quality test for amino acid

TITLE : QUALITATIVE TEST FOR AMINO ACIDS.

Introduction : Amino acids are building blocks for proteins. They share the same acid and amino group but the side chain differ. When amino acids are linked together to form an amide , the product is called a peptide , and the newly formed bond is called a peptide bond. If a few amino acids ( up to 20 ) are linked , the molecule is called a polypeptide. When many amino acids (20 – 1000 ) are in the peptide chain , it is called a protein. The α-amino acid can be detected using ninhydrin which forms a blue to blue-violet colour as positive indication. Other colour ( yellow, orange, red ) are negative . The test is very sensitive and often used for colorimetric determination of amino acid solutions or as a visualization agent in chromatography of amino acids . During the reaction the amino group will liberate ammonia , the ammonia will react with react with ninhydrin to form a coloured complex. Most protein give positive results in Xanthoprotein Test because of the presence of phenyl group (-C6H5 ) . The phenyl group procedure coloured nitro compounds in reaction with nitric acid. The amino acid with thiol group (-SH ), such as cysteine when heated in the presence of alkali will produced sulphide ions. These ions can be detected by reaction with lead cations a black precipitate plumbum sulphide ( PbS ) is formed. In Millon’s Test , if the hydroxyphenyl side group is present , a red colour will be observed . Tyrosine is the only amino acid which gives a positive test . However , any molecule with the phenolic –OH will react. In Hopkins-Cole test, heterocyclic side chain ( indole group) of tryptophan reacts to give a purple to violet ring at the interface of the two layers. In Sakaguchi test , ammonia and ammonium ions give positive reactions in this test.

Objective : To conduct a series of qualitative test for amino acids.

Procedure :

Ninhydrin Test 1. 2. 3. 4. 5.

1 ml of glycine was pipette in a test tube. The 5 drops of ninhydrin was add. The test mixture was heat in boiling water bath for 2 minutes. Record any changes. The experiment was repeated with stock solutions of tyrosine , tryptophan , phenylalanine and proline.

Xanthoproteic Test 1. 2. 3. 4. 5. 6. 7.

1 ml of glycine was pipette in a test tube. 1 ml of concentrated nitric acid was add. The test tube was swirl and immerse the test tube in cold water to cool the down. Any changes was record. 40 % of NaOH solution was add dropwise to change the pH solution to alkaline Record the changes. The experiment was repeated with stock solution if tyrosine , tryptophan , phenol and phenylalanine.

Millon Test 1. 2. 3. 4. 5. 6. 7.

1 ml of glycine was add in the test tube. 5 drops of Millon reagent was add. The test mixture was heat in the boiling water bath for 10 minutes. The test tube was cool it down in room temperature. 5 drops of natrium nitric was add. The changes was record in the table. The stock solutions was repeated by using tryrosine and phenylalanine.

Hopkins-Cole-Test 1. 1 ml of glysine was mix with 1 ml of glacial acetic acid in a clean test tube. 2. The test tube was incline and slowly add 1 ml of concentration sulphuric acid but do not mix. 3. The two layer was form. 4. The layers was stand and note the colour at the interface after 2-3 minutes. 5. The stock solutions was repeat using tryrosine and tryptophan.

Test for Presence of Cysteine

1. 2. 3. 4. 5. 6. 7. 8.

1 ml of cysteine was pipette in a test tube. 1 ml of 40% was add in NaOH. The test tube was swirl. The test mixture was heat in a boiling water bath for 2 minutes. The test tube was immerse in cold water to cool the them down. 1 ml of natrium plumbat was add. The changes was record in the table. The stock solutions was repeated with tyrosine and methionine.

Sakagushi Test 1. 2. 3. 4. 5. 6. 7.

1 ml of arginine was add in a test tube. 1 ml of 40 % NaOH was add in test tube. 2 drops of napthol was add in test tube. 5 drops of bromine was add in the test tube. The test tube was swirl. Any changes that we can see was record in the table. The procedure was repeated by using glycosiamine, methyl guanidine , creatine and urea.

Result : Ninhydrin Test

Sample Glysine Tyrosine Tryptophan Phenylalanine Proline

Observation

Conclusion

Its colourless at the initial and turn to blue at the final. Its cloudy at the initial and turn blue at the final. Its brown colour at the initial and blue at the final. Its colourless at the initial and turn to blue at the final. Its cloudy at the initial and final.

Presence of free amino group

Observation Its colour colourless at the initial and final. Its colour colourless at the initial and final. Its colour colourless at the initial and turn light orange yellowish at the final. Its colour colourless at the initial and final. Its colour colourless at the initial and turn light orange at the final.

Conclusion Negative Phenyl Group

Observation Its colourless at the initial and final. Its colourless at the initial and turn to red at the final . Its colourless at the initial and final.

Conclusion Absence of phenol group

Presence of free amino group Presence of free amino group Presence of free amino group Absence of free amino group

Xanthoprotein Test Sample Glysine Tyrosine Tryptophan

Phenylalanine Phenol

Negative Phenyl Group Positive Phenyl Group

Negative Phenyl Group Positive Phenyl Group

Millon Test Sample Glysine Tyrosine Tryptophan

Hopkins-Cole-Test

Presence of phenol group Absence of phenol group

Sample Glysine Tyrosine Tryptophan

Observation Its colourless at the initial and final Its colourless at the initial and final. Its colourless at the initial and form two layer with yellow ring.

Conclusion Negative Indole Group

Observation Its colourless at the initial and form black precipitate. Its colourless at the initial and final . Its colourless at the initial and final .

Conclusion Positive result Thiol group

Observation Its colourless at the initial and turn to dark maroon at final. Its colourless at the initial and turn to dark maroon at final. Its colourless at the initial and turn black at final. Its colourless at the initial and turn to dark brown at final.

Conclusion Presence of Guanadine ion

Negative Indole Group Positive Indole Group

Test for Presence of Cysteine Sample Cysteine Tyrosine Methionine

Negative result Thiol group Negative result Thiol group

Sakaguchi Test Sample Arginine

Methyl guanidine

Creatine Urea

Presence of Guanadine ion

Absence of Guanadine ion Absence of Guanadine ion

Discussion : Certain functional groups in amino acids and protein can react to produce characteristically coloured product. The colour intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional , or free , groups present. In this experiment, various colour-producing reagent (dyes) will be used to qualitatively detect the presence of certain functional groups in amino acids and protein. Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines . When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect fingerprints , as the terminal amines of lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin . It is a white solid which is soluble in ethanol and acetone at room temperature Ninhydrin can be considered as the hydrate of indane-1,2,3trione. In Xanthoproteic test some amino contains aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions that are characteristic of benzene and its derivatives. One such reactions is the nitration of a benzene ring with nitric acid. The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo nitration. The amino acid phenylalanine also contains a benzene ring but the ring is not activated and therefore does not undergo readily nitration. The principle nitric acid gives colour when heated with protein containing tyrosine ( yellow colour ) or tryptophan ( orange colour ), the colour is due to nitration. Millon’s test reagent gives positive results ( pink to dark red colour ) with proteins containing the phenolic amino acid “ tyrosine “. Some protein containing tyrosine will initially from a white precipitate that turns red when heated , while others from a red solution immediately. Hopkins- cole – test is the indole ring reacts with glyoxylic acid acid in the presence of a strong acid to form a violet cyclic product. A positive indole will give observation colourless at the initial and form two layer with yellow rings as a final results. Although classified as a non-essential amino acid, in rare cases, cysteine may be essential for infants, the elderly, and individuals with certain metabolic disease or who suffer from malabsorption syndromes. Cysteine can usually be synthesized by the human body under normal physiological conditions if a sufficient quantity of methionine is available. Cysteine is catabolized in the gastrointestinal tract and blood plasma . In contrast, cystine travels safely through the GI tract and blood plasma and is promptly reduced to the two cysteine molecules upon cell entry. The purpose for the Sakaguchi test used for the detection of a specific type of protein with the amino acid containing the guanadine ion group. The sample arginine and methyl guanidine was positive test and form final colour is dark maroon.

Conclusion : Protein also make up the second largest portion of cells after water. They are larger polymeric compounds that cells synthesize from various building blocks called amino acid. Twenty different amino acids, which differ only in the structure of their side chains, are used by human cells to build proteins. The side chain structure determine the class of the amino acid , non-polar, neutral , basic or acidic. Human cells can synthesize most the amino acids needed to build proteins. However, about nine amino acids called essential amino acids cannot be synthesized by human cells and must be obtained from food. For this experiment the objective is to conduct a series of qualitative test for amino acids. I think we manage to do the experiment successfully and get a good result with help from our lecture.

Refences : 1. Belitz , H.-D; Grosch , Werner; Schieberle , Peter (2009-02-27). Food Chemistry. ISBN 9783540699330. 2. www.wikipedia.com.

3. Kamsani Ngalip, Nordin Ali, 01/06/2003. Biochemistry Lab Manual. UITM Negeri

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