Characteristic tests for carbohydrates fats and proteins in pure samples and their detection in given food samples

Aim

To perform characteristic tests for identifying carbohydrates, fats, and proteins in pure samples and detect their presence in various food samples through qualitative analysis in a laboratory setting.

Apparatus Required

Equipment:

• Test tubes (15-20 pieces)
• Test tube stand
• Beakers (100ml and 250ml)
• Measuring cylinder (10ml)
• Glass rod
• Spirit lamp/Bunsen burner
• Wire gauze
• Tripod stand
• Dropper/pipette
• Test tube holder

Chemicals Required:

• Molisch's reagent (α-naphthol in ethanol)
• Concentrated sulfuric acid (H₂SO₄)
• Fehling's solution A and B
• Benedict's solution
• Iodine solution
• Sudan III stain
• Biuret reagent
• Ninhydrin solution
• Pure samples: Glucose, starch, sucrose, oil, egg albumin
• Food samples: Potato, rice, bread, butter, milk, pulses, egg white

Theory

Carbohydrate Tests

1. Molisch's Test Carbohydrates form a purple or reddish-purple ring with α-naphthol and concentrated H₂SO₄ due to dehydration of carbohydrates to furfural derivatives.

2. Fehling's Test Reducing sugars reduce Cu²⁺ ions in Fehling's solution to form red precipitate of Cu₂O.

3. Benedict's Test Similar to Fehling's test, reducing sugars form colored precipitate ranging from yellow to red.

4. Iodine Test Starch forms blue-black complex with iodine due to helical structure accommodating iodine molecules.

Fat Tests

1. Spot Test Fats leave translucent oily spots on filter paper due to their non-volatile nature.

2. Sudan III Test Fats stain red with Sudan III due to their lipophilic nature.

Protein Tests

1. Biuret Test Proteins containing two or more peptide bonds form violet color with CuSO₄ in alkaline solution.

2. Ninhydrin Test α-amino acids form purple or blue complex with ninhydrin.

Procedure

A. Tests on Pure Samples

I. Carbohydrates

Molisch's Test:

1. Take 2ml sample solution in test tube
2. Add 2-3 drops of Molisch's reagent
3. Incline tube and add 1ml concentrated H₂SO₄ carefully along sides
4. Observe color change at interface

Fehling's Test:

1. Mix equal volumes of Fehling's A and B solutions
2. Add 2ml mixture to 2ml sample solution
3. Heat for 2-3 minutes in water bath
4. Observe precipitate formation

Benedict's Test:

1. Add 2ml Benedict's solution to 2ml sample
2. Heat gently for 3-4 minutes
3. Note color changes from blue to green, yellow, orange, or red

Iodine Test (for starch):

1. Add 2-3 drops iodine solution to sample
2. Observe color change immediately

II. Fats

Spot Test:

1. Place 1-2 drops of fat on filter paper
2. Allow to dry
3. Observe translucent spot

Sudan III Test:

1. Add few drops of Sudan III to fat sample
2. Observe red coloration

III. Proteins

Biuret Test:

1. Add 2ml NaOH solution to sample
2. Add 2-3 drops CuSO₄ solution
3. Observe violet color formation

Ninhydrin Test:

1. Add 2-3 drops ninhydrin solution to sample
2. Heat gently
3. Observe blue or purple color

B. Tests on Food Samples

Follow identical procedures as above with appropriate dilutions or preparations:

• Grind solid food samples with distilled water
• Filter if necessary
• Adjust concentration before testing

Observation Table

Key: ppt = precipitate

Result

Pure Samples Characterization:

• Glucose: Confirmed as reducing sugar (positive Molisch's, Fehling's, Benedict's tests)
• Starch: Confirmed as polysaccharide (positive Molisch's, Iodine tests)
• Sucrose: Confirmed as non-reducing sugar (positive Molisch's, negative reducing tests)
• Oil: Confirmed as fat (positive spot, Sudan III tests)
• Egg Albumin: Confirmed as protein (positive Biuret, Ninhydrin tests)

Food Sample Analysis:

• Potato: Contains starch (blue-black with iodine)
• Rice/Bread: Contain carbohydrates (positive Molisch's test)
• Butter: Contains fats (positive spot and Sudan III tests)
• Milk: Contains lactose, fats, and proteins (multiple positive tests)
• Pulses: Contain proteins and carbohydrates
• Egg White: Rich in proteins (strong Biuret and Ninhydrin positive)

Precautions

1. Safety First: Handle concentrated H₂SO₄ with extreme care; always add acid to sample slowly
2. Cleanliness: Thoroughly clean all apparatus to avoid cross-contamination
3. Proper Mixing: Always mix reagents gently to prevent splashing
4. Temperature Control: Heat solutions gently to prevent violent reactions
5. Reagent Freshness: Use freshly prepared solutions for accurate results
6. Control Experiments: Perform tests with known samples alongside unknowns
7. Waste Disposal: Dispose chemical waste according to laboratory guidelines
8. Observation Timing: Note observations immediately as some colors fade quickly
9. Sample Preparation: Use appropriate dilutions to avoid false results
10. Storage: Store sensitive reagents like ninhydrin in dark bottles

Viva Questions and Answers

Q1: What is the principle behind Molisch's test?

A: Molisch's test is based on the dehydration of carbohydrates by concentrated sulfuric acid to form furfural or hydroxymethyl furfural, which condenses with α-naphthol to produce a purple-colored complex.

Q2: Why do we get red precipitate in Fehling's test?

A: Red precipitate of cuprous oxide (Cu₂O) forms because reducing sugars reduce the Cu²⁺ ions in Fehling's solution to Cu⁺ ions.

Q3: What is the difference between Fehling's and Benedict's test?

A: Both tests detect reducing sugars, but Benedict's test is more stable and can detect even weak reducing agents. The color change in Benedict's test is more gradual and ranges from blue to red.

Q4: Why does starch give blue-black color with iodine?

A: Starch has a helical structure that can trap iodine molecules within its coils, forming a blue-black complex. Amylose gives blue color while amylopectin gives red color.

Q5: Explain the Biuret test principle?

A: In alkaline medium, Cu²⁺ ions form a violet-colored complex with peptide bonds (‑CO‑NH‑) present in proteins, giving characteristic color change.

Q6: What precautions should be taken with concentrated H₂SO₄?

A: Always add acid slowly along the sides of the test tube to prevent splashing, work in fume hood, wear safety goggles, and never add water to concentrated acid.

Q7: Why do some carbohydrates not give positive Fehling's test?

A: Non-reducing sugars like sucrose lack free aldehyde or ketone groups required for reduction reaction, hence give negative Fehling's test.

Q8: How does Sudan III help in fat detection?

A: Sudan III is a lipophilic dye that dissolves in fats and oils, staining them red, making it easy to identify lipid content in samples.

Q9: What is the significance of ninhydrin test?

A: Ninhydrin specifically reacts with α-amino acids to form colored complex (Ruhemann's purple), making it highly specific for protein detection.

Q10: Why should food samples be properly prepared before testing?

A: Proper preparation ensures accurate results by removing interfering substances, achieving appropriate concentration, and making constituents accessible for chemical reactions.

Conclusion

This comprehensive laboratory approach for detecting carbohydrates, fats, and proteins through characteristic tests provides reliable identification of macronutrients in both pure samples and food materials. The systematic application of these qualitative tests forms the foundation for nutritional analysis and food quality assessment in analytical chemistry and food science laboratories.