tube" + Perform Benedict's test with sample in test tube ‘A’. Doyou observe any change in colour? If there is a change, itindicates the presence of simple sugar or monosaccharides. + Perform Fehling's test with saiiple in test tube '5'Do you observe any change in colour? If there is change. it indicates the presence of simple sugar or monosaccharides. C. Test for starch (lodine test) Principle: lodine specifically makes a blue-coloured complex with starch. Procedure * Add 1 or 2 drops of dilute iodine solution to 2 to Sm of extract. + Ablue-black colour shows the presence of starch. * The blue colour disappears on heating and reappears on cooling. Discussion ‘The composition of blue coloured substance is not well defined. This may be an adsorption complex of starch with iodine rather than a definite compound.Laboratory Manual: Biology Questions 1. Whatis the use of HClin the test for carbohydrate? 2. Why does the blue colour disappear on boiling and reappear on cooling in the test, for starch? 3. Sucrose solution gives a negative test with Benedict's test. Why? 4, Will Iodine test give a positive result with glucose, fructose or sucrose solution? 5. Why are monosaccharides called reducing sugars? 23.04.2018Exercise 21 ‘Aim: To detect the presence of proteins. Principle: Proteins respond to some colour reactions due to the presence of one or more radicals or groups of the complex protein molecule. All proteins do not contain the same amino acids, and hence they de not respond to all colour reactions. Nitrogen atoms in the peptide chain form a complex (violet colous) with copper ions in the Biuet test (Biuret tests for peptide bond in the molecule of a protein.) Xanthoproteic test is specific for protein containing aromatic amino acids. The benzene ring in the amino acids is nitrated by heating ‘with nitric acid and forms yellow nitro-compounds which turns to orange colour with alkali Requirement: Glasswares: test tube, spirit lamp; Chemicals: 40% NaOH, 1% CuSO, solution, Concentrated HNO, , 20% NaOH solution; Miscellaneous: test ube holder, test tube stand, Procedure | | (2) Biuret test + Take 2 mL of protein solution (milk, albumin of egg or gram seed extract) in a test tube. ‘+ Add 1 mL of 40% NaOH solution and 1 or 2 drops of 1% CuSO, solution, + Aviolet colour indicates the presence of proteins. Care must be taken that excess of copper sulphate is not added otherwise there will be blue colour instead of violet colour. (0) Xanthoproteic test + Add carefully ImL of concentrated HNO, to 2mL of protein solution (albumin of egg, milk or gram seed extract) + Awhite precipitate is formed. + Boil the solution and the colour changes to yellow, + Cool the test tube and add 2mL of 20% NaOH (or ammonia solution] to make it alkaline. + The colour changes to orange indicating the presence of proteins. 23.04.2018Laboratory Manual: Biology iscussion o Ayellow stain is often observed on skin when it comes in contact with nitric acid. The reason of yellow stain is xanthoproteic reaction. Questions 1. Why does the skin turn yellow when it inadvertently comes in contact with HNO,? 2. Why are only few drops of CuSO, solution added during the biuret test? 23.04.2018Exercise 22 Jim: To detect the presence of fats (lipid) indifferent plants and animal materials. Principle: Lipids (fas) are usually insoluble in water but soluble in organic solvent, Sudan IIL reagent gives a specific red colour with fats. Requirement: Glassware; test tubes; Chemicals: Sudan IIL, ethyl alcohol, chloroform, ether, benzene; Miscellaneous: test tube holder, test tube stand, oil/ghee/butter. Procedure (a) Solubility test Arrange five dry test tubes in a test tube stand. ‘Add 2 mL of water, ether, chloroform, benzene and ethyl aleohol to cach test tube followed by one drop of mustard oil, ghee or butter. Shake thoroughly. Oil, ghee or butter is broken into small droplets in water which float at the surface indicating that fat is insoluble in water. But in acetone, benzene and ethyl alcohol no such droplets are formed as the oil, ghee or butter dissolves in these organic solvents. (b) Sudan III test ‘Take 2mL of mustard oil in a test tube, Add equal volume of water to it and shake. Add a pinch of sudan Ill and shake again. ‘As the layers separate out, the lipid layer is seen to be stained red, whereas water layer remains uncoloured. This indicates the presence of lipids. Questions 1, What is the simplest form of fat? 2, Will fat dissolved in organic solvent give a positive result with sudan III? 23.04.2018