Biochemistry for Dental Students (Theory and Practical) Shreya Nigoskar
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151PRACTICAL152

CarbohydratesCHAPTER 12

 
 
Molisch's Test [Group Test for Carbohydrates]
Principle: Alcoholic alpha naphthol forms furfural derivatives, such as hydroxymethyl furfural, by the concentrated sulphuric acid acting on the sugar. This compound forms a reddish-violet colored ring at the junction of the two liquids.
Molisch's Reagent: A 5% solution of alpha naphthol in alcohol.
Procedure: Add 2 drops of Molisch's reagent to 2 ml of sugar solution in a test tube. Mix thoroughly. Add 2 ml of conc. H2SO4 by the side of the test tube slanting the tube. Then erect the test tube slowly. The formation of reddish-violet ring at the junction of the two liquids indicates the presence of carbohydrate.
Discussion: Concentrated solution of organic compounds may give a red instead of a violet colour due to charring action of the sulphuric acid. In case of doubt, the experiment should be repeated on a more diluted solution of the substance to be tested.
 
Iodine Test [Test for Polysaccharides]
Principle: The composition of the blue- or-red wine red-colored substance is not well defined. This may be an adsorption complex of starch or dextrin or glycogen with iodine rather than a definite compound.
Iodine Reagent: The 0.5 ml. of iodine diluted to 5 ml with distilled water.
Procedure: Add 1–2 drops of dilute iodine solution to 2–3 ml of dilute starch or dextrin or glycogen, respectively, in case of starch, the blue colour disappears on heating and reappears on cooling. But the red colour and the brown colour in cases of dextrin and glycogen, respectively, do not reappear on cooling as in the case of starch.
Reduction tests: Carbohydrates with free aldehyde or ketone groups have the ability to reduce solutions of various metallic ions. These properties are mentioned below.154
 
Fehling's Test
Principle: Carbohydrates with free aldehyde or ketone groups reduce copper sulphate to cuprous oxide forming a yellow or brownish—red coloured precipitate.
Fehling's reagent: Prepare freshly by mixing equal volumes of two stock solutions A and B.
Solution A: The 6.93 gm of CuSO4. 5H2O per 100 ml of water.
Solution B: The 20 gm of KOH and 34.6 gm of sodium potassium tartar ate (Rochelle salt) per 100 ml solution.
Procedure: Add a few drops of sugar solution at a time to 5 ml of Fehling's solution and heat the mixture after each addition. The production of yellow or brownish—red cuprous oxide precipitate indicates the presence of reducing sugars.
 
Benedict's Test
Principle: Carbohydrates with free aldehyde or ketone groups reduce copper sulphate of benedict's solution to cuprous oxide on boiling forming a red, yellow or green coloured precipitate depending on the concentration of the sugar.
Benedict's qualitative solution: Dissolve with heat 173 gm. of sodium citrate and 100 gms. of anhydrous sodium carbonate in 600 ml of water in a beaker. Into this with constant stirring, run slowly a solution of copper sulphate (CuSO4 5H2O) containing 17.3 gm dissolved in about 100 ml. of water. Cool and transfer to a litre flask and make to the mark with water.
Procedure: Add 8 drops of sugar solution to 5 ml of the Benedict's qualitative reagent in a test tube. Boil vigorously for 2 minutes or place in a water bath for 3 minutes. Allow to cool spontaneously (do not cool it by immersion in cold water). A red, yellow or green precipitate develops depending on the concentration of sugar present.
Colour
Approximate amount of reducing sugar
No change of blue colour
Absence of reducing sugar
Blue changes to green ppt.
0.1–0.5 gm% of reducing sugar
Blue changes to yellow ppt.
0.5–1 gm% of reducing sugar
Blue change orange-red ppt.
1–2.0 gm% of reducing sugar
Blue changes to brick-red ppt.
Over 2.0 gm% of reducing sugar
 
Barfoed's Test
Principle: The monosaccharides with free aldehyde or ketone groups can cause the reduction of copper sulphate to cuprous oxide with the development of a red precipitate.155
Barfoed's Solution: Dissolve 66.5 gm of neutral crystalline copper acetate in 1000 ml of distilled water, add 9.0 ml of glacial acetic acid.
Procedure: Add 8 drops of sugar solution to 5 ml of Barfoed's solution. Heat to boiling for 30 seconds. A red precipitate appears indicating the presence of monosaccharides.
Discussion: This test is a reduction test for mososacchardies and the reduction is brought about in an acid solution. If the solution with the sugar is boiled for a few minutes, the disaccharides also are hydrolysed giving a positive. This test is specific for monosaccharides.
 
Selivanoff's Test
Principle: Fructose is acted on by hydrochloric acid to from a derivative of furfuraldehyde which gives a red coloured compound when linked with resorcinol.
Seliwanoff's Reagent: Dissolve 50 mg of resorcinol in 33 ml of concentrated hydrochloric acid and dilute to 100 ml with water.
Procedure: Add a few drops of sugar solution (0.5ml) to 5 ml of Seliwanoff's reagent in a test tube. Heat to boiling for 30 seconds. Formation of red colour indicates the presence of fructose. The test may be positive for sucrose also if it is hydrolyzed during the course of the test.
Discussion: A similar colour may also develop in case of glucose or maltose if the boiling is prolonged due to the transformation of glucose into fructose by the catalytic action of the hydrochloric acid.
 
Osazone Test
Principle: A solution of reducing sugar when heated with phenylhydrazine, characteristic yellow crystalline compounds called osazone are fromed. Simple sugars like glucose, fructose and mannose produce the same osazone because of the similarities in their molecular structures.
Table 12.1   Characteristic features of osazone crystals
Osazones
Times of formation of crystals
Appearence of crystals
Glucosazones
5 minutes
Broomstick like
Fructosazone
2 minutes
Broomstick like
Maltosazone
10–15 minutes
Sunflower like
Galactosazone
7 minutes
Rhombic like
Lactosazones
10–12 minutes
Powder puff like
Procedure: Add 10 drops of glacial acetic acid to 5 ml of sugar solution in test tube. Then add a knife point of phenylhydrazine hydrochloride and double the amount of sodium acetate crystals. Mix and warm a little to see that the solids are dissolved.156
zoom view
Fig. 12.1: Osazones for glucose/fructos, lactose, maltose, galactose respectively
Filter the solution in another test tube and keep the filtrate in a boiling water bath for 20 minutes. Allow the tube to cool slowly in the water bath without cooing it hurriedly under the tap to have better crystals and examine the crystals under the microscope.
Discussion: Formation of osazone crystals of different sugars depends on the time schemed below (Table 12.1 and Fig. 12.1).
Sucrose does not produce osazone crystals when the solution is kept in the boiling water bath for 30 minutes because of the hydrolysis of sucrose to glucose and fructose.