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)

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.

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.

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.

Barfoed’s Reagent

Purpose: Differentiates between monosaccharides and disaccharides.

Procedure:

1. Dissolve 66.5 g copper(II) acetate in 1000 ml distilled water.

2. Add 9 ml glacial acetic acid.

3. Mix thoroughly and store in a dark bottle.

Molisch’s Reagent

Purpose: A general test for carbohydrates.

Procedure:

1. Dissolve 5 g of α-naphthol in 100 ml of ethanol (95%).

2. Store in a dark bottle.

Phosphate Buffer (pH 7.4)

Purpose: Used in biological experiments to maintain pH close to physiological conditions.

Procedure:

1. Dissolve 8.0 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, and 0.24 g KH₂PO₄ in 800 ml distilled water.

2. Adjust pH to 7.4 using HCl or NaOH.

3. Make up to 1000 ml with distilled water.

4. Store at 4°C if not used immediately.

Simulated Intestinal Fluid (SIF)

Purpose: Mimics the environment of the small intestine to test drug stability, dissolution, and release.

Procedure:

1. Weigh 6.8 g of monobasic potassium phosphate (KH₂PO₄).

2. Dissolve in 250 ml of distilled water.

3. Add 77 ml of 0.2 N NaOH.

4. Add 10 g of pancreatin.

5. Dilute to 1000 ml with distilled water.

6. Mix well and adjust pH to 6.8 ± 0.1.

7. Filter if necessary before use.