Biochem Lab For Revision

1. Qualitative and Quantitative Tests for Carbohydrates

1.1 Introduction to Tests

  • Different tests can determine the presence and concentration of carbohydrates like starch and sugars.

  • Key tests include:

    • Iodine Test for starch

    • Molisch Test for carbohydrates

    • Benedict's Test and Fehling's Test for reducing sugars.

1.2 Iodine Test

  • Substance Tested: Starch

  • Result: Blue coloration indicates presence of starch, reddish-brown indicates glycogen, and reddish indicates the presence of dextrose.

1.3 Molisch Test

  • Reagents Needed:

    1. Molisch reagent (contains alpha-naphthol) mixed with sulfuric acid

    2. 2% Lugol’s iodine solution

  • Procedure:

    1. Mix the sample with Molisch reagent, then add H2SO4 carefully to form a layer.

    2. Observe color change to develop results.

  • Interpretation: Presence of carbohydrates indicates a violet complex formation.

1.4 Benedict's and Fehling's Tests

  • Use: These are qualitative tests for reducing sugars.

  • Procedure:

    1. Mix the sample with Benedict’s or Fehling’s reagent.

    2. Heat the mixture for 2-5 minutes.

  • Results Interpretation: Formation of a colored precipitate indicates the presence of reducing sugars. Colors range from blue to brick red, indicating sugar concentration.

2. Applications and Results

2.1 Benedict's Test Interpretation

  • Results:

    • Color change corresponds to sugar concentration:

      • Yellow: 0-0.5 g%

      • Brick red: >0.5 g%

      • Color fails to form indicates no reducing sugar present.

2.2 Fehling's Test Interpretation

  • Similar principles as Benedict's Test, but specifically uses copper ions to evaluate the presence of reducing sugars by forming copper(I) oxide.

3. Sucrose and Other Carbohydrates

3.1 Sucrose Testing Principles

  • Must not exceed 12% as prolonged heating might convert it into glucose and fructose (hydrolysis).

3.2 Selivanoff's Test

  • Testing for ketoses and aldoses using phenylhydrazine.

  • Reagents Needed: Phenylhydrazine and resorcinol.

  • Procedure:

    1. Add sample to phenylhydrazine and heat.

    2. Formation of osazone crystals indicates presence of reducing sugars.

  • Interpretation: Specific shapes of crystals confirm specific sugars (e.g. glucose vs fructose).

4. Production of Ethanol through Fermentation

4.1 Fermentation Process

  • Definition: Conversion of carbohydrates to alcohol via yeast in an anaerobic environment.

  • Importance of Urea: Added as a nitrogen source for yeast growth.

5. Methods for Chemical Analysis

5.1 Sterilization Techniques

  • Various techniques for removal of microbes, including:

    • Chemical: Detergents and alcohol.

    • Physical: Autoclaving at 120°C.

5.2 Spectrophotometry

  • Used for quantitative analysis of unknown substances through measurement of absorbance at specific wavelengths.

  • Principle: Develop a standard curve for comparison, correlating concentration and absorbance.