Benedict’s Test: Principle, Procedure, Steps, Results, Uses

What is Benedict’s Test?

Definition of Benedict’s Test

Lowering sugar levels in a solution are determined using Benedict’s Test, a chemical analysis method. Benedict’s Test is a qualitative examination, often utilised to differentiate between both reducing and non-reducing types of carbohydrates (sugars and carbohydrates).

Reducing sugars are sugars that have free aldose or ketose groups and may donate electrons to other molecules that are oxidising them. At the end of their molecules is a free carbon atom. All monosaccharides and some disaccharides, oligosaccharides, and polysaccharides include reducing sugars.

It is often used to distinguish monosaccharides (simple sugars) from other reducing sugars. It is used in place of Fehling’s test. Identification is based on the appearance of a brick-red colour caused by the chemical reaction with Benedict’s reagent and reducing sugar. The sugar concentration can be determined based on the intensity of the reaction mixture, but its exact value cannot be anticipated. Consequently, this is a semi-quantitative as well as qualitative test.

As a possible diagnostic for diabetes mellitus, it is also used to detect glucose in urine.

Stanley Rossiter Benedict, an American scientist and biochemist, made the discovery.

Objectives of Benedict’s Test

  • to determine if reducing sugar is present in the sample solution.
  • Detecting glucose in a urine sample to determine whether a person has diabetic mellitus
  • to determine how much reducing sugar is present in the sample solution.
  • To differentiate and distinguish the extracted sugars from

Principle of Benedict’s Test

The sodium carbonate in the Benedict reagent elevates the pH of the sample and reagent mixture. Under alkaline and heated conditions, reducing sugars transform into strong reducing agents, enediols. These enediols transform the cupric ions (Cu2+) of the Benedict reagent into carbon dioxide and water, which are found as copper sulphate (CuSO4), into cuprous ions (Cu+). The cuprous particles are present as the red cuprous oxide (Cu2O), also known as insoluble copper (I) oxide. These copper oxides, which are red, precipitate.

The intensity and hue of the reaction mixture’s hue vary based on the sample’s reducing sugar content. Using the hue of this colour, the percentage of reduced sugar in the sample may be measured. The hue might vary from yellowish to orange-red to brick-red. As the concentration of reducing sugar grows, the colour gradually changes from green to yellow to orange to brick red.

Requirements of Benedict’s Test

  • sample of an unidentified carbohydrate solution (or urine sample)
  • Test tubes and holders for test tubes
  • Pipette
  • Bunsen burner
  • Benedict’s Reagent

Preparation of Benedict’s Reagent 

  • Incorporate 17.3 grams of copper sulphate (CuSO4), 173 grams of sodium citrate (Na3C6H5O7), and 100 grams of anhydrous sodium carbonate (Na2CO3) (or 270 grams of sodium carbonate decahydrate (Na2CO3.10H2O)) into your measurement.
  • Place all the calculated substances in a volumetric flask of 1000 mL.
  • Fill the container to the 1000 mL mark using distilled water.
  • Shake vigorously to mix all of the ingredients.

Procedure of Benedict’s Test

  • 1 mL of the sample solution should be added to a clean test tube (urine or carbohydrate solution).
  • Pour two millilitres of Benedict’s reagents over the sample.
  • Warm up the test tube either directly over a flame or over a pan of boiling water for three to five minutes.
  • Watch for any changes in colour.

Result Interpretation/Observation of Benedict’s Test

Any colour change from blue to green, yellow, orange, or red during three minutes indicates a positive Benedict test or the existence of reducing sugar in the specimen.

For semiquantitative analysis, the concentration of reducing sugar may be calculated based on the hue of the generated colour;

Shade of Color Approx. Concentration of Reducing Sugar (in g%) Indication
Blue 0 No reducing sugar
Green solution < 0.5 Trace reducing sugar
Green ppt. 0.5 – 1 Trace reducing sugar
Yellow ppt. 1 -1.5 Low reducing sugar
Orange-red ppt. 1.5 – 2 Moderate reducing sugar
Brick-red ppt. >2 High reducing sugar

Precautions for Benedict’s Test

  • Accurate measurement is required.
  • Do not immediately heat the mixture. The most effective method of heating is using a water bath.
  • When heating the liquid, take a test tube holder.
  • Avoid turning the test tube toward yourself or others while it is boiling.
  • At least three heatings should be performed before declaring anything unfavourable.

Applications of Benedict’s Test

  • in biochemistry, for the aim of analysing and detecting unidentified carbohydrate extracts
  • Utilize medical diagnosis for a rapid preliminary diagnosis of diabetes mellitus.
  • For the purpose of detecting and quantifying simple sugar in quality control.

Advantages of Benedict’s Test

  • This is a quick exam, needing little preparation and little time.
  • non-toxic chemicals
  • inexpensive
  • Both semi-quantitative and qualitative analysis

Limitations of Benedict’s Test

False-positive test results brought on by drug interactions with salicylates, p-aminosalicylic acid, isoniazid, penicillin, and streptomycin

Benedict’s reaction is slowed down by substances found in urine, such as creatinine, ascorbic acid, and urate.

Only an approximated semiquantitative number may be used to show the precise concentration of reducing sugar.

We need more testing to identify the carbohydrate.


  • Robert D. Simoni; Robert L. Hill & Martha Vaughan (2002). “Benedict’s Solution, a Reagent for Measuring Reducing Sugars: the Clinical Chemistry of Stanley R. Benedict”. J. Biol. Chem. 277 (16): 10–11. doi:10.1016/S0021-9258(19)61050-1.
  • National Institutes of Health, Testing for Lipids, Proteins and Carbohydrates – Benedict’s solution.
  • Northern Kentucky University- Benedict’s Reagent: A Test for Reducing Sugars.
  • Shrestha B (2002). Practical biochemistry and biotechnology. First edition. 99933-665-1-X.
  • Fayetteville State University- Biological Molecules: Carbohydrates, Lipids, Proteins.
  • KNUST Open Educational Resources.
  • Amrita Virtual Lab Collaborative Platform- Qualitative Analysis of Carbohydrates.
  • Benedict’s Test – Reagent Preparation, Principle, Procedure, Reaction (byjus.com)
  • Benedicts Test – Principle, Procedure, Result and Limitation (vedantu.com)
  • Benedict’s Test- Principle, Preparation, Procedure and Result Interpretation (microbiologyinfo.com)
  • Benedict’s test: Definition, Principle, Uses, and Reagent (chemistrylearner.com)
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