QUALITATIVE TESTS FOR CARBOHYDRATES Ben Paolo C. Rabara, Andrea Bernadette G. Reyes, Jhona Marie D. Sana, Suzette Pamela G. Santos, Patricia Marie D. Sigua, and Maria Jo Fatima J. Tabi Group 8 2FPharmacy Pharmaceutical Biochemistry ABSTRACT Each group of carbohydrates has been observed through different qualitative test for carbohydrates that exhibited changes in color based on certain reactions after reacting on specific functional groups in them. Benedict’s test, a test for reducing sugars, resulted in a red precipitate. Barfoed’s test formed rusty color which distinguished reducing monosaccharides from reducing disaccharides, Seliwanoff’s test produced cherry red color due to the condensation in its mechanism, and the Bial’s test gave out blue precipitate due to condensation with Orcinol.
INTRODUCTION Simple sugars are said to be the building blocks of all carbohydrates which are made up of organic compounds that have the approximate formula C(H2O)n, which accounts for the name carbohydrate (or hydrate of carbon) that is usually applied to this group of compounds. But in reality, carbohydrates are polyhydroxyaldehydes (aldoses) and polyhydroxyketones (ketoses). These give the carbohydrates some of their chemical properties.[3] Carbohydrates can be classified by the saccharide units present, namely, monosaccharides, which are considered as the simplest that cannot be hydrolyzed, disaccharides which are composed of two monosaccharide molecules, oligosaccharides which are composed of three to ten monosaccharides and lastly polysaccharides which are composed of more than ten monosaccharides. These criteria used in classifying compounds are very important in determining how they would behave during chemical reactions.[1] Monosaccharides or simple sugars are further classified by the number of carbon atoms they contain. Monosaccharides containing 5 carbons are called pentoses while those with 6 carbon atoms are known as hexoses. Ribose and xylose are common examples of aldopentoses, while glucose and galactose are aldohexoses. Meanwhile, fructose is an example of a ketohexose. Common examples of
disaccharides are sucrose, maltose, and lactose. Polysaccharides can be made up of hundreds of thousands of monosaccharide units connected in various patterns. Starch is one of the most important polysaccharides. A common characteristic of polysaccharides is that they can be hydrolyzed by heating in a slightly acidic solution.[2] These are the most important sources of energy. They are widely distributed in plants and are even found in certain animal tissues such as the liver and muscles. [2] The experiment aims to analyze the sugars, glucose, fructose, xylose, lactose and starch qualitatively. EXPERIMENTAL A. Compounds Tested Five drops each of 0.1M glucose, fructose, xylose, lactose, sucrose, and 1% starch, in five separate test tubes. Benedict’s reagent, Barfoed’s reagent, Seliwanoff’s reagent, Orcinol reagent B. Procedure Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s Orcinol Tests In separate test tubes, 5 drops of the carbohydrate solution (glucose, fructose, xylose, lactose, sucrose, and starch) was mixed
with 1 mL of the required reagent for each test. Different carbohydrate solutions on one test were performed at the same time. All the test tubes were placed into a boiling water bath at the same time and were all removed at once upon the appearance of a visible result for one test. The results and the time it took for the visible result to form in each test were noted. RESULTS AND DISCUSSION 1. Benedict’s Test Reducing sugars are usually detected using Benedict’s reagent which contains copper (II) ions in alkaline solution with sodium citrate added to keep the cupric ions in the solution. The alkaline condition of this test causes isomeric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion to cuprous oxide (Cu2O), a brick-red precipitate, by all monosaccharides and most disaccharides. A mechanism in Benedict’s test is illustrated in Figure 1.
Figure 1. Barfoed’s Test Reaction
2. Barfoed’s Test Barfoed’s solution contains cupric ions in an acidic medium. The milder condition allows only the oxidation of monosaccharides but not of disaccharides. If the time of heating is controlled, disaccharides do not react but reducing monosaccharides give the positive brick-red Cu2O result. Ketoses do not isomerizes with this reagent. Barfoed’s Test’s mechanism is illustrated in Figure 2.
Figure 2. Reaction in Barfoed’s Test
3. Seliwanoff’s Test Seliwanoff’s reagent is resorcinol in 6M HCl. Ketohexoses undergo dehydration to form hydroxymethylfurfural that condenses with resorcinol to form a cherry-red condensation product. Other sugars may produce yellow to faint pink colors. Seliwanoff reaction is illustrated in Figure 3.
Figure 3. Reaction in Seliwanoff’s Test
4. Bial’s Orcinol Test The reagent contains orcinol (5methylresorcinol) in concentrated HCl with a small amount of FeCl3. Pentoses undergo dehydration to form a furfural that condenses with orcinol to create a blue-green solution.Bial’s test mechanism is illustrated in Figure 4.
Figure 4. Bial’s Test Mechanism
Table 1. Results of the conducted qualitative tests Sugar
Benedict’s Test
Glucose
brick red precipitate (+)
Xylose
brick red precipitate (+)
Fructose
brick red precipitate (+)
Lactose
brick red precipitate (+)
Sucrose
blue solution (-)
Starch
Barfoed’s Test brick red precipitate (+) brick red precipitate (+) brick red precipitate (+) blue solution (-) blue solution (-)
Sugar
Seliwanoff’s Test
Glucose
faint pink (-)
Xylose
yellow solution (-)
Fructose
cherry red (+)
Lactose Sucrose Starch
faint pink (-) cherry red (+)
Bial’sOrcinol Test green (-) blue green (+) reddish brown (-) green (-) green (-)
Benedict's reagent is used as a test for the presence of all monosaccharides, and generally also reducing sugars. These include glucose, galactose, mannose, lactose and maltose. Even more generally, Benedict's test will detect the presence of aldehydes (except aromatic ones), and alpha-hydroxy-ketones, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxyketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.[4] Benedict’s test yields a positive result with reducing sugars and is used to distinguish non-reducing and reducing ones. Any carbohydrates that contain aldehydes, αhydroxymethyl ketones, or a hemiacetal are classified as reducing sugars. The Benedict’s reagent consists of CuSO4, NaOH, and tartaric acid. When heated, reducing sugars can be oxidized by the Cu+2 ions of CuSO4. In other words, reducing sugars can reduce the blue Cu+2 ions to brick-red Cu2O. This brick-red precipitate is the positive result for Benedict’s test and is observed only in reducing sugars. All monosaccharides are reducing sugars since they have either an aldehyde or an α-hydroxymethyl ketone. Therefore, glucose, galactose, xylose, and fructose are considered reducing sugars and were tested positive with Benedict’s test. Many disaccharides are also reducing sugars (if they have a hemiacetal). But since sucrose is an acetal, it is not a reducing sugar. It rather gave a negative Benedict’s test even though sucrose consists of glucose and fructose which are both reducing sugars. The reason for this is that the aldehyde of glucose and αhydroxymethyl ketone of fructose are
linked together in a glycosidic bond, thus there is no free reducing group available. Lactose tested positive in Benedict’s test and is therefore a reducing sugar.[5] Barfoed's Test is a chemical test used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brickred precipitate. (Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate. A number of other substances, including sodium chloride, may interfere. Seliwanoff’s test bears a positive result with ketohexoses and is used to distinguish between ketohexoses and aldohexoses. The test reagent consists of resorcinol in 6M HCl. The 6M HCl dehydrates ketohexoses to 5hydroxymethylfurfural which reacts with resorcinol to produce a cherry red condensation product within 2 min. Ketoses are more rapidly dehydrated than aldoses when heated. A positive result is indicated by a cherry-red precipitate within 2 min. Fructose, a ketohexose, was tested positive while xylose, an aldopentose, was tested negative. Aldohexoses react to form the same product but with a faint pink color instead and would do so more slowly. As a result, glucose and galactose were tested negative. 6M HCl hydrolyses sucrose and lactose into their monosaccharide subunits. Sucrose, a disaccharide consisting of fructose and glucose, gave a positive test while lactose, a disaccharide consisting of glucose and galactose, gave a negative test.[6] Bial’s orcinol test shows a positive result for pentoses and is used to differentiate pentoses from hexoses. Bial’s reagent consists of orcinol, conc. HCl, and FeCl3. The conc. HCl dehydrates pentoses to form furfural which further reacts with orcinol and Fe+3 ions to produce a blue-green condensation product. A positive result for Bial’s orcinol test is the immediate formation of a blue-green color. Xylose, a pentose, was tested positive. When dehydrated, hexoses give 5-hydroxymethylfufural which reacts with the reagent to yield colors such as green and reddish brown.
Glucose, galactose, and fructose which are hexoses were tested negative with Bial’s orcinol test suggesting that they are hexoses. Lactose and sucrose which do not even have a pentose subunit were also tested negative.[7]
REFERENCES [1] Seager, S., et. al. (2006). Chemistry for today. Australia: Thomson/Brooks/Cole. [2] Crisostomo, A.C., Daya, M.L., De Guia, R.M., Farrow, F.L., Gabona, M.G., Liu, I.D., Peña, G.T., Peña, L.L., Santiago, L.A., Santiago, M.R., Sarile, A.S., Torres, P.C., Vargas, A.G., Ysrael, M.C. (2010). Laboratory Manual in General Biochemistry. Quezon City: C&E Publishing, Inc. [3]http://www.phoenix.liu.edu./~nmatsun a/che4x/e7chos.pdf [4]http://en.wikipedia.org/wiki/Benedict% 27s_reagent [5]http://jchemed.chem.wisc.edu/jcesoft/ CCA/CCA5/MAIN/1ORGANIC/ORG18/TRAM 18/B/MENU.HTM [6] http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/seli/seli. htm 24 May 2001 [7] http://www.harpercollege.edu/tmps/chm/100/dgodambe/thedisk/carbo/bial/ bials.htm 24 May 2001
