-Definition and Color Reactions of Reducing Sugars-
PROCEDURE
A) Benedict Test
1) Sodium citrate and anhydrous
sodium carbonate are dissolved in
approximately 800 ml of water in a
separate container.
2) Copper(II) sulfate pentahydrate is
dissolved in 100 ml of water in
another container.
3) Both solutions are mixed together.
4) The volume is diluted to 1 L with
distilled water.
5) If necessary, the solution is filtered
to ensure clarity.
6) Benedict solution is taken into a test
tube.
7) 1 ml of the sample is added to the
Benedict solution.
8) The test tube is heated on a heater.
9) A reddish-brown precipitate of Cu2O
(copper II oxide) is observed.
B) Tollens Test
1) Solution A, consisting of a 5%
AgNO3 solution in distilled water,
and Solution B, a 5% KOH or NaOH
solution, are prepared.
2) Equal volumes of solutions A and B
are taken into separate test tubes.
3) Upon mixing, white AgOH
precipitate is immediately formed.
4) NH4OH is added dropwise to the
precipitate until complete
dissolution occurs.
5) The obtained silver diamine
solution is combined with 1 ml of
the sample to be examined.
6) The mixture is heated in a water
bath.
7) The formation of a silver mirror on
the inner surface of the tube is
observed.
 C) Picric Acid Test D) Methylene Blue Test

1) A 5% solution of picric acid 1) Into a test tube, pure water (1

in alcohol or a saturated ml) and 1 drop of 1%
solution of picric acid in methylene blue are added.
water is prepared.
2) Then, 1 ml of 0.1 N NaOH is
2) A 10% solution of NaOH in added to the test tube.
distilled water is prepared.

3) When heated, no color change

3) Into a test tube, the sample is observed.
is added (1 ml), and 3-4
drops of picric acid are
dripped.

4) The test tube is heated in a

water bath.

5) During heating, 3-4 drops of

NaOH are dripped into the
solution.

6) The formation of a red color

is observed.
DATA AND CALCULATION

In the Benedict trial, a reddish-brown precipitate of Cu2O (copper II oxide) was observed due to
the presence of reducing sugars in the sample solution.

In the Tollens trial, the formation of a silver mirror on the inner surface of the tube indicated the
presence of reducing sugar.

During the Picric Acid trial, the appearance of a red color in the tube indicated the presence of
reducing sugar.

In the Methylene Blue experiment, initially, a blue color was observed in the tube. This color
change occurred because methylene blue was reduced in the presence of reducing sugars in the
sample, leading to the disappearance of the blue color. However, upon depletion of reducing
sugars, shaking the tube open allowed the reduced form of methylene blue to be oxidized by
atmospheric oxygen, leading to the reappearance of the blue color.
RESULT AND DISCUSSION

The experiments conducted aimed to detect the presence of reducing sugars using four
different tests: Benedict test, Tollens test, Picric Acid test, and Methylene Blue test. In the
Benedict test, a brick-red precipitate of copper (I) oxide (Cu2O) was observed in the presence of
reducing sugars. This color change occurs due to the reduction of copper (II) ions to copper (I)
ions, which forms the insoluble Cu2O precipitate. The alkalinity required for this reaction is
provided by sodium carbonate, while sodium citrate prevents premature reduction of copper (II)
ions. In the Tollens test, the formation of a silver mirror on the inner surface of the test tube
indicated the presence of reducing sugars. Tollen's reagent reacts with reducing sugars or
aldehydes to produce free silver metal. The silver ions in Tollen's reagent are reduced to metallic
silver, resulting in the formation of a silver mirror. The Picric Acid test also confirmed the presence
of reducing sugars. When reducing sugars react with picric acid, a red-colored picramic acid is
formed. The appearance of a red color in the test tube indicated a positive result for the presence
of reducing sugars.Lastly, in the Methylene Blue test, the blue color of methylene blue
disappeared when reducing sugars were present in the sample due to their reduction. However,
when reducing sugars were exhausted, shaking the tube allowed the reduced form of methylene
blue to be oxidized by atmospheric oxygen, resulting in the reappearance of the blue color.
Overall, all four tests provided positive results for the presence of reducing sugars in the samples
tested. These tests are valuable tools for qualitative analysis of carbohydrates, allowing for the
identification of compounds with free ketone or aldehyde functional groups.

CONCLUSION

The experiments employing the Benedict, Tollens, Picric Acid, and Methylene Blue
tests effectively identified the presence of reducing sugars in the samples. Distinct
reactions such as precipitate formation or color changes confirmed the presence of
compounds with free ketone or aldehyde groups. These qualitative methods provide
valuable insights into sample composition, demonstrating their utility in carbohydrate
analysis and beyond.