Total soluble carbohydrate Reducing carbohydrate Aldo reducing carbohydrate

Total soluble carbohydrate Reducing carbohydrate Aldo reducing carbohydrate QUANTITATIVE ANALYSIS OF CARBOHYDRATE

Extraction of soluble carbohydrate � Accurately weighed sample � Boiling 80% neutral ethanol/6 h � Filter � Complete to 50 ml 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)→ RCOOH + Cu 2 O↓ (equivalent to the amount of sugars) H SO dil. ++ � Cu (the excess) + KI I 2 (equivalent to the amount of excess Cu++ ) + K 2 SO 4 � I 2 +Na 2 S 2 O 3 → 2 I−+ Na 2 S 4 O 6 (Titration from blue to colorless in presence of starch indicator) � 2 4

Total reducing sugars Colorimetric method � R-CHO + Cu++ → R-COO− + Cu 2 O↓ (weight) � Cu 2 O↓ + 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 � I 2 + Na. OH → Na. OI (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 + Na. OI → R-COONa � Na. OI (excess) + H 2 SO 4 → I 2 (equi. to the excess) � I 2 +Na 2 S 2 O 3 → 2 I−+ Na 2 S 4 O 6 (Titration from blue to colorless in presence of starch indicator)

Na 2 S 2 O 3 (0. 0901 N) flask Schoorl method 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 H 2 SO 4 dil Then 2 ml starch indicator Titration from blue to colorless

Na 2 S 2 O 3 (0. 0901 N) flask Iodine alkaline 10 ml sugar solution (or water in the blank) Then 2 drops Na. OH + 10 ml iodine solution Let in dark place for 2 min. Then adding 10 ml H 2 SO 4 dil. Then 2 ml starch indicator Titration from blue to colorless

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