Chemical Tests for Biomolecules

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

For amino acids and protein

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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