COLORIMETRIC IDENTIFICATION OF UNKNOWN SUGARS
Purpose:
Identify which samples contain real maple syrup, and which contain fake maple syrup, and determine which carbohydrates the imposters contain.
Qualitative Carbohydrate Assays (It is useful to run both negative and positive controls at the same time as the sample.)
Molisch Test (Carbohydrates)
Prepare Molisch's reagent by dissolving 0.5 g reagent grade a-naphthol in10 mL of 95% ethanol. Store the reagent, protected from light, at room temperature. To test for carbohydrates, add 0.02 mL of the reagent to 1 mL of 0.1% carbohydrate (1 mg/mL) solution in a small test tube. After mixing, tilt the tube and carefully add without mixing, 0.5 mL of concentrated sulfuric acid by pouring it down the side of the tube. (Use a glass Pasteur pipette to add the H2SO4: do not use a mechanical pipettor with concentrated acids.) A red-violet layer at the interface between the acid (bottom) and aqueous (upper) layers is a positive test for carbohydrates. (Reminder: Always add acid to water.)
Bial's Test (Pentoses)
Prepare Bial's reagent by dissolving 0.3 g reagent-grade orcinol and 0.05 g ferric chloride in 100 mL of concentrated (12 M) HCl. Store the reagent protected from light. To test for pentoses, add 0.05 mL of 0.1% carbohydrate solution in water to 1 mL of Bial's reagent, (Use a glass Pasteur pipette to add the Bial's reagent: do not use a mechanical pipettor with concentrated acids.) and heat the solution in a boiling water bath for 2 minutes.* A blue-green color indicates pentoses or nucleotides containing pentoses; a yellow-green color indicates hexoses, and disaccharides are yellow.
Diphenylamine ((f)2NH) Test (2-Deoxy sugars)
Prepare the reagent by dissolving 0.1 g of diphenylamine in 10 mL of glacial acetic acid and adding 0.25 mL of sulfuric acid. To test for 2-deoxy sugars, add 0.6 mL of a 0.01% carbohydrate solution to 1 mL of the diphenylamine reagent and heat in a boiling-water bath for 10 minutes.* If 2-deoxy sugars are present a blue-green color is produced.
Resorcinol (Seliwanoff's) Test (Ketohexoses)
Prepare Seliwanoff's reagent by dissolving 0.05 g of reagent-grade resorcinol in 100 mL of 3 M HCl. Store protected from light. To test for ketohexoses, add 0.1 mL of a 1% carbohydrate solution in water to 1 mL of the reagent, and heat the solution in a boiling water bath for 5 minutes.* A deep red colored precipitate within 5 minute indicates ketohexoses. Sucrose gives a positive ketohexose test because of partial hydrolysis to glucose and fructose. Other sugars give a red color upon prolonged heating.
Benedict's Test (Reducing sugars)
Dissolve 1.73 g trisodium citrate (dihydrate) and 1.0 g anhydrous sodium carbonate in 8 mL of warm dH2O (Solution A). Dissolve copper sulfate (pentahydrate) (1.73 g) separately in 20 mL of dH2O (Solution B). Immediately before using, prepare Benedict's reagent by mixing 0.8 mL of Solution A with 0.2 mL of Solution B. To test for reducing sugars, add 0.2 mL of a 1% carbohydrate solution to 1 mL of Benedict's reagent and heat in a boiling water bath for 5 minutes. A brick-red precipitate indicates a positive test for reducing sugars.
Barfoed's Test (Reducing monosaccharides)
Prepare Barfoed's reagent by dissolving 0.66 g cupric acetate (monohydrate) and 0.18 mL glacial acetic acid in 10 mL of dH2O. To test for reducing monosaccharides, add 0.3 mL of 1% carbohydrate solution to 0.6 mL of Barfoed's reagent and heat in a boiling-water bath for 5 minutes,* then cool to room temperature. A copious amount of brick-red precipitate indicates a reducing monosaccharide. Some hydrolysis of disaccharides may lead to trace precipitates.
Iodine Test (Starch/Amylose)
A few drops of 0.01 M iodine in 0.12 M KI are added to a 1% solution of the carbohydrate in question. The immediate formation of a vivid blue color indicates amylose.
Glucose Test
Prepare the assay reagent by dissolving a capsule (Sigma 510-6) containing 500 units of glucose oxidase and 100 units of horseradish peroxidase with buffer salts in 100 mL of distilled water, and adding 1.6 mL of 2.5 mg/mL o-dianisidine dihydrochloride (Sigma 510-50). Combine 0.1 mL of a sample solution containing 0.25 to 3 mg/mL of carbohydrate with 1 mL of the assay reagent. A brown color within 30 minutes indicates glucose.
* Heat in a fume hood.
Qualitative Tests for Carbohydrates
+ = positive; - = negative; bl = blue; br = brown; gr = green; r = red; v = violet; y = yellow; ppt = precipitate * To test for DNA, the sample is first hydrolyzed in 10% trichloroacetic acid at 95°C for 10 minutes then diluted with two volumes of water before assaying with the diphenylamine test. § GO = glucose oxidase
Identify which samples contain real maple syrup, and which contain fake maple syrup, and determine which carbohydrates the imposters contain.
Qualitative Carbohydrate Assays (It is useful to run both negative and positive controls at the same time as the sample.)
Molisch Test (Carbohydrates)
Prepare Molisch's reagent by dissolving 0.5 g reagent grade a-naphthol in10 mL of 95% ethanol. Store the reagent, protected from light, at room temperature. To test for carbohydrates, add 0.02 mL of the reagent to 1 mL of 0.1% carbohydrate (1 mg/mL) solution in a small test tube. After mixing, tilt the tube and carefully add without mixing, 0.5 mL of concentrated sulfuric acid by pouring it down the side of the tube. (Use a glass Pasteur pipette to add the H2SO4: do not use a mechanical pipettor with concentrated acids.) A red-violet layer at the interface between the acid (bottom) and aqueous (upper) layers is a positive test for carbohydrates. (Reminder: Always add acid to water.)
Bial's Test (Pentoses)
Prepare Bial's reagent by dissolving 0.3 g reagent-grade orcinol and 0.05 g ferric chloride in 100 mL of concentrated (12 M) HCl. Store the reagent protected from light. To test for pentoses, add 0.05 mL of 0.1% carbohydrate solution in water to 1 mL of Bial's reagent, (Use a glass Pasteur pipette to add the Bial's reagent: do not use a mechanical pipettor with concentrated acids.) and heat the solution in a boiling water bath for 2 minutes.* A blue-green color indicates pentoses or nucleotides containing pentoses; a yellow-green color indicates hexoses, and disaccharides are yellow.
Diphenylamine ((f)2NH) Test (2-Deoxy sugars)
Prepare the reagent by dissolving 0.1 g of diphenylamine in 10 mL of glacial acetic acid and adding 0.25 mL of sulfuric acid. To test for 2-deoxy sugars, add 0.6 mL of a 0.01% carbohydrate solution to 1 mL of the diphenylamine reagent and heat in a boiling-water bath for 10 minutes.* If 2-deoxy sugars are present a blue-green color is produced.
Resorcinol (Seliwanoff's) Test (Ketohexoses)
Prepare Seliwanoff's reagent by dissolving 0.05 g of reagent-grade resorcinol in 100 mL of 3 M HCl. Store protected from light. To test for ketohexoses, add 0.1 mL of a 1% carbohydrate solution in water to 1 mL of the reagent, and heat the solution in a boiling water bath for 5 minutes.* A deep red colored precipitate within 5 minute indicates ketohexoses. Sucrose gives a positive ketohexose test because of partial hydrolysis to glucose and fructose. Other sugars give a red color upon prolonged heating.
Benedict's Test (Reducing sugars)
Dissolve 1.73 g trisodium citrate (dihydrate) and 1.0 g anhydrous sodium carbonate in 8 mL of warm dH2O (Solution A). Dissolve copper sulfate (pentahydrate) (1.73 g) separately in 20 mL of dH2O (Solution B). Immediately before using, prepare Benedict's reagent by mixing 0.8 mL of Solution A with 0.2 mL of Solution B. To test for reducing sugars, add 0.2 mL of a 1% carbohydrate solution to 1 mL of Benedict's reagent and heat in a boiling water bath for 5 minutes. A brick-red precipitate indicates a positive test for reducing sugars.
Barfoed's Test (Reducing monosaccharides)
Prepare Barfoed's reagent by dissolving 0.66 g cupric acetate (monohydrate) and 0.18 mL glacial acetic acid in 10 mL of dH2O. To test for reducing monosaccharides, add 0.3 mL of 1% carbohydrate solution to 0.6 mL of Barfoed's reagent and heat in a boiling-water bath for 5 minutes,* then cool to room temperature. A copious amount of brick-red precipitate indicates a reducing monosaccharide. Some hydrolysis of disaccharides may lead to trace precipitates.
Iodine Test (Starch/Amylose)
A few drops of 0.01 M iodine in 0.12 M KI are added to a 1% solution of the carbohydrate in question. The immediate formation of a vivid blue color indicates amylose.
Glucose Test
Prepare the assay reagent by dissolving a capsule (Sigma 510-6) containing 500 units of glucose oxidase and 100 units of horseradish peroxidase with buffer salts in 100 mL of distilled water, and adding 1.6 mL of 2.5 mg/mL o-dianisidine dihydrochloride (Sigma 510-50). Combine 0.1 mL of a sample solution containing 0.25 to 3 mg/mL of carbohydrate with 1 mL of the assay reagent. A brown color within 30 minutes indicates glucose.
* Heat in a fume hood.
| Sugar | Molisch | Bial | (f)2NH | G.O.§ | Resorcinol | Benedict | Barfoed | Iodine |
| Ribose | + (r/v) | (bl/gr) | - | - | - | +(r ppt) | + (r ppt) | - |
| DNA* | + (r/v) | (y) | (bl/gr) | - | - | +(r ppt) | + (r ppt) | - |
| Fructose | + (r/v) | (y/gr) | - | - | + (r) | +(r ppt) | + (r ppt) | - |
| Galactose | + (r/v) | (y/gr) | - | - | - | +(r ppt) | + (r ppt) | - |
| Glucose | + (r/v) | (y/gr) | - | +(br) | - | +(r ppt) | + (r ppt) | - |
| Sucrose | + (r/v) | (y) | - | - | + (r) | - | - | - |
| Lactose | + (r/v) | (y) | - | - | - | +(r ppt) | - | - |
| Maltose | + (r/v) | (y) | - | - | - | +(r ppt) | - | - |
| Amylose | + (r/v) | (y) | - | - | - | - | - | + (bl) |
| Glycogen | + (r/v) | (y) | - | - | - | - | - | + (br) |
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