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

By Ashma Shrestha •  Updated: 05/15/22 •  3 min read

Benedict’s test is a test used to determine the presence of reducing sugar in any substance. Reducing sugars 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

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

Shades of colorReporting
BlueNegative; No reducing sugar
GreenTrace; Few amount of reducing sugar
OrangePositive (+); Moderate amount of reducing sugar
Brick redPositive (++); a Large amount of reducing sugar
Benedict’s Test with Positive and Negative Control

Uses and Significance of Benedict’s Test

Limitations of Benedict’s Test

Reference and Further Reading

Join 20,060 other subscribers

Ashma Shrestha

Hello, I am Ashma Shrestha. I am currently pursuing my Master's Degree in Microbiology. Passionate about writing and blogging. Key interest in virology and molecular biology

We love to get your feedback. Share your queries or comments

This site uses Akismet to reduce spam. Learn how your comment data is processed.