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Chemical Tests for Biomolecules

Paper Type: Free Essay Subject: Chemistry
Wordcount: 4233 words Published: 26th Jan 2018

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Kaneshanathan Kumaraguru

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

 

 

C:UsersSamanthaSudheeraLab reportsBiomolculeresultsiod.suc-lac-glu-fru.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsiod.sta.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsmol.lac-suc-sta.JPG

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

C:UsersSamanthaSudheeraLab reportsBiomolculeresultsbar.lac-suc-sta.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsben.fru.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsben.sta-suc.JPG

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.

C:UsersSamanthaSudheeraLab reportsBiomolculeresultstol.sta-suc.JPGC:UsersKaneshanathanDownloadsCompressedssfsdfdIMG-20141129-WA0003.jpgC:UsersSamanthaSudheeraLab reportsBiomolculeresultsbar.fru.JPG

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

C:UsersSamanthaSudheeraLab reportsBiomolculeresultsmill.egg.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsxen.trip.JPGC:UsersSamanthaSudheeraLab reportsBiomolculeresultsnin.tyr.JPG

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.

C:UsersSamanthaSudheeraLab reportsBiomolculeresultspaul.tyr-trip.JPG

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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