02.12 Practical: Food test

1. Reducing Sugars

• Definition: Sugars capable of acting as reducing agents (lose electrons).
• Includes: All monosaccharides and some disaccharides.
• Exception: Sucrose (a common non-reducing sugar).
• Benedict’s Test for Reducing Sugars:
  • Principle: Reducing sugars convert blue copper(II) sulfate (Cu²⁺) in Benedict’s reagent to red-brown copper(I) oxide (Cu⁺), which forms a precipitate.
  • Equation: Reducing sugar + Cu²⁺ → Oxidised sugar + Cu⁺ (blue) (red-brown)
• Procedure:

1. Add Benedict’s reagent to the test solution.
2. Heat in a water bath.
3. Observe colour change:
   • Sequence: Blue → Green → Yellow → Orange → Red-Brown (if reducing sugar is present).
4. Semi-quantitative analysis:
   • Intensity of red-brown indicates concentration.
   • Use colour standards or a colorimeter for more precise measurement.

2. Non-Reducing Sugars

• Definition: Sugars that do not reduce Benedict’s reagent and give a negative result in Benedict’s test.
• Example: Sucrose.
• Testing for Non-Reducing Sugars:
• Hydrolysis Step:
  1. Add hydrochloric acid to break down the non-reducing disaccharide into monosaccharide components (which are reducing sugars).
  2. Neutralise with an alkali (e.g., sodium hydroxide).
  3. Perform Benedict’s test again.
• Procedure:

1. Initial Benedict’s Test:
   • If negative, proceed with a fresh sample.
2. Hydrolysis:
   • Heat the solution with hydrochloric acid.
   • Neutralise by adding alkali.
3. Benedict’s Test (again):
   • Add Benedict’s reagent and heat.
   • Positive result (red precipitate): Confirms a non-reducing sugar was present.
   • Heavier precipitate: Indicates both reducing and non-reducing sugars are present.
   • No colour change: No sugar is present.

Key Terms

• Glycosidic Bond: The bond linking two sugar molecules in disaccharides.
• Benedict’s Test: A test for reducing sugars; a blue-to-yellow/red/brown colour change indicates a positive result.

Questions

(a) Why do you need to use excess Benedict’s reagent to find the concentration of a sugar solution?

Answer:

• Excess Reagent Ensures Complete Reaction:
• Using excess Benedict’s reagent means there’s more than enough copper(II) ions (Cu²⁺) available to react with all reducing sugar present in the solution.
• Ensures that the reaction reaches completion for all available reducing sugar molecules.
• Color Intensity and Concentration:
• The final intensity of the red-brown precipitate (copper(I) oxide, Cu⁺) reflects the total amount of reducing sugar present.
• More accurate concentration estimation can be made, as the intensity directly correlates with the sugar concentration when excess reagent is present.

(b) Outline how you could use the Benedict’s test to estimate the concentration of a solution of a reducing sugar.

Answer:

1. Prepare a Series of Known Concentration Standards:

• Prepare solutions of the reducing sugar at known concentrations (e.g., 0.1%, 0.2%, 0.5%, etc.).
• Perform the Benedict’s test on each concentration and record the color intensity or time to first color change.

1. Benedict’s Test Procedure:

• Add an excess of Benedict’s reagent to each sugar solution.
• Heat each sample in a water bath.
• Observe the color change through blue → green → yellow → orange → red-brown as the concentration of reducing sugar increases.

1. Compare Test Sample Against Standards:

• Test the unknown sugar solution with Benedict’s reagent using the same method.
• Match the color intensity of the unknown sample to the closest color standard.

1. Semi-Quantitative Estimation:

Alternatively, measure the time taken for the first color change of the test solution, comparing this against known concentration standards for an estimation.

If exact concentrations are needed, use a colorimeter to measure absorbance or color intensity, which provides a more precise comparison.

Background Information

• Structure of Starch:
  • Starch is a polysaccharide that forms long spiral chains. The spiral structure creates a hollow core along the length of the molecule.
  • Iodine molecules can fit into this hollow core, forming a starch-iodine complex with a blue-black color.
• Iodine Solution:
  • Iodine alone does not dissolve in water, so iodine solution is made by dissolving iodine in potassium iodide solution.
  • The iodine-potassium iodide complex remains orange-brown in color until it interacts with starch.
• Reaction Basis:
  • When iodine enters the spiral of starch, a starch-iodine complex forms, resulting in a distinctive blue-black color. This reaction is specific to starch, making it a reliable test.

Purpose

• To confirm the presence of starch in a sample by observing a color change using iodine solution.

Materials Needed

• Iodine solution (iodine dissolved in potassium iodide solution)
• Dropper
• Samples to test (solid or liquid substances containing or suspected to contain starch)
• White tile or plate (for better visibility of color change)
• Water bath or beaker of water (optional, for liquid samples)
• Paper towels (for cleanup)

Procedure

1. Prepare the Iodine Solution:

• Ensure you have iodine solution prepared in potassium iodide for the test.
• Iodine solution should appear orange-brown.

1. Place the Sample:

• Place a small amount of the sample on a white tile or plate to provide a clear background for observing any color change.

1. Add Iodine Solution:

• Use a dropper to add 1–2 drops of iodine solution directly onto the sample.
• For liquid samples:
  • Place a few drops of the liquid on the white tile or in a test tube.
  • Add iodine solution drop by drop to observe any color change.

1. Observe Color Change:

• Positive Result (Starch Present): A blue-black color appears almost immediately, indicating the formation of the starch-iodine complex.
• Negative Result (No Starch): The solution retains its orange-brown color.

1. Record Observations:

• Note the color change and the speed at which it occurs. A rapid color change typically indicates a higher starch concentration.

1. Cleanup:

• Dispose of any used materials safely.
• Clean the tile, dropper, and work area with water and paper towels.

Notes and Additional Information

Why This Test Works:

• The specific size and shape of the starch molecule’s spiral core allows iodine molecules to fit and interact, producing the blue-black complex.

Limitations of the Test:

• The test is specific for starch but may not work effectively in highly acidic or basic environments, which can alter starch structure.
• False Negatives can occur if the sample contains modified starches or very low concentrations of starch.

Further Applications:

This test is commonly used in botany and food science to verify starch content in plants, grains, and processed foods.

The biuret test is used to detect the presence of proteins by identifying peptide bonds, which contain nitrogen. When proteins are present, a purple color develops as a result of the reaction between copper(II) ions and nitrogen atoms in the peptide bonds.

Background Information

• Principle of the Test:
  • Proteins contain peptide bonds that link amino acids together. These bonds include nitrogen atoms.
  • When copper(II) ions in biuret reagent react with these nitrogen atoms, a purple complex forms, indicating the presence of protein.
  • The color change from blue to purple confirms the presence of proteins due to the formation of a copper-nitrogen complex.
• Biuret Reagent:
  • Composition:
    • Biuret reagent contains copper(II) sulfate (providing copper ions) and sodium or potassium hydroxide (alkaline medium).
    • A stabilizing agent like sodium potassium tartrate or sodium citrate is often included to prevent unwanted precipitation of copper hydroxide.
  • Options:
    • Ready-Mixed Biuret Reagent: Contains both copper(II) sulfate and hydroxide pre-mixed with stabilizing agents.
    • Separate Solutions: Alternatively, you can use separate solutions of potassium or sodium hydroxide and copper(II) sulfate.

Materials Needed

• Biuret reagent (ready-mixed or as separate solutions: copper(II) sulfate and potassium or sodium hydroxide)
• Test solution suspected of containing protein
• Test tubes
• Dropper or pipette
• Test tube rack

Procedure

1. Prepare the Test Sample:

• Place a small volume (1-2 ml) of the test solution into a clean, dry test tube.

1. Add Biuret Reagent:

• If using ready-made biuret reagent:
  • Add a few drops (1-2 ml) of biuret reagent to the test solution.
• If using separate solutions:
  • First, add an equal volume of potassium hydroxide or sodium hydroxide to the test solution.
  • Then add a few drops of copper(II) sulfate solution.

1. Observe the Color Change:

• Positive Result (Protein Present): The solution turns purple within several minutes, indicating protein presence.
• Negative Result (No Protein): The solution remains pale blue, as no copper-nitrogen complex forms in the absence of proteins.

1. Record Observations:

• Note the intensity and timing of the color change, as this can vary depending on protein concentration.

Explanation of Color Change

• Biuret Reaction:
  • The copper(II) ions (Cu²⁺) in the reagent react with peptide bonds (specifically, nitrogen atoms) in the protein under alkaline conditions.
  • This interaction creates a copper-nitrogen complex that produces a purple color.
• Why Alkaline Conditions Are Necessary:
  • The alkaline medium (potassium or sodium hydroxide) helps keep copper ions in a reactive state and promotes binding to nitrogen atoms in the peptide bonds.

Notes and Tips

• Time for Color Development:
  • The purple color may take a few minutes to develop fully. Be patient and observe the solution for several minutes to confirm the result.
• Avoiding Precipitation:
  • If using separate solutions, add them in the correct order (hydroxide before copper(II) sulfate) to avoid precipitation.
  • Ready-mixed biuret reagent includes stabilizers (e.g., sodium potassium tartrate) to prevent copper hydroxide precipitate formation.
• Sensitivity of the Test:
  • This test detects proteins and peptides, but it does not respond to free amino acids, as they lack peptide bonds.

Summary

• Purpose: To detect the presence of proteins by identifying peptide bonds.
• Procedure: Add biuret reagent to the test solution and look for a blue to purple color change.

Result Interpretation:

Purple Color: Protein is present.

Pale Blue: No protein detected.

The emulsion test for lipids takes advantage of lipids’ insolubility in water and solubility in ethanol to detect their presence in a sample.

Background Information

• Principle of the Test:
  • Lipids are insoluble in water but can dissolve in ethanol (especially absolute ethanol, which contains minimal water).
  • When a lipid-dissolved ethanol solution is mixed with water, the lipids are forced out of solution and form tiny droplets that scatter light, creating a white, cloudy suspension called an emulsion.
• What is an Emulsion?
  • An emulsion is a mixture where tiny droplets of one liquid are dispersed in another liquid with which it is not miscible (does not mix).
  • In this test, lipid droplets scatter light, causing the cloudy appearance.

Materials Needed

• Absolute ethanol (alcohol with little or no water)
• Distilled water
• Test tube(s)
• Sample substance suspected to contain lipids
• Dropper or pipette
• Stopper or lid for test tube (optional, for shaking)
• Test tube rack

Procedure

1. Prepare the Sample:

• Place a small amount of the test substance into a clean, dry test tube.
• If the sample is solid, grind it or crush it into a fine powder if possible, or use a small piece.

1. Add Ethanol:

• Add absolute ethanol (enough to cover the sample) to the test tube.
• Secure the test tube with a stopper or lid if available.

1. Dissolve the Lipids:

• Shake the tube vigorously for about 1 minute to ensure that any lipids present dissolve fully in the ethanol.
• If lipids are present, they should dissolve, as they are soluble in ethanol.

1. Add Water:

• Pour the ethanol-sample mixture carefully into a second test tube containing distilled water.

1. Observe for a Cloudy Emulsion:

• Positive Result (Lipids Present): A cloudy white suspension appears, indicating the presence of lipids. This cloudy appearance is due to lipid droplets forming an emulsion in the water, scattering light and making the mixture appear white and opaque.
• Negative Result (No Lipids): The solution remains clear and transparent, as the ethanol mixes into the water without forming droplets.

Additional Notes

• Why Absolute Ethanol?
  • Using absolute ethanol (ethanol with minimal water) ensures that any lipids present will dissolve completely in the ethanol before adding water.
  • If too much water is present in the ethanol, lipids may not dissolve effectively, leading to unreliable results.
• Explanation of Cloudiness:
  • Lipids do not remain dissolved when ethanol is added to water.
  • The lipid molecules precipitate out and form tiny droplets throughout the water, creating an emulsion that scatters light, which appears as a white, cloudy solution.
• Potential Sources of Error:
  • Ethanol Quality: Use ethanol with minimal water content for accuracy.
  • Sample Size: Too large a sample may interfere with the test; use a small, manageable amount.

Summary

• Purpose: To detect lipids in a sample by observing if a cloudy white emulsion forms.
• Procedure:

1. Dissolve the sample in absolute ethanol.
2. Add ethanol solution to water.
3. Observe for a cloudy suspension as a positive lipid result.

Result Interpretation:

• Cloudy White (Emulsion): Lipids are present.
• Clear Solution: No lipids are present.