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
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; Benedict’s test, a test for reducing sugars, positive for all standard sugars given except for sucrose; Barfoed’s test, test to identify monosaccharide, positive for glucose, xylose, fructose and galactose; Bial’s Orcinol test, test to distinguish pentoses, positive for xylose; and Seliwanoff’s 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.
Glucose is the most abundant monosaccharide in nature and most important component of human diet. monosaccharides cannot be broken down into simpler units by hydrolysis. the carbohydrates involved were glucose. is the most abundant of all disaccharides and is produced commercially from the juice of sugar cane and sugar beets (Stoker. fructose. the sweetest-tasting of all sugars. milk sugar. an aldose. Sucrose.Monosaccharides are the simplest carbohydrates for they contain a single polyhydroxy aldehyde or ketone. a sugar commonly found in lactose or milk sugar. is a natural sugar usually found in berries. Molisch test indicates the presence of a carbohydrate. galactose. xylose. As for polysaccharides. Fructose. maltose and sucrose. fructose. 2010). glucose. . is classified as a monosaccharide. and a reducing sugar. commonly known as malt sugar. Anthrone test. In the experiment done. 2010). a hexose. xylose and galactose. lactose. table sugar. Maltose. lactose. different reactions were completed in order to identify and classify each sugar given. the main carbohydrate found in milk. In the experiment. maltose and glucose. Because of its size. Meanwhile the disaccharides used in the experiment were lactose. Xylose. is made up of the monosaccharides galactose and lactose joined by a glycosidic linkage. spinach and pears. fructose and galactose are monosaccharide sugars. Sucrose. and maltose are examples of disaccharides. oligosaccharides are carbohydrates containing two to ten monosaccharide unit attached together by covalent bonds. As aforementioned. They are usually building blocks of disaccharides or polysaccharides for instance glucose. is a biochemically the most important ketohexose (Stoker. Whereas.
. Lactose. common table sugar. is formed by two glucose units. On the other hand. they contain many monosaccharide units bonded covalently. disaccharides are water-soluble carbohydrates having two monosaccharide units bonded covalently to each other. xylose. a polymeric carbohydrate. Galactose. It is known to be a great alternative to white sugar.
Barfoed’s test is test for monosaccharides having a muddy green suspension with brick red precipitate as a positive result. likewise. 10 drops of standard glycogen solution and ten drops of standard cellulose solution was placed in their respective test tubes. ten drops of Anthrone solution was dropped in three wells of the spot plate. was used for the remaining specific tests.
B. The color formed in the interphase was recorded. In the Anthrone test. there are specific reactions for carbohydrates to determine what exact sugar it is. Bial’s Orcinol test is test for pentoses giving a blue-green solution for xylose. General Tests for Carbohydrates In the Molisch test. On the other hand. Benedict’s test is a test for reducing sugars. Iodine test is used to see if starch is present. ten drops of standard amylose solution.determines non-reducing sugars and reducing sugars. the standard glycogen solution and the standard cellulose solution was dropped to the other two wells as well. and mixed well. 10 drops of concentrated H2SO4 was further placed running along the side of the test tube. also dissolved. Mucic Acid test is to test the presence of galactose. 2 drops of Molisch reagent was then added. 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. Preparation of the solution of the unknown sugar An unknown solid sugar was given to each member of the group. Seliwanoff’s test is to check for the presence of ketone sugar giving a cherry red solution for sucrose and fructose. and half of the unknown was dissolved in 5 grams of water and used for the Mucic Acid test whereas the other half.
A. These tests are general tests for carbohydrates. an indication of a positive outcome. The color formed right after was noted.
. One to two drops of the standard amylose solution was dropped on one well.
The test tubes were observed right after and the changes were recorded. The test tubes were then immersed in boiling water bath. Ten drops of concentrated were placed on each test tube one hour. The time when the test tubes changed appearance were noted and the results were also noted. ten drops of the sugar solutions sugars and the unknowns dissolved that were placed in test HNO3 were then added to each test tube. In the Barfoed’s test. The test tubes were then removed from the water bath and then cooled. If no color change happened within five minutes. the test tubes were then removed from the water bath and cooled. The test tubes were then subjected to heat in a water bath.In the Iodine test. The results were then recorded. If no color change occurred within five minutes the test tubes were then removed from the water bath. the test tubes were removed from the water bath. Specific Reactions for Carbohydrates In Mucic Acid test. The test tubes were subjected to boiling water bath until a blue-green solution was observed. The test tubes were heated until a cherry red solution was observed.
C. The time when the brick red precipitate formed was noted. then cooled. The observations seen were recorded. the test tubes were removed from the water bath and cooled. Cotton plugs subsequently placing each test tube in boiling water bath for noted after letting the test tubes stand for four days. The time when the solution turned to blue-green was noted. In the Bial’s Orcinol test. If no change in color happened within five minutes. *** were added to the eight standard tubes. The test tubes were then placed in a water bath until a brick red precipitate was observed. ten drops of the Seliwanoff’s reagent was added to five drops of the eight standard sugar solutions and unknown sugar solutions. the changes again were recorded. ten drops of standard glycogen solution and ten drops of standard cellulose solution was added to each test tube.
. if no color change happened after five minutes. five drops of the eight standard sugar solutions and the unknown sugar solutions were dropped in their respective test tubes. Ten drops of the Bial’s Orcinol reagent was then added to each test tube. The changes were then recorded. In the Seliwanoff’s test. ten drops of the Barfoed’s reagent was added to five drops of the eight standard sugars and the unknown sugar solutions placed in each test tube with same sizes. ten drops of the standard amylose solution. the changes produced were noted. The test tubes were then heated in a boiling water bath until a muddy green suspesion was observed. The changes were then
In the Benedict’s test. After standing for minutes to cool. A drop of iodine solution was then added to the test tubes. ten drops of Benedict’s reagent was added to the five drops of the eight standard sugars and the unknown sugar solutions placed in test tubes with similar sizes. and again.
a green solution was seen and for the standard cellulose solution. a test done with all carbohydrates. The three standards gave off positive results. and the one with the standard cellulose solution still has a clear liquid solution with white precipitate on the bottom. a positive result for this test. an insoluble mucic acid. the one with the standard amylose solution had a dark blue upper layer. a liquid aldehyde. by using concentrated H2SO4. the one with the standard glycogen solution had a yellow-orange surface. For the specific test. Starches. As for the standard glycogen solution. a dark yellow green color appeared on the interphase resulting to a negative outcome. fructose. lactose. the one with the standard glycogen solution remained as a light yellow solution. In an Anthrone test. In the experiment done. In the Mucic Acid test. The other standard sugar solutions formed acids that are soluble in water. 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 reddish violet ring at the junction of the two interphases is an indication of the presence of a carbohydrate. and sucrose. When a drop of polysaccharide was acidified with HCL. the sugar solutions undergoes dehydration with the use of H2SO4 forming furfural or furfural derivatives which then condensate by the use of α-naphthol giving a reddish violet ring. the standard sugar solutions used were xylose. it was then added to a solution of iodine. In an Iodine test. Crystals are produced by the oxidation of the primary functional group and the secondary alcoholic group to carboxylic groups in the presence of
. a light green one.Results and Discussion
In a Molisch test. the positive result of the formation of deep blue color is due to the starch-iodine complex. In the standard amylose solution. therefore the formation of insoluble crystals indicates the presence of galactose which denotes a positive result. after dropping iodine to the test tubes. 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. which condenses with anthrone forming a green complex. and the one with the standard cellulose solution had a yellowish upper layer. Examples are hexoses. different hues of green can be observed. These results tell that only the standard amylose solution had a positive outcome. carbohydrates are dehydrated forming furfural. This test both determines reducing and non-reducing sugars. glucose. In the experiment. As for the standard amylose solution. the test tubes containing standard cellulose solution and standard glycogen solution both gave a red violet to a violet color ring at the interphase. it will give off a red color solution. a dark green solution was noted. aldopentoses. In the experiment when the standard solutions were subjected to Anthrone test. When starch undergoes this test. it gives off a blue color whereas if glycogen undergoes this test. specifically a monosaccharide that denotes a positive result. and hexuronic acids. maltose. glycogens and dextrins can be distinguished from other carbohydrates by this test. galactose. After cooling: the one with the standard amylose solution returned to a blueviolet solution. oxidation of galactose and hydrolysis of lactose will yield a dicarboxylic acid. a change from a yellow-orange surface to a light yellow colored solution happened in the standard glycogen solution.
because of the acidic property of its medium. even after five minutes didn’t change into cherry red solutions. In the Barfoed’s test. 24 secs. xylose after 2 mins. ketoses were dehydrated into hydroxyl methyl furfural derivatives with the presence of concentrated HCl. 13 secs.. And 5 secs. In the experiment. The presence of a green muddy suspension indicates that there are up to 0. . and sucrose. turned to a blue-green solution indicating the absence of pentose sugar forming a blue-green solution. Barfoed’s test is a reduction test and its reagent is copper acetate in acetic acid. the cupric ions are reduced to cuprous ions by the enediols formed from sugars in the alkaline medium of the Benedict’s reagent. 15 secs. maltose and lactose had a brick red precipitate at the bottom of their tubes. Carbohydrates with free aldehyde or keto group have the capability to reduce various metallic ions. the time limit is important to keep for the reason that disaccharides will also respond to the test positively.
Below are the tables of results of the following tests performed.. only xylose after 1 min.
. The time given in this experiment was 5 minutes maximum therefore the disaccharides: sucrose. formed a cherry red solution after being subjected to heat. since lactose is a disaccharide of glucose and galactose. the mucic acid test indicates the presence of galactose. Selvan. Likewise. In the Bial’s Oricnol test. In the Benedict’s test.. xylose. produced brick red precipitates on the most bottom part of the tube. The other standard sugars produce a brown colored solution denoting a negative result. 2010). Thus the reaction inferred that glucose. maltose and lactose had the presence of reducing sugars. In the experiment. having a much lighter color that those two. In this test. In the experiment performed. 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. 12 secs. In the Seliwanoff’s test. fructose. The other standards. and fructose after 2 mins. galactose after 2 mins. because of the presence of monosaccharides. The hydroxyl methyl furfural derivatives react with resorcinol yielding a cherry red color. the outcome of the experiment done was positive. monosaccharides enolize much more readily than disaccharides and these enediols reduce cupric ions released by copper acetate of the Barfoed’s reagent giving a brick red precipitate. glucose after 2 mins. after 1 min. only sucrose remained blue after subjecting the solution to heat. On the other hand. galactose. only galactose and lactose had formation of crystals while the other standard sugar solutions had none. among the given standards. after 1 min.. On the contrary if the solution remained blue (the color of Benedict’s reagent) it indicates the absence of reducing sugars. fructose.concentrated HNO3. pentoses dehydrate into furfural because of the presence of concentrated HCl in the reagent. In the experiment.5 gm% of reducing sugars in the solution. confirming the absence of keto sugars. The formation of this color was said to be the positive outcome. & Shanmugam. the presence of brick red precipitate has 2 gm% of reducing sugars. As mentioned beforehand.
) Pinkish orange solution (5 mins.) Sucrose No crystals formed Blue-green suspension Clear blue solution (5 mins.) Lactose Crystals formed Brick red precipitate Clear blue solution (5 mins.) Brown Solution (5 mins.) Cherry red solution (1 min.) Orange solution (5 mins.) Brown Solution (5 mins.) Light Orange solution (5 mins.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
After boiling: Sudden transition from blue to clear. 12 secs) Galactose Crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.
After boiling: Clear liquid with white ppt.) Light orange solution (5 mins. 5 secs) Brown solution (5 mins.) Blue-green solution (1 min) Orange solution (5 mins.) Brown Solution (5 mins. 13 secs) Brown solution (5 mins. 15 secs) Brown solution (5 mins.) Maltose No crystals formed Brick red precipitate Clear blue solution (5 mins.) Cherry red solution (1 min.) Xylose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins. 24 secs)
. 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 Benedict’s Test Barfoed’s Test Bial’s Orcinol Test Seliwanoff’s Test STANDARDS Glucose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.) Fructose No crystals formed Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.
) Brown solution (5 mins.
.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
Muddy green suspension with brick red precipitate Brick red precipitate (2 mins.) Cherry red solution (1 min and 2 secs.) Pinkish orange solution (5 mins.)
Brick red precipitate Clear blue solution (5 mins.)
Brick red precipitate Clear blue solution (5 mins.)
Conclusion Identification of unknown sugars is conceivable by them undergoing different tests because of the uniqueness of sugar reactions.) Blue-green solution (51 secs.) Pinkish Orange solution (5 mins.)
Barfoed’s Test Bial’s Orcinol Test
Clear blue solution (5 mins. The result of the tests done with the standard sugars facilitated the determination of the unknown sugars given.) Orange solution (5 mins.) Brown solution (5 mins.) Brown solution (5 mins.
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