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Investigation of Unknown Carbohydrate

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From the name itself, Carbohydrates are hydrates of carbons that are polar in nature. The building blocks of carbohydrates are monosaccharides which are simple sugars due to their low molecular weight. Carbohydrates are the product of photosynthesis from the condensation of carbon dioxide that requires light and chlorophyll. Carbohydrates have a vital role in the nutrition of organisms since it is the major source of energy. ATP is energy released by plants and it is the needed by the body to function accordingly.

Carbohydrates have different structures thus it gives distinct reactions to various reagents depending on its chemical composition. It can be grouped into monosaccharides, disaccharides and polysaccharides. Monosaccharides could be classified as polyhrdoxy aldoses or ketoses. These are the simplest carbohydrates that cannot be broken down into smaller aggregates. These are aldehydes that contain two or more hydroxyl groups. Disaccharides are two simple sugars that are linked together by a glycosidic bond- an ether bond formed from the merging of two hydroxyl groups between monosaccharides. Polysaccharides, on the other hand, are made up of multiple sugar units attached to a group of disaccharides. They are formed by a glycosidic linkage.


For the identification of the unknown carbohydrates samples, 1.00 ml of two unknown samples were transferred in a test tube and 1.00 ml of Molisch reagents was added as well as 1.00 ml of concentrated . For each of the tests- Iodine test, Benedict's test, Barfoed's test, Seliwanoff's test and 2,4-DNP test, fresh samples were needed for each. Table 1 shows the needed amount of reagent for each test for a qualitative analysis.


The identity of the two unknown samples was then distinguished based on the reaction of the given set of carbohydrates.

For the hydrolysis of starch, 50.00 ml of 5% starch solution was placed in a 100 ml beaker. About 5.00 ml of concentrated sulfuric acid was added. Covering the beaker with aluminum foil, it was then heated until boiled in a water bath. About 1.00 ml of the sample was placed in two separate test tubes with the addition of 1.00 ml of iodine reagent to one and 1.00 ml of Benedict's reagent to the other. The sample was heated continuously. With an interval of 5 minutes, 1.00 ml of the sample was transferred into two separate tees tubes once again and with the addition of the iodine and Benedict's reagent until a blue-black precipitate is formed with the iodine reagent and a brick red color with the Benedict's reagent.



Table 2 shows the desired color change of the carbohydrates upon the addition of certain reagents. Molisch's test is a general test for carbohydrates that determines the presence of carbonyl groups, which gives off a deep purple colored substance. The Iodine test gives off a blue-black colored complex as a positive reaction towards iodine. Benedict's test determines the identity of the reducing sugars which results to an orange-rust color. Barfoed's test has the same purpose as Benedict's test for determining the reducing sugars, but this Barfoed's test gives off a positive test for reducing monossaccharides only. Seliwanoff's test determines the presence of aldoses and ketoses, only the ketoses give off a positive reaction resulting to a brick red color. The 2,4-dinitrophenylhydrazine or the 2.4-DNP test determines the reaction of monosaccharides that gives off yellow- black crystals of osazones that intensifies the color of the substance.



Molisch's test is a general test for carbohydrates. Concentrated sulfuric acid was added producing a deep purple colored substance. The carbohydrate undergoes dehydration wherein water was released upon the addition of sulfuric acid. Pentoses and hexoses react with the sulfuric acid resulting to the positive color change.

For the Iodine test, the only sugar that reacted was starch. Starch is a polysaccharide- a mixture of amylose and amylopectin. An amylose forms a helical structure in water. Iodine could easily penetrate through the helical structure, since monosaccharides and disaccharides aren't too small they do not react with iodine. Upon the penetration of the iodine to the core of the helix, it produces a blue-black colored substance. When heated, the blue color disappears because the helical ring of the amylose is disrupted. Iodine is does not have the capacity to bind itself back to helix. The blue color returns when the starch is cooled. The iodine can now bind back to the helix.

Benedict's test identifies the reducing sugars, the monosaccharides and the disaccharides. This reagent is a weak oxidizing reagent. Cuprous oxide was converted from cuprous hydroxide. The former determines the presence of the reducing sugar.

Seliwanoff's test differentiates ketoses from aldoses. The ketose yields a brick red color upon the addition of heat. Ketose undergoes dehydration when diluted in HCl and heated.

Barfoed's test identifies the reducing sugars as well, but this test is specific only for monosaccharides. Carbohydrates exposed to the Barfoed reagent, a mixture of copper acetate and glacial acetic acid, undergoes reduction. The reducing monosaccharide reduces the cupric ions to cuprous ions in acidic medium (4). The cuprous ions formed in turn, reduce the colorless phosphomolybdic acid to blue phosphomolybdous acid (4). The positive color change for monosaccharides was exhibited by a deep blue color, while the disaccharide exhibited a light blue color.

The 2,4- DNP test is a general test for carbohydrates. This determines the presence of aldehydes and ketones. The aldoses and ketoses are quite similar. The reducing sugars produce a positive test.

The identity of starch could be easily distinguished through the iodine test. When the starch is hydrolyzed it can have a positive result in the Benedict's test. The acetal linkages in starch are hydrolyzed in hot aqueous acid (6).

Benedict's test is a useful test in detecting the sugar concentration in the urine of a patient diagnosed with diabetes mellitus. The color of the precipitate gives an approximate percentage of sugar excreted in the urine (4). The color determines the percentage of sugar present in the urine. If the precipitate is blue, sugar is absent, green if there are 0-0.5% sugar, yellow if 1% sugar, orange if 1.5% sugar, and red if 2% sugar or more.

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