7

Activity 6-B

Qualitative Test for

Carbohydrates

Nylander’s Test

Procedure: Mix 1 ml of 5% glucose solution with

1 ml of Nylander’s reagent. Heat for 5 minutes in a boiling

water

bath and note the result. Do the same with galactose,

fructose, sucrose, lactose, maltose, starch and glycogen.

Nylander’s Test In the case of

alkaline solution bismuth subnitrate, the bismuth hydroxide formed is reduced to black metallic bismuth as shown in the following equation:

Bi(OH)2NO3 + KOH →Bi(OH)3 + KNO3

2Bi(OH)3 (with reducing sugar and heat) → 2Bi + 3O + 3H2O

a test for glucose in urine using a solution containing bismuth subnitrate which forms a black precipitate in a positive reaction.

Lactose and Maltose – light yellow but maltose is darker.

Glycogen and Starch – crystal clear but starch has a cloudy precipitate at the bottom.

Galactose – yellow orange

Glucose – dark olive green

Fructose – olive green

Sucrose – clear olive green

Lactose & Maltose (-)(-)(L-R)

Sucrose & Galactose (+)(-)(L-R)

Glycogen & Starch (-)(-)(L-R)

Fructose & Glucose (+)(+)(L-R)

Barfoed’s Test Procedure:

Mix 1 ml of Barfoed’s reagent with 1 ml of 5% glucose solution.

Heat for 30 seconds and allow to stand for 10 minutes.

Do the same with galactose, fructose, maltose, lactose, sucrose, starch and glycogen.

Add 5 drops of HCl to the 7 test tubes, heat for 5 minutes. Observe the changes.

Barfoed’s Test

POSITIVE ONLY FOR REDUCING

MONOSACCHARIDES

Solution of cupric acetate in weak acetic acid ( Barfoed’s ) is reduced to monosaccharides but not by disaccharides. This serves therefore, to differentiate the two sugars.

WHICH ARE: GLUCOSE, FRUCTOSE, GALACTOSE

In acid solution, the sugars reduce less vigorously. Barfoed’s test utilizes this fact for distinguishing monosaccharides from reducing disaccharides. The former react, whereas the latter do not.

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.

Galactose, Fructose, Glucose and Sucrose had red precipitate but after adding HCl to all of the samples except the glucose which wasn’t added HCl had faded back to its skyblue color without the red precipitate anymore.

Glucose

Picric Acid Test Procedure:

To 1 ml of 5% glucose solution add 1ml of saturated picric acid solution and about 5 drops of 10% sodium carbonate.

Heat to boiling. Do the same with all the different types of sugar solution.

Picric Acid Test / Jaffe Reaction

Some organic acids when in alkaline solutions are also reduced. Thus, picric acid ( yellow ) is reduced to picramic acid ( mahogany red ).

It is the basis for one of the quantitative blood sugar methods widely used for a time and still used to some extent.

C6H2OH(NO2)3 → C6H2OHNH2(NO2)2

Starch and Glucose– Colorless

Glucose, Fructose and Galactose – Red orange

Lactose and Sucrose – Light orange

Maltose - Orange

Seliwanoff’s Test Procedure:

Mix 1 ml of Seliwanoff’s reagent in each of 6 test tubes.

To the 1st test tube add 1 ml of 5% freshly prepared fructose solution. Glucose to the second, galactose to the third, sucrose to the fourth, maltose to the fifth, and lactose to the sixth.

Boil in water bath.

Note carefully the color produced and record the time required for the development of pink color in each of the test tubes.

Seliwanoff’s Test This involves the

action of resorcinol and HCl on sugar. In the presence of ketose sugar, like fructose, a red color is developed. This is a specific test for ketose sugar, which forms rapidly hydroxymethyl furfural as a result of dehydration. This condenses with resorcinol to form red colored compounds.

Positive only for sugars having potentially free ketone group, as in the case of fructose.

sucrose-46sec,fructose-55sec,glucose-2:29sec, galactose-2:43sec,lactose-3:30sec,maltose-3:50sec

Pink but after sucrose and fructose were very red

Iodine Test Procedure:

Prepare starch solution by mixing thoroughly 2 grams of starch in 10 ml of water and then pouring this mixture in 100 ml of boiling water.

Add 2 drops of a very dilute aqueous solution of iodine in potassium iodide to 1 ml of starch solution.

What is the color of the solution? Heat the colored solution to boiling and observe the effect.

Allow the solution to cool back down to room temperature. Now what do you observe?

Add a few drops of sodium thiosulfate solution to the cooled starch solution and note the result. Do the same with glycogen.

Iodine Test

iodine test: starch plus lugol's iodine solution produced purple-black color, upon heating it cleared and then upon cooling turned purple-black again and upon dropping sodium thiosulfate, it turned clear again. With glycogen it did not have a purple-black color, upon heating it was still the same until cooling when dropped with sodium thiosulfate it also cleared.

Starch

raw starch is insoluble in cold water. When heated in water the granules swell forming a viscous hydrophillic colloidal substance. Prolonged heating or grinding causes the disintegration of the granules liberating the amylose which is slightly soluble in water. This is responsible for the deep blue color produced with iodine due to the formation of starch iodine; amylopectin produces a purple color.

Glycogen

It differs from starch in being soluble in water, forming an opalescent solution, form which it can be precipitated by ethyl alcohol; and in giving with iodine a red instead of a blue color.

well the iodine is a STARCH test

so you put it in at first and there is starch right? so the positive test is blue/black..

then you heat it up it is not starch anymore, it is glucose starch is a long chain of glucose macromolecules. so that is why it tested negative (it was clear)

then it cooled so the glucose macromolecules bonded up together again in a long chain, called starch. that is why it tested positive for starch..

Then sodium thiosulfate is an "inhibitor", it doesn't let the glucose bond together. That is why it turned clear again

Mucic Acid Test Procedure:

To each test tube filled with 1 pinch of galactose for the first, glucose for the second, lactose for the third, fructose for the fourth, and sucrose for the fifth add 1 ml of 5N HNO3.

Heat the test tubes in boiling water for 1 hour, then allow to cool at room temperature.

If no crystals appear let stand until the next lab period.

Examine crystals under the microscope and draw as seen.

Confirm the solubility of the crystals by adding 1 ml of water to the tubes where crystals are formed.

Not carefully which tubes shows water insoluble crystals.

Mucic Acid Test Positive only for

galactose due to the formation of an insoluble mucic acid crystals when heated with concentrated nitric acid.

Galactose, on being oxidized with HNO3, forms mucic acid, an isomer of saccharic acid. Mucic is insoluble and forms characteristic sandy crystals which serve to identify galactose.

Water insoluble crystals

Galactose – white precipitate is present and the oil is floating

Water soluble crystals

Lactose - White precipitate settled.

END