Dr. Steinmeier Biochemistry April 27, 2003 Chemical Properties of Carbohydrates Introduction: The identity of carbohydrates can be determined through a series of chemical tests. The Molisch test differentiates between carbohydrates and non-carbohydrates. Carbohydrates react with -napthol in the following way:

The solubility test categorizes carbohydrates based on their solubility in water. Generally, polysaccharides arent as soluble as mono-, di-, or trisaccharides. Benedicts test is used to differentiate reducing and non-reducing sugars through the use of an alkaline reagent, citric acid, and copper. The Barfoeds test utilizes the different rates of reduction between mono- and disaccharides. Monosaccharides will reduce copper in three minutes, but disaccharides take twenty minutes. The Orcinol test (Bials test or Tollens Orcinol test) distinguishes hexoses from pentoses and uronic acids. Orcinol (see below) reacts with pentoses and uronic acids treated with non-oxidizing acids. The presence of ferric chloride intensifies the sensitvity of the reaction.

The aniline test reacts furfural (the product of the reaction of pentoses with nonoxidizing acids) with aniline (see below) to give a color change. Uronic acids do not react.

Xylose will react with benzaldehyde and methanol in the absence of water to give the following precipitant:

Resorcinol (see below) reacts with high concentrations of furfural formed by ketohexoses but not with lower concentrations formed by aldohexoses.

Starch, glycogen, agar, and xylan will react with iodine to give complexes of varying colors.

For a quantitative determination of carbohydrates, enzyme reagents are often used. A standard curve can be created based on the absorbance of known concentrations of the substrate. From the absorbance of the unknown solution, the concentration can be determined using the standard curve. The enzyme glucose oxidase reacts with D-glucose to give D-gluconic acid and H2O2.

The enzyme horseradish peroxidase reacts with H2O2 and o-dianiside (see below) to give a chromophoric product.

o-dianisidine

If percent transmittance is read from the spectrometer, absorbance can be determined by the equation: Absorbance = 2 log(% Transmittance). Procedure: A. Five to ten mg of the unknown were placed in a test tube containing 1 mL of

water and 2 drops of -napthol. After good mixing, 3 mL of concentrated sulfuric acid were poured down the side to form a layer at the bottom of the tube. A pink or violet color developed at the interface. Twenty mg of the unknown were added to 2 mL of cold water and mixed until dissolution.

Several mg of unknown were added to a test tube containing 2.5 mL of Benedicts reagent. The tube was heated for a few minutes in boiling water bath until color developed. One mg of unknown was added to a test tube containing 2.5 mL of Barfoeds reagent, and the tube was placed in a boiling water bath. Color appeared in 3 to 5 minutes. Three mL of Bials reagent were added to a test tube containing 1 mg of unknown. The tube was heated in a boiling water bath until a blue precipitate was formed. One mg of unknown is added to a test tube containing 2 mL of water, 2 mL of glacial acetic acid, and 5 drops of aniline. The test tube was heated to boiling, and then allowed to stand. A bright red color was formed. After a negative benzahydrazine test and a positive cadmium-brom-xylonate test, 200 mg of unknown were added to a test tube containing 6 mL of absolute methanol, 1 mL 2.4 M HCl in absolute methanol, and 2 mL of redistilled benzaldehyde. The tube was mixed thoroughly and stoppered. After one day, the side of the tube was scratched, and after one week, a precipitate formed. B. The following standard solutions of glucose were prepared:

Another tube was made using 1 mL of unknown glucose solution. The enzyme reagent that was used was composed as follows: 30 mg of glucose oxidase, 3 mg of horseradish peroxidase, 10 mg of orthodianiside hydrochloride dissolved in 100 mL of Trisphosphate-glycerol buffer, 400 mL of glycerol, and water was added to make the volume 1 L. Two mL of enzyme reagent was added to each of the 11 tubes. The test tubes were placed in a water bath at 37C for thirty minutes. Four mL of 5 M HCl were then added to each of the tubes. The percent transmittance and absorbance values were obtained for each of the solutions. A standard curve was prepared of absorbance versus concentration, and the concentration of the unknown was determined from the graph. Data: Test Molisch Solubility Benedicts Barfoeds Orcinol Aniline Test Benzhydazine Cadmium-brom-Xylonate Benzaldehyde Methanolic-HCl Result Positive Very Soluble Positive