Qualitative and Quantitative Analysis of Sugars – Experimental Procedures

1. Qualitative Analysis of Sugars (Molisch Test)

Procedure

  1. Transfer 5 mL of glucose sample into a clean test tube using a pipette.
  2. Add 2–3 drops of α-naphthol reagent to the sample.
  3. Mix the solution thoroughly by gently shaking the test tube.
  4. Carefully add 2 mL of concentrated sulfuric acid along the side of the test tube.
  5. Observe the formation of a purple-colored ring at the junction of the two layers.
  6. The appearance of the purple ring confirms the presence of carbohydrates in the sample.

2. Quantitative Estimation of Reducing Sugars (DNS Method)

A. Preparation of Sample Solutions

  1. Add the sample solution into Test Tube 1 as specified in the table.
  2. Click Run to dispense the sample into the remaining test tubes as per the table.
  3. Add distilled water to the blank test tube as specified in the table.
  4. Click Run to add distilled water to the remaining blank tubes.
  5. Add DNS reagent to all test tubes as per the table using the Run function.
  6. Proceed to the next step.

B. Color Development

  1. Place all test tubes in a boiling water bath.
  2. Boil the samples for 5–10 minutes for color development.
  3. Allow the samples to cool to room temperature.
  4. Add 1 mL of 40% Rochelle salt solution to the test tubes.
  5. Click Run to add the Rochelle salt solution to the remaining tubes.
  6. Proceed to the next step.

C. Measurement of Absorbance at 540 nm

  1. Select the micropipette and set the volume to 1 mL.
  2. Transfer 1 mL of blank solution into a cuvette.
  3. Open the spectrophotometer lid and insert the cuvette.
  4. Set the wavelength to 540 nm (A540).
  5. Press the Blank button and then press Enter to record the blank absorbance.
  6. Click Run to measure the absorbance of the remaining samples in the same manner.
  7. Proceed to the next step for result calculation.

3. Benedict Test for Reducing Sugars

Procedure

  1. Add 5 mL of Benedict’s reagent into a clean test tube.
  2. Add 5 mL of the sample solution into the same test tube.
  3. Mix the contents thoroughly.
  4. Heat the mixture over a flame or burner for up to 5 minutes.
  5. Click on Result Analysis to observe and interpret the color change.
  6. A brick-red, green, or yellow precipitate indicates the presence of reducing sugars.

4. Benedict Test for Non-Reducing Sugars

(Performed only if no color change is observed in the reducing sugar test)

Procedure

  1. Add 2 mL of sample solution into a clean test tube.
  2. Add 2 mL of hydrochloric acid (HCl) into the test tube.
  3. Mix thoroughly and place the test tube in a water bath for 5–10 minutes.
  4. Add a few drops of sodium hydroxide (NaOH) to neutralize the acid.
  5. Add 2 mL of Benedict’s reagent to the test tube.
  6. Mix thoroughly and place the test tube in a water bath for 3–5 minutes.
  7. Click on Result Analysis to observe and interpret the results.
  8. A color change confirms the presence of non-reducing sugars.