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Colour Reaction of Amino Acids Prepared by: Group#2 Quinto, Jasmine Sta. Isabel, Rizzalyn Yadao Y adao , Monique Monique Niazmand Niazman d Saravan Saravani, i, Mostafa Niazman Nia zmand d Sara Saravani vani,, Mojta Mojtaba ba Seong, Jo-eun Majidzadeh, Hossein
Proteins are complex, high molecular molecular weight weight biomolecul biomolecules es composed composed of amino acids joined together by peptide bonds. The twenty amino acids normally found found in proteins differ in their amino groups in the side chains. These respond to specific qualitative tests tests with certain cer tain chemical compounds chemical reagents and thus become the basis of their detection whether as free free amino acids or in combined form form as in peptides or proteins
Reactions of proteins may be divided into three categories: 1. Reaction due to the presence of specific chemical groups are linkages in the protein molecule 2. Reaction due to the acidity or basicity of the protein 3. Reactions due to the colloidal nature of the proteins
Colour Reactions of Amino Acids and Proteins
Biuret test This is the test indicates the presence of peptide linkages. The purplish to violet colour is apparently due to the cupric ions with the unshared electron pairs of four nitrogen atoms. All substance is proportional to the number of peptide bonds give the test and the intensity of the purple colour produced id proportional to the number of peptide bonds present.
Ninhydrin test Amino acids reacts with ninhydrin (tryketoninhydrindene hydrate) to yield CO2, NH3 and aldehyde containing one less carbon than the amino acid. The reaction is also yields a blue or purple colour useful for the colorimetric determination of amino acids.
X anthoproteic test This test is positive for proteins and amino acids containing an aromatic side chain (phenylalanine, tyrosine, and tryptophan). The benzene ring undergoes nitration with concentrated HNO3 giving nitro derivatives which are yellow in colour. Phenylalanine does not response readily to this test and requires H2SO4 as catalyst.
Millon-Nasse test The phenolic group of tyrosine reacts with Millon-Nasse reagent (HgSO4 in H2SO4) forming an old rose or pink to red complex upon heating. The complex is probably the mercury salt of the phenolic compound.
Bromine water test The pyrrole ring of tryptophan undergoes halogenation with Br2 water in the presence of amyl alcohol giving a pink or lavender colour in the alcohol layer.
Unoxidized sulfur
test This is given by the sulfur containing amino acids cysteine and cystine (methionine is quite stable alkaline lead acetate) whn boiled in strongly alkaline solutions. The liberated sulfur reacts with PbAc2 forming a brown to black precipitate of lead sulfide.
Sakaguchi
Reaction The guanidinium group of arginine in alkaline solution gives an orange or red colour with alphanaphthanol and sodium hypobromite
Hopkins-Cole test The indole ring of tryptophan reacts with Hopkins-Cole reagent (glyoxylic acid) and sulfuric acid to produce a violet or ed purple color.
Objectives 1. To carry out the different colour reaction testes for amino acids and proteins; 2. Ro characterize amino acids based on their reactions with specific chemical reagents; 3. Ro identify the functional group of the amino acid responsible for the positive colour reaction.
Materials 1%solution of egg albumin 0.1% solution of tryptophan Arginine Cysteine Tryrosine Phenylalanine 10% formic acid solution 0.1% ninhydrin 5% CuSO4
Saturated lead acetate
10% NaOH
Millon-Nasse reagent
Bromine water
N-amyl alcohol
Alpha-naphtanol
solution
20%NaOH
Hopkins-Cole reagent
Apparatus 1. Hot plate/ stove
1. 10mL graduated cylinder
2. Alcohol lamp
2. Pipets
3. 600mL beaker
3. Droppers
4. 50mL beaker
4. Litmus paper
5. Test tubes
5. Wire guaze
6. Test tube rack
Procedure
Preparation of Saliva Solution 1. Collect 10mL of saliva using a clean 50mL beaker 2. Dilute the saliva with an equal volume of distilled water
Colour Reaction of Amino Acids and Proteins The following tests will be performed on acid soluble proteins of saliva solution and 1% solution of egg albumin. Some or the tests may require to be performed on specific amino acids. Ain all of the following tests, perform a blank test with distilled water instead of the protein sample.
Biuret test 1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. To each test tube add 1mL of 10%NaOH 3. Add 2 drops of CuSO4 solution. Observe the result. 4. Run a blank test with distilled water.
Ninhydrin test 1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. Heat each of the protein solutions with 0.25mL of 0.1%ninhydrin 3. Let cool and observe 4. Run a blank test with distilled water.
X anthoproteic test 1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. To each protein solution, add 1mL of concentrated HNO3. 3. Note if a heavy white precipitate is formed. Heat carefully to boiling and observe the colour of the solution/precipitate. 4. Cool and make alkaline with 10% NaOH. Note the colour change 5. Cool and make alkaline with 1% solutions of tryptophan, tyrosine, and phenylalanine in different test tubes. 6. Run a blank test with distilled water.
Millon-Nasse test 1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin) 2. Add 2 to 3 drops of Millon¶s reagent to each of the protein solutions. 3. Boil in a boiling water bath for 5 minutes. Observe. 4. Perform an identical test with 0.1% tryptophan solution. 5. Run a blank test with distilled water.
Bromine water test 1. In 2 different test tubes, place1mL each of the protein samples (saliva solution and 1% solution of egg albumin). And add 3 drops of bromine water and 1mL of n-amyl alcohol. Shake well 2. Observe the colour of the alcohol layer. 3. Repeat the steps in with the 1% tryptophan solution 4. Run a blank test with distilled water.
Unoxidized sulfur
test 1. Mix 1mL each protein samples in 3 different tubes with 1mL each of 10% NaOH and saturated lead acetate, PbAc2. 2. Boil in a water bath for 3 minutes and observe the result. 3. Perform an identical test with 0.1% cystein and a bank test with distilled water.
Sakaguchi Reaction 1. Place 1mL each of protein samples in 3 different test tubes and add 2mL each of 20% NaOH and alpha naphtanol solution. 2. Mix thoroughly and add 0.5mL of freshly prepared bromine water. Observe the result. 3. Perform an identical test with 0.1% cystein and a bank test with distilled water.
Hopkins-Cole test 1. Place 2mL each of sample solution in 3 different test tubes and to each tube add 1mL of Hopkins-Cole reagent. 2. Incline the tube and carefully add along the side 2mL at concentrated H2SO4. 3. Observe the colour at the junction of two liquids. 4. Perform an identical test with 0.1% trytophan.