📘 𝐕𝐢𝐭𝐚𝐦𝐢𝐧 𝐄𝐬𝐭𝐢𝐦𝐚𝐭𝐢𝐨𝐧 𝐍𝐨𝐭𝐞𝐬 These notes are helpful for DMLT, BSc MLT, and other paramedical students. ✅ Vitamin C (Ascorbic Acid) Estimation 🔬 Principle: Vitamin C reduces 2,6-Dichlorophenol Indophenol (DCPIP) dye from blue to colorless in acidic medium. The amount of dye reduced is proportional to the Vitamin C present. 🧪 Reagents: ▪ DCPIP Solution ▪ Oxalic Acid (as diluent) ▪ Standard Ascorbic Acid Solution ⚗️ Procedure: ● Take sample in conical flask. ● Add 5 ml oxalic acid. ● Titrate against DCPIP until light ● pink color persists (30 seconds). ● Repeat with standard. 🧮 Calculation: Vit C (mg/100ml) = (Sample titre / Standard titre) × Standard concentration ✅ Vitamin A (Retinol) Estimation 🔬 Principle: Vitamin A reacts with Trichloroacetic acid (TCA) or Antimony trichloride forming a blue-colored complex, measured colorimetrically. 🧪 Reagents: ▪ TCA in chloroform ▪ Vitamin A standard ▪ Chloroform extract of sample ⚗️ Procedure: ● Mix sample with TCA reagent. ● Allow blue color to develop. ● Measure absorbance at 620 nm. 🧮 Calculation: Compare absorbance with standard curve. ✅ Vitamin D (Calciferol) Estimation 🔬 Principle: Vitamin D absorbs UV light at 265 nm. Estimation done via spectrophotometer or HPLC. 🧪 Reagents: ▪ Chloroform ▪ Standard Vitamin D ▪ UV Spectrophotometer ⚗️ Procedure: ● Extract sample using chloroform. ● Measure absorbance at 265 nm. ● Compare with standard curve. 🧮 Note: HPLC provides better accuracy in clinical labs. ✅ Vitamin B1 (Thiamine) Estimation 🔬 Principle: Thiamine is oxidized and produces a fluorescent compound measured fluorometrically. 🧪 Reagents: ▪ Potassium ferricyanide ▪ Alkaline solution ▪ Thiamine standard ⚗️ Procedure: ● Oxidize sample with ferricyanide. ● Measure fluorescence intensity. ● Compare with standard. ✅ Vitamin B2 (Riboflavin) Estimation 🔬 Principle: Riboflavin is naturally fluorescent and detected under UV after acid hydrolysis. 🧪 Reagents: ▪️ HCl (for hydrolysis) ▪️ Riboflavin standard ⚗️ Procedure: 🔸 Hydrolyze sample using acid. 🔸 Neutralize & extract riboflavin. 🔸 Measure fluorescence at 450 nm.