Procedure

Simulator-Guided Step-by-Step Procedure of the Experiment

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Molisch Test

Reagents

• Molisch reagent: A 10% solution of α-naphthol in ethanol.
• Concentrated sulfuric acid (H₂SO₄): Used for dehydration.

Procedure

1. Take 5 mL of the test solution in a clean test tube.
2. Add 2-3 drops of Molisch reagent and mix gently.
3. Carefully pour 2 mL of concentrated sulfuric acid along the walls of the test tube to form a layer beneath the mixture (do not mix).
4. Observe the interface between the two layers:

• A purple or violet ring indicates the presence of carbohydrates.

Interpretation

• Positive Test (Violet Ring): Presence of carbohydrates (mono-, di-, or polysaccharides).
• Negative Test: Absence of carbohydrates.

Applications

1. To confirm the presence of carbohydrates in food, biological samples, or chemical preparations.
2. To differentiate carbohydrates from other biomolecules, as proteins and lipids do not typically yield a positive Molisch test.

Limitations

• This test is non-specific; it only confirms the presence of carbohydrates but does not differentiate between types of carbohydrates.
• Glycoproteins and glycolipids may also give a positive result due to their carbohydrate moieties.

DNS (3,5-Dinitrosalicylic Acid) Method for Reducing Sugar

Reagents

• DNS reagent: Dissolve 1g DNS in 50 mL distilled water and dilute to 100 mL.

Procedure

1. Take 10 mL of sample solution (diluted appropriately as specified in the experimental table).
2. Add 3 mL of DNS reagent.
3. Boil the mixture in a water bath for 5-10 minutes.
4. Cool the mixture and add 10 mL of distilled water.
5. Add 1 mL of 40% Rochelle Salt (sodium potassium tartrate) solution.
6. Measure absorbance at 540 nm against a blank (sample without DNS reagent).

Calculation

• Prepare a standard curve using glucose or other reducing sugars.
• Compare the absorbance of samples to the standard curve to determine sugar concentration.

Benedict's Method for Reducing Sugars

Reagents

• Benedict's reagent: Mix 17.3g CuSO₄5H₂O, 100 g sodium citrate, and 70g sodium carbonate in 1 L of water.
• Preparation: Dissolve sodium carbonate and sodium citrate in ~800 mL of distilled water. Add copper sulfate solution slowly while stirring. Adjust the volume to 1 L with distilled water.

Sample Preparation

• If the sample is solid, dissolve it in distilled water to prepare a solution.
• If the sugar concentration is too high, dilute the sample appropriately.

Procedure

• Mix 5 mL of Benedict's reagent with 5 mL of the sample.
• Heat in a boiling water bath for 5 minutes.
• Cool and observe the color change (green, yellow, orange, or red, depending on sugar concentration).

Observation

A color change indicates the presence of reducing sugars.

Color scale for semi-quantitative analysis

• Blue: No reducing sugar
• Green: Trace amounts of reducing sugar
• Yellow: Low concentration of reducing sugar
• Orange: Moderate concentration of reducing sugar
• Brick red: High concentration of reducing sugar

Calculation

• Semi-quantitative based on the intensity of the color or quantitative with a spectrophotometer (read at 635 nm).

Interpretation

• Positive Result: Green, yellow, orange, or brick-red precipitate confirms the presence of reducing sugars.
• Negative Result: Blue solution indicates absence of reducing sugars.

Non-Reducing Sugars Test

Reagents

• 1 N HCl
• DNS reagent (as described above)
• 40% NaOH

Procedure

1. Hydrolysis: Mix 2 mL of the sample with 2 mL of 1 N HCl.
2. Heat in a boiling water bath for 10 minutes.
3. Neutralize with 1 mL of 1 N NaOH.
4. Proceed with the DNS method to measure reducing sugars as described earlier.

Calculation

• Subtract the initial reducing sugar content (pre-hydrolysis) from the total sugar content (post-hydrolysis) to determine the non-reducing sugar content.