35770653 Qualitative Tests

7/16/2019 35770653 Qualitative Tests

http://slidepdf.com/reader/full/35770653-qualitative-tests 1/36

Qualitative Tests

Proteins & Amino Acids in Saliva

Co. Cordero. Cruz.

De Jesus



Sakaguchi Test Test for the presence of guanidine



Sakaguchi Test Test for the presence of guanidine

ARGININE



Mechanism:Sakaguchi Test Reagents:

α -naphthol

NaOH *NaOCl

+ NaOH, pH

zwitterionic form



Sakaguchi Test

condensation reaction with

red/ wine-colored

solution



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



Millon-Nasse Reaction Millon’s Reagent: Mercuric ion in acid

Mechanism:

Tyrosine + Millon’s Reagent = complex



Millon-Nasse Reaction Complex treated with Nitrous Acid (NaNO2) yields a

pink-red solution



XANTHOPROTEIC REACTION



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.



XANTHOPROTEIC REACTION



(A) NITRATED TYROSINE AND

TRYPTOPHAN (B)



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.



NINHYDRIN

REACTION



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



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.



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).



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.



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.



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.







It yielded a positive result for

saliva thus, saliva containsproteins.

It yielded a negative result forglycine.



Saliva Glycine



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



Alkenes are able to undergoaddition reactions becausethey contain a double bond.They decolourise because

they are unsaturated andhave a carbon=carbon doublebond



Tryptophan



Formation of pinkishlayer is the positiveresult.

Tryptophan is positiveunder the bromine water

test



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



Samples

Saliva : +

: contains histidine and tyrosine

Histidine : +

Tyrosine : +

* Theoretical result



Saliva

Histidine

Tyrosine



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

http://en.wikipedia.org/wiki/File:Methionin_-_Methionine.svg

http://en.wikipedia.org/wiki/File:Cysteine.svg

http://en.wikipedia.org/wiki/File:Cysteine.svg



POSITIVE TEST :gray

: cysteine (cys)



cys

saliva

hair



* Theoretical result

Samples

Saliva : (-)

Hair : (-)

: high amount of sulfur due to cysteine

Cysteine: (+)

: sulfur was liberated



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

Date post:	30-Oct-2015
Category:	Documents
Upload:	anirbanmanna88320
View:	44 times
Download:	0 times

35770653 Qualitative Tests

Documents