Quantitative Analysis - Chemical Method

Nelson-somogyi, folin-wu, neocuproine method benedicts method,

Alkaline copper reduction method (4)

Alkaline copper reduction method

All reducing sugars will be measured, except Sucrose and Trehalose.

Nelson-somogyi

Alkaline copper reduction method

• Copper reduction method that uses BaSO4 to remove saccharoids.

• Based on the absorbance at 540 nm of a coloured complex between a copper oxidized sugar and arsenomolybdate.

• The amount of carbohydrate present is determined by comparision with a calibration curve using a colorimeter, under the proper conditions. This method is accurate up to 10 µg of glucose.

REAGENT: (Remember)

• Arsenomolybdic Acid

Folin-wu

Alkaline copper reduction method

• Serum/plasma sample is treated with sodium nitroprusside solution and then ferrous sulfate.

• After incubation at 37°C for 30 min, the yellow colour intensity produced is measured spectrophotometrically at 540 nm using Folin reagent (a mixture of phosphoric acid 6M + ammonium molybdate 4H2O

REAGENT: (Remember)

• Phosphomolybdate

Neocuproine method

Alkaline copper reduction method

REAGENT: (Remember)

• Neocuproine

Benedict’s Method (Modification of Folin-Wu)

Alkaline copper reduction method

• Detection and quantitation of reducing substances in body fluids like blood and urine

• Use citrate or tartrate as stabilizing agents, same reagents as Folin-Wu

Hagedorn Jensen

Ferric Reduction Method, Inverse colorimetry (1)

Hagedorn Jensen

Ferric Reduction Method, Inverse colorimetry (1)

• The standard sodium is treated with potassium ferricyanide.

• A part of ferricyanide is reduced by glucose to ferrocyanide. The remaining ferricyanide is determined from the amount of iodine liberated.

REAGENT:

• Ferrycyanide (Yellow)

Orthro Toluidine

Condensation Method (1)

• The proteins are first precipitated by tricholoroacetic acid.

• The glucose present in a protein free filtrate react with O-toluidine (primary aromatic amine) in a hot acidic medium to form a stable green colored complex, namely N-glycosamine.

• The intensity of the color developed is measured photometrically at 630nm, which is directly proportional to the concentration of the glucose present in the fluid.

• Condensation of Carbohydrates with aromatic amines producing Schiff’s Bases (green)

  • Cyanide reagent - Sir Marky

Noted

Note all the chemical methods that are mentioned from nelson - hegedor jensen, they are already considered as obsolete, they not used in the laboratory.