lab 1 qualitative tests 2-2-2014

8/19/2019 Lab 1 Qualitative Tests 2-2-2014

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Introduction

• The food we consume is divided into three mainclasses:

1.Carbohydrates: the body’s most readily availableenergy source.

2.Lipids: the body’s principal energy reserve..!roteins: the body’s source of energy for growth

and cellular maintenance.

• !roteins also ma"e up the second largest portionof cells# after water.

• !roteins consists of amino acid lin"ed together by

peptide bond.


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• $mino acids incorporated into proteins are covalently

lin"ed by peptide bonds.

• !eptide bonds are amide bonds formed between thecarbo%ylic acid group of one amino acid and the amino

group of a second amino acid.


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• &ote: each amino acids consists of:

Carbo%ylic acid groups '(C))*+.

$mino groups '(&*2+.,ubstituent# or replaceable.

,ide chains '(-+.

• ifferent side

chain result in

various amino

acid .


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• The α carbon on all amino acids# e%cept glycine# is a

chiral carbon because it has four different groups

bonded to it.• Glycine: is simple amino acid because - chain is *.


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• Twenty different amino acids# which differ onlyin the structure of their side chains# are used byhuman cells to build proteins.

• The side chain structure determines the class ofthe amino acid: non(polar# neutral# basic# or

acidic.• *uman cells can synthesi/e most the aminoacids needed to build proteins. *owever# about10 amino acids# called essential amino acids

cannot be synthesi/ed by human cells and must be obtained from food.


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Essential Amino Acids in Humans

1. $rginine

2. *istidine

. soleucine

. Leucine

3. 4aline

5. Lysine

6. 7ethionine

8. Threonine

9. !henylalanine

10. Tryptophan


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Non-Essential Amino Acids in Humans

1. $lanine

2. $sparagine

. $spartate

. lutamate3. lutamine

5. lycine

6. !roline8. ,erine

9. Cysteine

10. Tyrosine


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Chemical Reactions of Amino Acids and Protein

Functional Groups

• Certain functional groups in amino acids and

proteins can react to produce characteristically

colored products.

• The color intensity of the product formed by a

particular group varies among proteins in

proportion to the number of reacting functional# or

free# groups present.

• n this part of e%periment# various color(producing

reagents 'dyes+ will be used to ;ualitatively detect

the presence of certain functional groups in amino

acids and proteins.


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E !"#$ %illon Reaction

• Principle: 7illontyrosine?

• Purpose: To detect the amino acid that have phenol group oftyrosin.

Note: some proteins containing tyrosine will initially form a

white precipitate that turns red when heated# while others forma red solution immediately. &ote that any compound with a

phenol group will yield a positive test# so one should be certain

that the sample being tested does not contain any phenols other

than those present in tyrosine.


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E !$ 'anthoprotic Reaction

• ,ome amino acids contain aromatic groups that are

derivatives of ben/ene.

• These aromatic groups can undergo reactions that are

characteristic of ben/ene and its derivatives.

• )ne such reaction is the nitration of a ben/ene ring with nitric

acid.

• The amino acids tyrosine and tryptophan contain activated

ben/ene rings and readily undergo nitration.

• The amino acid phenylalanine also contains a ben/ene ring#

but the ring is not activated and therefore does not undergo

readily nitration.


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• Principle: &itric acid gives color when heated

with proteins containing tyrosine 'yellow color+

or tryptophan 'orange color+B the color is due tonitration.

• Purpose: used to identify the presence of anactivated ben/ene ring.

Note: f one spills a concentrated solution of nitric

acid onto someone’s s"in. The proteins in s"in

contain tyrosine and tryptophan# which become

nitrated and turn yellow.


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Procedure

1. $dd 2 ml of protein solution in atest tube and add 2 drops of

concentrated nitric acid.

2. The formed white precipitate# willturn yellow upon heating# and

finally will dissolve giving a

yellow color to the solution.

. Cool the solution down. Carefully

add ml of 5 & &a)*. &ote that

the yellow color turns orange.


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E !(#$ Glyoylic Acid Reaction

!Hop)ins-Col* test#:

• Principle: The indole ring reacts with glyo%ylic

acid in the presence of a strong acid to form a

violet cyclic product.

• Purpose: The *op"ins(Cole test is specific for

tryptophan# the only amino acid containing indole

group. .


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Procedure:

• $dd 1 ml of protein solution in a test tube# add 1 ml

of *op"ins(Col reagent and mi% well.

• ncline the test tube and slowly add 1 ml of

concentrated *2,) on the inner wall of the test tube

thus forming a reddish ( violet ring at the interface of

the two layers.


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Result :

• $ reddish violet ring is

formed at the Dunction

between the 2 layers with

albumin and casein.

• elatin gives negative

results.


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E !+#$ Reduced sulfur test !,est for !-H# .roup#

• Principle: !roteins containing sulfur 'li"e:

7ethionine and cystine+ give a blac" deposit of

lead sulfide '!b,+ when heated with lead acetatein al"aline medium.

• Purpose: To detect amino acid which containe

sulfer group.

• Sulfur-containing protein ----> NaOH----> S2- ----Pb2+----> PbS


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Procedure:

1. $dd 1 ml of protein solution in a test tube# add 2 drops

of 10E sodium hydro%ide solution and 2 drops 'or few+

of lead acetate.

2. ,topper the tubes and sha"e them. -emove the stoppers

and heat in a boiling water bath for 3 minutes.

. Cool and record the results.


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Result :

• $ blac" deposit is formed with albumin• while a slight blac" turbidity is obtained with

casein due to its lower content of sulfur.

• elatin gives negative result.


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E !/#$ a)a.uchi ,est for Ar.inine:

• Principle: F( naphthol and sodium

hypobromite=chlorite react with the above mentioned

compound to form red orange comple%es.

• Purpose: used for the detection of a specific type of

protein with the amino acid containing the

guanidinium group 'e.g. arginine+.


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Procedure

1. To 1 ml of the protein solution add 1 ml of & &a)* solution and 0.3 ml of 0.1 E F( naphtholsolution# and a few drops of 2 E hypobromitesolution.

2. The presence of a guanidinium group in thecompound under e%amination will be confirmed

by the formation of a red color.


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Ninhydrin ,est

• &inhydrin is a chemical used to detect free amino acidand proteins

• $mino acids'&*2+ also react with ninhydrin at p*G.

• The reduction product obtained from ninhydrin thenreacts with &* and e%cess ninhydrin to yield a blue

colored substance.

• This reaction provides an e%tremely sensitive test for

amino acids.


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Procedure

• To 1 mL solution add 3 drops of 0.3E ninhydrinesolution

• Hoil over a water bath for 2 min.

• $llow to cool and observe the blue color formed.

• -esult: all amino acid will give purple or deep blue

with e%ception !roline gives yellow not violet.


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lab 1 qualitative tests 2-2-2014

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