General Color Tests for Carbohydrates

Carbohydrates are dehydrated when treated with strong mineral acids under non-oxidizing conditions. The free or potentially free aldehyde or ketonic groups of monosaccharides and certain disaccharides are readily oxidized by mildly alkaline solutions of cupric ions; such as Benedict's reagent ( copper sulfate, sodium carbonate, and sodium citrate ). These carbohydrates are called reducing sugars because they reduce cupric (Cu+2)ions to form cuprous (Cu+1) oxide, Cu2O, a yellow to brick-red precipitate. The aldoses are oxidized to aldonic acids (glucose forms gluconic acid), as well as other products. The ketoses yield a mixture of lower molecular weight oxidation products.


Bring in different samples of carbohydrates, such as sugars, starches, fruits, vegetables, cookies rice, pasta, beans etc.

When performing the following test, run a control tube for each.

Molisch test: a general for carbohydrates.

To three separate test tubes, add 10 drops of 1% solutions of glucose, sucrose and starch, and dilute each sugar solution with 2 ml. of water. Add 2 drops of alpha naphthol solution (dissolve 10g alpha naphthol in 100 ml. 95% ethyl alcohol ) to each tube and mix. Incline the test tube and slowly and carefully add 3 ml. of concentrated sulfuric acid down the side of the tube to form a layer below the sugar solution. A purple ring at the interface is indicative of a carbohydrate. Additional tests can be performed on apples, celery petioles, carrots, grapes, and onions.

Barfoed's test: a general test to distinguish between monosaccharides and disaccharides.

To 5 ml. of Barfoed's reagent (dissolve 66.5g of neutral, crystallized copper acetate in 800 ml. of water and add 6 ml. of glacial acetic acid, and make to one liter ), in separate test tubes, add 5 ml. of the 1% carbohydrate solutions, shake the contents of each tube well, and place all of the tubes at the same time in an actively boiling water bath. Heat for 3.5 min. after the water starts boiling again. Timing is important since a false positive test can be obtained for monosaccharides with disaccharides, if the disaccharides are heated for more than 3.5 minutes thereby breaking down ( hydrolyzing ) to monosaccharides. During this period observe the tubes closely and note any change or clarity of the solutions. A positive test for monosaccharides is the appearance of a red precipitate of Cu2O within 1 or 2 minutes, if no precipitate forms it indicates the presence of a disaccharide. Record the time if a reaction ( turbidity )precipitate occurs. For purposes of comparison, it is helpful to make a control tube with 5 ml. of water in place of the sugar.

Benedict's test for Reducing sugars:

Perform the tests on the 1% carbohydrate solutions. Add 5 ml. of Benedict's reagent and 2 ml. of carbohydrate to a test tube and shake each tube thoroughly. Place all tubes in a boiling water bath at the same time. Heat in the water bath for 5-6 min. and observe what happens. Record any changes in color, in the transparencies and in the formation and color of any precipitate.

Add 4-5 drops of 3M HCl to 5 ml. of 1% sucrose solution and heat in the boiling water bath for 5 min. Treat the 1% starch solution in the same way but extend the heating period to 25-30 min. Apply Benedict's test to 1-2 ml. of each solution in the same manner as before. Compare the results with those obtained without acid treatment.

Bial's Orcinol test distiguishes a pentose ( 5 carbons ) from a hexose ( 6 carbons ) monosaccharide.

Pipet 4 ml. of each carbohydrate ( 1% solutions ) into separate test tubes. Add 2 ml. of Bial's reagent ( dissolve 6g orcinol in 200 ml. 95% ethanol. Then add 40 drops of 10% FeCl3.6H2O solution ) followed by 5 ml. of concentrated HCl to all the tubes. Mix and stopper the tubes with cotton and heat in a boiling water bath for 10 min. A positive reaction for a pentos is indicated by the development of a green to deep blue color; hexoses give a muddy brown to gray color. In addition try several pieces of shredded wheat.

Seliwanoff's Resorcinol test distinguishes ketose from aldose monosaccharises.

Place 2-3 drops of 1% solutions to be tested in separate test tubes. Add 5ml. of the resorcinol ( dissolve 0.05g resorcinol in 100 ml. diluted HCl, concentrated HCl:H2O; 1:2 ). Set all the tubes in a beaker two-thirds filled with boiling water. Record the color you observe in each tube at the end of 1 min. and after 4 min. The red color from ketoses after 4 min. constitutes a positive test. Aldoses react more slowly.

Ketohexoses- cherry red solution.

Ketopentoses- blue-green solution.

Aldose- no color change.

Disaccharides- no color change.







Bial's Orcinol

Seliwanoff's Resorcinol































































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