Biochemical Test

Benedict’s Test Principle, Reagent Preparation, Procedure, Result, Limitation

Benedict's test is a chemical test that is used to test for the presence of reduced sugars within an analytical test. Thus,...

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This article writter by MN Editors on November 30, 2021

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Benedict’s Test Principle, Reagent Preparation, Procedure, Result, Limitation
Benedict’s Test Principle, Reagent Preparation, Procedure, Result, Limitation

Benedict’s Test

Benedict’s test is a chemical test that is used to test for the presence of reduced sugars within an analytical test. Thus, simple carbohydrates that contain an aldehyde or free ketone functional group are detected using this test. The test is basing itself upon Benedict’s Reagent (also called Benedict’s solution) which is a complex mix of sodium carbonate, sodium citrate, and the pentahydrate of copper(II) Sulfate.

If exposed to sugars that are reduced, the reactions will be triggered by Benedict’s reagent which causes a brick-red-colored precipitate that is an indication of Benedict’s test is positive. 

It is worth noting that Benedict’s test may be used to test for sugar levels in urine sample. Because this test can detect ketones, a-hydroxy ketones, and aldehydes. Since the glucose aldose, whose open-chain creates an aldehyde-aldehyde group it gives a positive result when glucose is detected inside the test. But, a positive reaction could also be caused due to the presence of homogentisic acid, ascorbic acid, or other reducing substances in urine. Thus that a positive Benedict’s test does not mean that the subject in question is diabetic.

Benedict’s quantitative reagent has potassium thiocyanate. It’s used to determine the amount of reduction sugar exists. This solution produces a copper thiocyanate crystal that is white and may be utilized as a Titration. The titration process should be repeated using glucose solution 1% in place of the test sample to ensure calibration.

Objectives of Benedict’s Test

  • To find the amount or presence of reducing sugar present in the mixture.
  • To determine the concentration of glucose in the solution. It is quantifiable.

Principle of Benedict’s Test

The solution is heated when simple carbohydrates and Benedict’s solution are combined. transforms into brick red. This is due to the reducing properties in simple carbohydrates. It is believed that the copper (II) Ions in Benedict’s solution get reduced into Copper (I) Ions and cause the change in color.

It is believed that the red copper(I) oxide that forms is not soluble in water, and it is precipitated out of the solution. This is why the precipitate is formed. The more the concentration of reduced sugar rises and the more close the final colour will be brick-red, and the more precipitate is formed. Sometimes, a brick-red solid, copper oxide, crystallizes from the solution and is deposited in the bottom of the tube.

The alkaline properties of sodium carbonate provide the conditions that are needed to trigger the Redox reaction. The Sodium citrate complexes with copper (II) Ions so that they don’t degrade to copper(I) Ions during storage.

Complex carbohydrates, such as starches will not react positively to the Benedict’s test unless they’re broken down via digestion or heating (try chewing on crackers before conducting this test). Table Sugar (disaccharide) is non-reducing sugar, and will not react with Iodine or the Benedict Reagent. Sugar must be broken down into its constituents fructose and glucose. The glucose test will be positive, but the test for starch will be negative.

Benedict’s Solution Composition

One liter of Benedict’s Solution is made by combining 100 grams of anhydrous sodium carbonate, 173 grams of sodium citrate and 17.3 grams of copper(II) pentahydrate of sulfate.

Quality Checking: enedict’s solution has a violet in hue. To determine the quality of Benedict’s solution, put 5ml of Benedict’s mixture in a test tube, and then the temperature. If it does not change color, that means that it is pure.

Procedure of Benedict’s Test

Preparation of Benedict’s Reagent

One liter of Benedict’s Reagent can be made using a mixture of 17.3 grams of copper pentahydrate sulfate (CuSO4.5H2O), 100 grams sodium carbonate (Na2CO3) as well as 173 grams sodium citrate, in distillated water (required amount). This is because the copper(II) sulfurate functions as a source for Cu2+ ions. The sodium carbonate acts as an alkaline medium as well as the sodium citrate form complexes when it comes in contact with the Cu2+ ions. Distilled water is utilized as solvent.

Pureness of Benedict’s reagent may be verified through heating in the test tube. The absence of any change in the blue colour of the solution after heating indicates that the reagent is of pure.

Testing for Reducing Sugars

  1. Approximately 1 ml of sample is placed into a clean test tube.
  2. 2 milliliters (10 drops) of Benedict’s Reagent (CuSO4) is put into the testing tube.
  3. After that, the solution will be heated by a water bath for about 3-5 minutes.
  4. Check for changes in color inside the the test tube or the formation of precipitate.

Observation and Results of Benedict’s Test

Benedict’s Test Principle, Reagent Preparation, Procedure, Result, Limitation
Benedict’s Test Principle, Reagent Preparation, Procedure, Result, Limitation | Source: https://www.chemistrylearner.com/benedicts-test.html
  • If the color after boiling turns green, then there will there be 0.1 or 0.5 percent sugar in the solution.
  • If the color changes to yellow Then 0.5 to 1 percent sugar levels are present.
  • If it turns orange, it indicates that between 1 and 1.5 percent sugar has been present.
  • If the color changes from red to black, 1.5 or 2.0 percent sugar levels are present.
  • If the color changes from brick red to red, it signifies there is more sugar than in the solution.

Positive Benedict’s Test: Develops reddish precipitate within three minutes. Reducing sugars present. Examples: Glucose

Negative Benedict’s Test: no change in color (Remains in blue). Sugars that reduce sugars are not present. Example: Sucrose.

Appearance of solutionThe concentration of reducing sugar (g%)Interpretation
Brick red with heavy precipitate2% or >2%A large amount of reducing sugar is present
Brownish orange with red precipitate1.5%A moderate amount of reducing sugar is present
Yellow with precipitate1%A small amount of reducing sugar is present
Greenish blue and cloudy0.5%Traceable amount of reducing sugar is present
Greenish blue with yellow precipitate0.25%Traceable amount of reducing sugar is present
Green with no precipitate0.1%Traceable amount of reducing sugar is present
Blue color or cloudyNilNo reducing sugar is present

Limitation of Benedict’s Test

The weaknesses of Benedict’s test can be seen as follows:

  • False-positive reactions during the test may be observed when certain drugs are such as streptomycin, isoniazid and salicylates penicillin, P-aminosalicylic acid are obtained.
  • The chemical compounds present in concentrated urine can decrease Benedict’s reaction, which comprises creatinine, urate, and ascorbic acid (the reduction is only slight).

Uses of Benedict’s Test

  • It can be utilized in the search for unknown carbohydrate in biochemistry analysis.
  • It is possible to detect diabetes mellitus by a clinical diagnosis.
  • Because the procedure for preparing the benedict reagent is easy and straightforward, executing the test is inexpensive and easy to conduct.
  • The amount of time needed is extremely minimal.
  • The test may be both quantitative and qualitative.
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