Quantitative Analysis - Chemical Method

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10 Terms

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

Alkaline copper reduction method (4)

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Alkaline copper reduction method

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

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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

<p>Alkaline copper reduction method</p><ul><li><p>Copper reduction method that uses <span style="color: yellow">BaSO4 </span>to remove saccharoids.</p></li><li><p>Based on the<span style="color: yellow"> absorbance at </span><span style="color: red">540 nm</span> of a coloured complex <span style="color: yellow">between a copper oxidized sugar and arsenomolybdate.</span></p></li><li><p>The amount of carbohydrate present is <span style="color: yellow">determined by comparision with a calibration curve using a colorimeter,</span> under the proper conditions. This method is accurate up to 10 µg of glucose.</p></li></ul><p>REAGENT: (Remember)</p><ul><li><p><span style="color: red">Arsenomolybdic Acid </span></p></li></ul><p></p>
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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

<p>Alkaline copper reduction method</p><ul><li><p>Serum/plasma sample is <span style="color: yellow">treated with sodium nitroprusside solution</span> and then <span style="color: yellow">ferrous sulfate.</span></p></li><li><p>After incubation at 37°C for 30 min, the <span style="color: yellow">yellow colour intensity produced is measured spectrophotometrically at </span><span style="color: red">540 nm</span> using Folin reagent (a mixture of phosphoric acid 6M + ammonium molybdate 4H2O</p></li></ul><p>REAGENT: (Remember)</p><ul><li><p><span style="color: red">Phosphomolybdate</span> </p></li></ul><p></p>
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Neocuproine method

Alkaline copper reduction method

REAGENT: (Remember)

  • Neocuproine

<p>Alkaline copper reduction method</p><p>REAGENT: (Remember)</p><ul><li><p><span style="color: red">Neocuproine </span></p></li></ul><p></p>
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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

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Hagedorn Jensen

Ferric Reduction Method, Inverse colorimetry (1)

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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)

<p>Ferric Reduction Method, Inverse colorimetry (1)</p><ul><li><p>The standard <span style="color: yellow">sodium is treated with potassium ferricyanide.</span></p></li><li><p>A part of ferricyanide is reduced by glucose to <span style="color: yellow">ferrocyanide</span>. The remaining ferricyanide is determined from the amount of iodine liberated.</p></li></ul><p>REAGENT:</p><ul><li><p><span style="color: red">Ferrycyanide (Yellow)</span></p></li></ul><p></p>
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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

<p>Condensation Method (1)</p><ul><li><p>The proteins are first precipitated by <span style="color: yellow">tricholoroacetic acid.</span></p></li><li><p>The glucose present in a protein free filtrate react with O-toluidine (primary aromatic amine) in a hot acidic medium to form a <span style="color: red">stable green colored complex, </span>namely <span style="color: red">N-glycosamine.</span></p></li><li><p>The intensity of the color developed is measured photometrically at <span style="color: red">630nm,</span> which is <span style="color: yellow">directly proportional</span> to the concentration of the glucose present in the fluid.</p></li><li><p>Condensation of Carbohydrates with aromatic amines producing<span style="color: yellow"> Schiff’s Bases (green)</span></p><ul><li><p><span style="color: green">Cyanide reagent - Sir Marky </span></p></li></ul></li></ul><p></p>
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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.