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Name MUKARAM, SHAIRA R. Rating ____________________ The word carbohydrate is formed from the words carbon and hydrogen. Carbohydrates are combinations of the chemical elements carbon and hydrogen plus oxygen. Carbohydrates make up the bulk of organic substances on earth and perform numerous roles in living things. I.OBJECTIVES :
- To detect the presence of Monosaccharide, Disaccharides and Polysaccharides using different tests. II.PROCEDURES: a. Using the internet visit an educational website and search for the procedure, chemical composition and positive results of the following test; b. Write the procedure and attach an image for the positive results. A. MONOSACCHARIDES AND DISACCHARIDES
1. Moore’s Test – Influence of concentrated alkali
Procedures:
- Mix 1ml of 5% glucose sol’n with 1ml concentrated NaOH.
- Boil and note the change of color and color produced.
CARBOHYDRATES
Activity No. 2
Principles: This test is based on the liberation of aldehydes which subsequently polymerizes to form a resinous substance, caramel. That is, when a solution of reducing sugar is heated with alkali (NaOH), it turns yellow to orange and finally dark brown, liberating the odor of caramel. Chemical Composition: Positive Results: The solution turns yellow in the beginning and reddish brown later due to formation of caramel (a condensed product of glucose). Conc. NaOH : White solid ionic compound consisting of sodium cations Na+^ and hydroxide anions OH− . Reddish brown solution
interface. This is a sensitive but a non- specific test and is given positive by all types of carbohydrates. If the oligosaccharides or polysaccharides are present they are first hydrolysed to mono saccharides which are then dehydrated to give the test positive. Chemical Composition: Positive Results: An appearance of reddish violet or purple colored ring at the junction of two liquids is observed in a positive Molisch test. Purple colored ring Molisch reagent : 5% a – naphthol in alcohol
B. REDUCTION TEST
1. Fehling’s Test
Procedures:
Place 5ml each of Fehling’s A and B in a test tube.
Dilute it with 4 cc of water.
Boil 1ml of this mixture in a water bath (if change in color is observed, it indicates contamination. Discard and get a freshly prepared solution).
If color is unchanged after boiling, add 5% glucose sol’n drop by drop heating the mixture after each dilution.
Note the changes produced Principles: When aldehydes are added to Fehling’s solution, they are easily oxidized by the bistartratocuprate (II) complex. The reducing sugar glucose reduce the copper (II) ions in the test reagent to form red precipitate of cuprous oxide. The copper ions present in fehling’s solution in +3 state is reduced to +2 oxidation state and in alkaline medium it is precipitated as red cuprous oxide. Why the solutions are kept separate? It is made initially as two separate solutions, known as Fehling's A and Fehling's B. Fehling's A is a blue aqueous solution of copper(II) sulphate pentahydrate
2. Benedict’s Test
Procedures:
- Mix 1ml of Benedict’s reagent with 2 drops of glucose sol’n.
- Boil for 2 minutes and allow to cool.
- Observe for any change in color. Principles: Cupric ions, which in an alkaline environment, oxidize the aldehyde group to a carboxylic acid. Free aldehyde or keto group in reducing sugars reduce cupric hydroxide in alkaline medium to red colored cuprous oxide. Depending on the concentration of sugars, yellow to green color is developed. All monosaccharides are reducing sugars as they all have a free reactive carbonyl group. Some disaccharides, like maltose, have exposed carbonyl groups and are also reducing sugars, but less reactive than monosaccharides. Chemical Composition: Benedict’s Reagent - CuSO4, Sodium citrate, anhydrous sodium carbonate and water
Positive Results: Benedict's reagent starts out aqua-blue. As it is heated in the presence of reducing sugars, it turns yellow to orange. The "hotter" the final color of the reagent, the higher the concentration of reducing sugar. In general, blue to blue-green or yellow-green is negative, yellowish to bright yellow is a moderate positive, and bright orange is a very strong positive. Changes occur while exposed to heat. Green color - Up to 0.5 G% (+) Green precipitate - 0.5-1.0 G% (++) • Yellow precipitate - 1.0-1.5 G% (+++) Orange precipitate- 1.5-2.0 G% (++++) Brick red precipitate- > 2.0 G %(+++++)
3. Nylander’s Test
Procedures:
- Mix 1ml of 5% glucose w/ 0.1 ml of Nylander’s reagent.
- Heat for 5 minutes in a water bath and note the result.
4. Barfoed’s Test
Procedures:
- Mix 1ml of Barfoed’s reagent with 0.1 ml of 5% glucose sol’n.
- Heat for 30 seconds and allow to stand for 15 minutes. Principles: Barfoed’s test used copper (II) ions in a slightly acidic medium. Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes. Reducing disaccharides undergo the same reaction, but do so at a slower rate. This test is used to distinguish reducing monosaccharides from disaccharides by controlling pH and time of heating. Monosaccharides usually react in about 1-2 minute while the reducing disaccharides take much longer time between 7-12 minutes to react with the reagent. Similar to Benedict's but lower pH and shorter time. Chemical Composition: Barfoed’s Reagent: Copper (II) acetate in the dilute acetic acid with a pH of 4.6.
Positive Results:
5. Picric Acid Test
Procedures:
- To 1ml of 5% glucose sol’n add 0.5 ml of saturated picric acid sol’n and 0.2mlof 10% Na2CO3.
- Warm and note the result. Principles: The picric acid test for carbohydrates is a very sensitive chemical test for the presence of reducing sugars. The reducing sugars react with Picric Acid (toxic yellow crystalline solid) also chemically known as 2,4,6-trinitrophenol (TNP) to form a red coloured Picramic Acid. All monosaccharides and disaccharides containing the potentially free aldehyde or ketone group possess reducing properties. Red precipitate of cuprous oxide
6. Seliwanoff’s Test (Resorcinol HCL Reaction)
Procedures:
- Place 1ml of Seliwanoff’s reagent in each of 6 test tubes.
- To the first tube, add 3 drops of 2% freshly prepared sol’n of fructose, to the second add glucose, to the third, galactose, to the fourth sucrose, to the fifth maltose, and to the sixth, xylose.
- Boil in a water bath.
- Note the color produced and record the time required for the development of pink in each of the tubes.
- Filter and dissolve the precipitate in ethyl alcohol. Principles: This test is useful for detecting the presence of Keto – hexose. Keto hexoses on treatment with hydrochloric acid form 5 - hydroxy methyl furfural which on condensation with resorcinol gives a cherry red colored complex. The test will be answered by fructose, sucrose and other keto containing carbohydrates. Other sugars, especially sucrose will also give a positive test, but usually require a longer time. Color turns red fastest if the sugar is a ketose:
Red < 1 min (fastest) is a monosaccharide ketose Red ~ 1 min. (slightly slower) is a disaccharide ketose Red > 1 min. (longest) is aldose Chemical Composition: Positive Results: Cherry red solution Seliwanoff’s reagent: Resorcinol, Conc. hydrochloric acid Sucrose (+) Fructose (+) Glucose Galactose Xylose Maltose
Chemical Composition: Positive Results:
8. Mucic Acid Test
Procedures:
- Prepare 3 test tubes and label as follows: “glucose”, “galactose” and “lactose”.
- Add 1 mL (15 drops) of each of the test sugars respectively.
- Carefully place 1 mL (15 drops) of concentrated nitric acid into each tube and mix thoroughly for a minute.
- Heat the tubes in a boiling water bath for about 1hr.
- Observe crystals. For arabinose: Blue-green solution (positive) For galactose: Muddy-brown to grey solution (negative) Bial’s reagent : 50% saturated orcinol solution in alcohol
Principles: The mucic acid test is used to identify the presence of the sugar galactose. When galactose solution is heated with concentrated nitric acid, a dicarboxylic acid called mucic acid or galactaric acid is formed as a white precipitate, which counts as a positive result. Chemical Composition: Positive Results: Crystal precipitate Concentrated nitric acid: Nitrogen dioxide absorbed in water
Chemical Composition: Positive Results:
2. Hydrolysis Test
Procedures:
- Place 20ml of starch solution in a beaker.
- Add two drops of concentrated HCl.
- Boil.
- At intervals of 3 minutes, test a drop with Lugol’s and a small portion with Benedict’s reagent until iodine test becomes negative and the Benedict’s test becomes positive. Blue-black solution Lugol’s Solution: Potassium iodide with iodine in water
Principles: Starch is basically a complex carbohydrate containing large number of sugar molecules which are bonded together with glyocsidic linkage. HCL is added to starch to change it into simpler monomers of glucose. The mechanism behind the working of HCL on starch is called acid hydrolysis. If the HCl hasn’t yet broken down the starch into monosaccharides, the reaction with Benedict’s reagent is negative but positive with the Lugol’s reagent. While boiling, the process is repeated until the solution becomes positive for the Benedict’s test and negative for the iodine test, indicating the presence of monosaccharides. *Chemical Composition: Positive Results: *shown in a spot plate Lugol’s solution* : iodine (5%) and potassium iodide (KI, 10%) together with distilled water Benedict’s test Iodine^ test Positive for starch Positive for^ Negative for starch monosaccharides Negative for monosaccharides
