BIOSCIENCE EDUCATION

 Froehde’s Reagent

Purpose: Spot test for alkaloids and some phenols.

Procedure:

1. Dissolve 0.5 g molybdic acid (or MoO₃) in 100 ml concentrated sulfuric acid.

2. Prepare freshly before use.

3. Apply dropwise onto the test substance.

Liebermann’s Reagent

Purpose: Colorimetric spot test for alkaloids, especially morphine and codeine.

Procedure:

1. Slowly add 1 ml concentrated sulfuric acid to 1 ml of cold fuming nitric acid in a chilled container.

2. Mix well and use immediately.

⚠️ Use extreme caution due to strong acids and fumes.

  Dragendorff’s Reagent

Purpose: Detection of alkaloids in plant extracts or formulations.

Procedure (stock solution method):

1. Stock A: Dissolve 1.7 g bismuth subnitrate in 20 ml glacial acetic acid.

2. Stock B: Dissolve 40 g potassium iodide in 100 ml distilled water.

3. Mix 10 ml of A and 20 ml of B.

4. Dilute with distilled water to make 100 ml.

5. Store in a dark bottle; stable for weeks.

DETERMINATION OF DISSOLVED OXYGEN IN WATER (WINKLER METHOD)

AIM:
To determine the dissolved oxygen (DO) content in water.

APPARATUS:
BOD bottles, burette, pipette.

REAGENT:
Manganese sulfate, alkaline iodide, starch, sodium thiosulphate.

PRINCIPLE:
DO oxidizes Mn²⁺ to Mn⁴⁺, which liberates iodine that is titrated.

PROCEDURE:

• Add MnSO₄ and alkaline iodide to water sample.

• Allow precipitate to form and settle.

• Add H₂SO₄ to dissolve.

• Titrate liberated iodine with sodium thiosulphate using starch as indicator.

CALCULATION:
DO (mg/L) = Volume of thiosulfate used × Normality × 8

DETERMINATION OF CITRIC ACID IN FRUIT JUICE

AIM:
To estimate citric acid content in fruit juice.

APPARATUS:
Burette, conical flask, pipette.

REAGENT:
0.1M NaOH, phenolphthalein.

PRINCIPLE:
Citric acid neutralized by NaOH; endpoint indicated by pink color.

PROCEDURE:

• Pipette 10ml of juice into a flask.

• Add 2–3 drops phenolphthalein.

• Titrate with NaOH to pink endpoint.

CALCULATION:
% Citric Acid = Titre × 0.064 × 100 / Volume of sample

DETERMINATION OF CHLOROPHYLL IN LEAF EXTRACT

AIM:
To estimate chlorophyll content in a leaf sample.

APPARATUS:
Spectrophotometer, centrifuge, test tubes.

REAGENT:
80% acetone.

PRINCIPLE:
Chlorophyll is extracted and measured at specific absorbance values.

PROCEDURE:

• Grind 1g of leaf in 10ml acetone.

• Centrifuge and collect supernatant.

• Measure absorbance at 645 nm and 663 nm.

CALCULATION:
Use Arnon's formula:
Chlorophyll a = 12.7(A663) – 2.69(A645)
Chlorophyll b = 22.9(A645) – 4.68(A663)

DETECTION OF BORIC ACID IN FOOD

AIM:
To detect boric acid in food samples (e.g., milk, jaggery).

APPARATUS:
Test tubes, turmeric paper.

REAGENT:
None (turmeric paper acts as indicator).

PRINCIPLE:
Boric acid turns turmeric paper red, which turns green on ammonia exposure.

PROCEDURE:

• Dip turmeric paper in food extract.

• Dry and observe color.

• Expose to ammonia vapor for confirmation.

RESULT:
Red to green color change confirms boric acid.

TEST FOR SULPHUR DIOXIDE IN DRIED FRUITS

AIM:
To detect presence of SO₂ preservative in food.

APPARATUS:
Test tubes, beaker.

REAGENT:
Pararosaniline hydrochloride, formaldehyde.

PRINCIPLE:
SO₂ reacts with dye and formaldehyde to form pink/magenta color.

PROCEDURE:

• Boil fruit sample in water, cool extract.

• Add 2ml dye reagent + formaldehyde.

• Observe color change.

RESULT:
Pink color indicates presence of sulphur dioxide.

ALCOHOL DETECTION IN HAND SANITIZERS

AIM:
To determine the presence and concentration of alcohol in hand sanitizer.

APPARATUS:
Test tube, alcohol hydrometer or colorimeter.

REAGENT:
Potassium dichromate + sulfuric acid.

PRINCIPLE:
Alcohol reduces orange dichromate to green chromium ions.

PROCEDURE:

• Add 2ml sanitizer to test tube.

• Add dichromate reagent.

• Shake and observe color change from orange to green.

RESULT:
Color change confirms alcohol; intensity can indicate concentration.

TEST FOR FORMALIN IN MILK

AIM:
To detect formalin (formaldehyde) in milk.

APPARATUS:
Test tube, ice bath.

REAGENT:
Sulfuric acid, ferric chloride.

PRINCIPLE:
Formalin reacts with ferric chloride and acid to give a violet ring.

PROCEDURE:

• Take 5ml milk in a test tube.

• Add 5ml sulfuric acid with FeCl₃ carefully along the wall.

• Observe for a violet ring at the interface.

RESULT:
Presence of violet ring indicates formalin.

TEST FOR PRESENCE OF UREA IN MILK

AIM:
To detect adulteration of milk with urea.

APPARATUS:
Test tubes, dropper.

REAGENT:
Para-dimethylaminobenzaldehyde (PDAB) reagent.

PRINCIPLE:
Urea reacts with PDAB to form a yellow color.

PROCEDURE:

• Add 5ml milk sample in a test tube.

• Add 2ml of PDAB reagent.

• Observe for yellow color development.

RESULT:
Yellow color indicates presence of urea.

DETERMINATION OF PHOSPHATE IN FOOD (VANADOMOLYBDATE METHOD)

AIM:
To determine phosphate content in a food sample.

APPARATUS:
Test tubes, colorimeter.

REAGENT:
Ammonium molybdate, ammonium vanadate, nitric acid.

PRINCIPLE:
Phosphate reacts with vanadomolybdate reagent to form a yellow complex.

PROCEDURE:

• Prepare sample extract.

• Add reagent and incubate for 10 minutes.

• Measure absorbance at 400–420 nm.

CALCULATION:
Compare with phosphate standard curve.

DETERMINATION OF VITAMIN A (COLORIMETRIC METHOD)

AIM:
To determine the vitamin A content in margarine or food sample.

APPARATUS:
Test tubes, colorimeter, pipettes.

REAGENT:
Chloroform, antimony trichloride reagent (Carr-Price reagent).

PRINCIPLE:
Vitamin A reacts with antimony trichloride to form a blue complex, measurable colorimetrically.

PROCEDURE:

• Extract sample with chloroform.

• Add 2ml of Carr-Price reagent.

• Measure absorbance at 620 nm.

• Compare with vitamin A standard.

CALCULATION:
Use standard calibration curve to estimate µg vitamin A.

DETERMINATION OF SUGAR IN HONEY (FEHLING’S TEST)

AIM:
To detect reducing sugars in honey.

APPARATUS:
Test tubes, burette, water bath.

REAGENT:
Fehling’s A and B solutions.

PRINCIPLE:
Reducing sugars reduce Cu²⁺ to Cu₂O (red precipitate).

PROCEDURE:

• Mix equal volumes of Fehling A and B.

• Add honey solution gradually while boiling.

• Observe brick-red precipitate.

CALCULATION:
Compare against known glucose standards.

Tollen’s Reagent

Purpose: Detects aldehydes (reducing agents).

Procedure:

1. Prepare fresh before use:

   • Mix 2 ml of 0.1 M silver nitrate solution.

   • Add a few drops of dilute NaOH to form Ag₂O precipitate.

   • Add dilute ammonia dropwise until the precipitate just dissolves.

DETERMINATION OF PROTEIN (KJELDAHL METHOD)

AIM:
To determine total protein content in a sample.

APPARATUS:
Digestion flask, distillation unit, titration setup.

REAGENT:
Concentrated H₂SO₄, NaOH, boric acid, indicator.

PRINCIPLE:
Nitrogen is converted to ammonia, distilled, and titrated to estimate protein.

PROCEDURE:

• Digest sample with H₂SO₄ + catalyst.

• Neutralize, distill ammonia into boric acid.

• Titrate with HCl.

CALCULATION:
% Protein = Titre × 1.4 × 6.25 / Sample weight

Seliwanoff’s Reagent

Purpose: Differentiates between aldose and ketose sugars.

Procedure:

1. Dissolve 0.05 g resorcinol in 100 ml of 3M HCl.

2. Store in an amber bottle.

DETERMINATION OF ALCOHOL IN WINE (DISTILLATION + DENSITY)

AIM:
To measure ethanol content in wine.

APPARATUS:
Distillation setup, pycnometer or alcohol hydrometer.

REAGENT:
None.

PRINCIPLE:
Ethanol is distilled and measured based on density.

PROCEDURE:

• Distill 100ml of wine.

• Collect distillate and measure density at 20°C.

• Refer to alcohol tables.

CALCULATION:
% Alcohol = Based on density from standard chart.

Biuret Reagent

Purpose: Detects proteins (peptide bonds).

Procedure:

1. Dissolve 1.5 g CuSO₄·5H₂O in 500 ml distilled water.

2. Add 6 g sodium potassium tartrate and 375 ml of 10% NaOH.

3. Make up to 1000 ml with distilled water.

DETERMINATION OF IRON IN FOOD (COLORIMETRY)

AIM:
To estimate iron content in food using colorimetric method.

APPARATUS:
Colorimeter, test tubes, pipettes.

REAGENT:
1,10-phenanthroline, hydroxylamine HCl, acetate buffer.

PRINCIPLE:
Iron reacts to form a red-orange complex, whose absorbance is measured.

PROCEDURE:

• Prepare food extract and react with reagents.

• Measure absorbance at 510 nm.

• Compare with standard iron curve.

CALCULATION:
Use standard curve to determine ppm of iron.