Quantitative analysis of Carbohydrate Total soluble carbohydrate Reducing carbohydrate Aldo reducing carbohydrate Quantitative analysis of Carbohydrate

Extraction of soluble carbohydrate Accurately weighed sample Boiling 80% neutral ethanol/6h Filter Complete to 50ml in volumetric flask

Phenol-sulfuric acid method 1 ml extract + 1 ml 5%phenol then 5 ml sulfuric acid conc. Distilled water as blank sample Standard curve is done by pure glucose. Measuring in spectrophotometer at 490 nm.

Total reducing sugars Schoorl method (volumetric method) Reducing sugars + Cu++ (more than enough to oxidize all available reducing sugars)→ R-COOH + Cu2O↓ (equivalent to the amount of sugars) Cu++ (the excess) + KI I2 (equivalent to the amount of excess Cu++ ) + K2SO4 I2 +Na2 S2O3 → 2I−+ Na2S4O6 (Titration from blue to colorless in presence of starch indicator) H2SO4 dil.

Total reducing sugars Colorimetric method R-CHO + Cu++ → R-COO− + Cu2O↓ (weight) Cu2O↓ + molybdic → blue color The intensity of the color is related to the concentration of the sugars. Ferricyanide (yellow; measured at 420 nm) is reduced by sugars to Ferrocyanide (colorless) in relation with the concentration

Aldo reducing sugars Iodine in alkaline medium I2 + NaOH → NaOI (Hypoiodide; a weak oxidizing agent that can oxidize only aldosugar but not ketosugars; should be more than enough to oxidize all available aldosugars) R-CHO + NaOI → R-COONa NaOI (excess) + H2SO4 → I2 (equi. to the excess) I2 +Na2 S2O3 → 2I−+ Na2S4O6 (Titration from blue to colorless in presence of starch indicator)

Schoorl method Na2S2O3 (0.0901N) 10 ml sugar solution (or water in the blank) Then 10 ml Fehling’s mixture Then heating 2 min. then cooling Then adding 10 ml KI + 10 ml H2SO4 dil Then 2 ml starch indicator Titration from blue to colorless flask

Iodine alkaline Na2S2O3 (0.0901N) 10 ml sugar solution (or water in the blank) Then 2 drops NaOH + 10 ml iodine solution Let in dark place for 2 min. Then adding 10 ml H2SO4 dil. Then 2 ml starch indicator Titration from blue to colorless flask

Calculation mg/l = (Vb-Vs) х N х 10 х 9 х 100