Benedict actually worked on the development and modifications of the Fehling’s test and then he actually came up with a new method or test which is more convenient and stable. The Benedict’s test is a basic qualitative biochemical test which enables us to ascertain the presence or absence of reducing sugars (sugars or carbohydrates with a free aldehyde or ketone group) or if the anomeric carbon of a sugar consists of a free aldehyde or ketone group the sugar is said to be reducing .
Some disaccharides too have free carbonyl groups and are reducing sugars, disaccharides like lactose and maltose. In case If the anomeric carbon of both the monomer unit of disaccharides are being involved in glycosidic bonding , then that disaccharide will become non-reducing sugar and thus it will not react with Benedict’s solution. Starches are also non-reducing sugars.
The reduction reaction is much more proficient in alkaline medium than in the acidic medium. An American chemist, Stanley Rossiter Benedict ,has been Given credit for this test as the test is named after him. The Benedict’s reagent used in this qualitative test is often also called Benedict’s qualitative solution or Benedict’s solution.
Objectives of Benedict’s Test
- It helps in the determination of the presence or absence of reducing sugar in the given test solution.
- It also helps in the quantitative analysis or determination of glucose concentration in the test solution.
Benedict’s reagent is quantitative and consists of potassium thiocyanate which is used to estimate the amount of reducing sugar present. This reagent solution forms a copper thiocyanate precipitate which is white in colour and can be useful in a titration. The titration process should be performed with 1% glucose solution instead of the calibrated sample.
Principle of Benedict’s Test
The Benedict’s test is carried out by heating the reducing sugar with the Benedict‘s reagent which is specific for this test. The alkaline sodium carbonate in the reagent is responsible for converting the sugar into enediols which is a strong reducing agent.
In an alkaline condition the process of producing enediols process by shifting of hydrogen atom from one carbon atom to another is known as Tautomerization or Enolization. The Reducing sugars in the presence of alkaline condition tautomerize and form enediols. This whole process only is the basis of producing enediols test.The enediols produced in the test reaction are extremely reactive, consequently the sugars in the alkaline environment are powerful reducing agents. Hence ,they actually can reduce cupric ions (Cu2+) to cuprous form (Cu+), which is responsible for the change in the color of the test reaction. In the course of test reaction, enediols performs the reduction of the cupric II ions (Cu2+) of CuSO4 from Benedict’s reagent to cuprous I
ions (Cu+) which give rise to a yellow precipitate of cuprous hydroxide or a red precipitate of cuprous oxide which segregates from the solution. This implies that the red-colored cuprous oxide is insoluble in water and hence, isolates out from the solution
The final color of the solution depends on how much of this precipitate was formed, and therefore the color gives an indication of how much reducing sugar was present.This implies that
When there is high concentration of the reducing sugar in the solution, then the color as a result becomes more intense (reddish) and also the volume of precipitate increases in the test tube making it clearly visible.
Requirements:
1. Reagent
- Sugar solutions (test solutions): 5 % Glucose, 5 % Sucrose
- Distilled Water (control)
- Benedict’s reagent: Benedict’s reagent is an alkaline solution which has a deep blue color, it is prepared by mixing copper sulfate pentahydrate, sodium citrate, and sodium carbonate in distilled water. The alkaline condition has been rendered by Sodium carbonate which is required for carrying out the redox reaction, and the other component of Reagent is a complexing agent which is sodium citrate ,to avoid degradation into copper (I) ions during storage, it actually forms complexes with the copper (II) ions.
2. Materials Required
- 1 Glass stirring rod
- 1 Spatula, micro double ended
- 4 Test tubes, 30 x 200 mm
- 1 Dropping pipette, 7 x 150mm
- 1Test tube stand
- 2 Volumetric flask Boro 3.3,250 ml and 100 ml
- 1 Measuring cylinder 100 ml
- 1 Beaker, Boro 3.3, 600 ml
- 1 Boiling stones 250 g
- Water bath
- 1 Magnetic stirrer with hotplate
- 1 Thermometer, -10…+150 °C/1 K.
Procedure of Benedict’s Test:
- Take clean and dry test tubes .Add 1-3 ml of given test samples of various sugars.The concentration of the test samples should be 5% (w/v).
- In another test tube 1 ml of distilled water is taken as a control.
- In Next step ,about 2-3 drops of Benedict’s reagent is added to all test tubes using a dropper or a dropping pipette and then mixed within a vortex.
- The Beaker of 600 ml is filled with water and placed on the magnetic stirrer as a water bath with a heating plate.
- Put the water bath to get heated up to 80 °C with boiling stones. To track the temperature use a thermometer.
- When the temperature is reached to the desired value, the water bath is removed from the heating plate.
- The test tubes are kept in the water bath for 1-2 minutes.
- Observations are noted for the appearance of color in the test tubes.
Observation and Result Interpretation:
Positive Benedict’s test: the positive result is indicated by the change in color from blue to brick red precipitate (positive for glucose).
Negative Benedict’s test: it is indicated by no change in color. For sucrose and water the results are negative.
With the concentration of reducing sugar in the solution ,the magnitude or intensity of red colour and the volume of precipitate changes. Hence , further interpretation of the result can be done as:
- When the solution appears Brick red with heavy precipitate, the concentration of reducing sugar is – 2% or >2% , this interprets that Large amount of reducing sugar is present in the test solution.
- If the solution appears as Brownish orange with red precipitate then the concentration of reducing sugar is 1.5%, it shows that moderate amount of reducing sugar is present.
- Suppose the solution appears Yellow with precipitate it represents the concentration of reducing sugar as 1%,it is interpreted as Small amount of reducing sugar is present.
- If appears solution Greenish blue and cloudy ,the concentration of reducing sugar is 0.5% And it implies that Traceable amount of reducing sugar is present.
- In case the solution appears Greenish blue with yellow precipitate ,the Concentration Of reducing sugar is 0.25%,and it interprets that Traceable amount of reducing sugar is present.
- If appears Blue in color ,the concentration of reducing sugar is 0%, and it is interpreted as there is Absence of reducing sugar.
Precautions:
- While heating the test tube in the water bath ,be careful and keep your distance from the water bath.
- Use dropping pipette or a dropper to put the reagent in the corresponding test tubes.
- The remaining Benedict’s Reagent can be kept in a sealed vessel for further analysis. As it is a quite stable reagent.
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