EXPERIMENT NO. 4: ISOLATIONAND CHARACTERIZATION OF CARBOHYDRATES

ABSTRACT

Carbohydrates are the most abundant class of organic compounds found in living organisms. It is defined as any of a group of organic compounds that includes sugars, starches, celluloses, and gums and serves as a major energy source in the diet. The objective of this experiment is to isolate the polysaccharide glycogen from chicken liver and explain the principle involved in it and in the general tests done to determine the polysaccharide content of the sample. Some of the other goals of the experiment are to prepare a dialyzing bag, to perform TLC properly, to microscopically examine the different osazone and mucic acid crystals, and to classify unknown carbohydrates. Initially, the glycogen from chicken liver is isolated by heating and adding 0.1% acetic acid and then adding 5-10 drops of ethanol. It then undergoes the general tests for polysaccharides, including Molisch’s Test which uses 5% α – naphthol in 95% ethanol ( + : blue-violet colored ring) and I2 Reaction involving 0.01 M I2 ( + : bluish purple). The sample is also hydrolysed via acidic and enzymatic hydrolysis, after which it undergoes the qualitative tests for carbohydrates, namely Benedict’s Test ( + : brick-red precipitate), Barfoed’s Test ( + : brick-red precipitate), Seliwanoff’S Test ( + : yellow to faint pink solution), Bial’s-Orcinol Test ( + : blue-green solution), Muric Acid Test, and Phenylhydrazone Test. TLC or thin layer chromatography is then performed, followed by a quantitative analysis of the samples using Nelson’s method of measurement.

INTRODUCTION

Carbohydrates, also known as saccharides, are carbon compounds that contain large quantities of hydroxyl groups, and are also the most important sources of energy. They have the basic general formula Cn(H2O)n and they are the most commonly found organic compounds in living organisms. They are classified into several groups, namely monosaccharides, disaccharides, and polysaccharides, depending on the number of their monosaccharide units.

Monosaccharides are further divided with regards to the number of carbons they have – pentoses and hexoses. Pentoses contain five carbon atoms while hexoses contain six carbon atoms. They can also be classified as aldoses or ketoses. Aldoses contain one aldehyde group while ketoses contain one ketone group within the molecule.

An oligosaccharide’s monosaccharide units, on the other hand, range from two to ten, all linked by glycosidic bonds (a covalent bond which binds between the hemiacetal group of a saccharide). It is different from polysaccharides because it contains multiple but few carbon atoms, whereas polysaccharides may contain up to hundreds of monosaccharide units. Oligosaccharides and polysaccharides are similar, however, in the fact that both of them can be hydrolysed by heating in a slightly acidic solution.

Glycogen, the major glucose storage polymer in animals, has a highly branched structure which permits rapid release of glucose from glycogen stores, e.g., in muscle cells during exercise. The ability to rapidly mobilize glucose is more essential to animals than to plants. Glycogen is a very compact structure that results from the coiling of the polymer chains. This compactness allows large amounts of carbon energy to be stored in a small volume, with little effect on cellular osmolarity. In this experiment, glycogen was isolated from the chicken liver via precipitation.

Chicken liver is used in this experiment because it is a good source to isolate glycogen from. Since glycogen is

used in movement of body structures, several other good sources from which it may be isolated are muscle tissues, beef or pork liver.

The isolation of glycogen from the chicken liver is attained by using the mechanism of precipitation. By mincing, grinding, and boiling the liver, separation of the proteins from the glycogen found in the sample is elicited.

The carbohydrate tests used in this experiment can be divided into two classifications based on the mechanism of action which takes place and on the reagents used in the tests. The first involves the use of dehydrating acids followed by condensation reagents. This is called the two-step analysis, which often than not yield highly coloured results. The second classification is that which makes use of copper (II) ion-containing reagents. The copper (II) ions are reduced to cuprous oxide – copper (I) oxide – by the carbohydrates present in the samples.

The Molisch’s Test shows positive results (purple interface) for all carbohydrates, with monosaccharides reacting much faster than disaccharides and polysaccharides. The Iodine Test, on the other hand, is used to identify glycogen and starch. Polysaccharides combine with iodine to form a positive result (a blue-black colour).

Acid hydrolysis is performed on the isolate using concentrated hydrochloric acid while enzymatic hydrolysis is done using saliva and is to be stored in a dialyzing bag.

The qualitative tests for carbohydrates which were performed for this experiment include Benedict’s Test, Barfoed’s Test, Seliwanoff’s Test, Bial’s-Orcinol Test, most of which yield highly coloured and visible results, and Mucic Acid Test, and Phenylhydrazone Test, the products of which are to be microscopically examined and tested for solubility.

The Benedict's test allows us to detect the presence of reducing sugars or sugars with a free aldehyde or ketone group. Barfoed’s Test also detects the presence

of reducing sugars. Seliwanoff’s Test is used to detect ketoses – sugars containing one ketone group per molecule – while Bial’s-Orcinol Test is used to detect pentoses. Mucic Acid Test, named after the mucic acid, dicarboxylic, or galactaric acid it produces after the oxidation reaction takes place, is specifically useful in identifying galactose. Lastly, Phenylhydrazone Test differentiates reducing sugars via microscopic examination of the phenylhydrazones (osazones) formed from the reactions with phenylhydrazine and solubility in hot water.

EXPERIMENTALA. Compounds Tested (or Samples Used)

For Isolation of GlycogenChicken liverBoiling water0.1% acetic acid

For Glycogen Precipitation by EthanolEthanol

For General Tests for Carbohydrates5% α – naphthol in 95% ethanolConc. H2SO4

0.01 M I2

For Hydrolysis of PolysaccharidesConc. HClSaliva

For Qualitative Tests for CarbohydratesBenedict’s reagentBarfoed’s reagentSeliwanoff’s reagentOrcinol reagentConc. HNO3

Phenylhydrazine HClCH3COONaDistilled water0.1 M glucose0.1 M fructose0.1 M xylose0.1 M galactose0.1 M lactose0.1 M sucrose1% starch

B. ProcedureFor Isolation of Glycogen

Weigh 3 g of chicken liver using an analytical balance and place on a Petri dish. Cut the sample with the use of scissors. Transfer sample to a beaker and pour 12 mL of distilled boiling water. Stir contents with a glass rod and boil for two minutes using a hot plate to precipitate the proteins in the sample. Pour the mixture into a mortar and use pestle to grind the sample thoroughly until no lumps are visible. Add 3 mL distilled water and transfer the mixture into a beaker. Heat the mixture in a boiling water bath for thirty minutes and, if necessary, add distilled water to the mixture to avoid evaporation since glycogen goes to the solution when heating. Filter solution using filter paper and separate

glycogen samples into four portions and transfer to test tubes.

*0.1% acetic acid may be added to improve precipitation of proteins during heating of sample in boiling water bath.

For Glycogen Precipitation by EthanolAdd five to ten drops of ethanol to 1 mL glycogen

solution and observe precipitation.

For General Tests for PolysaccharidesMOLISCH’S TESTAdd a few drops of Molisch’s Reagent into 1 mL

glycogen solution. Carefully pour 2 mL conc. H2SO4

down the side of a tube using a glass rod to form a purple interface.

I2 REACTIONAdd a few drops of 0.01 M I2 into the sample

solution measuring 1 mL. A red colour produced indicates the presence of glycogen. Warm the mixture in a water bath and observe if there is any change in colour. Cool and note the result.

For Preparation of a Dialyzing BagPour collodion solution into a clean and dry hard

glass (ignition) tube. With the tube in a horizontal position, completely and carefully coat its inside by slowly rotating it while pouring off the excess collodion solution back into its container. Suspend the ignition tube so the inner coating of collodion solution will dry. When dried, loosen the coat from inside and slowly peel of the membrane.

For Hydrolysis of PolysaccharidesACID HYDROLYSISAdd 5 drops conc. HCl into 5 mL of the isolate.

Cover the test tube with a marble and boil it in water bath for thirty minutes. Keep the hydrolysate for Benedict’s Test.

*Store the hydrolysate in a refrigerator if the test cannot be performed on the same day.

ENZYMATIC HYDROLYSISPlace 10 mL isolated carbohydrate in a beaker.

Add 2.3 mL saliva. Allow it to stand at room temperature for about thirty minutes and take note of any changes in the hydrolysate’s viscosity. Pour the solution into a dialyzing bag and suspend the bag overnight in a small flask or beaker with 50 mL distilled water. Remove and discard the dialyzing bag. Concentrate the solution inside the flask using an open flame to the volume of 10 mL. Test for the presence of reducing sugar in the hydrolysate by performing Benedict’s Test.

For Qualitative Tests for CarbohydratesBENEDICT’S, BERFOED’S, SELIWANOFF’S, AND BIAL’S TESTSIn separate test tubes, mix 5 drops of the 0.1 M

carbohydrate solutions (glucose, fructose, xylose, lactose, sucrose, and starch) and 1 mL of the required reagent for each test. Perform one test on the different carbohydrate solutions at the same time. Place all the test tubes at the same time into a boiling water bath. Remove the tubes from the water bath when solutions for one test give visible colour results. Note the result and the time it took for the visible result to form for each test.

MURIC ACID TEST

Mix 3 drops of the carbohydrate solution (galactose, lactose) and 3 drops of HNO3 on a glass slide. Pass the mixture over a small flame from an alcohol lamp until it is almost dry. Cool at room temperature. Examine the crystals under the microscope and draw the mucic acid crystals. If no crystals appear, let the glass slide stand until the next period.

PHENYLHYDRAZONE TESTPrepare the phenylhydrazine reagent by mixing 2

g phenylhydrazine hydrochloride, 3 g CH3COONa, and 10 mL distilled water. Place reagent in a warm water bath. Stir until solution clears. In different test tubes, mix 2 drops of carbohydrate solution (glucose, fructose, xylose, lactose, sucrose, and starch) with 4 drops of freshly prepared phenylhydrazine reagent. Mix well and cover the tubes with cotton. Heat in a boiling water bath for 30 minutes and record the time when yellow crystals first appear. Cool the tubes and observe the crystals under the microscope. Draw the different phenylhydrazones (osazones).

RESULTS AND DISCUSSIONSTable 1. Results for General Tests for Polysaccharides

The glycogen elicited a positive result upon the addition of Molisch’s Reagent and conc. sulfuric acid because the Molisch’s Test is a test for carbohydrates. It also produced a positive result for I2 Reaction because it will react with glycogen found in a polysaccharide or in a solution.

Table 2. Results for Qualitative Tests for Carbohydrates

Carbohydrate Solution

Visible Results

Ben

ed

ict’

s

Barf

oed

’s

Seliw

an

off

’s

Bia

l’s

Glucose (+) (+) ( – ) (+)Fructose (+) (+) (+) ( – )Xylose (+) (+) (+) (+)Lactose (+) ( – ) (+) ( – )Sucrose ( – ) ( – ) (+) ( – )Starch ( – ) ( – ) (+) ( – )

Glycogen Hydrolysate

( – ) ( – ) ( – ) (+)

Benedict’s Test is a test to detect the presence of reducing sugars. The reagent used contains copper (II) ions in alkaline solution with sodium citrate to keep the cupric ions in the solution. The alkalinity of the solution causes ketoses to form isomers and become aldoses,

reducing the cupric ions to cuprous oxide. Barfoed’s Test shows positive for reducing monosaccharides. Its mechanism of action relies on the mildness of the condition of the environment which elicits the oxidation of the sugars. Seliwanoff’s Test is used to distinguish aldoses from ketoses. This is because ketoses undergo dehydration to form hydroxymethylfurfural which forms a cherry red, pale pink or yellow condensate when reacted with the resorcinol from the reagent. Lastly, Bial’s Test detects the presence of pentoses found within the sample. Pentoses dehydrate to form furfural which condenses with orcinol to form a blue-green solution.

Figure 1. Positive Result for Molisch’s Test and Iodide Test Respectively

The purple interface is shown as the product of the Molisch Test, a test for carbohydrates. A deep red colour, indicating a positive reaction, is meanwhile seen as the product of the Iodide test which detects the presence of glycogen.

Figure 2. Positive Result for Benedict’s, Barfoed’s, Seliwanoff’s, and Bial’s Tests Respectively

Figure 3. Microscopic Observations of the Osazones of Sucrose, Xylose, Fructose, Lactose, Starch, and Glucose Respectively

Test Results

Molisch’s Test (+) Purple interface

I2 Reaction (+) Deep red colour

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