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7/16/2019 35770653 Qualitative Tests
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Qualitative Tests
Proteins & Amino Acids in Saliva
Co. Cordero. Cruz.
De Jesus
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Sakaguchi Test Test for the presence of guanidine
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Sakaguchi Test Test for the presence of guanidine
ARGININE
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Mechanism:Sakaguchi Test Reagents:
α -naphthol
NaOH *NaOCl
+ NaOH, pH
zwitterionic form
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Sakaguchi Test
condensation reaction with
red/ wine-colored
solution
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For compounds containing a phenolic hydroxy group
Amino acid: Tyrosine
Compound must be
validated as protein/
amino acid to confirm
presence of tyrosine
Millon-Nasse Reaction
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Millon-Nasse Reaction Millon’s Reagent: Mercuric ion in acid
Mechanism:
Tyrosine + Millon’s Reagent = complex
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Millon-Nasse Reaction Complex treated with Nitrous Acid (NaNO2) yields a
pink-red solution
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XANTHOPROTEIC REACTION
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XANTHOPROTEIC REACTION Boiling concentrated nitric acid reacts with tyr, trp
and phe to yield yellow products.
This reaction involves the nitration of benzene nucleusin alkaline medium. As a result, amino acids thatcontain aromatic nucleus undergo this reaction.
Aromatic AAs form yellow nitro derivative on heating
with concentrated nitric acid, the salts of thisderivative are orange.
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XANTHOPROTEIC REACTION
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(A) NITRATED TYROSINE AND
TRYPTOPHAN (B)
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XANTHOPROTEIC REACTION Using 65% nitric acid the aromatic rings of amino
acids like tyrosine and tryptophan are nitrated. Thenitro derivate show an intensely yellow color. Becausenearly all proteins contain aromatics it is taken as aprotein-test either.
The yellow stains on the skin caused by nitric acid arethe result of the xanthoproteic reaction. The epidermiscells of the skin contain aromatic proteins.
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NINHYDRIN
REACTION
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NINHYDRIN REACTION Triketohydrindene hydrate, commonly
known as ninhydrin reacts with aminoacids to form a purple colored iminoderivative. This derivative forms a
useful test for amino acids, most of which are colorless.
Ninhydrin is a powerful oxidizingagent which reacts with all amino acidsbetween pH 4-8 to produce a purple-colored compund
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NINHYDRIN REACTION A color reaction given by amino acids
and peptides on heating with thechemical ninhydrin
The amino acids proline andhydroxyroline also reacts but producesa yellow color.
Ninhydrin (triketohydrindene
hydrate) is an oxidating agent whichleads to the oxidative deamination of alpha-amino groups. It is very important for the detection and thequantitative analysis of amino acids.
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NINHYDRIN REACTION Ninhydrin also reacts with primary
amines however the formation of
carbon dioxide is quite diagnostic foramino acids.
Alpha amino acids yield a purplesubstance that absorbs maximally at
570 nm. Imino acids (proline) yield a yellow product (absorption maximum440 nm).
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NINHYDRIN REACTIONα -amino acid + 2ninhydrin
CO2 + aldehyde + final complex(purple) +3H2O
Ninhydrin, which is originally yellow,reacts with amino acid and turns deeppurple color that is detected in thismethod.
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Biuret Test Presence of peptide bonds is detected by performing a
chemical test named biuret test.
The Biuret Reagent is made of sodium hydroxide andcopper sulfate. The blue reagent turns violet in thepresence of proteins, and changes to pink when
combined with short-chain polypeptides.
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Cupric ion in an alkaline medium forms a violetcoloured complex with peptide bond nitrogens of peptides and proteins.
The reaction is so named biuret(NH2CONHCONH2)formed by condensation of two molecules of urea,
when heated at 180C, also answers this test. Theminimum requirement for a positive test is thepresence of 2 peptide bonds in the molecule.
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It yielded a positive result for
saliva thus, saliva containsproteins.
It yielded a negative result forglycine.
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Saliva Glycine
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Bromine Water test The bromine water test is an example of an addition
reaction.(A reaction in which a small molecule adds onacross a double bond).
The decoloration of a solution of bromine in water isan analytical test for the presence of alkenes:
CH2=CH2 + Br2 → BrCH2-CH2Br
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Alkenes are able to undergoaddition reactions becausethey contain a double bond.They decolourise because
they are unsaturated andhave a carbon=carbon doublebond
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Tryptophan
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Formation of pinkishlayer is the positiveresult.
Tryptophan is positiveunder the bromine water
test
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Pauly Reaction
Diazotization
General Mechanism:
Sulfanilic acid gets diazotized in the presence of
sodium nitrite (NaNO2) + sample + sodium carbonate(Na2CO3)
POSITIVE TEST : dark yellow or orange
: histidine and tyrosine residues
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Samples
Saliva : +
: contains histidine and tyrosine
Histidine : +
Tyrosine : +
* Theoretical result
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Saliva
Histidine
Tyrosine
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Lead Acetate Reaction
Liberates sulfur content to detect (cys)
Sulfur group of cysteine is liberated through heating with
strong alkali
Treatment with alkali does not liberate sulfur from
methionine
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POSITIVE TEST :gray
: cysteine (cys)
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cys
saliva
hair
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* Theoretical result
Samples
Saliva : (-)
Hair : (-)
: high amount of sulfur due to cysteine
Cysteine: (+)
: sulfur was liberated
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Referencesn.a.(n.d.). Amino Acids. Date Retrieved August 2,2010,from
http://home.earthlink.net/~dwyerg/HL%20Labs/proteins%20and
%20amino%20acids.htm
n.a.(n.d.). Diazotization. Date Retrieved August 2,2010,from
http://www.ecompound.com/Reaction%20reference/reactions/D
iazotization%20related%20reactions.gif
n.a.(n.d.). Hair fibers. Date Retrieved August 3,2010,from
http://www.keratin.com/aa/aa012.shtml