General and Specific Tests for Carbohydrates

General and Specific Tests for Selected Carbohydrates Ariana Isabel T. Rebosa, Wulfrano C. Ricafort III, Anndrey Mae M. Role, Marielle D.S. San Miguel* College of Science, University of Santo Tomas, Espana Blvd., Manila Abstract The chemical properties of carbohydrates differ with respect to the presence or absence of aldehyde and ketone groups as well as the number of hydroxyl groups it contains. Molisch’s, Anthrone, and Iodine tests were used to characterize standard solutions of amylose, glycogen, and cellulose. Specific color reaction tests were then used to identify given unknown solutions in comparison with the standard sugars xylose, fructose, glucose, galactose, lactose, maltose, and sucrose.

Introduction Carbohydrates are polyhydroxy aldehydes or ketones or compounds that produced the mentioned functional groups upon hydrolysis. They are also commonly known as sugars. Carbohydrates are very abundant in nature as key nutrients and readily available energy sources in plants and animals. They also function in cell-to-cell recognition, signaling, binding, and some provide structural support to cells like the cellulose in plants. Carbohydrates may be classified according to the number of saccharide units they contain and they may be monosaccharides, oligosaccharides, or polysaccharides. Monosaccharides are the most basic units of carbohydrates. These units could not be split by hydrolysis into simpler sugars. Monosaccharides are categorized based on the functional groups present in them, or based on the number of carbon atoms they have. Based on functional groups, a monosaccharide could be an aldose which has an aldehyde like glucose and glyceraldehyde or a ketose which has a ketone like dihydroxyacetone and fructose. Based on the number of carbon atoms, monosaccharides could be trioses (3), tetroses (4), pentoses (5), hexoses (6), and heptoses (7). Meaawhile, oligosaccharides are composed of 2-10 monosaccharides. They are further classified

into disaccharides, trisaccharides, and tetrasaccharides. Disaccharides have 2 monosaccharide units and could be a reducing or non-reducing sugar depending on the presence of free aldehyde/ketone group. The most common disaccharides are maltose or malt sugar from 2 glucose units, lactose or milk sugar from glucose and galactose, and sucrose or table sugar from glucose and fructose. Polysaccharides, on the other hand, are polymers of monosaccharides with high molecular weights and could be homopolysaccharides which have the same monosaccharides all throughout the molecule or a heteropolysaccharide which is made up of two or more different units. Examples of homopolysaccharides are starch, glycogen, inulin, cellulose, and dextrins while for heteropolysaccharides are hyaluronic acid, chondroitin sulfate, dermatan sulfate, kerato sulfate, and heparin. The chemical properties of the sugars differ depending on the number of hydroxyl groups and the presence or absence of aldehyde and ketone groups. These variations serve as the basis in the different color reaction tests in the experiment which aim to characterize carbohydrates, and to identify an unknown sugar based on the results of the different chemical tests in comparison with the standard sugars. Methodology A. General Tests for Carbohydrates Standard solutions of amylose, glycogen, and cellulose were prepared beforehand. For the Molisch’s test, 10 drops of each standard solution were placed in separate small test tubes. Two drops of Molisch’s reagent were then added to each solution and they were mixed thoroughly. After mixing, 10 drops of concentrated sulfuric acid were added to the test tubes

which were inclined at 450 angles. The color formed at the interphase of each solution was then noted. For the Anthrone test, 5 drops of each standard solution were placed in three separate wells of a spot plate after which 10 drops of Anthrone solution were administered to each well. The color formed per well was noted. For the Iodine test, 10 drops of each standard solution was placed in three separate small test tubes. A drop of Iodine solution was mixed per test tube and the appearance of each solution was noted. The test tubes were heated in a boiling water bath for 3 minutes and the appearances of the solutions during heating were noted as well. The test tubes were then removed from the water bath and were allowed to cool in the rack. The appearances of the solutions after cooling were then noted. B. Specific Reactions of Carbohydrates Four unknown sugars were placed in big, dry, and clean test tubes. Half of the unknown sugars were transferred to separate large test tubes and were kept for the last three tests. The remaining portions of the sugars were dissolved in 5 mL of distilled water each. These solutions were then mixed in a vortex mixer for 5-10 seconds for the sugars that easily dissolved and 1 minute for the ones that are not easily dissolved. These solutions will be the ones used for the specific chemical tests together with standard solutions of xylose, fructose, glucose, galactose, lactose, maltose, and sucrose. In the Mucic acid test, 10 drops per sugar solution was placed in medium-sized test tubes. To these solutions, 10 drops of concentrated HNO 3 were added. The test tubes were plugged

with cotton and heated in a boiling water bath for an hour. The appearances were noted at the next laboratory meeting. In the Benedict’s test, 10 drops of Benedict’s reagent was placed to each of the eleven medium-sized test tubes. After that, 5 drops of the sugar solutions were placed in their respective test tubes. The solutions were heated in a hot water bath until a brick red precipitate forms and they were immediately removed from the beaker. The test tubes were removed from the water bath if no brick red precipitate formed after 5 minutes of heating. The appearances of the solutions were then noted. The remaining halves of the unknowns were dissolved in 5 mL of distilled water each then mixed in the vortex mixer for the same time they were subjected to the vortex mixer before. In the Barfoed’s test, 10 drops of the Barfoed’s reagent was placed to each of the 11 large test tubes. Then, 5 drops of the sugar solutions was added to their corresponding tubes. The solutions were heated in a boiling water bath until a brick red precipitate formed and the test tubes were removed from the water bath. The time for the precipitate to form was noted for each sugar. The test tubes were removed from the water bath if no brick red precipitate formed after 5 minutes of heating. The appearances of the solutions were then noted. In the Bial’s Orcinol test, 5 drops of per sugar solution was placed in separate small test tubes then 10 drops of Bial’s Orcinol reagent was added per solution. The solutions were mixed thoroughly then placed in a boiling water bath until a blue-green solution formed and they were immediately removed from the water bath and the time for the blue-green solution to appear was noted. The test tubes were removed from the water bath if no blue-green solution formed after 5 minutes of heating. The appearances of the solutions were then noted.

In the Seliwanoff’s test, 10 drops of the Seliwanoff’s reagent were placed in medium-sized test tubes then 5 drops of the sugar solutions were placed in their respective test tubes. The solutions were heated in a boiling water bath until a cherry-red solution appeared and they were immediately removed from the water bath and the time for the formation of the cherryred color was noted for each sugar. The appearances of the solutions were then noted. Results and Discussion The general tests can be used to qualitatively characterize carbohydrates. Molisch’s test is a test for the presence of carbohydrates. It operates on the principle that alpha-naphthol present in the Molisch’s reagent reacts with furfural of pentoses or to 5-hydroxymethyl furfural of hexoses or other derivatives of furfural which were formed by the dehydration of sugars using the concentrated sulfuric acid to form a purple interphase. Sulfuric acid serves as the dehydrating agent while the α-naphthol serves as the condensation agent. The amylose, glycogen, and cellulose were all expected to give a positive result to the Molisch’s test however, they gave incorrect results as seen in table 1. Possible sources of error for these are inadequate mixing of the Molisch’s reagent before addition of the concentrated sulfuric acid and/or contamination of the solutions. The next test, Anthrone test, runs on the principle of carbohydrates being hydrolysed first into simple sugars by dilute HCl and is dehydrated to hydroxymethyl furfural which forms a green to blue-green color with anthrone. It is a general test for determining the concentration of sugars in the sample. The glycogen was the only one that remained with a blue colored solution with the anthrone reagent. Amylose and cellulose originally had green colors but after a few minutes the colors disappeared and the solutions became yellow.

The Iodine test is a general test for the presence of amylose component of starch. Amylose has a helical structure and when it is treated with iodine, the iodine gets trapped in the helix giving the solution a blue color. In the experiment, amylose resulted to a blue solution while glycogen and cellulose were negative for the iodine test having only yellow colors. During heating, the blue color of amylose disappeared because its helical structure is disrupted thus losing its ability to bind iodine. After heating then allowing the solution to cool, the blue color was expected to reappear however, in the experiment it did not. This error was possibly a result of not letting the solution to cool sufficiently enough before recording the results. Table 1: Color Reactions of Carbohydrates to General Tests

TEST

SAMPLE Amylose yellow-green

Molisch’s Test

Glycogen

Cellulose dark-green

green interphase interphase

interphase light blue liquid in light yellow

Anthrone Test

yellow solution

light yellow solution

Iodine Test a. appearance before heating b. appearance during heating c. appearance after cooling *ppt=precipitate

solution clear yellow

turbid yellow

blue solution solution colorless solution colorless solution

solution colorless solution

colorless solution colorless solution

with white ppt colorless solution with white ppt

The Mucic acid test is a specific test for galactose and lactose which is a disaccharide of glucose and galactose. This test operates on the principle of oxidation of galactose or the hydrolysis of

the glycosidic linkage between the glucose and galactose of lactose to form insoluble mucic acid crystals. In the experiment, concentrated nitric acid was added. This reagent oxidizes galactose to mucic acid that crystallizes out from the solution after the acid fumes were removed from the solution by the 60-minute water bath and allowing the mucic acid to crystallize overnight at the least. Heating the solutions in a boiling water bath for an hour allowed all the acidic fumes of the concentrated HNO3 to be expelled and also to increase the rate of reaction. Galactose and lactose produced insoluble crystals while the other sugar solutions as well as all the unknowns did not. Meanwhile, the Benedict’s test detects the presence of reducing sugars. The Benedict’s reagent is composed of copper sulphate which provides cupric ions, sodium carbonate that provides an alkaline medium, and sodium citrate that serves as a chelating agent by preventing the precipitation of cupric ions. This test uses the principle of reduction. The reducing sugars reduce cupric hydroxide in basic solutions to a red-coloured cuprous hydroxide due to them having free aldehyde or ketone groups. The boiling water bath increased the rate of reaction between the reactants. All sugars except sucrose and unknown sample 39 had brick red precipitates in red solutions. Sucrose yielded a negative result of a blue-green solution. Unknown sample 39 also produced the same results as sucrose and thus, there is a high possibility that unknown sample 39 is sucrose. Barfoed’s test detects reducing monosaccharides. Its reagent contains copper acetate in dilute acetic acid. The copper ions are used to detect the reducing sugars in an acidic medium provided by the acetic acid. A positive result (compound is monosaccharide) is indicated by formation of brick red precipitates within 5 minutes. If after 5 minutes there is still no precipitate, the compound is a disaccharide. The monosaccharide sugars xylose, fructose, glucose, and galactose produced brick red precipitates after less than 5 minutes as expected while the disaccharides

lactose, maltose, and sucrose did not produce precipitates. Unknown sample 29 produced precipitates in 1 minute and 44 s which is the same for glucose, sample 30 produced precipitates within 4 minutes which did not correspond to any of the standard sugars, sample 31 produced precipitates within 1 minute and it may be xylose, and sample 32 did not produce precipitates. Bial’s-Orcinol test is specific for pentoses. Bial’s reagent contains orcinol, HCl, and FeCl 3. The orcinol in the reagent forms colored condensation products with furfural produced by dehydration of pentoses. A positive result is shown by the appearance of a blue-green color or precipitate within 5 minutes. As expected, xylose which is a pentose was the only sugar that produced the blue-green color. The other standard sugars yielded negative results as seen in table 2. The unknown samples did not produce the blue-green color thus they are all hexoses. However, an error might have possibly occurred in the testing of the unknowns. They might have been overheated in the boiling water bath and the rapid changes were not properly observed. The unknowns would still be subjected to further testing but unknown sample 29 could only be either fructose or glucose, 30 could be maltose, 31 could be xylose, and 32 sucrose. Seliwanoff’s test is a timed color-reaction specific for ketoses. The principle involves the ketoses undergoing dehydration in concentrated HCl medium to yield furfural derivatives more quickly than aldoses. The derivatives, specifically 4-hydroxymethylfurfural react with resorcinol to form cherry-red or deep red colored compound. Pentoses also react but more slowly compared to ketoses. Fructose and sucrose produced the cherry red color within 5 minutes therefore they are ketoses. Lactose yielded the cherry red color in 14 min, 41s while maltose was in 11 min, 44s. Xylose had a moss-green colored solution, glucose, and galactose had red-orange solutions after 15 minutes of heating. Xylose, glucose, galactose, lactose, and maltose are all aldoses. Unknown sample 29 gave a red-orange colored solution therefore it is an aldose and from the previous

tests, it may be narrowed down to only glucose. Sample 30 produced a cherry-red solution in 4 min, 57s, sample 31 in 1 min, 5s, and sample 32 in 1 min, 14 s all of which do not correspond exactly to any of the standard sugars’ reaction time. Samples 31 and 32 are near the time of sucrose. Unknown samples 29 and 31 are both glucose, a reducing monosaccharide and a hexo-aldose. Sample 30 is maltose, a reducing disaccharide and a hexo-aldose. Sample 32 is sucrose, a nonreducing disaccharide and a hexo-ketose. Table 2: Results of Standard Carbohydrate Solutions to Specific Tests STANDARD SOLUTIONS TEST

Fructos Xylose

Glucose Galactose

Lactose

Maltose

Sucrose

e white white ppt colorles

colorles colorless

Mucic acid test

s

ppt in in

s solution

solution

colorles colorless

colorles colorless

solution

s solution

s

solution

solution

Benedict’s test

brick

brick

brick

red ppt

red ppt

red ppt

in light-

in light-

in light-

red

red

red

solution

solution

solution

Barfoed’s test

1

a. Time for

min,8s

1 min,

brick

44s brick

32s reaction b. appearance

brick

solution dark brick red

brick

brick red

ppt in

red ppt

ppt in

light-red

in brick-

light-red

solution

red

solution

bluegreen solution

solution

1 min

brick red

∞

∞

∞

blue-

blue-

blue-

red ppt

red ppt

red ppt

ppt in

green

green

green

in blue-

in blue-

in blue-

blue-

solution

solution

solution

green

green

green

green

solution

solution

solution

solution

46 s

∞

∞

∞

∞

∞

∞

Bial’s Orcinol test a. time for reaction

dark-

very

dark

dark

moss

very

green

brownish-

brownis

green

dark

yellow

h-green

solution

moss

solution

solution

w/ dark

green

brown

green

solution

ppt

ppt

brown

b. appearance

green

solution solution

solution

w/

Seliwanoff’s test

14

11 mins,

1 min,

2 mins, a. time for

∞

∞

∞

mins,

44 s

56 s

17 s reaction b. appearance

moss-

cherry-

red

red

41 s red

red

cherry

green

red

orange

orange

orange

orange

red

solution

solution

solution

solution

solution

solution

solution *ppt=precipitate

Table 2: Results of Specific Tests for Unknown Carbohydrate Solutions TEST Mucic acid test Benedict’s test

29 colorless solution light red solution

UNKNOWN SOLUTIONS 30 31 colorless solution colorless solution brick red solution red solution with

32 colorless solution blue-green solution

with brick red ppt

with brick red ppt

dark brick red ppt

1 min, 44s

4 mins

1 min

∞

blue-green solution

blue-green solution

blue-green solution

blue-green

with brick red ppt

with brick red ppt

with brick red ppt

solution

∞

∞

∞

∞

dark brown

dark moss-green

solution with dark

solution w/ dark

green ppt

green ppt

1 min, 5 s

1 min, 14 s

Barfoed’s test a. Time for reaction b. appearance Bial’s Orcinol test a. time for reaction brown solution b. appearance

with dark brown

dark brown solution

ppt Seliwanoff’s test a. time for

6 mins, 14 s

4 mins, 57 s

reaction cherry red b. appearance

red orange solution

cherry red solution

cherry red solution solution

Conclusion Generally, the presence of carbohydrates can be detected using the Molisch’s test with the appearance of a purple interphase and the sugar concentrations can be determined using the Anthrone test which produces a green to blue-green colored compound. Amylose component of starch ise detected by the iodine test and will yield a blue solution. Using the specific tests, the unknown samples were determined. Unknown samples 29 and 31 are both glucose, a reducing

monosaccharide and a hexo-aldose. Sample 30 is maltose, a reducing disaccharide and a hexoaldose. Sample 32 is sucrose, a non-reducing disaccharide and a hexo-ketose. References Bial’s test

(n.d.).

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http://www.harpercollege.edu/tm-

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