Pharmaceutical Biochemistry Laboratory PH BIOCHEM (2012 2013) 2B-Ph Group 3 Experiment 3

Isolation, Hydrolysis and Color Reactions of Starch from Potato

Colleen Ann Caragay, Rizzalaine Caringal, Bett Shannen Carpio*, Ancell Julienn Cruz, John Matthew Cruz Department of Pharmacy, Faculty of Pharmacy University of Santo Tomas, Espana Street, Manila 1008
Date Submitted: February 13, 2013

Abstract In this experiment, potato was used as a sample to extract starch. Starch solution was successfully extracted from the potato. Several tests were done to help prove the extraction of starch solution from the potato. Molisch's test, iodine reaction and Benedict's test gave a positive result that indicates the presence of starch. On the other hand, general tests for carbohydrates including Benedict's test, Barfoed's, Seliwanoff's and Bial's - Orcinol tests confirmed that glucose and galactose is an aldohexose, xylose is an aldepentose, fructose is a ketohexose and lactose and sucrose are disaccharides. ______________________________________________________________________ INTRODUCTION Carbohydrates are organic molecules with the general formula of CHO in 1:2:1 ratio. They provide the raw fuel for cellular energy production. Carbohydrates are classified according to the number of molecules they contain. Monosaccharides are simple sugars which cannot be hydrolyzed. On the other hand, oligosaccharides contain 2-10 monosaccharide units, while polysaccharides contain more than 10. Monosaccharides are further classified into the number of carbons that they contain: pentoses (5 carbons) and hexoses (6 carbons). Examples of monosaccharides are glucose, fructose, galactose, deoxyribose and ribose. Oligosaccharides are subdivided into disaccharides (2 monosaccharide subunits), trisaccharides (3 monosaccharide subunits), and so on. Examples of disaccharides are sucrose maltose and lactose. Polysaccharides can be composed of hundreds of thousands of monosaccharide units connected in various patterns. Common examples of polysaccharides are starch, cellulose amd glycogen. Starch, which was isolated in this experiment, is a polymer of glucose, found in roots, rhizomes, seeds, stems, tubers and corms of plants, as microscopic granules having characteristic shapes and sizes. Most animals, including humans, depend on these plant starches for nourishment. The structure of starch is more complex than that of cellulose. This experiment aims to isolate polysaccharides from the plant source, perform general tests for carbohydrates and explain the principles involved in the processes. METHODOLOGY A. Extraction of Starch from Potato The potato was comminuted and grinded using a mortar and pestle. The mixture was transferred in a small beaker, added with 100 mL of water, and mixed. It was then strained using a cheesecloth. The starch was allowed to settle. After the extraction, the general tests and hydrolysis of polysaccharides were performed on the isolate. B. General Tests for Polysaccharides 1. Molischs Test

Molischs reagent is composed of 5% -naphthol in 95% ethanol. A few drops of it was added into a 1 mL starch solution. Down the side of the tube, 2 mL of concentrated H2SO4 was carefully poured to form a layer. The color at the junction of the two liquids was then observed. 2. Iodine Reaction A few drops of 0.01M iodine were added into a 1 mL starch solution. Then the mixture was warmed in a water bath. The change of color was observed. It was cooled and the result was noted. C. Hydrolysis of Polysaccharides 1. Acid Hydrolysis Five drops of concentrated HCl was added to 5 mL of the isolate. It was covered with a marble and boiled in a water bath for 30 minutes. The hydrolysate was kept for Benedicts test. The hydrolysate must be stored in the ref if the test cannot be performed on the same date. D. Qualitative Tests for Carbohydrates 1. Benedicts, Barfoed's, Seliwanoff's and Bial's Orcinol Test. In separate test tubes, five drops of each of the carbohydrate sample solutions 0.1M glucose, xylose, fructose, lactose,sucrose and 1% starch were mixed with 1 mL of Benedict's reagent. (One test on the different carbohydrate solutions must be performed at the same time.) The test tubes were then placed into a boiling water bath and were removed whenever the solutions for one test gave a visible result. The result and the time it took for the visible result to form in each test was noted. The same procedure was applied with Benedicts, Barfoed's, Seliwanoff's and Bial's - Orcinol test. 2. Mucic Acid Test. In the mucic acid test of galactose and lactose, 3 drops of the carbohydrate samples were mixed with 3 drops of concentrated HNO3. The mixture was placed on a glass slide which was passed over a

small flame until almost dry. (Use an alcohol lamp and do not scorch.) It was cooled at room temperature. The crystals were examined under the microscope. If no crystals appeared, let the glass slide stand until the next period. 3. Phenylhydrazone Test The phenylhydrazine reagent was prepared by mixing 2 grams of phenylhydrazine hydrochloride, 3 grams sodium acetate and 10 mL distilled water. The reagent was placed in a warm water bath and stirred until it was clear. In different test tubes, 2 drops of the carbohydrate samples were mixed with 4 drops of freshly prepared phenylhydrazine. It was mixed well and then covered with a cotton. The test tubes were later heated in a boiling water bath for 30 minutes. The time when yellow crystals first appeared were recorded. The test tubes were cooled and the crystals were observed.

RESULTS AND DISCUSSION The isolated starch was a light yellow, turbid solution. The successful extraction of starch was proved by acquiring positive results from the following tests which tested the presence of starch in the extracted solution Molischs test, iodine reaction, and Benedicts test. Table 1. Results of the General Tests for Polysaccharides TEST RESULT Molischs Test purple ring (+) Iodine Reaction red color (+) Molischs test is a general test for carbohydrates. The positive result is a violet interphase which is due to the dehydration of carbohydrate by sulfuric acid to produce an aldehyde, which then condenses with two molecules of phenol (-naphtol) to form a purple colored product. The extracted starch from the potato produced this positive result.

A positive iodine test indicates the presence of starch in a solution. A positive result of iodine test for starch is a violet solution. This is due to the coiling of starch which will trap the iodine ion and iodide to form starchiodine complex (triiodide).

The starch isolate underwent

acid hydrolysis and produced a turbid, viscous solution. Acid hydrolysis is a chemical process in which acid is used to convert cellulose or starch to sugar. It implies a chemical mechanism of hydrolysis catalyzed by a BrnstedLowry or Arrhenius acid. By contrast, it does not usually imply hydrolysis by direct electrophilic attackas may originate from a Lewis acid. After performing the qualitative tests, namely, Benedicts, Barfoeds, Seliwanoffs, and Bials orcinol tests, on glucose, xylose, fructose, lactose, and sucrose, the following results were obtained:
Table 1: Benedicts Test Results Sugar Visible result Fructose Brick red precipitate Lactose Blue solution Glucose Brick red precipitate Xylose Brick red precipitate Sucrose Blue solution Starch Blue solution Benedicts test is used to distinguish non-reducing and reducing ones in carbohydrates.. The Benedicts reagent uses the reagents (cupric sulfate) CuSO 4, NaOH, and tartaric acid. All monosaccharides can reduce the blue +2 cupric ion Cu ions to brick-red Cu2O. The color brick red precipitate is the positive result for Benedicts test. When heated by the mixture in a boiling water, it gives the reaction a higher rate.

Barfoeds is more specific than Benedicts test because it shows visible results. Barfoeds test is used to distinguish whether a carbohydrate is a monosaccharide or a disaccharide. Its reagent consists of (Cupric acetate) Cu(CH3COO)2 and (acetic acid) CH3COOH. All monosaccharides are reducing sugars, +2 they reduce Cu ions to a brick-red Cu2O when heated. A positive result for Barfoeds test is indicated by the formation of this brick-red precipitate within 5 minutes time. All Monosaccharides were tested positive. Ketoses here do not isomerize. Table 3: Seliwanoffs Test Results Sugar Visible result Fructose Cherry red product Lactose Pale yellow solution Glucose Pale yellow solution Xylose Pale yellow solution Sucrose Cherry red product Starch Pale yellow solution Seliwanoffs test is used for ketohexoses. It consists of resorcinol (C6H4(OH)2) in 6M HCl. The concentreated hydrochloric acid allows ketoses to undergo dehydration than aldoses which further condenses forming a cherry red product. Ketose containing compounds can also yield a positive result Frustose and sucrose are ketoses while lactose, glucose, xylose, and galactose are aldoses. Table 4: Bials Orcinol Test Results Sugar Visible result Fructose Turbid yellow solution Clear yello solution Lactose Clear yellow solution Glucose Blue-green solution Xylose Brown solution Sucrose Starch Clear yellow solution Bials orcinol test shows a positive result for pentoses and is used to differentiate pentoses from hexoses. Bials reagent consists of orcinol, conc. (Hydrochloric acid)HCl, and (ferric chloride)FeCl3. Pentoses are allowed to dehydrate yielding to furfural that condenses orcinol and ferric ion forming blue green solution. Mucic acid test is used for galactose. Oxidation of monosaccharides by conc. Nitric acid HNO3 yields soluble dicarboxylic acids. Mucic acids are insoluble with the said oxidizing agent. A positive result for mucic acid test is the formation of an insoluble crystalline precipitate and would indicate the presence of galactose. Lactose and galactose were tested positive with mucic acid test. The phenylhydrazone test detects reducing sugars like monosaccharides and

Table 2: Barfoeds Test Results Sugar Visible result Frustose Brick red precipitate Lactose Blue solution Glucose Brick red precipitate Xylose Brick red precipitate Sucrose Blue solution Starch Blue solution

disaccharides. Phenylhydrazone reagent consists of phenylhydrazine hydrochloride and NaCH3COO. Aldoses and ketoses when they react to phenyl hydrazine, they form crystalline product which is known as the (osazones). SUGAR Phenylhydrazone Crystals of Fructose Phenylhydrazone Crystals of Lactose Phenylhydrazone Crystals of Glucose DESCRIPTION yellow needle shaped badminton ball shaped yellow needle shaped

REFERENCES [1] Bayquen, A.V., Cruz, C.T., de Guia,R. M., Lampa, F.F., Pena, G.T., Sarile,A.S., T orres, P.C. (2009). LaboratoryManual in Organic Chemistry. C&Epublishing, Inc Online sources [2]http://himedialabs.com/TD/HTBC002. pdf [3]http://www.pua.edu.eg/Version2/Cour ses2/Dentistry%20Courses/Freshmen/S pring/BCM101/Practical/Week%202%20 practical%20_Chemistry%20of%20carb ohydrates_.pdf [4]http://www.wisconline.com/Objects/ViewObject.aspx?ID =AP13104 [5]http://www2.chemistry.msu.edu/facult y/reusch/VirtTxtJml/carbhyd.htm