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Chemical Tests for Biomolecules
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Contents (jump to)
Introduction
Objective
Materials
Methodology
Results
Discussion
Conclusion
References
Introduction
A macromolecule is “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 H2SO4, 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 α–naphthol 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 α–amino 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=BmPTDAnsUb0C&printsec=frontcover&dq=carbohydrates&hl=en&sa=X&ei=bXlKVavSGImTuAS7jYG4CQ&sqi=2&ved=0CCMQuwUwAQ#v=onepage&q=carbohydrates&f=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=X9ZTg47alJkC&pg=PA5&dq=Tollens+test&hl=en&sa=X&ei=2GhKVY3HOI2QuATD1YF4&ved=0CDEQuwUwAw#v=onepage&q=Tollens%20test&f=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=LHa1G131MuYC&pg=PA23&dq=Ninhydrin+test&hl=en&sa=X&ei=GGxKVavVMMSSuATEsYDADw&ved=0CB4QuwUwAA#v=onepage&q=Ninhydrin%20test&f=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=Ws570Ql8krAC&pg=PA25&dq=Barfoed%E2%80%99s+test&hl=en&sa=X&ei=i19KVbD7EJWmuQT5joHADA&ved=0CCEQuwUwAA#v=onepage&q=Barfoed%E2%80%99s%20test&f=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=Ws570Ql8krAC&pg=PA27&dq=Seliwanoff%E2%80%99s+test&hl=en&sa=X&ei=pWhKVcjcDoyouwSMj4HYCA&ved=0CB4QuwUwAA#v=onepage&q=Seliwanoff%E2%80%99s%20test&f=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=Ws570Ql8krAC&pg=PA41&dq=Millon%E2%80%99s+test&hl=en&sa=X&ei=ymxKVeXJH9GKuATY6IGwDQ&ved=0CCoQuwUwAg#v=onepage&q=Millon%E2%80%99s%20test&f=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=ega5c11VHvkC&pg=PA446&dq=Molisch%E2%80%99s+test&hl=en&sa=X&ei=rF5KVa39HtHguQSrvIGwCQ&ved=0CCUQuwUwAQ#v=onepage&q=Molisch%E2%80%99s%20test&f=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=ega5c11VHvkC&pg=PA451&dq=Iodine+test&hl=en&sa=X&ei=Dl9KVfTcHMuxuAT-roCIDg&ved=0CB4QuwUwAA#v=onepage&q=Iodine%20test&f=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=iIltMoHUtJUC&pg=RA1-PA344&dq=Benedict%E2%80%99s+test&hl=en&sa=X&ei=NF9KVcvTOMmxuASL9YH4Cw&ved=0CCcQuwUwAQ#v=onepage&q=Benedict%E2%80%99s%20test&f=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_4TYC&pg=PA611&dq=Xanthoproteic+test&hl=en&sa=X&ei=gGxKVY3yA9CbuQSa74CwAw&ved=0CCMQuwUwAQ#v=onepage&q=Xanthoproteic%20test&f=false (Accessed: 7 May 2015).
Walsh, G. (2004) Proteins: Biochemistry and Biotechnology. Google Books [Online]. Available at: https://books.google.lk/books?id=EXTEjL2wTnYC&printsec=frontcover&dq=proteins&hl=en&sa=X&ei=M3pKVdGXJIfGuATTgoCQAQ&ved=0CB4QuwUwAA#v=onepage&q=proteins&f=false (Accessed: 7 May 2015).
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