ISOLATION, HYDROLYSIS, AND QUANTITATIVE ANALYSIS OF CARBOHYDRATES Group 1 2G-Medical Technology Biochemistry Laboratory
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. Initially, the glycogen from chicken liver is isolated by heating and adding 0.1% acetic acid and then adding 5-10 drops of ethanol. The successful isolation of starch was proven from the Molisch's Test and I 2 Reaction. The sample is also hydrolyzed via acidic hydrolysis. Quantitative analysis of carbohydrates was also performed in this experiment by mixing 12.5 mL of Nelson’s A with 0.5 mL Nelson’s B and transferring the measured amounts of glucose solution to the test tubes. The concentration of the unknown in mg/tube and mg/mL was determined after constructing a glucose standard curve by plotting absorbance readings against concentrations of standard solutions. In this determination, the amount of free reducing sugars in the sample is directly related to the molybdenum blue formed via a series of oxidation/reductions reactions, and is measured colorimetrically.
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.
Polysaccharides and oligosaccharides can be further hydrolyzed to form monosaccharide unit. The enzyme α-amylase can be used to hydrolyze the polysaccharide. This enzyme classified as an endoglycosidase, hydrolyze a glycosidic linkage anywhere along the chain to produce the monosaccharide unit of the polysaccharide.
Fig. 2 (Structure of Glycogen)
Fig. 1 (Structure of Carbohydrates) Monosaccharide is usually the monomers of carbohydrate because it only contains one sugar unit which cannot be hydrolyze. Examples of monosaccharide are glucose, fructose, and galactose. Oligosaccharides are composed of two to ten monosaccharide units. Sucrose, lactose, and maltose are classified as oligosaccharides. Polysaccharides contain more than ten monosaccharide units. Glycogen and starch are examples of polysaccharides.
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.
EXPERIMENTAL A. Compounds tested ( or Samples used )
For Isolation of Glycogen Chicken liver Boiling water 0.1% acetic acid For Acid Hydrolysis of Polysaccharides 5 mL isolate conc. HCL For Quantitative Analysis of Carbohydrate sample Nelson's reagent A Nelson's reagent B Arsenomolybdate reagent Glucose standard Distilled water B. Procedure 1. Extraction of Glycogen from Chicken Liver 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. 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. 2. General Test for Polysaccharides 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 Few drops of Molisch's reagent were added into 1 mL glycogen solution. 2 mL concentrated H 2SO4 was poured down the side of the tube to form a layer. The color at the junction of the two liquids was observed.
B. I2 Reaction Few drops of 0.01 M I2 was added into 1 ml sample solution. The red color indicates the presence of glycogen. The mixture was warmed in a water bath and observed if there was any change in color. The result was then noted. 3. Acid Hydrolysis of Polysaccharides 5 drops of conc. HCl was added to 5 mL of the glycogen solution contained in a test tube. The tube was covered with a cotton boil and was boiled in a water bath for 30 minutes. The viscosity of the solution before and after heating was noted. The solution was then neutralized using dilute NaOH and the hydrolysate was then kept in a refrigerator until it was subjected for Benedict’s Test
4. Quantitative Analysis The Nelson's reagent was prepared by mixing 12.5 mL Nelson's A with 0.5 Nelson's B. 8 test tubes were labeled and measured amounts of standard glucose solution were transferred into the test tubes according to the following protocol. Tube No.
Glucose Distille Unknow Standar d n Sample d Water (mL) (mL) (mL) 1 0 1.0 0 2 0.1 0.9 0 3 0.2 0.8 0 4 0.4 0.6 0 5 0.6 0.4 0 6 0.8 0.2 0 7 1.0 0 0 8 0 0.6 0.4 Fig. 2 (Glucose standard, Distilled water, Unkown sample amounts) 1.0 mL Nelson's reagent was added to each test tube and shaken well. The tubes were heated simultaneously in a boiling water bath for 20 minutes. Then, the tubes were removed and cooled in a beaker of water. 1.0 mL arsenomolybdate reagent was added to each tube and shaken occasionally for 5 minutes or until the Cu2O precipitate dissolves. After that, the absorbance of the standards and unknown were read against a reagent blank at 480 nm. A glucose standard curve was then constructed by plotting absorbance readings against concentrations of standard solutions. The concentration of the unknown was determined in mg/tube and mg/mL.
RESULTS AND DISCUSSIONS The isolated polysaccharide from the chicken liver was glycogen and it was yellow in color and is water soluble. The successful isolation of glycogen was proven by positive results from the following tests which tested the presence of glycogen in the extracted solution - Molisch's test and I2 reaction. The glycogen isolate underwent acid hydrolysis and produced a viscous hydrolysate. Acid hydrolysis is a process in which a protic acid is used to catalyze the cleavage of a chemical bond via a nucleophilic substitution reaction, with the addition of the elements of water. The amount of carbohydrates present in a given sample can be measured by Nelson's method which is based on the capacity of the free reducing groups of sugars in a carbohydrate sample to reduce Cu+2 in an alkaline solution. This method is sensitive and the data obtained are reproducible. The results of the Quantitative Analysis are shown in the figure below.
Fig. 4 (Absorbance reading 1-7) The total volume of the solution was determined by adding the volume of Nelson's reagent, distilled water, and arsenomolybdate reagent in which the volume of each test tube is 3.00 mL. The absorbance of the standards and unknown were able to be measured at 480 nm. As the concentration of glucose increases, the absorbance also increases because the absorbance is directly proportional to the concentration of the standard glucose solution.
REFERENCES From books University of Santo Tomas Faculty of Pharmacy. (2014). Laboratory Manual in Organic Chemistry Revised Edition. Manila: Author From the internet (on-line) [1]Retrieved : May 10 2016
http://oregonstate.edu/instruct/bb450/450ma terial/stryer7/21/figure_21_02.jpg [2]Retrieved : May 10 2016 https://d2gne97vdumgn3.cloudfront.net/api/file/ 0QFD9Ud8RfKt2fdMGkJd
