Benedict’s Test- Objectives, Principle, Procedure, Results

There are generally two types of sugar namely reducing and non-reducing sugar based on their reducing property. All themonosaccharidesare reducing sugars as they have free anomeric carbon in their structure (free aldehyde group or a free ketone group) that can reduce cupric salt. However, in the disaccharides, the monosaccharide unit is linked together by glycosidic linkage formed between OH hydroxyl group of one and anomeric carbon of others. Some disaccharides cannot reduce cupric salt due to no availability of free anomeric carbon in their structure and hence they are termed as non-reducing sugars. Monosaccharides can be detected based on their oxidation in alkaline solution by Cu⁺⁺ or Ag⁺⁺ or ferricyanide, the mild oxidizing agent. The reducing property can be determined and demonstrated using Benedict’s solution.

Objectives of Benedict’s Test

• To determine the presence or absence of reducing sugar in the solution.
• To determine the glucose concentration in the solution quantitatively.

Principle of Benedict’s Test

Benedict’s test is performed by heating the reducing sugar solution with Benedict‘s reagent. The presence of the alkaline sodium carbonate converts the sugar into a strong reducing agent called enediols. During the reaction, enediols decrease the cupric particles (Cu²⁺) present in Benedict’s reagent to cuprous particles (Cu⁺) which appear as red copper oxide (Cu₂O) which is insoluble in water. When Benedict’s reagent solution and reducing sugars are heated together, the solution changes its color to orange-red/ brick red precipitate. The red-colored cuprous oxide is insoluble in water and hence, separate out from the solution. When the concentration of the reducing sugar is high in the solution, then the color becomes more intense (reddish) and the volume of the precipitate increases in the test tube making it clearly visible.

Figure: Principle of Benedict’s Test. Image Source:Chemistry Learner

Benedict’s solution is a deep-blue alkaline chemical reagent used to test for the presence of the aldehyde functional group -CHO which consists of copper sulfate pentahydrate (CuSO₄. 5H₂O), sodium carbonate (Na₂CO₃), sodium citrate (Na₃C₆H₅O₇) and distilled water. Sodium carbonate renders alkaline conditions which are required for the redox reaction, while sodium citrate is a complexing agent which complexes with the copper (II) ions to avoid degradation into copper (I) ions during storage.

The procedure of Benedict’s Test

1. Pipette out 2 ml (10 drops) of Benedict’s reagent and placed it in the clean test tube
2. Approximately 1 ml of sample (urine) is added to Benedict’s reagent.
3. 试管放置在沸腾ing water bath for 3-5 minutes (can be heated directly over flame).
4. Observe for color change in the solution of test tubes or precipitate formation.

Observation (Results) of Benedict’s Test

Figure: Observation (Results) of Benedict’s Test. Image Source:Chemistry Learner

Appearance of solution	The concentration of reducing sugar (g%)	Interpretation
Brick red with heavy precipitate	2% or >2%	A large amount of reducing sugar is present
Brownish orange with red precipitate	1.5%	A moderate amount of reducing sugar is present
Yellow with precipitate	1%	A small amount of reducing sugar is present
Greenish blue and cloudy	0.5%	Traceable amount of reducing sugar is present
Greenish blue with yellow precipitate	0.25%	Traceable amount of reducing sugar is present
Green with no precipitate	0.1%	Traceable amount of reducing sugar is present
Blue color or cloudy	Nil	No reducing sugar is present

References

1. Shrestha B (2002). Practical biochemistry and biotechnology. First edition. 99933-665-1-X
2. National Institutes of Health, Testing for Lipids, Proteins and Carbohydrates- Benedict’s solution.
3. Fayetteville State University- Biological Molecules: Carbohydrates, Lipids, Proteins.
4. Harper College- Benedict’s Test.
5. Chemistry learner- Benedict’s Test.
6. Northern Kentucky University- Benedict’s Reagent: A Test for Reducing Sugars.
7. KNUST Open Educational Resources, Benedict’s Test – Qualitative Test in Carbohydrates.
8. Amrita Virtual Lab Collaborative Platform- Qualitative Analysis of Carbohydrates.
9. https://laboratoryinfo.com/benedicts-test-principle-reagent-preparation-procedure-interpretation/

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