Advanced Analytical Chemistry Lab Experiments

Determination of Fat Content in Milk

Chemicals: Concentrated H₂SO₄, Amyl alcohol, Butyrometer
Apparatus: Gerber butyrometer, Centrifuge, Water bath
Procedure: Add 10ml H₂SO₄ to butyrometer, carefully add 11ml milk sample, add 1ml amyl alcohol. Centrifuge at 1100 rpm for 5 minutes at 65°C.
Calculation Formula:
Fat % = (Reading on butyrometer scale × 100) / Volume of milk sample
Sample Problem:

If the butyrometer reading is 3.2 for 11ml milk sample, calculate fat percentage.

Solution: Fat % = (3.2 × 100) / 11 = 29.09%

Quantification of Proteins (Lowry Method)

Chemicals: Folin-Ciocalteu reagent, Copper sulfate, Sodium carbonate, BSA standard
Apparatus: Spectrophotometer, Test tubes, Pipettes, Vortex mixer
Procedure: Prepare protein standards (0-100 μg/ml BSA). Add Lowry reagent, incubate 10 min. Add Folin reagent, measure absorbance at 750nm after 30 min.
Calculation Formula:
Protein concentration = (Absorbance – y-intercept) / Slope × Dilution factor
Sample Problem:

Sample absorbance = 0.45, Standard curve: y = 0.008x + 0.02, Dilution = 1:10

Solution: Protein = (0.45 – 0.02) / 0.008 × 10 = 537.5 μg/ml

Determination of Cholesterol in Food

Chemicals: Chloroform, Methanol, Cholesterol oxidase, Peroxidase, ABTS
Apparatus: Homogenizer, Separatory funnel, Rotary evaporator, UV-Vis spectrophotometer
Procedure: Extract lipids using Folch method. Saponify with KOH. Extract unsaponifiable matter. Analyze using enzymatic method at 405nm.
Calculation Formula:
Cholesterol (mg/100g) = (Absorbance × Standard factor × Volume) / Sample weight
Sample Problem:

Absorbance = 0.32, Standard factor = 0.156, Volume = 25ml, Sample = 5g

Solution: Cholesterol = (0.32 × 0.156 × 25) / 5 = 0.25 mg/100g

Quantification of Reducing Sugars (DNS Method)

Chemicals: DNS reagent, Glucose standards, Sodium potassium tartrate
Apparatus: Water bath, Spectrophotometer, Test tubes, Pipettes
Procedure: Mix sample with DNS reagent. Heat in boiling water bath for 5 minutes. Cool and measure absorbance at 540nm.
Calculation Formula:
Sugar concentration = (Absorbance – intercept) / Slope × Dilution factor
Sample Problem:

Absorbance = 0.68, Calibration: y = 1.2x + 0.05, Dilution = 1:5

Solution: Sugar = (0.68 – 0.05) / 1.2 × 5 = 2.625 mg/ml

Water Analysis for Drinking Purpose

Parameters: pH, TDS, Hardness, Chloride, Fluoride, Heavy metals
Apparatus: pH meter, Conductivity meter, Burette, Atomic absorption spectrophotometer
Procedure: Test pH using calibrated meter. Measure TDS by conductivity. Determine hardness by EDTA titration. Analyze metals by AAS.
Hardness Calculation:
Total Hardness (mg/L as CaCO₃) = (EDTA volume × EDTA normality × 50000) / Sample volume
Sample Problem:

EDTA used = 12.5ml, EDTA normality = 0.01N, Sample = 100ml

Solution: Hardness = (12.5 × 0.01 × 50000) / 100 = 62.5 mg/L

Determination of Caffeine Content

Chemicals: Chloroform, Sodium carbonate, Caffeine standards
Apparatus: HPLC system, C18 column, UV detector, Separatory funnel
Procedure: Extract caffeine with chloroform after alkalinization. Analyze by HPLC using mobile phase acetonitrile:water (30:70). Detect at 280nm.
HPLC Calculation:
Caffeine (mg/L) = (Peak area sample / Peak area standard) × Standard concentration × Dilution factor
Sample Problem:

Sample peak area = 2500, Standard peak area = 3000, Standard = 50mg/L, Dilution = 1:2

Solution: Caffeine = (2500/3000) × 50 × 2 = 83.33 mg/L

Determination of Heavy Metals in Food

Chemicals: Nitric acid, Hydrogen peroxide, Metal standards (Pb, Cd, Hg, As)
Apparatus: Microwave digestion system, Atomic absorption spectrophotometer, Graphite furnace
Procedure: Digest samples with HNO₃ and H₂O₂ in microwave. Analyze by AAS-GF. Use standard addition method for matrix interference.
AAS Calculation:
Metal concentration (mg/kg) = (Absorbance × Slope factor × Dilution × Volume) / Sample weight
Sample Problem:

Lead absorbance = 0.045, Slope = 0.0025, Dilution = 50, Volume = 25ml, Sample = 2g

Solution: Pb = (0.045 × 0.0025 × 50 × 25) / 2 = 0.70 mg/kg

Determination of Citric Acid in Juices

Chemicals: Pyridine, Acetic anhydride, NaOH solution, Phenolphthalein
Apparatus: Burette, Conical flask, Pipettes, Water bath
Procedure: Neutralize sample to pH 8.1. Add pyridine-acetic anhydride reagent. Heat at 70°C for 30 min. Titrate excess acetic anhydride with NaOH.
Titration Calculation:
Citric acid % = [(Blank – Sample) × NaOH normality × 0.064 × 100] / Sample weight
Sample Problem:

Blank = 25ml, Sample = 18ml, NaOH = 0.1N, Sample weight = 10g

Solution: Citric acid = [(25-18) × 0.1 × 0.064 × 100] / 10 = 0.448%

Determination of Ascorbic Acid in Fruit Juices

Chemicals: 2,6-dichlorophenolindophenol, Metaphosphoric acid, Ascorbic acid standard
Apparatus: Burette, Conical flask, Pipettes, Measuring cylinder
Procedure: Extract vitamin C with metaphosphoric acid. Titrate with DCPIP solution until pink endpoint persists for 15 seconds.
Vitamin C Calculation:
Ascorbic acid (mg/100ml) = (DCPIP volume × DCPIP factor × 100) / Sample volume
Sample Problem:

DCPIP used = 8.5ml, DCPIP factor = 0.5mg/ml, Sample = 10ml

Solution: Vitamin C = (8.5 × 0.5 × 100) / 10 = 42.5 mg/100ml

Evaluation of Rancidity – Acid Value

Chemicals: Ethanol, Diethyl ether, KOH solution, Phenolphthalein
Apparatus: Burette, Conical flask, Pipettes, Measuring cylinder
Procedure: Dissolve oil sample in ethanol-ether mixture (1:1). Add phenolphthalein indicator. Titrate with 0.1N KOH until pink endpoint.
Acid Value Formula:
Acid Value = (KOH volume × KOH normality × 56.1) / Sample weight
Sample Problem:

KOH used = 3.2ml, KOH normality = 0.1N, Oil sample = 5g

Solution: Acid Value = (3.2 × 0.1 × 56.1) / 5 = 3.59 mg KOH/g

HPLC Analysis of Pharmaceutical Binary Mixture

System: HPLC with UV detector, C18 column (250×4.6mm, 5μm)
Mobile Phase: Acetonitrile:Water (60:40), Flow rate 1.0 ml/min
Validation: Linearity, Precision, Accuracy, LOD, LOQ, Resolution, Tailing factor
Statistical Parameters:
RSD% = (Standard deviation / Mean) × 100
LOD = 3.3 × (σ/S), LOQ = 10 × (σ/S)
Resolution = 2(t₂-t₁)/(W₁+W₂)
Sample Calculation:

Peak areas: 2500, 2480, 2520, 2510, 2490

Mean: 2500, SD: 16.73, RSD: 0.67%

DNA Extraction from Biological Samples

Chemicals: Lysis buffer, Proteinase K, Ethanol, TE buffer
Apparatus: Centrifuge, Micropipettes, Vortex mixer, Water bath
Procedure: Lyse cells with buffer. Add proteinase K, incubate at 56°C. Precipitate DNA with ethanol. Wash and resuspend in TE buffer.
DNA Quantification:
DNA concentration (μg/ml) = A₂₆₀ × 50 × Dilution factor
Purity ratio = A₂₆₀/A₂₈₀ (should be 1.8-2.0)
Sample Problem:

A₂₆₀ = 0.45, A₂₈₀ = 0.25, Dilution = 1:100

Solution: DNA = 0.45 × 50 × 100 = 2250 μg/ml, Purity = 1.8

Scientific References & Standards