HYDROLYSIS OF POLYSACCHARIDES AND QUALITATIVE TESTS FOR CARBOHYDRATES

John Roel R. Tatad, Goldie Ann T. Tejada, Allain Joseph S. Templo, Kristinlyn C. Trajano, Kevin Christopher A. Viray, Quennie Bien Bien C. Yu Group 8 2F Pharmacy Biochemistry Laboratory

ABSTRACT
In Enzymatic hydrolysis, the solution was not that viscous after it was allowed to stand for 30 minutes. In this experiment, the polysaccharides starch and glycogen are extracted from potato and chicken liver respectively. These are then subjected to hydrolysis and are tested for the presence of reducing sugars. Another set of monosaccharides and disaccharides which include glucose, fructose, xylose, laltose and sucrose were also subjected to qualitative tests. The tests include Benedicts, Barfoeds, Seliwanoffs, Bials tests, mucic acid test and phenylhydrazone test. Visible results are later discussed.

INTRODUCTION
Carbohydrates together with proteins and fats are the bodys source of energy. Among these, carbohydrates are the most important and this act as the bodys primary source of energy. Carbohydrates can be found both in plants tissues as well as in animal tissues. In animals, they are mostly found in liver and in muscles. Carbohydrates that are water soluble often impart a sweet taste, therefore are also called sugars. Carbohydrates are also known as saccharides. Carbohydrates can either have aldehyde or ketone groups. Polyhydroxyaldehydes are known as aldose and polyhydroxyketones are known as ketose. (3) Carbohydrates can be divided further into three general classes according to the number of carbohydrate molecules they have. Monosaccharides are termed for single sugars, oligosaccharides for sugars containing 2-10 monosaccharide units and polysaccharides for sugars containing more than 10 monosaccharide units. (Schreck and Loffredo 1) Monosaccharides are further classified by the number of carbon atoms they contain. Monosaccharides with 5 carbons are termed pentose while the ones having 6 carbons are termed hexose. Examples include ribose, a fivecarbon aldose known as aldopentose; glucose and galactose, six-carbon aldoses known as aldohexose; and fructose, a ketohexose. (2) Oligosaccharides are sugars containing more that 1 sugar. This can be further divided into disaccharides or trisaccharides etc. depending on the number of saccharide units they contain. Examples of disaccharides are sucrose (glucosefructose), maltose (glucose-glucose) and lactose (galactose-glucose). (Schreck and Loffredo 1) Polysaccharides are carbohydrates exceeding 10 monosaccharide units. Examples of these include starch (which was used in the experiment as potato), glycogen (from chicken liver) and cellulose.

Both oligosaccharides and polysaccharides can be hydrolyzed by heating in slightly acidic solution to yield their monosaccharide forms. Monosaccharides are the simplest form of sugars and therefore cannot be hydrolyzed. The different tests made were benedicts test, barfoeds test, seliwanoffs test, bials-orcinol test, mucic acid test and pehylhydrazone test. Benedicts test detects reducing sugars using benedicts reagent which contain copper (II) ions in alkaline solution with sodium citrate added to keep the cupric ions in the solution. It causes isomeric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion to cuprous oxide due to the alkaline condition of this test. Carbohydrate test reagents can be divided into 2 general classes based on the type of reaction involved. The first one involves identifying products of carbohydrate condensations which consists of Bials Orcinol and Seliwanoffs Test. The second class involves reducing sugars which include the Barfoeds and Benedicts Test.

Experimental A. Compounds tested (or samples used)

Isolated carbohydrate from the potato, saliva, benedicts reagent, barfoeds reagent, seliwanoffs reagent, orcinol reagent, concentrated HNO3, phenylhydrazone HCl, CH3COONa, distilled water, 0.1M glucose, fructose, xylose, lactose and sucrose.

B. Procedure 1.) Enzymatic hydrolysis

10mL isolated carbohydrate was placed in a beaker then add 2.3mL of saliva, allow it to stand at room temperature for 30 minutes. Introduce this solution into a dialyzing bag( colloidon solution into a clean and dry hard glass test tube.) and suspend the bag overnight in a small flask with 50mL water. Remove and discard the dialyzing bag and concentrate the solution inside

the flask using an open flame to the volume of 10mL.

lactose

2.) Benedicts barfoeds, seliwanoffs and bials orcinol tests.

In separate test tubes, 5 drops of the carbohydrate solution (glucose, fructose, xylose, lactose and sucrose) were mixed with 1 ml of the required reagent for each test. One test per different carbohydrate solution was performed at the same time. All of the test tubes were place into a boiling water bath. The test tubes were immediately removed after one of the carbohydrates for each test showed visible results. The visible results and order of appearance were noted.

sucrose

ppt Red orange ppt Orange ppt

red ppt 5 no ppt

Yellow soln

soln Brown soln Olive green soln black ppt

2 dark red ppt

Dark red soln

3.) Mucic acid test

Mix 3 drops of the carbohydrate solution (galactose, lactose) and 3 drops concentrated HNO3 on a test tube. Pass the mixture over a small flame until almost dry. Cool at room temperature and examine the crystal under the microscope.

4.) Phenylhydrazone test

Prepare the phenylhydrazone reagent by mizing 2 g phenylhydrazone hydrochloride, 3 g CH3COONa and 10 mL distilled water. Place the reagent in a warm water bath, stir until solution clears. In different test tubes, mix 2 drops carbohydrate solution ( glucose, fructose, xylose, lactose and sucrose) with 4 drops of freshly prepared phenylhydrazone reagent. Mix well and cover the rubes with cotton. Heat in a boiling water for 30 minutes. RESULTS AND DISCUSSION

Figure 1. Dialyzing bag Table 1. Results in benedicts test, barfoeds test, seliwanoffs test and bials test.
glucose fructose benedicts Red ppt Orange ppt barfoeds 4 dark red ppt 1 dark red ppt seliwanoffs Yellow soln Red soln bials Brown soln Olive green soln black ppt Green

Benedicts Test uses a mixture of copper(II) sulfate, sodium citrate, and sodium carbonate in a mildly basic solution. The alkaline condition of this test causes isomeric transformation of ketoses to aldoses resulting in the reduction of blue cupric ion. This reagent is used as a general test for detecting reducing sugars. If the saccharide is a reducing sugar, it will reduce copper (II) ions to copper (I) oxide which will give a red precipitate. (Schreck and Loffredo 4) The test is positive for all monosaccharides and for most disaccharides. In this case, glucose xylose and fructose, all of which are monosaccharides exhibited a brick red precipitate. Maltose also imparted a brick red precipitate and as for sucrose, a disaccharide, a blue green solution was produced. This means that all except sucrose are reducing sugars. Barfoeds Test is a test for reducing sugars that are further classified into monosaccharide or disaccharide sugars. They both exhibit brick red precipitates but in different time frames. Monosaccharides produce the positive brick red precipitate within 2-3 minutes and disaccharides produce the positive precipitate in approximately 10 minutes. Barfoeds Test uses copper (II) ions in slightly acidic medium. (Schreck and Loffredo 4) In the experiment, glucose, fructose and xylose which are monosaccharides exhibited the brick red precipitate within three minutes while maltose and sucrose exhibited a blue solution. The ideal result for this is that disaccharides should have brick red precipitate in about 10 minutes. Seliwanoffs Test is a test that identifies ketose from aldose sugars. When reacted with the Seliwanoff reagent, the ideal result is that ketose would produce brick red or cherry red solution and blue-green solution for aldose sugars. Seliwanoffs reagent is composed of 6M hydrochloric acid as dehydrating agent and resorcinol as condensation agent. (Schreck and Loffredo 4) In the experiment, fructose, which is a ketose produced a brick red precipitate. Glucose and xylose, both aldoses, produced an orange solution and a green solution respectively. Aldoses which impart blue green solutions may further change to peach color, which explains the orange color for glucose.

xylose

Orange

3 dark

Yellow soln

Bials Test is used to distinguish pentose from hexoses. Pentose after mixing with Bials reagent would impart a blue or green condensation product while hexose would give a brown to gray product. Bials Test uses concentrated hydrochloric acid as dehydrating acid and orcinol with a trace of iron (III) chloride is the condensation reagent. (Schreck and Loffredo 3) In the experiment, glucose and fructose, both hexoses yield brown solutions. Xylose, a pentose, exhibited a dark green solution. Figure 5. Bials-orcinol test

Figure 2. Benedicts test results Figure 6. Mucic acid test using galactose as the carbohydrate solution.

Figure 3. Barfoeds test results. Figure 7. Mucic acid test using lactose as the carbohydrate solution.

Figure 4. Seliwanoffs test results

Figure 8. Results in phenylhydrazone test.

Figure 9. Phenylhydrazone test using glucose as carbohydrate solution.

Figure 13. Phenylhydrazone test using sucrose as carbohydrate solution. REFERENCES From books
Crisostomo, A., Daya M., DeGuia R., et al. (2010) Isolation and Characterization of Carbohydrates. Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing House. From internet Schreck, J.O. and William M. Loffredo. Qualitative Testing for Carbohydrates. Chemical Education Resources Inc. http://www.cerlabs.com/experiments/10875404464.pd p 2/28/11 King, M.W. Biochemistry of Carbohydrates - The Medical Biochemistry Page http://themedicalbiochemistrypage.org/carbohydrates. html 2/28/11

Figure 10. Phenylhydrazone test using fructose as carbohydrate solution.

Figure 11. Phenylhydrazone test using xylose as carbohydrate solution.

Figure 12. Phenylhydrazone test using lactose as carbohydrate solution.