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ISOLATION AND GENERAL TESTS OF POTATO STARCH Joe Renz N. Joya, Patrick Angelo D. Juacalla, Katherine Joy C.

Kalalo, Donna Marie S. Ledesma and Nenet V. Licuanan Group 6 2F-Pharmacy Biochemistry Laboratory ABSTRACT
Starch was settled at the bottom of the container after refrigeration of the resulting mixture of adding 100 mL water to the ground potato sample. The sample solution was then subjected to two (2) general tests for carbohydrates, namely the Molischs and Iodine reaction test. A violet layer formed upon addition of the Molischs reagent to the sample solution, which indicated a positive result or presence of carbohydrate. As for iodine reaction test, a purpleblack complex was observed, indicating presence of starch.

Carbohydrates are known to be the most abundantly found biomolecules in nature, classified as monosaccharides, disaccharides and polysaccharides. They serve as a principle source of energy. The chemical properties of sugars depend on the presence of hydroxyl (OH), aldehyde (CHO), and keto (C=O) groups, thus, those carbohydrates with an aldehyde are called aldoses as those with a keto group are called ketoses. These groups can reduce the ions of certain metals and in turn get oxidized to sugar acids. They may be either reducing or nonreducing in nature. [1] There are some simple and rapid tests that help to qualitatively detect the basic properties of carbohydrates, such as their reducing nature, furfural formation, and their conversion to other specific derivatives. Different saccharides can be easily identified in a biological fluid by studying their properties. Simple carbohydrates are watersoluble, giving a characteristic smell of burnt sugar on dry heating. Their melting points are of little value in the identification as they generally melt over a range of temperature and often decompose on heating. [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. Polysaccharides are carbohydrate polymers consisting of tens to hundreds to several thousand monosaccharide units. All of the common polysaccharides contain glucose as the monosaccharide unit.

Figure 1: Chemical structure of starch Polysaccharides are synthesized by plants, animals, and humans to be stored for food, structural support, or metabolized for energy. In this experiment, starch is the polysaccharide subjected to different tests. Plants store glucose as the polysaccharide starch. The cereal grains (wheat, rice, corn, oats, and barley) as well as tubers such as potatoes are rich in starch. Starch can be separated into two fractions, namely amylose and amylopectin. Natural starches are mixtures of 10 to 20% amylose and 80-90% amylopectin. [6] Amylose forms a colloidal dispersion in hot water whereas amylopectin is completely insoluble. The structure of amylose consists of long polymer chains of glucose units linked by an alpha-acetal. All of the monomer units are alphaD-glucose, and all alpha-acetal links connect carbon no. 1 (C1) of one glucose to carbon no. 4 (C4) of the next glucose. The experiment aims to isolate polysaccharides (specifically starch) from a plant source (potato), perform the general tests for carbohydrates and explain the principles involved in all procedures.

EXPERIMENTAL A. Samples used

For extraction or isolation of starch: potato sample, mortar, pestle, beaker, 100 mL water, cheesecloth. For Molischs test: Molischs reagent, 1 mL starch solution, two (2) mL concentrated H2SO4 solution, test tube. For I2 reaction: 0.01 M I2 solution, 1 mL sample solution, test tube, hot water bath.

After the mixture resulting from addition of one hundred (100) mL water to ground potato was refrigerated, the potato starch was settled at the bottom of the container. The starch obtained can be described as a white substance. Molischs test is a sensitive, general test for all carbohydrates. It detects or confirms the presence of a carbohydrate in a given solution. The principle involved is that carbohydrates form furfural derivatives (pentoses) upon dehydration (or 5-hydroxymethylfurfural for hexoses) by concentrated H2SO4, which form colored compounds when condensed with ethanolic naphthol (Molischs reagent).

B. Procedure 1. Extraction of Starch from Potato The potato sample was placed on a mortar and was ground with the aid of a pestle. The ground sample was then transferred to a small beaker and one hundred (100) mL water was added and mixed. The resulting mixture was then filtered or strained using cheesecloth. The filtrate collected was refrigerated, allowing the starch to settle below the beaker. 2. Molischs Test for Carbohydrates One (1) mL of the sample solution (starch) in a test tube was added with 5 drops of Molischs reagent (5% -naphthol in 95% ethanol). Then, two (2) mL of concentrated H2SO4 was carefully poured down the side of the tube until a layer is formed. 3. I2 Reaction/ Iodine Test One (1) mL of the sample solution in a test tube was added with a few drops of 0.01 M I2 solution. Then, the mixture was warmed in a water bath and any change in color was observed. Subsequently, the mixture was cooled, and any change was also observed.

Figure 3: Violet coloration indicates presence of a carbohydrate in Molischs test In other words, the reagent contains concentrated H2SO4, which hydrolyzes glycosidic bonds present in a polysaccharide to yield monosaccharides, which in the presence of an acid get dehydrated to form a five member ring called furfural and its derivatives. The positive result is a formation of a violet color ring at the junction of two layers. [4] Trioses and tetroses do not give this reaction as they do not possess the necessary minimum 5 carbon atoms for furfural formation [5]. The group was able to observe a positive result for this test.


Figure 2: Isolated starch (white part) from potato after refrigeration.

Figure 4: Reaction involved in Molischs test

Starch is a polysaccharide that can be easily identified by the iodine test. The principle involved is complexation, and the purpose of this test is to distinguish starch and glycogen from the other polysaccharides. The many glucose units in starch trap the iodine (I2) molecules and form a dark blue-black adsorption complex. Monosaccharides and disaccharides are too small and are unable to form a complex with I2. Cellulose, a polysaccharide does not form colored complexes with I2. The partially-hydrolyzed starch, glycogen forms red-brown or brown-blue colored complex. [6]

y [1] Alhuwalia, V.K., Dhingra, S. (2004). Comprehensive Practical Organic Chemistry: Qualitative Analysis. India: Universities Press. [2] Properties of Starch. Retrieved from ok/547/starch.html. 02/23/2012 [3] Dandekar, S.D., Rane S.A. (2005). Practicals and Viva in Medical Biochemistry. New York: Elsevier. [4] Nigam, A., Ayyagari, A. (2007). Lab Manual of Biochemistry, Immunology, and Biotechnology. New Delhi: Tata McGrawHill Publishing Company. [5] Shankara, S. Laboratory Manual for Practical Biochemistry. New Delhi: Jaypee Brothers Medical Publishers. [6] Sharma, R.K., Sangha, S.P.S. (2009). Basic Techniques in Biochemistry and Molecular Biology. I.K International Publishers.

Figure 5: Blue-black complex coloration indicates presence of starch. The amylase component of starch has helical structure and when it is treated with iodine solution, the iodine gets trapped inside the helical structure, giving the solution blue color, indicating the presence of starch. The blue color disappears on heating, because the helical structure of amylase is disrupted and it loses its iodine-binding capacity. The blue color reappears on cooling due to the regaining of helical structure and the iodine-binding capacity is also recovered. As additional information, dextrin solution gives red color with iodine as glycogen solution gives brown color. [5]

Figure 6: Reaction involved in Iodine test