Identification of Unknown Carbohydrates through Standard Sugars by General and Specific Tests Orense, Sharlaine Joi Ann, Palma,

Leo Karl, Pineda, Christelle, Ragodon, Eloisah Vin* Group 8, 3BIO6 Department of Biological Science, University of Santo Tomas, Manila, Philippines

Abstract
Carbohydrates are aldehydes or ketones of higher polyhydric alcohols components that yield derivatives on hydrolysis. Unknown sugars were given to each members of the group for identification. Different tests, general and specific reactions, are performed to each standard sugar solutions: xylose, glucose, fructose, galactose, lactose, maltose and sucrose; likewise to given unknowns. General tests for carbohydrates includes: Molisch test, a general test for carbohydrates; Anthrone test, a test for determining non-reducing sugars and reducing sugars; and Iodine test, which determines the presence of starch. On the other hand, specific reactions for carbohydrates are as follows: Mucic acid test, a test for the the presence of galactose, forming crystals as a positive outcome; Benedicts test, a test for reducing sugars, positive for all standard sugars given except for sucrose; Barfoeds test, test to identify monosaccharide, positive for glucose, xylose, fructose and galactose; Bials Orcinol test, test to distinguish pentoses, positive for xylose; and Seliwanoffs test, a test for the presence of ketone sugars, positive for fructose and sucrose.

Introduction Carbohydrates are naturally occurring gases in plants (where they are produced photosynthetically), animals, and microorganisms that has different roles involving structures and metabolism. Carbohydrates are aldehydes or ketones of higher polyhydric alcohols or components that yield these derivatives on hydrolysis (Prasad, 2010). Carbohydrates is also known as hydrate carbon since it contain carbon, oxygen and hydrogen with the ratio (CH2O)n. One feature of carbohydrate is the presence of large number of functional groups. Carbohydrates are classified according to the number of simple sugar units they comprise.

Monosaccharides are the simplest carbohydrates for they contain a single polyhydroxy aldehyde or ketone. Because of its size, monosaccharides cannot be broken down into simpler units by hydrolysis. They are usually building blocks of disaccharides or polysaccharides for instance glucose, fructose, xylose and galactose. Whereas, disaccharides are water-soluble carbohydrates having two monosaccharide units bonded covalently to each other. Sucrose, table sugar, lactose, milk sugar, and maltose are examples of disaccharides. On the other hand, oligosaccharides are carbohydrates containing two to ten monosaccharide unit attached together by covalent bonds. As for polysaccharides, a polymeric carbohydrate, they contain many monosaccharide units bonded covalently. In the experiment done, the carbohydrates involved were glucose, xylose, fructose, galactose, lactose, maltose and sucrose. As aforementioned, glucose, xylose, fructose and galactose are monosaccharide sugars. Glucose is the most abundant monosaccharide in nature and most important component of human diet. Xylose, is a natural sugar usually found in berries, spinach and pears. It is known to be a great alternative to white sugar. . Fructose, the sweetest-tasting of all sugars, is a biochemically the most important ketohexose (Stoker, 2010). Galactose, a sugar commonly found in lactose or milk sugar, is classified as a monosaccharide, an aldose, a hexose, and a reducing sugar. Meanwhile the disaccharides used in the experiment were lactose, maltose and glucose. Lactose, the main carbohydrate found in milk, is made up of the monosaccharides galactose and lactose joined by a glycosidic linkage. Maltose, commonly known as malt sugar, is formed by two glucose units. Sucrose, common table sugar, is the most abundant of all disaccharides and is produced commercially from the juice of sugar cane and sugar beets (Stoker, 2010). In the experiment, different reactions were completed in order to identify and classify each sugar given. Molisch test indicates the presence of a carbohydrate. Anthrone test,

determines non-reducing sugars and reducing sugars. Iodine test is used to see if starch is present. These tests are general tests for carbohydrates. On the other hand, there are specific reactions for carbohydrates to determine what exact sugar it is. Mucic Acid test is to test the presence of galactose. Benedicts test is a test for reducing sugars. Barfoeds test is test for monosaccharides having a muddy green suspension with brick red precipitate as a positive result. Bials Orcinol test is test for pentoses giving a blue-green solution for xylose, an indication of a positive outcome. Seliwanoffs test is to check for the presence of ketone sugar giving a cherry red solution for sucrose and fructose. This experiment aims to (1) identify an unknown sugar with several standard sugars and (2) accomplish identification of unknown sugars using a number of specific chemical tests.

Methodology

A. Preparation of the solution of the unknown sugar An unknown solid sugar was given to each member of the group, and half of the unknown was dissolved in 5 grams of water and used for the Mucic Acid test whereas the other half, also dissolved, was used for the remaining specific tests.

B. General Tests for Carbohydrates In the Molisch test, ten drops of standard amylose solution, 10 drops of standard glycogen solution and ten drops of standard cellulose solution was placed in their respective test tubes. 2 drops of Molisch reagent was then added, and mixed well. 10 drops of concentrated H2SO4 was further placed running along the side of the test tube. The color formed in the interphase was recorded. In the Anthrone test, ten drops of Anthrone solution was dropped in three wells of the spot plate. One to two drops of the standard amylose solution was dropped on one well, likewise, the standard glycogen solution and the standard cellulose solution was dropped to the other two wells as well. The color formed right after was noted.

In the Iodine test, ten drops of the standard amylose solution, ten drops of standard glycogen solution and ten drops of standard cellulose solution was added to each test tube. A drop of iodine solution was then added to the test tubes. The test tubes were observed right after and the changes were recorded. The test tubes were then subjected to heat in a water bath, and again, the changes produced were noted. The test tubes were then removed from the water bath and then cooled. After standing for minutes to cool, the changes again were recorded.

C. Specific Reactions for Carbohydrates In Mucic Acid test, ten drops of the sugar solutions sugars and the unknowns dissolved that were placed in test HNO3 were then added to each test tube. Cotton plugs subsequently placing each test tube in boiling water bath for noted after letting the test tubes stand for four days. *** were added to the eight standard tubes. Ten drops of concentrated were placed on each test tube one hour. The changes were then

In the Benedicts test, ten drops of Benedicts reagent was added to the five drops of the eight standard sugars and the unknown sugar solutions placed in test tubes with similar sizes. The test tubes were then heated in a boiling water bath until a muddy green suspesion was observed. If no color change happened within five minutes, the test tubes were then removed from the water bath and cooled. The observations seen were recorded. In the Barfoeds test, ten drops of the Barfoeds reagent was added to five drops of the eight standard sugars and the unknown sugar solutions placed in each test tube with same sizes. The test tubes were then placed in a water bath until a brick red precipitate was observed. The time when the brick red precipitate formed was noted. If no color change occurred within five minutes the test tubes were then removed from the water bath, then cooled. The results were then recorded. In the Bials Orcinol test, five drops of the eight standard sugar solutions and the unknown sugar solutions were dropped in their respective test tubes. Ten drops of the Bials Orcinol reagent was then added to each test tube. The test tubes were subjected to boiling water bath until a blue-green solution was observed. The time when the solution turned to blue-green was noted. If no change in color happened within five minutes, the test tubes were removed from the water bath and cooled. The changes were then recorded. In the Seliwanoffs test, ten drops of the Seliwanoffs reagent was added to five drops of the eight standard sugar solutions and unknown sugar solutions. The test tubes were then immersed in boiling water bath. The test tubes were heated until a cherry red solution was observed, if no color change happened after five minutes, the test tubes were removed from the water bath. The time when the test tubes changed appearance were noted and the results were also noted.

Results and Discussion

In a Molisch test, a test done with all carbohydrates, the sugar solutions undergoes dehydration with the use of H2SO4 forming furfural or furfural derivatives which then condensate by the use of -naphthol giving a reddish violet ring. A reddish violet ring at the junction of the two interphases is an indication of the presence of a carbohydrate, specifically a monosaccharide that denotes a positive result. In the experiment done, the test tubes containing standard cellulose solution and standard glycogen solution both gave a red violet to a violet color ring at the interphase. As for the standard amylose solution, a dark yellow green color appeared on the interphase resulting to a negative outcome. In an Anthrone test, by using concentrated H2SO4, carbohydrates are dehydrated forming furfural, a liquid aldehyde, which condenses with anthrone forming a green complex, a positive result for this test. This test both determines reducing and non-reducing sugars. Examples are hexoses, aldopentoses, and hexuronic acids. In the experiment when the standard solutions were subjected to Anthrone test, different hues of green can be observed. In the standard amylose solution, a dark green solution was noted. As for the standard glycogen solution, a green solution was seen and for the standard cellulose solution, a light green one. The three standards gave off positive results. In an Iodine test, the positive result of the formation of deep blue color is due to the starch-iodine complex. When a drop of polysaccharide was acidified with HCL, it was then added to a solution of iodine. When starch undergoes this test, it gives off a blue color whereas if glycogen undergoes this test, it will give off a red color solution. Starches, glycogens and dextrins can be distinguished from other carbohydrates by this test. In the experiment, after dropping iodine to the test tubes, the one with the standard amylose solution had a dark blue upper layer; the one with the standard glycogen solution had a yellow-orange surface; and the one with the standard cellulose solution had a yellowish upper layer. After boiling: a sudden transition from dark blue to blue and formation of bubbles occurred in the test tube with the standard amylose solution; a change from a yellow-orange surface to a light yellow colored solution happened in the standard glycogen solution; and a change from yellowish upper layer to a clear liquid with white particles formed on the bottom of the tube occurred in the standard cellulose solution. After cooling: the one with the standard amylose solution returned to a blueviolet solution; the one with the standard glycogen solution remained as a light yellow solution; and the one with the standard cellulose solution still has a clear liquid solution with white precipitate on the bottom. These results tell that only the standard amylose solution had a positive outcome. For the specific test, the standard sugar solutions used were xylose, fructose, glucose, galactose, lactose, maltose, and sucrose. In the Mucic Acid test, oxidation of galactose and hydrolysis of lactose will yield a dicarboxylic acid, an insoluble mucic acid. The other standard sugar solutions formed acids that are soluble in water; therefore the formation of insoluble crystals indicates the presence of galactose which denotes a positive result. Crystals are produced by the oxidation of the primary functional group and the secondary alcoholic group to carboxylic groups in the presence of

concentrated HNO3. In the experiment, only galactose and lactose had formation of crystals while the other standard sugar solutions had none. As mentioned beforehand, the mucic acid test indicates the presence of galactose, since lactose is a disaccharide of glucose and galactose, the outcome of the experiment done was positive. In the Benedicts test, the cupric ions are reduced to cuprous ions by the enediols formed from sugars in the alkaline medium of the Benedicts reagent. Carbohydrates with free aldehyde or keto group have the capability to reduce various metallic ions. The presence of a green muddy suspension indicates that there are up to 0.5 gm% of reducing sugars in the solution. Likewise, the presence of brick red precipitate has 2 gm% of reducing sugars. On the contrary if the solution remained blue (the color of Benedicts reagent) it indicates the absence of reducing sugars. In the experiment performed, among the given standards, only sucrose remained blue after subjecting the solution to heat. Thus the reaction inferred that glucose, xylose, fructose, galactose, maltose and lactose had the presence of reducing sugars. In the Barfoeds test, because of the acidic property of its medium, monosaccharides enolize much more readily than disaccharides and these enediols reduce cupric ions released by copper acetate of the Barfoeds reagent giving a brick red precipitate. Barfoeds test is a reduction test and its reagent is copper acetate in acetic acid. In this test, the time limit is important to keep for the reason that disaccharides will also respond to the test positively. The time given in this experiment was 5 minutes maximum therefore the disaccharides: sucrose, maltose and lactose had a brick red precipitate at the bottom of their tubes. On the other hand, because of the presence of monosaccharides, glucose after 2 mins. 13 secs. , xylose after 2 mins., galactose after 2 mins. 15 secs., and fructose after 2 mins. And 5 secs., produced brick red precipitates on the most bottom part of the tube. In the Bials Oricnol test, pentoses dehydrate into furfural because of the presence of concentrated HCl in the reagent. The formed furfural then condense with orcinol in the presence of ferric ion to yield a blue-green colored product and confirms the absence of pentose sugars (Kumar, Selvan, & Shanmugam, 2010). In the experiment, only xylose after 1 min. turned to a blue-green solution indicating the absence of pentose sugar forming a blue-green solution. The formation of this color was said to be the positive outcome. The other standard sugars produce a brown colored solution denoting a negative result. In the Seliwanoffs test, ketoses were dehydrated into hydroxyl methyl furfural derivatives with the presence of concentrated HCl. The hydroxyl methyl furfural derivatives react with resorcinol yielding a cherry red color, confirming the absence of keto sugars. In the experiment, fructose, after 1 min. 12 secs. and sucrose, after 1 min. 24 secs., formed a cherry red solution after being subjected to heat. The other standards, even after five minutes didnt change into cherry red solutions, having a much lighter color that those two.

Below are the tables of results of the following tests performed.

Table 1: Results of the General tests for Carbohydrates

General Tests for Carbohydrates Molisch Test Antrhone Test Standard Glycogen Solution Red violet color at interphase Green solution After drop of iodine: Yellow orange surface

Standard Amylose Solution Dark yellow green at interphase Dark green solution After drop of iodine: Dark blue upper layer

Standard Cellulose Solution

Violet color at interphase Light green solution After drop of iodine: Yellowish upper layer

Iodine Test

After boiling: Sudden transition from blue to clear;

After boiling: Clear liquid with white ppt. formed at the bottom

After boiling: Clear Light yellow colored solution

After Cooling: Blue-violet solution

After Cooling: Clear colorless solution with white precipitate

After Cooling: Light yellow solution

Table 2: Results of the Specific Reactions for Carbohydrates

SPECIFIC TESTS Mucic Acid Test Benedicts Test Barfoeds Test Bials Orcinol Test Seliwanoffs Test STANDARDS Glucose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins. 13 secs) Brown solution (5 mins.) Light Orange solution (5 mins.) Xylose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.) Blue-green solution (1 min) Orange solution (5 mins.) Fructose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins. 5 secs) Brown solution (5 mins.) Cherry red solution (1 min. 12 secs) Galactose Crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins. 15 secs) Brown solution (5 mins.) Light orange solution (5 mins.) Lactose Crystals formed Brick red precipitate Clear blue solution (5 mins.) Brown Solution (5 mins.) Orange solution (5 mins.) Maltose No crystals formed Brick red precipitate Clear blue solution (5 mins.) Brown Solution (5 mins.) Pinkish orange solution (5 mins.) Sucrose No crystals formed Blue-green suspension Clear blue solution (5 mins.) Brown Solution (5 mins.) Cherry red solution (1 min. 24 secs)

Table 3: The results of the unknowns given

SPECIFIC TESTS 29 Mucic Acid Test No crystals formed 30 No crystals formed 31 No crystals formed 32 No crystals formed

Benedicts Test

Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.) Blue-green solution (51 secs.) Orange solution (5 mins.)

Brick red precipitate Clear blue solution (5 mins.) Brown solution (5 mins.) Pinkish orange solution (5 mins.)

Blue-green solution

Brick red precipitate Clear blue solution (5 mins.) Brown solution (5 mins.) Pinkish Orange solution (5 mins.)

Barfoeds Test Bials Orcinol Test

Clear blue solution (5 mins.) Brown solution (5 mins.) Cherry red solution (1 min and 2 secs.)

Seliwanoffs Test

Conclusion Identification of unknown sugars is conceivable by them undergoing different tests because of the uniqueness of sugar reactions. The result of the tests done with the standard sugars facilitated the determination of the unknown sugars given.

References

Damodaran, G. (2011). Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd. Kumar, T. S., Selvam, K. P., & Shanmugam, N. S. (2010). Laboratory Handbook on Biochemistry. New Dehli: Asole K. Gosh Patil, U. K., & Muskan, K. (2009). Essentials of Biotechnology. New Delhi: L.K. Publishing House (P) Ltd. Prasad, S.K. (2010). Biochemistry of Carbohydrates. New Delhi: Discovery Publishing House (P) Ltd. Stoker, S. (2010). Exploring general, organic, and biological chemistry. Philippines: ESP Printers Inc. Talwar, G. P., & Srivastava, L. M. (2006). Textbook of Biochemistry 3rd Edition, New Delhi: Asole K. Gosh