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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