Detective tests for amino

acids

A report submitted to the

Department of dentistry
University of Duhok

Student name:Mahsum muhsin avdal

Moodle Email:[email protected]
Year: 1st
Course:basic biochemistry
Course code:TNT121
Instructor:Dr.lina Y. Mohammad
Date:26/6/2020
Introduction
Detectives use ninhydrin to reveal fingerprints left at crime scenes. Ninhydrin reacts with
amino acids found in the natural oils on our skin to produce a purple product. The intensity of
the color may also be used as quantitative test for the amount of amino acids in a sample.
Amino acids are the building blocks of proteins

Amino acids are a vital part of diets for both humans and animals. Several of the amino
acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan, and valine) can not be produced by the human body and are therefore
considered “essential”. A lack of any of the essential amino acids in the diet can lead to
poor health and death. In addition, what constitutes essential amino acids can vary. For
example, cats cannot synthesize taurine and lack of taurine causes eye-problems, hair
loss, and tooth decay. For that reason, amino acid testing is important to determine the
quality of protein in food, pet food, and animal feeds.

Eurofins is the leader in amino acid analysis offering a variety of methods that can
quickly and accurately quantify the amino acids present in almost any sample. Eurofins
has established methods in testing for free amino acids as well as the bound molecules
in peptide form.

Our technical expertise and market knowledge allow our clients to verify compliance
with the latest regulations in each country and market, both for raw materials and
finished goods.
Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-
CONH-) to form the primary structure of a protein. Amino acids possess an amine group, a
carboxylic acid group and a varying side chain that differs between different amino acids.

There are 20 naturally occurring amino acids, which vary from one another with respect to their
side chains. Their melting points are extremely high (usually exceeding 200°C), and at their pI,
they exist as zwitterions, rather than as unionized molecules.

Amino acids respond to all typical chemical reactions associated with compounds that contain
carboxylic acid and amino groups, usually under conditions where the zwitter ions form is
present in only small quantities. All amino acids (except glycine) exhibit optical activity due to
the presence of an asymmetric α – Carbon atom. Amino acids with an L – configuration are
present in all naturally occurring proteins, whereas those with D – forms are found in antibiotics
and in bacterial cell walls.

                                                
                                                                        FIG 1.  STRUCTURE OF AMINO ACID
Materials Required:

1) Glasswares

2)    Testtubes

3)     Testtubeholder

4)     Waterbath

5)     Spatula

6)     Dropper

7)     Bunsenburner

8)     Icebox

9)     Vortexmixer

10)  Filterpaperstrip

11)   Hair drier

Materials Required:

1) Glass wares

2) Test tubes

3) Test tube holder

4) Water bath

5) Spatula

6) Dropper

7) Bunsen burner

8) Icebox
‫ـ‬
9) Vortex mixer

10) Filter paper strip

11) Hair drier

Reagents Required:

1. Dihydric reagent: Ninhydrin (2%W/V) in acetone

2. Sodium hydroxide (40%W/V)

3. Conc. Nitric acid

4. Hydrochloric acid (6N)

5. Sulphanilic acid (1%W/V) in 1N HCl

6. Sodium nitrite (5%W/V) in distilled water ( to be freshly prepared)

7. Sodium carbonate (10%W/V) in distilled water

8. Millon’s reagent ( 15%W/V mercuric sulphate in 6N sulphuric acid)

9. Sodium nitrite solution(5%) [To be freshly prepared]

10. Bromine (5%) in acetic acid(33%) solution

11. Amonium carbonate(5%)

12. Acetic acid – Glyoxilic acid reagent – Glacial acetic acid is exposed to sun light ( for
5 – 6 hours) for the formation of small amounts of glyoxilic acid)

13. Con. Sulphuric acid

14. Sodium hydroxide (10%W/V)

15. α Naphthol reagent (1%W/V in ethyl alcohol)

16. Hypobromite solution ( To be freshly prepared) : -Take 100 of 5%(W/V) sodium

hydroxide solution in a glass reagent bottle and add 1ml of pre chilled liquid bromine,
using a pro pipette. Shake the contents till bromine dissolves)

17. Urea solution 5% (W/V)

18. Lead acetate solution(10%W/V)

19. 5N NaOH

20. 1% Glycine solution

21. 10% Sodium nitroprusside solution

22. Isatin reagent : Isatin (1% W/V) in acetic acid

Abstract
We describe a method to identify and quantify amino acids using capillary
electrophoresis‐electrospray ionization‐triple‐quadrupole tandem mass spectrometry
(CE‐ESI‐MS/MS). Amino acids, including physiological amino acids, were first
separated by CE under acidic pH conditions and then detected by MS/MS. To efficiently
introduce the whole sample into the capillary, no electrical potential was applied to the
electrospray probe until running electrophoresis. The position of the electrosprayer with
respect to the MS capillary entrance drastically affected sensitivity and generation of
cluster ions. MS/MS with multiple reaction monitoring (MRM) detection was performed
to obtain sufficient selectivity and sensitivity. Under optimized CE‐MS/MS conditions,
the minimum detectable levels for 32 free amino acids normally found in proteins and
other physiological amino acids were between 0.1 and 14 μmol/L with pressure injection
of 50 mbar for 3 s (3 nL) at a signal‐to‐noise ratio of 3. For most amino acids, this
constitutes a severalfold increase in sensitivity compared to CE‐MS. The relative
standard deviations (% RSD) for all amino acids were better than 0.4% for migration
times and between 1.4% and 8.6% for peak areas (n = 10). Since amino acids exhibited
characteristic MS/MS spectra, this approach is useful for the simultaneous, selective,
quantitative, and reproducible analysis of amino acids in physiological and biological
samples that contain various kinds of matrices. The power of the method was
demonstrated by analyzing amino acids in human urine.

Amino acids are molecules containing an amine group, a carboxylic acid group and a
side chain that varies betwen different amino acids. Amino acids of the general formula
RCH(NH2)COOH are amphoteric, behaving as amines in some reactions and as
carboxylic acids in others. At a certain pH known as the isoelectric point an amino acid
has no overall charge, since the number of protonated ammonium groups (positive
charges) and deprotonated carboxylate groups (negative charges) are equal. Since the
amino acids at their isoelectric points have both negative and positive charges, they are
known as zwitterions.
Amino acids are critical to life. They have particularly important functions like being the
building blocks of proteins and being the intermediates in metabolism.
Amino acids are generally classified by the properties of their side chain into four
groups. The side chain can make an amino acid a weak acid or a weak base, and a
hydrophile if the side chain is polar or a hydrophobe if it is nonpolar.
TESTS ON AMINO ACIDS:

1) Solubility Tests:

The solubility of amino acids and proteins is largely dependent on the solution pH. The
structural changes in an amino acid or protein that take place at different pH values alter
the relative solubility of the molecule. In acidic solutions, both amino and carboxylic
groups are protonated. In basic solutions, both groups are deprotonated.
Amino acids are essentially soluble in water. Their solubilities in water, dilute alkali and
dilute acid vary from one compound to the other depending on the structure of their
side chains. Apply this test to glycine, tyrosine, glutamic acid and cysteine.

Procedure::
- Note the solubility of amino acids in water and alcohol by placing a small amount
in a test tube, adding a few mL of solvent and warming if necessary.
- Determine the amino acid solution is acidic or basic by using a litmus paper while
- testing the solubility in water.
- Repeat the solubility test using dilute HCl and dilute NaOH.s
2) Ninhydrin Test:

Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammoniaor

primary
and secondary amines. Amino acids also react with ninhydrin at pH=4. The
reduction
product obtained from ninhydrin then reacts with NH3 and excess ninhydrin to
yield a
blue colored substance. This reaction provides an extremely sensitive test for
amino
acids. Apply this test to any of the amino acids you choose.

WARNING: Avoid spilling ninhydrin solutions on your skin, as the resulting stains
are
difficult to remove. (Ninhydrin is the most commonly used method to detect
fingerprints, as the terminal amines or lysine residues in peptides and proteins
sloughed
off in fingerprints react with ninhydrin).

Procedure:

- To 1 mL amino acid solution add 5 drops of 0.2% ninhydrine solution in

acetone.
- Boil over a water bath for 2 min.
- Allow to cool and observe the blue color formed.
3) Stability to Alkali

Amino acids, unlike amides and volatile amines, do not evolve NH3 or alkaline
vapor
when boiled with alkali. This method can be used to differentiate amino acids
from
amines and amides. Apply this test to the provided amine or amide and also to
glycine.

Procedure:

- Pipette 1 mL 1% glycine and the amide or amine solution into separate test
tubes.
- Add 1 mL dilute NaOH to each test tube and boil.
- Test the vapor from each boiling tube with wet litmus paper.
4) Specific Reactions for Individual Amino Acids:
WARNING: Please DO NOT use vast amounts of solution for these tests, since
most of the
amino acids are very expensive!!

a) Xanthoproteic Test:

Some amino acids contain aromatic groups that are derivatives of benzene.
These
aromatic groups can undergo reactions that are characteristics of benzene and
benzene
derivatives. One such reaction is the nitration of a benzene ring with nitric acid.
The
amino acids that have activated benzene ring can readily undergo nitration. This
nitration reaction, in the presence of activated benzene ring, forms yellow
product.
Apply this test to tyrosine, tryptophan, phenylalanine and glutamic acid.

Procedure
:
- To 2 mL amino acid solution in a boiling test tube, add equal volume of
concentrated HNO3.
- Heat over a flame for 2 min and observe the color.
- Now COOL THOROUGHLY under the tap and CAUTIOSLY run in sufficient
40%
NaOH to make the solution strongly alkaline.
- Observe the color of the nitro derivativative of aromatic nucleus.
 b)Millon’s Test

Millon’s test is specific to phenol containing structures (tyrosine is the only

common
phenolic amino acid). Millon’s reagent is concentrated HNO3, in which mercury is
dissolved. As a result of the reaction a red precipitate or a red solution is
considered
as positive test. A yellow precipitate of HgO is NOT a positive reaction but
usually
indicates that the solution is too alkaline. Apply this test to tyrosine,
phenylalanine,
glycine and β-naphtol.

Procedure:

- To 2 mL amino acid solution in a test tube, add 1-2 drops of Millon2s reagent.
- Warm the tube in a boiling water bath for 10 min.
o A brick red color is a positive reaction.
o Note that this is a test for phenols, and the ninhydrin test should also be
positive if it is to be concluded that the substance is a phenolic amino acid.

c)Hopkin’s Cole Test:

The indole group of tryptophan reacts with glyoxylic acid (glacial acetic acid, which has
been exposed to light, always contains glyoxylic acid CHOCOOH as an impurity) in the
presence of concentrated H2SO4 to give a purple color. Apply this test to glycine,
tryptophan and tyrosine.

Procedure:
- To a few mL of glacial acetic acid containing glyoxylic acid, add 1-2 drops of the
amino acid solution.
- Pour 1-2 mL H2SO4 down the side of the sloping test tube to form a layer
underneath the acetic acid.
- The development of a purple color at the interface proves a positive reaction.

d)Lead-Sulfide Test:

When cystine is boiled with 40% NaOH, some of sulfur in its structure is coverted to
sodium sulfide (Na2S). The Na2S can be detected by using sodium plumbate solution
which causes the precipitation of PbS from an alkaline solution. In order to apply this
test, first the sodium plumbate solution should be prepared. Apply this test to cysteine
and cystine.

Procedure:

- Sodium Plumbate Solution Preparation:

o Add 5 mL dilute NaOH to 2 mL dilute lead acetate.
o A white precipitate of lead hydroxide forms.
o Boil until the precipitate dissolves with the formation of sodium plumbate.
- Boil 2 mL amino acid solution with a few drops of 40% NaOH for 2 min.
- Cool and add a few drops of the sodium plumbate solution.
- A brown color or precipitate is a positive test for sulfides.
e) Ehrlich Test:
Aromatic amines and many organic compounds (indole and urea) give a colored
complex with this test. Apply this test to tryptophan, urea and glycine.

Procedure:

- Put 0.5 mL of the amino acid solution to a test tube.

- Add 2 mL Ehrlich reagent and observe the color changes

f) Sakaguchi Test:

The Sakaguchi reagent is used to test for a certain amino acid and proteins. The amino
acid that is detected in this test is arginine. Since arginine has a guanidine group in its
side chain, it gives a red color with α-naphthol in the presence of an oxidizing agent like
bromine solution. Apply this test to arginine.

Procedure:

- 1 mL NaOH and 3 mL arginine solution is mixed and 2 drops of α-naphthol is

added.
- Mix thoroughly and add 4-5 drops of bromine solution UNDER THE HOOD!!
- Observe the color change.
g) Nitroprusside Test:

The nitroprusside test is specific for cysteine, the only amino acid containing sulfhydryl
group (-SH). This group reacts with nitroprusside in the presence of excess ammonia.
Apply this test cysteine, cystine and methionin.

Procedure:

- Put 2 mL amino acid solution into the test tube.

- Add 0.5 mL nitroprusside solution and shake thoroughly.
- Add 0.5 mL ammonium hydroxide.
- Observe the color change