Qualitative tests to identify functional groups of carbohydrates

test for carbohydrates, molisch's test, benedict's test, iodine test, seliwanoff's test, osazone tests

1. Qualitative tests to identify functional groups of carbohydrates in given solutions (Glucose, Fructose, Sucrose, Lactose) DR. UDITA MUKHERJEE UNIVERSITY OF DELHI

2. MATERIALS REQUIRED Test tubes, test tube stands, test tube holders, water bath, droppers, cotton, slides, coverslips etc. Conc. Sulphuric acid, a- Naphthol , iodine solution, Barfoed’s reagent, Benedict’s reagent, Seliwanoff’s reagent, glacial acetic acid, phenylhydrazine , sodium acetate.

3. INTRODUCTION Carbohydrate , class of naturally occurring  compounds  and derivatives formed from them. In the early part of the 19th century, substances such as  wood ,  starch , and  linen  were found to be composed mainly of molecules containing  atoms  of  carbon  (C),  hydrogen  (H), and  oxygen  (O) and to have the general formula C 6 H 12 O 6 ; other organic molecules with similar formulas were found to have a similar ratio of hydrogen to oxygen. The general formula C x (H 2 O) y  is commonly used to represent many carbohydrates, which means “watered carbon.”

4. CLASSIFICATION OF CARBOHYDRATES Although a number of classification schemes have been devised for carbohydrates, the division into four major groups— monosaccharides ,  disaccharides ,  oligosaccharides , and  polysaccharides —used here is among the most common . Most  monosaccharides , or simple  sugars , are found in grapes, other fruits, and honey. Although they can contain from three to nine carbon atoms, the most common representatives consist of five or six joined together to form a chainlike  molecule . Three of the most important simple sugars— glucose  (also known as dextrose, grape sugar, and corn sugar),  fructose  ( fruit sugar ), and  galactose —have the same molecular formula, (C 6 H 12 O 6 ), but, because their atoms have different structural arrangements, the sugars have different characteristics; i.e., they are  isomers .

5. Two molecules of a simple sugar that are linked to each other form a  disaccharide , or double sugar. The disaccharide  sucrose , or table sugar, consists of one molecule of glucose and one molecule of fructose; the most familiar sources of sucrose are  sugar beets  and cane sugar. Milk sugar, or  lactose , and maltose are also disaccharides. Before the energy in disaccharides can be utilized by living things, the molecules must be broken down into their respective monosaccharides.  Oligosaccharides , which consist of three to six monosaccharide units, are rather infrequently found in natural sources, although a few plant derivatives have been identified.

6. Polysaccharides  (the term means many sugars) represent most of the structural and energy-reserve carbohydrates found in nature. Large molecules that may consist of as many as 10,000 monosaccharide units linked together, polysaccharides vary considerably in size, in structural complexity, and in sugar content; several hundred distinct types have thus far been identified.  Cellulose , the principal structural component of plants, is a complex   polysaccharide   comprising  many glucose units linked together; it is the most common polysaccharide. The starch found in plants and the  glycogen  found in animals also are complex glucose polysaccharides. Starch (from the Old English word  stercan , meaning “to stiffen”) is found mostly in seeds, roots, and stems, where it is stored as an available energy source for plants.

7. QUALITATIVE TESTS FOR CARBOHYDRATES Molisch’s test: General for carbohydrates. Iodine test: For glycans (starch, glycogen). Barfoed’s test: Distinguishing between reducing sugars and disaccharides. Seliwanoff’s test: Distinguishing between aldoses and ketoses. Benedict’s test: For reducing sugars. Osazone Test: For crystal formation.

8. MOLISCH’S TEST Molisch's test  (named after Austrian botanist  Hans Molisch ) is a sensitive  chemical test  for the presence of  carbohydrates , based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with two molecules of phenol (usually  α- naphthol , though other phenols (e.g.  resorcinol ,  thymol ) also give colored products), resulting in a red- or purple-colored compound.

9. PROCEDURE FOR MOLISCH’S TEST Chemicals required: Concentrated sulphuric acid, freshly prepared a- Naphthol (5% w/v) in ethanol. Add 2 to 3 drops of a- Naphthol to 2ml of the testube containing the given solution. Gently pippette out 2ml concentrated sulphuric acid and add along the sides of the test tube, so that two distinct layers are formed. Appearance of a purple ring at the junction, indicates the presence of carbohydrates.

10. EXPECTED RESULTS Glucose, fructose, lactose, maltose and starch are all positive for Molisch’s test.

11. IODINE TEST Iodine forms colored adsorption complexes with polysaccharides like starch. The reaction of starch with iodine to give a blue- black color while glycogen reacts with iodine to form a reddish brown complex. Used as a convenient and a ready test for amylose, amylopectin and glycogen.

12. PROCEDURE FOR IODINE TEST Chemicals required: Iodine solution (Prepare 0.005N Iodine solution in 3% w/v of potassium iodide solution) Take 1 ml of the sample extract or the test solution in a test tube and 4-5 drops of iodine solution. Mix the contents gently and note the formation of a colored product.

13. EXPECTED RESULTS Glucose, fructose, lactose and maltose do not react with iodine whereas starch reacts with iodine to give a blue black compound, indicating that starch is a polysaccharide.

14. BARFOED’S TEST This test is used for distinguishing between reducing monosaccharides and reducing disaccharides. Monosaccharides usually react in 1-2 minutes, while disaccharides take between 7- 12 minutes to get hydrolyzed and then react with the reagent. Brick red color is obtained due to formation of Cu 2 O (cuprous oxide).

15. PROCEDURE FOR BARFOED’S TEST Chemicals required: Barfoed’s reagent: Dissolve 13.3 gm of copper acetate in 200ml water and add 1.8ml of glacial acetic acid. Take 2ml of Barfoed’s reagent in a test tube and add 1ml of sample solution to it. Keep the test tube in a boiling water bath. Appearance of brick red color should be observed. Note the time taken for the color to appear.

16. EXPECTED RESULTS Reducing monosaccharides give brick red color while starch and sucrose remain unaffected.

17. SELIWANOFF’S TEST This test is used to distinguish aldoses from ketoses. Ketoses undergo dehydration to give furfural derivatives which then condense with resorcinol to give a red colored complex. In case of disaccharides, prolonged heating should be done, so that they are converted into monosaccharides and eventually produce the red colored complex.

18. PROCEDURE FOR SELIWANOFF’S TEST Chemicals required: Seliwanoff’s reagent 0.05% w/v, resorcinol in 3N HCl . Add 1 ml of test solution to 2ml of Seliwanoff’s reagent and warm in boiling water bath for 1 minute. Note the appearance of a deep red color, which indicates that the sample contains a ketose sugar.

19. EXPECTED RESULTS Only sucrose and fructose give a red complex, indicating the presence of ketones, whereas the others remain unaffected.

20. BENEDICT’S TEST Benedict's reagent is a chemical  reagent  named after American chemist  Stanley Rossiter Benedict . It is a complex mixture of  sodium carbonate ,  sodium citrate  and  copper(II) sulfate  pentahydrate .  It is often used in place of  Fehling's solution  to detect the presence of  reducing sugars . The presence of other reducing substances also gives a positive reaction. Such tests that use this reagent are called the  Benedict's tests . A positive test with Benedict's reagent is shown by a color change from clear blue to a brick-red precipitate. Generally, Benedict's test detects the presence of  aldehydes  and  alpha- hydroxy -ketones , also by  hemiacetal , including those that occur in certain ketoses. Thus, although the ketose  fructose  is not strictly a reducing sugar, it is an alpha- hydroxy -ketone, and gives a positive test because it is converted to the aldoses  glucose  and  mannose  by the base in the reagent. The principle of Benedict's test is that when reducing sugars are heated in the presence of an alkali they are converted to powerful reducing species known as  enediols . Enediols reduce the cupric compounds (Cu 2+ ) present in the Benedict's reagent to cuprous compounds (Cu + ) which are  precipitated  as insoluble red  copper(I) oxide (Cu 2 O).

21. PROCEDURE FOR BENEDICT’S TEST Chemicals required: (1) Dissolve 173g of sodium citrate and 100g of anhydrous sodium carbonate in 600ml of hot water, which is to be diluted to 800ml with hot water. (2) Dissolve 17.3g of copper sulphate pentahydrate in 100ml of fresh water. Cool and dilute to 100ml. Add reagent number 2 to reagent number 3, slowly with constant stirring making the final volume to 1l.

22. Add 0.5ml to 1ml of test solution to 2ml of Benedict’s reagent. Keep test tubes in boiling water bath. Observe the formation of red precipitate whose appearance would suggest the presence of reducing sugars in the given solution.

23. EXPECTED RESULTS Sucrose and starch do not give the test, while the rest do. The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution, hence the test is semi-quantitative. A greenish precipitate indicates about 0.5 g% concentration; yellow precipitate indicates 1 g% concentration; orange indicates 1.5 g% and red indicates 2 g% or higher concentration.

24. OSAZONE TEST This test is also known as the phenylhydrazine test. It involves the reaction of a monosaccharide with phenylhydrazine . All reducing sugars form osazones with excess of phenylhydrazine when kept at boiling temperatures.

25. PROCEDURE FOR OSAZONE TEST Chemicals required: Glacial acetic acid, phenylhydrazine , sodium acetate. Take 5ml of test solution and add 10 drops of glacial acetic acid. Add a pinch of phenylhydrazine to it, and twice the amount of sodium acetate. Heat the solution to dissolve the solute. Keep the solution in boiling water bath with a cotton plug for 30- 40 minutes. Observe the change of color. Once the color changes, take out the test tube and cool it under tap water. Note the shape of the crystals and the time needed for its formation.

26. EXPECTED RESULTS Glucose and fructose containing tubes shall turn cloudy in approximately 10 minutes, while the other sugars would take longer. Fructose Glucose Sucrosee Lactose