Benedict’s reagent is a group of chemical solutions used in Benedict’s test, a test used to identify the presence of reducing sugars in a sample.

The most common Benedict’s reagent is a copper(II) sulfate solution, which is mixed with a reducing sugar and then heated. If the sugar is present, it will reduce the copper(II) ions to copper(I) ions, causing a color change from blue to green, yellow, orange, brown, or red, depending on the concentration of sugar present.

Benedict’s reagents are commonly used in clinical and research settings to detect the presence of glucose and other reducing sugars in blood and urine samples.

Principle of Benedict’s Test

  • When reducing sugars are heated with Benedict’s reagents (an alkaline solution of copper (ΙΙ) sulfate), they form precipitates of copper (Ι) oxide.
  • The precipitate’s color changes from green to yellow, orange, brown, and red, depending upon the quantity of reducing sugar used up in the reaction.

Preparation of Benedict’s Reagent

  1. Take 4.25 g of copper sulfate (CuSO4.5H2O) in a flask and dissolve 25 mL of distilled water in it. Shake well to make a homogeneous solution.
  2. Take 400 mL of distilled water in a large flask and add 43.5 g of sodium citrate (Na3C6H5O7) and 25 g of sodium carbonate (Na2CO3). Heat the mixture to dissolve both compounds.
  3. Transfer both solutions to a large beaker with constant stirring. Add more distilled water to make the volume of the whole solution 250 mL.

The reaction to prepare Benedict's reagent

The procedure of Benedict’s Test

  1. Take 4-5 mL of Benedict’s reagent in a test tube.
  2. Add 8-10 drops of 1-2% sugar solution in it.
  3. Heat this solution in a water bath for 3-5 minutes.
  4. Allow the test tube to stand and cool to room temperature for a few minutes.
  5. Observe the color change of the mixture in the test tube.

Net Reaction of Benedict’s Reagent

Reaction of benedict's reagentMechanism

CuSO→ Cu+2 + SO4-2

Reducing sugar + alkali → Enediol

Cu+2 + Na3C6H5O7 → CuNaC6H5O7

Enediol + CuNaC6H5O→ Cu+ + sugar acid

Cu+ OH– → CuOH

CuOH → Cu2O (colored ppt) 


  • Na3C6H5O= sodium citrate
  • CuNaC6H5O= cupric sodium citrate
  • R-C(OH)=CH(OH) = Enediol

Results Interpretation of Benedict’s Test

  1. If red, brown, orange, yellow, or green color precipitates are developed, the reducing sugar is detected. Reducing sugars give a positive Benedict’s test.
  2. And if no color change takes place, the reducing sugar is not detected. Non-reducing sugars give negative Benedict’s test.

Color change linked to concentration

Precipitates colors in Benedict's test


Different precipitates in Benedict's reagent testing

Semi-Quantitative Nature of Benedict’s Reagent

Benedict’s test is semi-quantitative due to a wide range of colors (green, yellow, orange, brown, and red). It can be used to estimate the approximate concentration of reducing sugar in a sample.

Prepare a range of color standards of known concentration. Make solutions of different concentrations of the same reducing sugar. Take an equal volume of each solution and add the same amount of Benedict’s reagent in all samples. Heat all the solutions in test tubes for an equal length of time before cooling them at room temperature. Take an unknown sample and treat it in the same way. Compare the results with the following table.

Concentrations determination by Benedict's reagent

Furthermore, filter the precipitates, dry and then weigh them. The greater the weight of precipitates, the higher the concentration of reducing sugars.

Applications of Benedict’s Reagent

  • It is an excellent test for detecting the presence of all monosaccharides (e.g glucose, fructose galactose) and certain disaccharides (e.g maltose).
  • It is a semi-quantitative test.
  • It can be used to calculate the approximate amount of reducing sugar in an unknown sample.
  • It can be used to detect glucose in urine samples.

Related content:

Concept Bergs

What is Benedict’s test used for?

Benedict’s test is used for the detection of monosaccharides and some disaccharides in the given sample. Aldose sugars give positive Benedict’s test.

What is Benedict’s reagent formula?

RCHO + 2Cu+2 + 5OH → RCOO + Cu2O + 3H2O

What is Benedict’s reagent made of?

CuSO4.5H2O(aq) + Na2CO3(aq) + Na3C6H5O7(aq)Benedict’s Reagent

What is the basic principle of Benedict’s test?

When reducing sugars are heated with an alkaline solution of copper (ΙΙ) sulfate (Benedict’s reagents), they form insoluble precipitates of copper (Ι) oxide. The color of the precipitate varies from green to red depending upon the concentration of reducing sugar used.

Why is it called Benedict’s test?

Benedict’s reagent and test are named after an American chemist, S.R. Benedict.

What is the difference between Fehling’s test and Benedict’s test?

Both Fehling’s and Benedict’s tests are based on the same principle. The main difference is that Fehling’s test uses sodium potassium tartrate solution, while sodium citrate is used for Benedict’s solution.

References Books

  • General Organic and Biological Chemistry By Kenneth W. Raymond
  • Textbook of Medical Biochemistry By S. Ramakrishnan
  • Essential AS Biology for OCR By Glenn Toole, Susan Toole
  • Practical Advanced Biology By Tim King, Michael Reiss, Michael Roberts
  • Experimental Animal Physiology And Biochemistry By S. C. Nigam
  • Practicals and Viva in Medical Biochemistry By Dandekar

Reference links