Benedict’s Test: Principle, Procedure, Uses, and Limitation

Benedict’s test is a test used to determine the presence of reducing sugar in any substance. Reducing sugar is a simple carbohydrate with a free aldehyde or ketone group and acts as a reducing agent. Reducing sugar includes glucose, fructose, glyceraldehyde, lactose, arabinose, maltose, etc.

Principle of Benedict’s Test 

Benedict’s test is based on the principle that under alkaline conditions reducing sugar forms enediols which are powerful reducing agents. The benedict solution contains milder alkali, Na₂CO₃ to maintain alkaline conditions. Reducing sugar which is changed into enediols reduces the cupric ions to cuprous ions. Copper sulfate is the source of cupric ions but once dissociated the cupric ion may form copper hydroxide which is insoluble.

Sodium citrate is added to the benedict solution as a chelating agent that inhibits the precipitation of cupric hydroxide by forming a loosely bound cupric sodium citrate complex. This bond gives a continuous supply of cupric ions on dissociation. 

Thus obtained cuprous ions forms cuprous hydroxide which on heating gives cuprous oxide resulting in the formation of the bright red colored precipitate.

CuSO₄→Cu⁺⁺ +SO₄^–   (CuSO₄ serves as a source of cupric ions.) 

Cu⁺⁺ + Sodium citrate→ Cupric sodium citrate complex

Reducing sugar→ Enediols

(In the presence of Na₂CO₃ which makes the medium alkaline.)

Enediols + cupric sodium citrate complex→Cu⁺ + Mixture of sugar acids

Cu⁺ +OH^– → CuOH

CuOH→Cu₂O + H₂O (On heating)

Preparation of benedict reagent: Dissolve 173 grams of sodium citrate and 100 grams of anhydrous sodium carbonate in 800ml hot water. 7.3 grams of cupric sulfate pentahydrate is mixed in 200ml hot water. Mix the first solution with the second with constant stirring.

Materials Required

• Benedict’s reagent
• Water bath
• Dry test tubes
• Pipettes
• Sample
• Positive control (5% glucose)
• Negative control (distilled water)

Procedure of Benedict’s Test

1. Take 1ml of sample in a dry test tube. 
2. Take 1ml of 5% glucose and 1ml distilled water in two separate dry test tubes.
3. Add 2ml of benedict’s reagent to all the test tubes. 
4. The test tubes are placed in a water bath for about 5 minutes.
5. The development of the brick red color precipitate indicates a positive result.

Result Interpretation

Uses and Significance of Benedict’s Test

• It could be used in the detection of unknown carbohydrates in biochemistry analysis.
• Diabetes mellitus could be identified in clinical diagnosis. 
• Since the preparation of benedict reagent is simple and easy, performing the test is cheap and easy to carry out.
• The time required is very low.
• The test could be quantitative as well as qualitative.

Limitations of Benedict’s Test

• Even though it is a quantitative test, the exact amount of sugar cannot be determined.
• Chemicals like creatinine, urates maybe slow the reaction in urine.
• The false-positive results may be seen in the presence of substances like penicillin, streptolysin, and P-aminosalicylates.

Reference and Further Reading

• (2022). Retrieved 4 May 2022, from https://www.vedantu.com/chemistry/benedicts-test.
• Vodopich, D., & Moore, R. (1996). Biology (9th ed., pp. 57-59). WCB/McGraw-Hill.