ISOLATION AND CHARACTERIZATION OF CARBOHYDRATES Angelie Cuyugan, Ysaac Lance De Ocampo, D-vona Dexy Diwa, Justine Esguerra, and Jearweine Formaran Group 4 2C Medical Technology Biological Chemistry Laboratory
ABSTRACT Carbohydrates are a common class of simple organic compounds. A carbohydrate is an aldehyde or a ketone that has additional hydroxyl groups. Carbohydrates serve several different biochemical functions such as being fuel for cellular metabolism and several other biochemical functions. In this experiment, Glycogen and starch was extracted from Chicken Liver and Potatoes respectively. Then general tests were administered to both isolates. Hydrolysis was also done on both glycogen and starch. After the said tests were done and recorded, the qualitative tests were done as well. The carbohydrates were subjected to thin layer chromatography and quantitative analysis. The objectives of this experiment are to (i) isolate polysaccharides from plant and animal sources and explain the principle involved, (ii) perform the general tests for carbohydrates and explain the principles involved, (iii) perform thin-layer chromatography on the carbohydrate hydrolysates, (iv) correlate the data obtained from the color tests and thin-layer chromatography of the carbohydrate hydrolysates and identify the monosaccharide present in the polysaccharide sample, (v) perform qualitative tests for carbohydrates based on furfural formation and oxidation, (vi) examine the different osazone and mucic acid crystals microscopically, (vii) classify an unknown carbohydrate based on the results of the different qualitative tests. The results obtained from the tests shows that glucose, fructose, and xylose are simple carbohydrates while lactose, sucrose, and starch are complex carbohydrates.
INTRODUCTION Carbohydrates are classified aldose or ketones. Therefore, they will exhibit chemical properties associated with both alcohols and carbonyl compounds. In the following series of activities, then we will be examining the reactivity of selected monosaccharides, disacchardies, polysaccharides, and other samples as well. In the Benedict’s test a reducing sugar reacts with the Cu2+ ion in the presence of a base. The copper (II) ion is reduced to a redishorange precipitate whereas the aldehyde group is oxidized to the carboxylic acid. The Barfoed’s test’s Copper (II) acetate in acetic acid is not as reactive as the Cu 2+ Benedict’s reagent. Thus, one can distinguish monosaccharides from disaccharides based on how fast the red-orange precipitate forms. Typically, monosaccharides react within 2-3 minutes, whereas disaccharides take longer. The Seliwanoff test is used to distinguish ketoses from aldoses using the aromatic alcohol in the presence of concentrated hydrochloric acid. Bial’s test can be used to distinguish hexoses from pentoses. Pentoses react with orcinol in the presence of FeCl 3 and concentrated HCl to give a characteristic blue-green color. Nonreacting sugars may produce a brown precipitate but the solution usually remains the yellow color of the FeCl3.
Molisch’s test is a sensitive chemical test for all carbohydrates, and some compounds containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde. Thin layer chromatography is a chromatographic technique used to separate the components of a mixture using a thin stationary phase. One of the processes used to determine glucose is the Nelsons method, which is also called Nelson-Somongyi method. Nelsons method is a widely used classical method for quantitative determination of reducing sugars. In summary, the objectives of this experiment are to (i) isolate polysaccharides from plant and animal sources and explain the principle involved, (ii) perform the general tests for carbohydrates and explain the principles involved, (iii) perform thin-layer chromatography on the carbohydrate hydrolysates, (iv) correlate the data obtained from the color tests and thinlayer chromatography of the carbohydrate hydrolysates and identify the monosaccharide present in the polysaccharide sample, (v) perform qualitative tests for carbohydrates based on furfural formation and oxidation, (vi) examine the different osazone and mucic acid crystals microscopically, (vii) classify an unknown carbohydrate based on the results of the different qualitative tests.
Add 3 mL of distilled water into the mixture and transfer the mixture into a beaker. Heat the mixture in a boiling water bath for 30 minutes. Add water to the mixture if necessary to avoid drying. Add 1 mL 0.1% acetic acid to improve the precipitation of proteins. Filter the mixture using cheesecloth and divide the glycogen sample into 4 test tubes. Use the glycogen extracted for the remaining tests. You can also precipitate glycogen using ethanol by adding 5-10 drops of ethanol to 1 mL of glycogen solution. Precipitation is induced by the loss of the water shell of the glycogen molecules.
EXPERIMENTAL A. Compounds Tested The materials used in this experiment are Iodine Solution, Concentrated Hydrochloric acid, Saliva, Acetic acid, Molisch’s reagent (5% alphanaphtol in 95% ethanol), Concentrated Sulfuric acid, Distilled water, Benedict’s reagent, Barfoed’s reagent, Seliwanoff’s reagent, Orcinol reagent, Concentrated Nitric acid, Phenylhydrazine Hydrochloric acid, Sodium Acetate, Glucose Fructose, Xylose, Galactose, Lactose, Sucrose, Acid hydrolysate, Enzymatic hydrolysate, Nbutyl alcohol:Acetic acid:ether:water (9:6:3:1), Dextrin, Maltose, anisaldehyde, ethanol, Nelson’s reagent A, Nelson’s reagent B, Arsenomolybdate reagent, & Glucose standard.
B. Procedure This experiment can be split into 6 parts, namely: (i). Extraction of Glycogen from Chicken Liver (ii). General tests for Polysaccharides (iii). Acid Hydrolysis of Polysaccharides (iv). Qualitative tests for carbohydrates (v). Thin Layer Chromatography (vi). Quantitative analysis of carbohydrates I.
Extraction of Chicken Liver
Glycogen
from
3.00 grams of chicken liver was weighed and placed on a Petri dish. The sample was then minced using a pair of scissors. 12 mL of boiling water is poured onto the minced sample and stirred using a glass rod. The mixture is then transferred into a small beaker and boiled for 2 minutes to let the proteins precipitate. After the proteins have precipitated, pour the mixture into a mortar and grind it thoroughly until no lumps are visible.
II.
General Tests Polysaccharides
for
There are 2 tests that are considered to be part of the general tests for polysaccharides as they are able to test for the presence of carbohydrates. The 2 tests are Molisch’s test and Iodine Reaction test. A. Molisch’s test Add a few drops of Molisch’s reagent into 1 mL of a known carbohydrate solution. Pour 2 mL of concentrated H2SO4 down the side of the tube to form a layer. A positive result would lead to a red or purple colored compound. B. I2 Reaction Add a few drops of 0.01 M I2 into 1 mL of the sample solution. Warm the mixture in a water bath and observe the results. The positive result is a bluish-black solution.
III.
Acid Hydrolysis Polysaccharides
of
Add 5 drops of concentrated HCl to 5 mL of the glycogen isolate. Cover it with a marble and boil in a water bath for 30 minutes and keep it for the qualitative tests. IV.
Qualitative Carbohydrates
test
for
The qualitative tests can be divided into 6 tests. Namely, Benedict’s, Barfoed’s, Seliwanoff’s, Bial’s-Orcinol, Mucic Acid, and Phenylhydrazone test. These tests are capable of differentiating carbohydrates based on their different types such as simplicity, number of saccharides, type of agent, type of side chain, etc. A. Benedict’s Test Prepare a boiling water bath and label eight clean small test tubes. In separate test tubes, add 1 mL of the Benedict’s reagent. Add 5 drops of each carbohydrate to the test tubes with Benedict’s reagent. Mix the samples and place all of them into the water bath at the same time. Note how long it takes for the red precipitate to form. The positive result is a brick red precipitate. B. Barfoed’s Test Prepare a boiling water bath and label eight clean small test tubes. In separate test tubes, add 1 mL Barfoed’s reagent. Add 5 drops of each carbohydrate to the test tubes with Barfoed’s reagent. Mix the samples and place all of them in the water bath at the same time. Note how long it takes for the red precipitate to form. The positive result is a brick red precipitate.
C. Seliwanoff’s Test Prepare a boiling water bath and label eight clean small test tubes. In separate test tubes, add 1 mL of the Seliwanoff’s reagent. Add 5 drops of each carbohydrate to the test tubes with Seliwanoff’s reagent. Mix the samples and place all of them into the water bath at the same time. The positive result is a cherry-red solution. D. Bial’s-Orcinol Test Prepare a boiling water bath and label eight clean small test tubes. In separate test tubes, add 1 mL of the Bial’s-Orcinol reagent. Add 5 drops of each carbohydrate to the test tubes with Bial’s-Orcinol reagent. Mix the samples and place all of them into the water bath at the same time. The positive result is a blue-green solution. E. Mucic Acid Test Mix 3 drops of the carbohydrate solution and 3 drops of concentrated HNO3 on a glass slide. Pass the mixture over a small flame until the mixture is almost dry, cool the mixture at room temperature and examine the crystals under a microscope. F. Phenylhydrazone Test In different test tubes, mix 2 drops of carbohydrate solution with 4 drops of freshly prepared phenyl hydrazine reagent. Mix them well and cover the test tubes with cotton. Heat them in a boiling water bath for 30 minutes and record the time when yellow crystals appear. Cool the tubes and observe the crystals under a microscope.
Sereth, E. (n.d.). Metabolism: Carbohydrates. Retrieved April 21, 2015, from
http://www.fastbleep.com/biolog y-notes/40/116/750 Helmenstine, A. (n.d.). Chemistry of Carbohydrates. Retrieved April 21, 2015, from http://chemistry.about.com/od/biochem istry/a/carbohydrates.htm
Anonymou (n.d.). Thin Layer Chromatography. Retreived April 21, 2015, from http://chemwiki.ucdavis.edu/Refer ence/Lab_Techniques/Thin_Layer_ Chromatography
