Chemical Tests for Biomolecules

Chemical Tests for Biomolecules Kaneshanathan Kumaraguru Contents (jump to) Introduction Objective Materials Methodology Results Discussion Conclusion References Introduction A macromolecule is â€Å"a molecule of high relative molecular mass, the structure of which essentially comprises the multiple repetitions of units derived, actually or conceptually, from molecules of low relative molecular mass† (Jenkins et al., 1996, p.2289). Carbohydrates are made of monosaccharides, which typically consist 5 or 6 carbon straight saturated chain (Bochkov, Zaikov and Afanasiev, 1991, p.2). Proteins are made up of one or more polypeptides which consist of chains of amino acids connected by peptide bonds (Walsh, 2004, p.2). Objective To identify the macromolecules (carbohydrates and proteins) by using various chemical assays. Materials Samples – Glucose, lactose, fructose, starch, sucrose, tyrosin, tryptophan and egg albumin. Reagents – Molish’s reagent, iodine solution, benedict’s solution, barfoed’s reagent, seliwanoff’s reagent, ninhydrin solution and millon’s reagent. Other chemicals – Concentrated H ­2SO4, AgNO3, dilute NaOH, dilute NH4OH, concentrated HNO3, NaNO2/dilute HCl and sulpanilic acid. Equipments – Bunsen burner, pipett, beakers and test tubes. Methodology Tests for carbohydrates (glucose, fructose, lactose, sucrose and starch). Molisch’s test Using a transfer pipette, 1ml of each carbohydrate solution was poured into five test tubes. Then few drops of Molisch’s reagent was added into each test tube using a transfer pipette and mixed well. Then 2ml of concentrated H2SO4 was added down the sides of the test tubes. Iodine test Using a transfer pipette, 1ml of each carbohydrate solution was poured into five test tubes. Then 3 drops of diluted I2 was added into each test tube using a transfer pipette. Benadict’s test Using a transfer pipette, 5ml of Benadict’s solution was poured into five test tubes. Then 1ml of each carbohydrate solution was added into each test tube using a transfer pipette and was mixed well. Test tubes were then heated in a water bath for 3 minutes. Barfoerd’s test Using a transfer pipette, 1ml of each carbohydrate solution was poured into five test tubes. Then 5ml of Barfoerd’s solution was added into each test tube using a transfer pipette and was mixed well. Test tubes were then heated in a water bath for 3-4 minutes. Seliwanoff’s test Using a transfer pipette, 5ml of Seliwanoff’s reagent was poured into five test tubes. Then 5-6 drops of each carbohydrate solution was added into each test tube using a transfer pipette and was mixed. Test tubes were then heated in a water bath for exactly 30 seconds. Tollen’s test Using a transfer pipette, 1ml of AgNO3 was poured into five test tubes. Then using a transfer pipette, dilute NaOH was added until a slight precipitate was formed. Then dilute NH4OH was added until the precipitate just dissolved. 1ml of each carbohydrate solution was then added into each test tube using a transfer pipette. Test tubes were then heated in a water bath for 5 minutes. Tests for amino acids (tyrosine and tryptophan) and protein (egg albumin). Ninhydrin test Using a transfer pipette, 0.5ml of 0.02% amino acid solutions and protein was poured into three test tubes. Then 1ml of Ninhydrin solution was added into each test tube using a transfer pipette and was heated in a water bath for 3-4 minutes. Xanthoproteic test Using a transfer pipette, 2ml of 0.02% amino acid solutions and protein was poured into three test tubes. Then 2ml of concentrated HNO3 was added into each test tube using a transfer pipette and was heated in a water bath for 1-2 minutes. Millon’s test Using a transfer pipette, 2ml of 0.02% amino acid solutions and protein was poured into three test tubes. Then 3-4 drops of millon’s reagent was added into each test tube using a transfer pipette and was heated in a water bath for 3-4 minutes. Pauly’s test Using a transfer pipette, 1ml of 0.02% amino acid solutions and protein was poured into three test tubes. Then 1ml of sulphanilic acid was added into each test tube using a transfer pipette. 1ml of dilute HCl was then added into three separate test tubes. All six test tubes were kept in ice bath for 3 minutes. Then the amino acids / protein solutions were mixed with 1ml of dilute HCl in the test tubes and were kept in the ice bath again. Few drops of NaOH were then added to the test tubes in the ice bath. Results For carbohydrates Test Compounds Observation Inference Molisch’s test Starch Presence of purple colour ring The compound is a carbohydrate Glucose Presence of purple colour ring The compound is a carbohydrate Fructose Presence of purple colour ring The compound is a carbohydrate Lactose Presence of purple colour ring The compound is a carbohydrate Sucrose Presence of purple colour ring The compound is a carbohydrate Iodine test Starch Presence of blue-black complex The compound is a polysaccharide Glucose Absence of blue-black complex The compound is not a polysaccharide Fructose Absence of blue-black complex The compound is not a polysaccharide Lactose Absence of blue-black complex The compound is not a polysaccharide Sucrose Absence of blue-black complex The compound is not a polysaccharide Benadict’s test Starch Absence of brick-red colour precipitate The compound is a non- reducing sugar Glucose Presence of brick-red colour precipitate The compound is a reducing sugar Fructose Presence of brick-red colour precipitate The compound is a reducing sugar Lactose Presence of brick-red colour precipitate The compound is a reducing sugar Sucrose Absence of brick-red colour precipitate The compound is a non- reducing sugar Barfoerd’s test Starch Absence of red colour precipitate The compound is not a monosaccharide Glucose Presence of red colour precipitate The compound is a monosaccharide Fructose Presence of red colour precipitate The compound is a monosaccharide Lactose Absence of red colour precipitate The compound is not a monosaccharide Sucrose Absence of red colour precipitate The compound is not a monosaccharide Seliwanoff’s test Starch Absence of red colour complex The compound contains an aldehyde group Glucose Absence of red colour complex The compound contains an aldehyde group Fructose Presence of red colour complex The compound contains a ketone group Lactose Absence of red colour complex The compound contains an aldehyde group Sucrose Presence of red colour complex The compound contains a ketone group Tollen’s test Starch Absence of silver mirror The compound is a non- reducing sugar Glucose Presence of silver mirror The compound is a reducing sugar Fructose Presence of silver mirror The compound is a non- reducing sugar Lactose Presence of silver mirror The compound is a reducing sugar Sucrose Absence of silver mirror The compound is a non- reducing sugar For amino acids and protein Test Compounds Observation Inference Ninhydrin test Egg albumin Absence of purple colour complex The compound is not an amino acid Tryptophan Presence of purple colour complex The compound is an amino acid Tyrosin Presence of purple colour complex The compound is an amino acid Xanthoproteic test Egg albumin Absence of yellow colour complex The compound is a protein Tryptophan Presence of bright yellow colour complex The compound is an amino acid Tyrosin Presence of pale yellow colour complex The compound is an amino acid Millon’s test Egg albumin Absence of pink colour precipitate Tryptophan Absence of pink colour precipitate Presence of tyrosin Tyrosin Presence of pink colour precipitate Pauly’s test Egg albumin Absence of red azo dye Tryptophan Presence of red azo dye Presence of tryptophan/ tyrosin/ histodine Tyrosin Presence of red azo dye Figure 1: Molisch’s test: Figure 2: Iodine test: Figure 3: Iodine test: Presence of purple colour ring Presence of blue-black complex Absence of blue-black complex Figure 4: Benedict’s test: Figure 5: Benedict’s test: Figure 6: Barfoerd’s test: Absence of brick red ppt. Presence of brick red ppt. Absence of red ppt. Figure 7: Barfoerd’s test: Figure 8: Seliwanoff’s test: Figure 9: Tollen’s test   Presence of red ppt. Presence of red colour complex Presence of silver mirror Figure 10: Ninhydrin test: Figure 11: Xanthoproteic test: Figure 12: Millon’s test: Presence of purple colour complex Presence of yellow colour complex Presence of pink colour ppt. Figure 13: Pauly’s test: Presence of red azo dye Discussion The principles of each test: In molisch’s test, concentrated sulfuric acid is used to dehydrate the carbohydrates to form 5-hydroxymethylfurfural, which reacts with the ÃŽ ±Ã¢â‚¬â€œnaphthol to give a purple result (Pavia, 2005, p.446). In iodine test, a blue colour is formed when the iodine is absorbed into the open spaces of amylose molecules in starch (Pavia, 2005. p.451). In benedict’s test, the sugar (reducing sugar) gets oxidized and reduces Cu2+ present in the reagent (Raymond, 2010, p.344). Barfoerd’s test is a test unique for monosaccharide, where cupric hydroxide is reduced in acidic medium to give red colour cuprous oxide (Nigam and Ayyagari, 2008, p.25). In seliwanoff’s test, the ketoses are dehydrated to form furfural derivatives which then condense with resorcinol to give a red colour complex (Nigam and Ayyagari, 2008, p.27). In tollen’s test, silver ammonium salt oxidizes the aldehyde to give glucuronide ammonium salt and metallic silver, which gives the silver mirror effect (Brito-Arias, 2007, p.5). In Ninhydrin test, free ÃŽ ±Ã¢â‚¬â€œamino acid radical reacts with ninhydrin to give a blue-violet complex (Malhotra, 2003, p.23). In xanthoproteic test, benzene ring is nitrated with nitric acid which produces a yellow compound (Sim et al., 2008, p.611). In Millon’s test, hydroxybenzene radical of phenolic amino acids (tyrosine) react with millon’s reagent to form a red colour complex (Nigam and Ayyagari, 2008, p.41). In pauly’s test, sulfanilic acid in the reagent gives a diazonium compound in the presence of nitrous acid and hydrochloric acid, which combines with amines and phenols to form coloured azo-compounds (Nigam and Ayyagari, 2008, p.41). Conclusion Macro molecules presence in the given samples was successfully identified by using the given chemical assays. References Bochkov, A.F., Zaikov, G.E. and Afanasiev, V.A (1991) Carbohydrates. Google Books [Online]. Available at: https://books.google.lk/books?id=BmPTDAnsUb0Cprintsec=frontcoverdq=carbohydrateshl=ensa=Xei=bXlKVavSGImTuAS7jYG4CQsqi=2ved=0CCMQuwUwAQ#v=onepageq=carbohydratesf=false (Accessed: 7 May 2015). Brito-Arias, M. (2007) Synthesis and Characterization of Glycosides. Google Books [Online]. Available at: https://books.google.lk/books?id=X9ZTg47alJkCpg=PA5dq=Tollens+testhl=ensa=Xei=2GhKVY3HOI2QuATD1YF4ved=0CDEQuwUwAw#v=onepageq=Tollens%20testf=false (Accessed: 7 May 2015). Jenkins, A.D, Kratochvil, P., Stepto, R.F.T. and Suter, U.W. (1996) `Glossary of basic terms in polymer science`, Pure and Applied Chemistry, 68(12), pp. 2287–2311, ISSN [Online]. Available at: http://www.degruyter.com/view/j/pac.1996.68.issue-12/pac199668122287/pac199668122287.xml (Accessed: 6 May 2015). Malhotra, V.K. (2003) Practical Biochemistry for Students. Google Books [Online]. Available at: https://books.google.lk/books?id=LHa1G131MuYCpg=PA23dq=Ninhydrin+testhl=ensa=Xei=GGxKVavVMMSSuATEsYDADwved=0CB4QuwUwAA#v=onepageq=Ninhydrin%20testf=false (Accessed: 7 May 2015). Nigam, A. and Ayyagari, A. (2008) Lab Manual in Biochemistry: Immunology and Biotechnology. Google Books [Online]. Available at: https://books.google.lk/books?id=Ws570Ql8krACpg=PA25dq=Barfoed%E2%80%99s+testhl=ensa=Xei=i19KVbD7EJWmuQT5joHADAved=0CCEQuwUwAA#v=onepageq=Barfoed%E2%80%99s%20testf=false (Accessed: 7 May 2015). Nigam and Ayyagari (2008) Lab Manual in Biochemistry: Immunology and Biotechnology. Google Books [Online]. Available at: https://books.google.lk/books?id=Ws570Ql8krACpg=PA27dq=Seliwanoff%E2%80%99s+testhl=ensa=Xei=pWhKVcjcDoyouwSMj4HYCAved=0CB4QuwUwAA#v=onepageq=Seliwanoff%E2%80%99s%20testf=false (Accessed: 7 May 2015). Nigam, A. and Ayyagari, A. (2008) Lab Manual in Biochemistry: Immunology and Biotechnology. Google Books [Online]. Available at: https://books.google.lk/books?id=Ws570Ql8krACpg=PA41dq=Millon%E2%80%99s+testhl=ensa=Xei=ymxKVeXJH9GKuATY6IGwDQved=0CCoQuwUwAg#v=onepageq=Millon%E2%80%99s%20testf=false (Accessed: 7 May 2015). Pavia, D.L. (2005) Introduction to organic laboratory techniques: A small scale approach. Google Books [Online]. Available at: https://books.google.lk/books?id=ega5c11VHvkCpg=PA446dq=Molisch%E2%80%99s+testhl=ensa=Xei=rF5KVa39HtHguQSrvIGwCQved=0CCUQuwUwAQ#v=onepageq=Molisch%E2%80%99s%20testf=false (Accessed: 7 May 2015). Pavia, D.L. (2005) Introduction to organic laboratory techniques: A small scale approach. Google Books [Online]. Available at: https://books.google.lk/books?id=ega5c11VHvkCpg=PA451dq=Iodine+testhl=ensa=Xei=Dl9KVfTcHMuxuAT-roCIDgved=0CB4QuwUwAA#v=onepageq=Iodine%20testf=false (Accessed: 7 May 2015). Raymond, K.W. (2010) General Organic and Biological Chemistry. Google Books [Online]. Available at: https://books.google.lk/books?id=iIltMoHUtJUCpg=RA1-PA344dq=Benedict%E2%80%99s+testhl=ensa=Xei=NF9KVcvTOMmxuASL9YH4Cwved=0CCcQuwUwAQ#v=onepageq=Benedict%E2%80%99s%20testf=false (Accessed: 7 May 2015). Sim, K.S., Chin, F.S., Tso, C.P. and Thong, L.W (2008) `Protein identification in latex gloves for bio-compatibility using maximum minimal variation test`, in Osman, N.A.A., Ibrahim, F., Abas, W.A.B.W., Rahman, H.S.A. and Ting, H.N. (ed.) 4th Kuala Lumpur International Conference on Biomedical Engineering 2008. Google Books [Online]. Available at: https://books.google.lk/books?id=sdG-1hN_4TYCpg=PA611dq=Xanthoproteic+testhl=ensa=Xei=gGxKVY3yA9CbuQSa74CwAwved=0CCMQuwUwAQ#v=onepageq=Xanthoproteic%20testf=false (Accessed: 7 May 2015). Walsh, G. (2004) Proteins: Biochemistry and Biotechnology. Google Books [Online]. Available at: https://books.google.lk/books?id=EXTEjL2wTnYCprintsec=frontcoverdq=proteinshl=ensa=Xei=M3pKVdGXJIfGuATTgoCQAQved=0CB4QuwUwAA#v=onepageq=proteinsf=false (Accessed: 7 May 2015). 1 | Page Chemical Tests for Biomolecules Chemical Tests for Biomolecules INTRODUCTION Biomolecules are complex organic molecules. Carbon, hydrogen, oxygen, nitrogen and phosphorus are the atoms that make up most of the biomolecules. These molecules form the basic structure of a living cell. The compounds such as amino acids, nucleotides and monosaccharide’s serve as the building blocks of complex biomolecules. The important biomolecules are proteins, carbohydrates, fats, hormones and nucleic acids (Kimball, 2012). Carbohydrates Carbohydrates are substances which containing the elements carbon hydrogen and oxygen and they have the general formula of Cx (H2O) y. Simple carbohydrates or the entire carbohydrate family may also be called saccharides .They are the most abundant biomolecules belonging to class of organic compounds found in living organisms. The major source of metabolic energy for both animals and plants are carbohydrates (Churms, 1982). Carbohydrates link to with proteins forming glycoproteins and with lipids forming glycolipids. Moreover they are present in DNA and RNA, which are essentially polymers. More than 75% of the dry weight of the plant world is carbohydrate in nature mainly cellulose, hemicelluloses and lignin (Reed, 2005). Carbohydrates are classified on the basis of their behavior on hydrolysis. They have been broadly divided into following three groups: Monosaccharide’s, Disaccharides, Oligosaccharides, and Polysaccharides. Monosaccharide A carbohydrate that cannot be hydrolyzed further to give simpler unit of polyhydroxy aldehyde or ketone is called a monosaccharide. Monosaccharides are single sugars units and there general formula is (CH20) n. Moreover they are colorless, crystalline solids that are freely soluble in water but insoluble in nonpolar solvents. The backbone of monosaccharide is an unbranched carbon chain in which all the carbon atoms are linked by single bonds (GyoÃÅ'ˆrgydeaÃÅ' k and PelyvaÃÅ' s, 1998). One of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group each of the other carbon atoms has a hydroxyl group. If the carbonyl group is at an end of the carbon chain, the monosaccharide is an aldehyde and is called an aldose, furthermore if the carbonyl group is at any other position the monosaccharide is a ketone and is called ketoses. Glucose, fructose, galactose, and ribose are some examples of monosaccharide. The building blocks of disaccharides like sucrose an d polysaccharides such as cellulose and starch and hemicelluloses are monosaccharide (Ferrier, 1999). Figure 1.1.1 ring structure of monosaccharide molecules. https://www.google.lk/search Figure 1.1.2 monosaccharide molecule showing the aldehyde and ketone group http://academic.brooklyn.cuny.edu/biology/bio4fv/page/monosacchrides.html Disaccharides A Disaccharide is two monosaccharide units linked by an oxide linkage formed by the loss of a water molecule. Such a linkage between two monosaccharide units through oxygen atom is called glycoside linkage. Three most abundant disaccharides are sucrose, lactose, and maltose. In Maltose ÃŽ ± (1→4) glycosidic linkage joins two glucose units, this occurs mainly as a breakdown product during digestion of starch by enzymes called amylases (Owusu-Apenten, 2005). Sucrose is the most abundant disaccharide in nature and it’s mostly found in plants which acts a good transport sugar since it is very soluble and can move in very high concentration. In Sucrose the anomeric carbon atoms of a glucose unit and fructose unit are joined. Moreover lactose the disaccharide of milk consists of galactose joined to glucose by ÃŽ ² (1→4) glycosidic linkage (Denniston, Topping and Caret, 2004). In additionally Sucrose and lactose are heterosaccharides and maltose is homosaccharides as we ll as maltose and lactose are reducing sugars. Sucrose is the only common non reducing sugar. Figure 1.3.1 disaccharides are formed by condensation of two monosaccharide. https://www.google.lk/search?q=disaccharideses_sm=122source Polysaccharides Polysaccharides are complex carbohydrates made up of many monosaccharide joined together by glycosidic bond. They are large, often branched, macromolecules. Their large sizes make them more or less insoluble in water and have no sweet taste (Aspinall, 1982). When all the monosaccharide in a polysaccharide is of the same type, the polysaccharide is called a homopolysaccharide and when more than one type of monosaccharide is present, they are called heteropolysaccharides. Polysaccharides have a general formula Cn (H2O) n-1 where n can be any number between 200 and 2500. Starch glycogen and cellulose are the examples of polysaccharides (Tombs and Harding, 1998). Figure 1.4.1 ring structure of polysaccharides molecules. https://www.google.lk/search?q=polysaccahrideses_sm=122source=lnmstbm=ischsa Proteins Cells are made of protein. Proteins are the most versatile class of molecules in living organisms. All proteins contain C, H, N, O some S, P, Fe, Zn, Cu. Proteins contains 20 different amino acids which are encoded by the genetic code and which constitute the building blocks of the proteins in all living organisms (Walsh, 2002). Each protein species contains one or several polypeptide chains of defined amino acid sequence. Their functions are catalysis, transport, hormones and structure. Amino acids are molecules containing an amine group carboxylic acid group and a side chain. Simple proteins contain only polypeptide chains Proteins can be soluble (globular proteins) and insoluble (myosin, fibrinogen) (Whitford, 2005). Figure 1.5.1 classification of proteins and there structures. https://www.google.lk/search?q=protein structurerevid=120848340tbm OBJECTIVES To distinguish between monosaccharide’s and disaccharides. To differentiate between different types of amino acids. To identify an unknown sample of carbohydrate and amino acid. MATERIALS Albumin solution Arginine solution Barfoed reagent Beakers Benedict’s solution Bunsen burner Burner stand Concentrated sulphuric acid Concentrated nitric acid Copper sulphate Fructose solution Glucose solution Glysin solution Iodine solution Lactose solution Molisch’s reagent Ninhydrin solution Pipettes Seliwanoff’s reagent Sodium hydroxide Starch Sucrose solution Test tubes Tyrosine solution Unknown solutions Water bath TEST FOR CARBOHYDRATES METHODOLOGY Molisch’s Test Five test tubes were taken with 1ml of carbohydrate solutions. Few drops of Molisch’s reagent were added to the testubes following with concen.sulphuric acid down the slide of the test tube. The colour change was observed. Iodine test Three drops of Iodine solution was added to each test tube with 1ml of each of the carbohydrate solutions. The colour change was observed. Benedict’s test 1ml of each carbohydrate solutions was taken in five test tubes.5ml of Benedict’s reagent was added to all three test tubes. All five test tubes were placed in a water bath and heated for two minutes. The colour change was observed. Barfoed test 5ml of Barfoed reagent was added with 1 ml of carbohydrate solutions. Test tubes were placed in water bath and heated for five minutes. The colour change was observed. Seliwanoff test 1ml of each carbohydrate solution was added to the test tubes following with 4ml of Seliwanoff reagent. The test tubes were placed in the water bath and heated to two to three minutes. The colour change was observed. Two unknown samples were taken in a test tubes and labeled A and B. Sample A was added to two test tubes. To the sample A the Iodine reagent was added and the colour change was observed. The Benedict’s reagent was added to the sample A of another test tube and was heated in general flame for two minutes and the colour change was observed. The sample B was added to four test tubes. One drop of Iodine reagent was added to the sample B test tube and colour change was observed following with Benedict’s reagent, Barfoed reagent and the Seliwanoff reagent were added to the remaining test tubes with sample B and was heated in the water bath for three minutes and the colour change was observed. TEST FOR AMINO ACID METHODOLOGY Ninhydrin test 1ml of Ninhydrin solution was added into 0.5 ml of 0.02 % amino acid solution in four test tubes. The test tubes were placed in water bath and heated for three to four minutes. The colour change was observed. Xanthoproteic Test 2ml of conc. Nitric acid was added to 2ml of 0.02% amino acid solution in four test tubes. The test tubes were placed in water bath for two minutes and the colour change was observed. Millon’s Test Four drops of Millon’s reagent was added into 2ml of 0.02% of amino acid solution in four test tubes. The test tubes were placed in water bath for four minutes and the colour change was observed. Biurete Test 3ml of 10% of sodium hydroxide was added drop wise to 1% of copper sulphate. The colour change was observed. Two unknown samples were taken in test tubes and labeled C and D. Sample C was added into two test tubes. To the sample C the Biurete reagent was added and the colour change was observed. The Millon’s reagent was added to the sample C of another test tube and was heated in general flame for two minutes and the colour change was observed. The sample D was also added into two test tubes. Biurete reagent was added to the sample B test tube and colour change was observed. Besides Millon’s reagent were added to the remaining test tube with sample B and was heated in the water bath for three minutes and the colour change was observed. RESULTS Test for carbohydrates Test for amino acids DISCUSSION In Molisch’s test all the carbohydrate solution gave a positive result, so as it’s a general test to confirm the molecule is carbohydrate. Iodine test is performed to separate the polysaccharide from monosaccharide and disaccharide as a result in this test only starch gave a positive result since its unbranched molecule. Glucose has a free aldehyde group and fructose has a free ketone group. Thus they react with Benedicts reagent and reduce it to form a reddish orange colour, which is a positive indication of Benedicts reaction .The copper (II) ions in the Benedicts solution are reduced to Copper (I) ions, which causes the colour change. Complex carbohydrates such as starches do not react positive with the Benedicts test. Buiret solution is a blue liquid that changes to purple when proteins are present and to pink in the presence of short chains of polypeptides. The cause of this colour change is because of the copper atom of the Biuret solution reacts with the peptide bonds. Avoid spilling Ninhydrin solutions on your skin, as the resulting stains are difficult to remove. When handling with Concentrated Sulphuric acid wear safety garments to avoid Sulphuric acid getting on self. Do not over heat the amino solutions in water bath since all the proteins may denature moreover colour change cannot be observed. CONCLUSION The unknown solution A is sucrose and it’s a non reducing sugar since in Iodine and Benedict’s test it showed a negative result where there was no colour change in addition to unknown solution B is glucose which is a reducing sugar because in Iodine and Seliwanoff test it gave a negative result remaining colourless and in Benedict’s and Barfoed test it gave a positive result changing its colour from green precipitate to reddish colour solution concluding solution B is glucose. The unknown solution C is protein since positive result was obtained and the solution turned pink in Biurete and Millon’s reagent along with the solution D is an amino acid because it remained colourless in Millon’s test and turned light blue in Biurete test resulting both in negative. References Aspinall, G. (1982). The Polysaccharides. 1st ed. New York: Academic Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Churms, S. (1982). Carbohydrates. 1st ed. Boca Raton, Fla.: CRC Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Denniston, K., Topping, J. and Caret, R. (2004). General, organic, and biochemistry. 1st ed. Boston: McGraw-Hill Higher Education. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Ferrier, R. (1999). Carbohydrate chemistry. 1st ed. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). GyoÃÅ'ˆrgydeaÃÅ' k, Z. and PelyvaÃÅ' s, I. (1998). Monosaccharide sugars. 1st ed. San Diego: Academic Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Kimball, L. (2012). Biomolecules. 1st ed. Delhi: Research World. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Owusu-Apenten, R. (2005). Introduction to food chemistry. 1st ed. Boca Raton, Fla.: CRC Press. Google books [Online books] Available at: http://books.google.lk (Accessed: 4th July 2014). Reed, D. (2005). Biomolecular archaeology. 1st ed. Carbondale: Center for Archaeological Investigations, Southern Illinois University, Carbondale. Google books [Online books] Available at: http://books.google.lk (Accessed: 3rd July 2014). Tombs, M. and Harding, S. (1998). An introduction to polysaccharide biotechnology. 1st ed. London: Taylor Francis. Google books [Online books] Available at: http://books.google.lk (Accessed: 4th july2014). Walsh, G. (2002). Proteins. 1st ed. Chichester: J. Wiley. Google books [Online books] Available at: http://books.google.lk (Accessed: 6th July 2014). Whitford, D. (2005). Proteins. 1st ed. Hoboken, NJ: J. Wiley Sons. Google books [Online books] Available at: http://books.google.lk (Accessed: 6th July 2014).