Experiment 1:
Objective :
To estimate the total protein content in a milk sample by determining the nitrogen content
using the Kjeldahl method.
Principle: The Kjeldahl method involves:
1. Digestion: Milk proteins are digested with concentrated sulfuric acid in the presence of a
catalyst (K₂SO₄ + CuSO₄), converting nitrogen to ammonium sulfate.
2. Distillation: Upon addition of alkali (NaOH), ammonia is released, distilled, and
collected in boric acid.
3. Titration: The amount of ammonia (hence nitrogen) is determined by titrating the trapped
ammonia with standard HCl.
4. Protein Content: Nitrogen % is multiplied by 6.38, the specific conversion factor for milk
proteins.
Chemical Reactions:
Digestion:
Protein-N → (NH₄)₂SO₄ (in acidic medium with catalyst)
Distillation (with NaOH):
(NH₄)₂SO₄ + 2NaOH → 2NH₃ + Na₂SO₄ + 2H₂O
Absorption and Titration:
NH₃ + H₂SO₄ → NH₄HSO₄
H₂SO₄ + NaOH → Na₂SO₄ + H₂O
Materials Required:
• Milk sample
• Concentrated H₂SO₄
• K₂SO₄ and CuSO₄ (catalyst mixture)
• NaOH (40%)
• N/10 H₂SO₄
• N/10 NaOH
• Methyl red indicator
• Digestion and distillation apparatus
• Burette, pipette, conical flask, Kjeldahl flask
Procedure:
1. Digestion:
• Weigh 5.00 g of milk into a Kjeldahl flask.
• Add 10 g of K₂SO₄-CuSO₄ catalyst mixture.
• Add 15–20 mL of concentrated H₂SO₄.
• Heat on digestion block until the solution turns clear blue/green.
• Cool and dilute the digest to 100 mL with distilled water.
2. Distillation:
• Transfer 10 mL of digested solution into a distillation flask.
• Place 25 mL of 4% boric acid + mixed indicator in a receiving flask.
• Add excess NaOH (~25 mL of 40%) to liberate ammonia.
• Distill until all ammonia is collected (usually 60–70 mL distillate).
3. Titration:
• Titrate the contents of the receiving flask with N/100 or N/50 HCl until the green
color turns to pink.
• Perform a blank distillation and titration for accuracy.
Observations:
Parameter Value
Volume of milk taken 5.00 g
Volume of digest taken for distillation 10 mL
Volume of HCl used (sample) 13.4 mL
Volume of HCl used (blank) 0.4 mL
Normality of HCl 0.02 N
Calculations:
Result:
The total protein content in the milk sample was found to be 4.64%.
Experiment 2:
Objective:
To determine the titratable acidity of milk (expressed as % lactic acid) using standard sodium
hydroxide solution and phenolphthalein indicator.
Theory:
Milk contains natural acids—primarily lactic acid—as well as phosphates, casein, and
dissolved CO₂ that contribute to overall acidity. The titratable acidity measures the total acid
concentration, not just lactic acid.
This acidity is neutralized by titration with a standard solution of sodium hydroxide (NaOH).
The endpoint of the titration is identified by the appearance of a faint pink color, using
phenolphthalein as an indicator. The amount of alkali used is used to calculate the acidity,
which is expressed as the percentage of lactic acid equivalent.
Apparatus Required:
• Burette (50 mL)
• Pipette (10 mL)
• Conical flask (100 or 250 mL)
• White tile
• Beaker, funnel, wash bottle
Reagents Required:
• 0.1 N Sodium hydroxide solution (standardized)
• Phenolphthalein indicator (1% in ethanol)
• Distilled water
Sample Preparation:
• Use fresh, well-mixed milk.
• If necessary, filter milk to remove visible particles.
• Ensure milk is at room temperature (20–25°C) before analysis.
Procedure:
1. Rinse the burette with 0.1 N NaOH and fill it.
2. Pipette 10.0 mL of milk into a clean conical flask.
3. Add 10 mL of distilled water (optional, for easier endpoint detection).
4. Add 2–3 drops of phenolphthalein indicator. The solution will remain colorless initially.
5. Titrate with 0.1 N NaOH, swirling continuously, until a faint pink color appears and
persists for 30 seconds.
6. Record the volume of NaOH used.
7. Repeat the titration to obtain two concordant readings (≤ 0.1 mL apart).
Observations:
Trial Volume of Milk (mL) Volume of 0.1 N NaOH used (mL)
1 10.0 1.8
2 10.0 1.9
Average volume of NaOH used =
1.8 + 1.9
= 1.85 𝑚𝐿
2
Calculation:
The formula to calculate titratable acidity as % lactic acid is:
𝑉 × 𝑁 × 0.09 × 100
𝑇𝑖𝑡𝑟𝑎𝑡𝑎𝑏𝑙𝑒 𝐴𝑐𝑖𝑑𝑖𝑡𝑦 ( % 𝑙𝑎𝑐𝑡𝑖𝑐 𝑎𝑐𝑖𝑑) =
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑚𝑖𝑙𝑘 𝑠𝑎𝑚𝑝𝑙𝑒 (𝑚𝐿)
Where:
• V = Average volume of NaOH used (1.85 mL)
• N = Normality of NaOH (0.1 N)
• 0.09 = Equivalent weight of lactic acid per mL of 1N NaOH
• Volume of milk = 10.0 mL
1.85 × 0.1 × 0.09 × 100 1.665
𝑇𝑖𝑡𝑟𝑎𝑡𝑎𝑏𝑙𝑒 𝐴𝑐𝑖𝑑𝑖𝑡𝑦 = = = 0.1665 %
10 10
Result:
The titratable acidity of the given milk sample = 0.17% lactic acid
Experiment 3:
Objective:
To determine the percentage of lactose (milk sugar) in a given milk sample by titration with
Fehling’s solution using methylene blue as an internal indicator.
Principle:
Lactose, a disaccharide composed of glucose and galactose, is the major carbohydrate in milk
and functions as a reducing sugar. It reduces cupric ions (Cu²⁺) present in Fehling’s solution
to cuprous oxide (Cu₂O) under alkaline and heated conditions, resulting in a brick-red
precipitate.
The milk must first be deproteinized to remove casein and other proteins, which may interfere
with the titration. This is achieved using lead acetate, followed by potassium oxalate to
precipitate excess lead as insoluble lead oxalate.
Fehling’s solution is composed of:
• Fehling A: Copper(II) sulfate solution
• Fehling B: A solution of sodium potassium tartrate (Rochelle salt) and sodium hydroxide
The titration proceeds until the blue color disappears, indicating complete reduction. The
endpoint is typically aided by the internal indicator methylene blue, which becomes colorless
once all Cu²⁺ is reduced.
The volume of sugar solution required to completely reduce a known quantity of Fehling’s
solution is used to calculate the amount of lactose present in the milk.
Chemical Reaction:
Apparatus Required:
• Burette (50 mL)
• Pipette (10 mL, 20 mL)
• Conical flask (250 mL)
• Beakers (100 mL, 250 mL)
• Measuring cylinders
• Filter paper and funnel
• Heating apparatus (burner or water bath)
• White tile
• Glass rod, test tubes, wash bottle
Reagents:
• Fehling’s A: Copper(II) sulfate solution
• Fehling’s B: Alkaline Rochelle salt solution (sodium potassium tartrate + NaOH)
• Methylene blue (0.1% aqueous solution)
• Lead acetate (10%): For protein precipitation
• Potassium oxalate (solid): To precipitate excess lead
• Distilled water
Preparation of Sugar Solution (Deproteinization):
1. Measure 10.0 mL of milk into a 100 mL beaker.
2. Add 5.0 mL of 10% lead acetate while stirring.
3. Add about 0.5 g of potassium oxalate to remove the excess lead as lead oxalate (white
precipitate).
4. Stir thoroughly and filter through Whatman No. 1 filter paper.
5. Collect the clear filtrate in a clean beaker — this is your lactose-containing solution.
Titration Procedure:
1. Pipette 5.0 mL of Fehling’s A and 5.0 mL of Fehling’s B into a 250 mL conical flask.
2. Add about 20 mL of distilled water to the flask.
3. Add 2–3 drops of methylene blue (indicator). The solution will be deep blue due to
Cu²⁺ ions.
4. Boil the Fehling’s mixture gently over a flame or water bath.
5. Titrate with the prepared milk sugar filtrate drop by drop while maintaining boiling.
6. Continue adding and swirling until the blue color disappears, leaving a brick-red
precipitate and no blue tint.
7. The endpoint is the first permanent disappearance of blue color, indicating complete
reduction of Cu²⁺.
8. Repeat the titration for concordant values (difference ≤ 0.1 mL).
Observations Table:
Trial Volume of Fehling’s Solution (mL) Volume of Milk Filtrate Required (mL)
1 10.0 11.5
2 10.0 11.6
Average volume of filtrate used = (11.5 + 11.6) / 2 = 11.55 mL
Calculation:
• From standard tables, 10 mL Fehling’s solution (5 mL A + 5 mL B) is equivalent to 0.067
g lactose.
• 11.55 mL of milk filtrate contains 0.067 g lactose.
To find lactose per 100 mL of filtrate:
If 10 mL of milk was taken for deproteinization, then:
Lactose in milk sample=5.81%
Result:
The lactose content in the given milk sample is approximately 5.81%.
Experiment 4:
Objective:
To determine the ash content of a milk sample by incinerating it at high temperature in a muffle
furnace and weighing the residue.
Principle:
Milk contains minerals such as calcium, phosphorus, potassium, sodium, and magnesium.
These do not volatilize during heating and remain as inorganic residue (ash) when milk is fully
incinerated. By weighing this residue and comparing it to the original milk weight, we calculate
the ash content as a percentage of the total milk.
Apparatus Required:
• Silica crucibles
• Muffle furnace (up to 600°C)
• Hot plate or water bath
• Desiccator
• Analytical balance
• Tongs, crucible holder
Reagents:
• No chemicals required (distilled water may be used for rinsing)
Procedure:
1. Crucible Preparation
• Heat a clean, dry silica crucible in the muffle furnace at 550–600°C for 30 minutes.
• Cool it in a desiccator, then weigh accurately (Weight = W₁).
2. Sample Addition
• Measure 10.0 g of well-mixed milk into the crucible.
• Evaporate water gently on a hot plate or water bath to avoid splashing. Continue until a
dry mass remains.
3. Ashing
• Place the crucible in a preheated muffle furnace at 550–600°C.
• Incinerate for 3–4 hours, or until the residue becomes light grey or white (no black
particles).
• Remove crucible using tongs, cool in a desiccator, and weigh (Weight = W₂).
• If the ash weight fluctuates, repeat heating and weighing until constant weight is
achieved.
Observations:
Description Weight (g)
Weight of empty crucible (W₁) 30.245 g
Weight of crucible + milk 40.245 g
Weight of crucible + ash (W₂) 30.945 g
Milk taken = 10.000 g
Ash = 30.945 – 30.245 = 0.700 g
0.700
Ash content (%)= × 100= 0.70%
10.00
Result:
Ash content of the given milk sample = 0.70%
Expected Ash Content:
Milk Type Ash (%)
Cow milk 0.70–0.75%
Buffalo milk 0.80–0.85%
Human milk ~0.20%
The result is consistent with cow milk mineral content.
Experiment 5:
Objective:
To determine the total, temporary, and permanent hardness of a given water sample using the
EDTA complexometric titration method.
Principle:
Water hardness is primarily caused by the presence of calcium (Ca²⁺) and magnesium (Mg²⁺)
ions. These ions form stable complexes with EDTA (ethylenediaminetetraacetic acid) under
alkaline conditions.
• Total hardness is determined by titrating the sample with EDTA at pH 10 using
Eriochrome Black T (EBT) as the indicator.
• Temporary hardness (due to bicarbonates of Ca and Mg) is removed by boiling and
filtering the sample.
• Permanent hardness is calculated by subtracting temporary hardness from total
hardness.
Reactions Involved:
Apparatus Required:
• Burette (50 mL)
• Pipette (50 mL)
• Conical flask (250 mL)
• Beakers, funnel, measuring cylinders
• Water bath or heating apparatus
• Filter paper and funnel
Reagents Required:
• 0.01 M EDTA solution (standardized)
• Buffer solution (pH 10): NH₄Cl + NH₄OH
• Eriochrome Black T indicator (EBT in ethanol)
• Distilled water
• Water sample (unknown hardness)
Procedure:
A. Determination of Total Hardness
1. Pipette 50.0 mL of water sample into a conical flask.
2. Add 2 mL of buffer solution (pH 10) to maintain alkaline conditions.
3. Add 2–3 drops of Eriochrome Black T indicator. The solution turns wine red due to
Ca/Mg–EBT complex.
4. Titrate with 0.01 M EDTA solution from a burette.
5. The endpoint is the color change from wine red to pure blue.
6. Record the volume of EDTA used (V₁).
7. Repeat for concordant readings.
B. Determination of Permanent Hardness
8. Take 100.0 mL of water sample in a beaker.
9. Boil gently for 30 minutes to precipitate temporary hardness (as CaCO₃ and Mg(OH)₂).
10. Cool, filter through filter paper into a clean flask, and make up to 100.0 mL with distilled
water.
11. Pipette 50.0 mL of this filtrate into a conical flask.
12. Repeat the titration steps as in Part A. Record EDTA volume used (V₂).
Observations and Calculations:
• M = Molarity of EDTA = 0.01 mol/L
• V₁ = Volume of EDTA used for total hardness = 12.5 mL
• V₂ = Volume of EDTA used for permanent hardness = 7.0 mL
• Vsample = 50.0 mL
Total Hardness:
Permanent Hardness:
Temporary Hardness:
Result:
Type of Hardness Value (mg/L as CaCO₃)
Total Hardness 250.2 mg/L
Permanent Hardness 140.2 mg/L
Temporary Hardness 110.0 mg/L
