Names: Bryle Kristiann Camarote Date Performed: February 26, 2014

Nimrod Romelo Date Submitted: March 12, 2014

Sarah Jane Valdon
Group#3

EXPERIMENT #10
Amines, Amino Acids, Proteins

I. INTRODUCTION

Amines are any organic derivative of ammonia (NH3) formed by the replacing one or more
hydrogen atoms by alkyl or aryl groups. Amines are stronger bases and better nucleophiles than
other neutral compounds. Amines are organic nitrogen compounds. formed by replacing one or
more hydrogen atoms of ammonia (NH3) with alkyl groups. They are classified as 1°, 2°, Like
ammonia, the amine nitrogen atom has a non-bonded electron pair, making it both a base and
a nucleophile. As a result, amines react with electrophiles to form ammonium salts—
compounds with four bonds to nitrogen, or 3° by the number of alkyl groups bonded to the
nitrogen atom. The chemistry of amines is dominated by the non-bonded electron pair on the
nitrogen atom. An NH2 group named as a substituent is called an amino group. There are
many different nitrogen heterocycles, and each ring type is named differently depending on the
number of N atoms in the ring, the ring size, and whether it is aromatic or not. Amines exhibit
dipole–dipole interactions because of the polar C – N and N – H bonds. Primary and secondary
amines are also capable of intermolecular hydrogen bonding, because they contain N – H
bonds. Because nitrogen is less electronegative than oxygen, however, intermolecular
hydrogen bonds between N and H are weaker than those between O and H.
The compounds called amino acids are more precisely call α-aminocarboxylic acids. There
are 20 naturally occurring amino acids. All except the proline are primary amines. Proline is a
secondary amine because its amino group is incorporated into a five-membered ring. The
amino acids differ only in the substituent (R) attached to the α- carbon. The large variation in
these side chains is what gives protein their great structural diversity and as a consequence,
their great functional diversity.
Peptides and proteins are polymers of amino acids linked together by amide bonds. Peptide
bonds and disulphide bonds are the only covalent bonds that hold amino acids together in
peptide or protein. The monomeric units are called amino acid residues. Protein molecules
assume several levels of structure. The primary structure if a protein is the sequence of the
amino acids in the chain and the location of all the disulphide bridges. Secondary structure is a
description of the conformation of the backbone of the protein.
 Tertiary structure is a description of the three-dimensional structure of the entire
polypeptide. If a protein has more than one polypeptide chain, it has quaternary structure. It is
the way the individual chains are arranged with respect to each other.
Proteins can be divided into two classes. Fibrous proteins contain long chains of
polypeptides that occur in bundles. These proteins are insoluble in in water. All the structural
proteins are fibrous proteins. Globular proteins are soluble in water and tend to have roughly
spherical shapes. Essentially all enzymes are globular proteins.
Diazonium compounds or diazonium salts are a group of organic compounds sharing a
common functional group R-N2+ X- where R can be any organic residue such alkyl or aryl and X is
an inorganic or organic anion such as a halogen. The process of forming diazonium compounds
is called "diazotation", "diazoniation", or "diazotization". The most important method for the
preparation of diazonium salts is treatment of aromatic amines such as aniline with nitrous
acid. Usually the nitrous acid is generated in situ (in the same flask) from sodium
nitrite and mineral acid. In aqueous solution diazonium salts are unstable at temperatures
above +5 °C; the -N+≡N group tends to be lost as N2 (nitrogen gas).
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or
primary and secondary amines. When reacting with these free amines, a deep blue or purple
color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect
fingerprints, as the terminal amines or lysine residues in peptides and proteins sloughed off in
fingerprints react with ninhydrin
Biuret test is used for detecting the presence of peptide bonds. It relies on the reduction
of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide
bonds in an alkaline solution. A violet color indicates the presence of proteins. It is possible to
use the biuret reaction to determine the concentration of proteins because (for most proteins)
peptide bonds occur with approximately the same frequency per gram of material.
In Heavy Metal Ions Test, heavy metal ions precipitate proteins from their solutions by
cross-linking free amino groups and carboxylate groups. Ions commonly used for testing for the
presence of protein include Zn2+, Fe 3+, Cu2+, Sb3+, Ag2+, Cd2+, and Pb2+. Among the metal ions,
Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious damage to proteins (especially
enzymes) by denaturing them.
Lead(II) acetate is a chemical compound, a white crystalline substance with
a sweetish taste. It is made by treating lead(II) oxide with acetic acid. Like other lead
compounds, it is toxic. Lead acetate is soluble in water and glycerin. With water it forms the
trihydrate, Pb(CH3COO)2·3H2O, a colorless or
white efflorescent monoclinic crystalline substance.
Aniline, phenylamine or aminobenzene is an organic compound with the formula C6H5NH2.
Consisting of a phenyl group attached to an amino group, aniline is the prototypical aromatic
amine.
 Being a precursor to many industrial chemicals, its main use is in the manufacture of
precursors to polyurethane. Like most volatile amines, it possesses the somewhat unpleasant
odour of rotten fish. It ignites readily, burning with a smoky flame characteristic of aromatic
compounds. Aniline is colorless, but it slowly oxidizes and resinifies in air, giving a red-brown
tint to aged samples.
Albumin refers generally to any protein that is water soluble, which is moderately soluble in
concentrated salt solutions, and experiences heat denaturation. They are commonly found in
blood plasma, and are unique to other plasma proteins in that they are not glycosylated.
Substances containing albumin, such as egg white, are called albuminoids.
Alanine (abbreviated as Ala or A) is an α-amino acid with the chemical
formula CH3CH(NH2)COOH. The L-isomer is one of the 22proteinogenic amino acids, i.e., the
building blocks of proteins. Its codons are GCU, GCC, GCA, and GCG. It is classified as
a nonpolaramino acid. L-Alanine is second only to leucine in rate of occurrence, accounting for
7.8% of the primary structure in a sample of 1,150proteins. D-Alanine occurs in bacterial cell
walls and in some peptide antibiotics.

II. DATA AND DISCUSSION

In this experiment, we investigate the different properties of Amines, Amino acids and
Proteins. We also investigate their solubilities in water and in aqueous acid; how are they
prepared and the different chemical reactions they undergo.

A. Solubility in Water

In this part of the experiment, ten drops of liquid samples were placed in a test tube
together with 2 ml distilled water. The mixture was the observed and was tested with red and
blue litmus paper. The results of this part of the experiment are summarized in Table 1 below.

COMPOUND OBSERVATIONS LITMUS PAPER TEST

Diethyl amine Brown solution; miscible Red to Blue
Dimethyl amine Clear solution; miscible Red to Blue
Aniline Immiscible Red to Blue
Table1. Solubility in Water of Amine Compounds

Amines are organic derivatives of ammonia (NH3) formed by replacing one or more
hydrogen atoms by alkyl or aryl groups. Amines are stronger bases and are better nucleophiles
among any other organic compounds.
 They are also classified as primary, secondary or tertiary based on the number of alkyls
groups bonded to the nitrogen atom. Because the nitrogen atom is more electronegative than
carbon or hydrogen, C-N and N-H bonds are all polar, with the N atom electron rich and the C
and H atoms electron poor.

Amines are highly polar and those with fewer carbon atoms are generally water-soluble.
The Hydrophobic R group present in amines makes them insoluble in water; whereas their
conversion in ammonium salts converts them into hydrophilic species due to the positive
charge on the nitrogen atom.

Depending on the number of carbon atoms bonded directly to nitrogen, amines are
classified as either primary (one carbon atom), secondary (two carbon atoms), or tertiary (three
carbon atoms). In general, if the total number of carbons attached to nitrogen is four or less,
the amine is water soluble; amines with carbon content greater than four are water insoluble.
Compared to organic amines and carboxylic acids of similar molecular weight, amino acids have
much lower melting points and are highly soluble in polar organic solvents, but only slightly
soluble in water. The amino and carboxylic acid groups of constituent amino acids, as well as
the nature of various side chains, allow proteins to possess some of these same properties. And
unlike that of the amines, solubility of amino acids and proteins is largely dependent on the
solution pH.

Based from the results of the experiment, Dimethyl amine and Diethyl amine were
found to be soluble in water, while Aniline was not. Dimethyl and Diethyl amines were polar
compounds because they contain one amine group attached to a methyl radical making it
soluble in polar water. Aniline (BP: 184 ) on the other hand, is a type of an arylamine. The
nitrogen lone-pair electrons in arylamines are shared by orbital overlap with the pi orbitals of
the aromatic ring. Therefore, they are less available for bonding making them insoluble in polar
water.
Amines are stronger bases than alcohols, ethers and water. When an amine is dissolved in
water, equilibrium is established in which water acts as an acid and donates H+ to the amine.
Based from the results above, Aniline, dimethyl and diethyl amines were found to be
basic.
B. Solubility in Aqueous Acid

In this part of the experiment, the aqueous solutions obtained in the Part A were
utilized here. The solutions were just added with 5 ml of concentrated HCl. The observations
form this experiment were summarized in the Table 2 below.

COMPOUNDS OBSERVATIONS
Dimethyl amine Clear/Miscible
Diethyl amine Clear/Miscible
Aniline Clear/Miscible/Gas Evolved
Table2. Solubility in Aqueous Acid of Amine Compounds

‘
As you can observe from the results of the experiment above, all amine compounds
were found to be all soluble in the aqueous acid. The insoluble Aniline in part A turned out to
be soluble due to the presence of HCl. The chemistry of amines is dominated by the lone pair
of electrons on nitrogen which accounts for their alkalinity. Like ammonia, most amines are
Brønsted and Lewis bases, but their base strength can be changed enormously by substituents.
It is common to compare alkalinities quantitatively by using the pKa's of their conjugate
acids rather than their pKb's. The higher the pKa is the stronger the base. As we all know, the
solubility of amino acids and proteins is dependent on the pH of the solution. In acidic
solutions, both amino and carboxylic groups are protonated while in basic solutions, both
groups are unprotonated. The ionizing power of concentrated HCl attracted the lone pairs from
the N-containing group which made the Aniline soluble.

In general, the differences in pH of amines are due to the availability of lone pairs of
nitrogen and various properties of substituents. The inductive effect raises the energy of lone
pairs and thus elevating the basicity. Also, delocalization affects the basicity. The strength of the
pKa of conjugate acid affects the alkalinity. The higher the pKa of amine, the more acidic it is.

One application of the knowledge about Amines is of the importance of milk and egg
used as antidotes in heavy metal poisoning. Proteins can be denatured (distorted in shape) by
heat, alcohol, acids, bases, or the salts of heavy metals. Many well-known poisons are salts of
heavy metals such as mercury and silver; these denature the protein strands wherever they
touch them. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals
with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction
of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.
The common first aid antidote for swallowing a heavy-metal poison is to drink milk or eat raw
egg. The poison then acts on the protein of the milk/egg rather than on the protein sites and
tissues of the mouth, esophagus, and stomach. Vomiting can be induced to expel the poison
that has combined with the milk/egg. In this sense, the egg white absorbs the metal, while the
milk is used to neutralize a part of the stomach acid, so the metals are less soluble and on the
other hand, slows down absorption in the digestive system.

C. Preparation and Reactions of Diazonium Salts

1. Preparation of Diazonium salt

In this part of the experiment, 1 gram of Sulfanilic acid was added to the mixture of
8 ml 1% NaNO2 and 8 mL 2% HCl.
The mixture was allowed to stand for 1 minute in an ice bath, keeping the mixture
for further usage in parts 2 and 3. The reactions and the mechanisms involved in the
Preparation of Diazonium salt are shown below.
 2. Replacement Reaction of Diazonium salt

In this part, 0.5 ml of 3 M H2SO4 was added to the Diazonium salt solution prepared in
part 1. This was heated for several minutes in a hot water bath. The observations obtained from
this part were summarized in table 3 below.

OBSERVATIONS (for Diazonium salt + H2SO4)

Before heating, solution was almost colorless. But after several minutes of heating, the solution
started to change to a transparent pale orange solution.
Table3. Observations for the Replacement Reaction of Diazonium Salt

In the replacement reaction of diazonium salt, the diazonium group being a very good
leaving group is lost as N2. The positive charge in N= N causes it to be a withdrawing group of
substituent of the aromatic ring. Some other atom or group gets attached to the aromatic ring
system in its place through the same mechanism as electrophilic aromatic substitution.
Furthermore, in the presence of sulfuric acid, the diazonium group leaves the ring and is
replaced by –SO3H (Sulfonic Acid).

Diazonium salts - in aqueous solution, are unstable at temperatures higher than 5°C
since this temperature can decompose them to give phenol and gaseous N2.

One can isolate diazonium compounds as the crystalline tetrafluoroborate (BF4) salt,
which is stable at room temperature. However, diazonium compounds are typically not
isolated. After they have been prepared, they are used immediately in further reactions.

Otherwise, rapid decomposition proceeds on standing even when cold. Therefore, to

prevent immediate decomposition, as it was done in the experiment, the salt was kept in an ice
bath until its usage in the next parts of the experiment.

In this case, the purpose of cooling the system thoroughly is that in aqueous solutions,
the diazonium salts are unstable at temperatures higher than 5 ºC. The N +≡N group tends to be
lost as N2 so it is important to cool the system.
3. Coupling Reaction of Diazonium Salt

After preparing and conducting a replacement reaction to Diazonium salt, 4 drops of

N,N-dimethylaniline was prepared in the test tube together with 2 ml of water. 6M HCl was
then added dropwise until the amine just dissolves. 3 ml of the Diazonium salt was also added
together with a slight excess of 50% sodium acetate solution. The results and observations
obtained from the experiment were summarized in Table 4 below.

OBSERVATIONS (for Diazonium salt + dimethylaniline + HCl + CH3COONa)

After the 3 ml of the Diazonium salt was added, the solution became transparent red. Addition
of the slight excess of 50% NaOAc solution produced an even redder solution. Finally, after
excessive addition of 50% NaOAc solution, it formed a dark red-orange precipitate and the
solution became opaque, bloody-red.
Table4. Observations for the Coupling Reaction of Diazonium Salt

In the coupling reaction of diazonium salt, the reaction between diazonium salt and
dimethylaniline produces an azo compound. This coupling reaction is often referred as Azo
coupling; It is the most widely used industrial reaction in the production of dyes, lakes and
pigments. Aromatic diazonium ions act as electrophiles in coupling reactions with activated
aromatics such as anilines or phenols. The substitution normally occurs at the para position,
except when this position is already occupied, in which case ortho position is favored. The pH of
solution is quite important; it must be mildly acidic or neutral, since no reaction takes place if
the pH is too low.
In this reaction, the diazonium salt is an electrophile and the dimethylaniline is a
nucleophile in electrophilic aromatic substitution. Only high activated benzene rings such as
dimethylaniline can undergo electrophilic aromatic substitution reaction with aryl diazonium
electrophiles.
The important method in the preparation of diazonium salts is the treatment of
aromatic amines, such as aniline, with a nitrous acid (sodium nitrite). This is done in the
presence of a mineral acid (HCl) making the addition of some HCl necessary until the N,N-
dimethylamine dissolves. The solution must be mildly acidic or neutral, since no reaction takes
place if the pH is too low.
Afterwards, some of the previously prepared diazonium salt was added to the solution
as well as a slight excess of sodium acetate. This resulted to a deep red solution with yellow
precipitate settling at the bottom. In the diazonium coupling reactions, they follow the typical
electrophilic aromatic substitutions in which the positively charged diazonium ion is the
electrophile that reacts with the electron-rich ring of a phenol or an arylamine. Shown below
are the reaction mechanisms involved in the Azo Coupling Reaction:
General Mechanism:

Reaction of aniline and the Diazonium salt Mechanism:

An Azo Dye

D. Color Reaction of Amino Acids and Proteins

1. Ninhydrin test (Perform using aniline, alanine, and casein)

In this part of the experiment, a small amount of sample was spotted on a strip of filter
paper. This was allowed to dry and was later dipped with 2% Ninhydrin solution in acetone. This
strip was allowed to stand for 30 minutes until a color development is observed. The presence
of a violet coloration indicates a positive result of this test. Table 5 below summarizes the
results of this part of the experiment.

COMPOUND OBSERVATIONS
Aniline No blue-violet coloration (Yellow only) (-)
Alanine Blue-violet coloration appeared (+)
Casein Blue-violet coloration appeared (+)
Table5. Ninhydrin Test Result
 The Ninhydrin test is a test for detecting the presence of -amino acids wherein a blue
to purple product denotes a positive result. Spots of the samples in a filter paper were dipped
on a Ninhydrin solution and the solvent was allowed to evaporate to develop color. When
heated with Ninhydrin, these molecules give characteristic deep blue colors. The results show
that alanine and casein contain or is an amino acid whereas aniline does or is not. The reaction
mechanisms are shown below.

Blue-ion Complex

*General Reaction Mechanisms:

 2. Biuret test (Using Alanine, Casein and Albumin)

In this part of the experiment, 2ml of aqueous samples were placed in a test tube. 2ml
of 10% NaOH together with 1 drop of 2% CuSO4 and then the mixture was examined. Water
blank was run also for comparison. The formation of a blue-violet complex signifies a positive
result of this test. Table 6 summarizes all the results from this part of the experiment.

COMPOUND OBSERVATIONS
Albumin Blue-violet complex seen (+)
Casein Blue-violet complex seen (+)
Alanine None (-)
Water Blank Faint Blue-green
Table6. Results of the Biuret Test

The biuret test is a chemical test used for detecting the presence of peptide bonds or
proteins in solution. In the presence of peptides, a Copper (II) ion forms violet-colored
coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the
concentration of proteins because peptide bonds occur with the same frequency per amino
acid in the peptide. The intensity of the color, and hence the absorption at 540 nm, is directly
proportional to the protein concentration, according to the Beer-Lambert law.
Despite its name, the reagent does not in fact contain biuret ((H2NCO-)2NH). The test is
so named because it also gives a positive reaction to the peptide-like bonds in the biuret
molecule.
A positive result is denoted by a deep blue-violet color. The Biuret reagent is made of
sodium hydroxide (NaOH) and hydrated Copper (II) sulfate, together with potassium sodium
tartrate. Potassium sodium tartrate is added to complex to stabilize the cupric ions. Proteins in
the alkaline environment reduce Cu2+ to Cu+, which forms a coordination complex with
proteins, leading to a blue color change.
Based from the results shown above, Alanine produced a negative result which was
confirmed by the water blank in which their colors were actually close to each other
qualitatively. On the other hand, a deep blue-violet or purple was seen in both the albumin and
casein which confirms the positive result of this test, signifying that they are both proteins and
that they contain peptide bonds.
Biuret test does not, however, indicate the presence of proteins or polypeptides. Other
tests must be used to indicate whether the substance testing positive during the Biuret test is
either a polypeptide or a protein. Also, it is possible to obtain a false result even if there are
peptide bonds in a substance because the resulting purplish color is too faint. This could have
been the case in the experiment. Phosphoprotein casein and alanine have the formation of
fainted purple solution, which in our case a blue solution with insoluble precipitates. The said α-
amino acid, a building block of protein, may have contained peptide bonds yet it had a
seemingly negative result.
 The Blue-violet colored Complex

3. Reaction with Lead acetate [Pb(CH3COO)2]

In the last part of the experiment, two strands of hair were placed in a test tube. 1ml of
20% NaOH together with 2 drops of 10% Lead acetate was added. The mixture was then boiled
for 5 minutes and was examined. The formation of a black precipitate indicates a positive result
for this test. Summary of the results and observations obtained in the experiment are shown in
Table 7 below.

OBSERVATIONS (for two strands of Hair)

Black solution resulted (+)
Table7. Observations for the Reaction with Lead acetate

Lead (II) acetate is a substance used as a fixative for some dyes asides from being a
reagent to make other lead compounds, but is made by treating litharge (lead (II) oxide, PbO)
with acetic acid. Lead acetate, in low concentrations, is the principal active ingredient in
progressive types of hair coloring dyes. These products are applied over a period of time to
achieve a gradual coloring effect. Structural proteins present stiffness and rigidity to otherwise-
fluid biological components. Most structural proteins are fibrous proteins but these polymerize
to form long, stiff fibers that comprise the cytoskeleton, which allows the cell to maintain its
shape and size. Also, the hair integration shown in the reaction proves that Lead acetate reacts
with the protein.

In this experiment, reaction with Lead acetate was a test for Sulfur-containing Amino
acids. They also combine with the protein in the hair in a sufficiently alkaline and hot solution
to give a dark, almost black color.
III. CONCLUSIONS

Among all the amines, amino acids, and proteins, dimethylamine was the only
insoluble compound. Aniline, casein, methylamine, n,n-dimethylaniline, and albumin
were all soluble although the proteins were not tested, they were expected to be
soluble in water because the amino acids, which are contained in proteins, were
completely soluble in water.
In the reactions undergone by diazonium salt, diazonium group being a best
leaving group is lost as N2 in the spare reaction of diazonium salt. The positive charge in
N≡N roots it to be a withdrawing group. The mechanism that it undergoes is
electrophilic aromatic substitution. The diazonium group leaves the ring and is replaced
by –SO3H In the presence of sulfuric acid. In the coupling reaction of diazonium salt, the
reaction between diazonium salt and dimethylaniline produces an azo compound.
Diazonium salts, in aqueous solution, are unstable at temperatures higher than 5°C
since this can decompose to give phenol and gaseous N2. Therefore, as it was done in
the experiment, the salt was kept in an ice bath until its usage in the next parts of the
experiment.
Alanine and casein maybe an amino acid or a protein as confirmed by the
Ninhydrin test, which is used to detect amino acid or a group of amino acids (proteins).
Albumin and casein are protein which is confirmed by the biuret test, or the test for
presence of proteins. Thus, we can infer from these tests that alanine is only an amino
acid and not a protein, and that casein and albumin are proteins that contain amino
acids.
Therefore several tests allowed us to identify somehow among compounds if
they are amines or amino acids or even proteins.

IV. REFERENCES

Smith, Michael B.; March, Jerry (2007), Advanced Organic Chemistry: Reactions, Mechanisms,
and Structure (6th ed.), New York: Wiley-Interscience, ISBN 0-471-72091-7 Klaus Hunger, Peter
Mischke, Wolfgang Rieper, Roderich Raue, Klaus Kunde, Aloys Engel "Azo Dyes” in Ullmann’s
Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH,
Weinheim.doi:10.1002/14356007.a03_245.
J. L. Hartwell and Louis F. Fieser, "Coupling of o-tolidine and Chicago acid", Org. Synth.; Coll. Vol.
2: 145
H. T. Clarke and W. R. Kirner, "Methyl red", Org. Synth.; Coll. Vol. 1: 374
McMurry, et.al, 7th Philippine Edition, Organic Chemistry, 2004.