Rogeni Misaelle M.

Petinglay Date performed: April 24, 2017

B.S. in Chemistry II Date submitted: May 8, 2017

Experiment No. 12
AMINES, AMINO ACIDS AND PROTEINS

I. Summary of Results

Table 1. Solubility Test

Water solubilty
Test w/ Test w/
Sample blue red Aqueous acid solubility
Obsevations
litmus litmus
paper paper
Forms gas, exothermic,
Soluble, homogenous and
Methylamine B -> B R -> B colorless and oily solution,
colorless solution
soluble
Forms gas, exothermic,
Soluble, homogenous and
Dimethylamine B -> B R -> B colorless and oily solution,
yellowish solution
soluble
Soluble, homogenous and Forms gas, clear dark brown
Aniline B -> B R -> R
colorless solution solution, exothermic,soluble
Insoluble, heterogenous and Forms gas, exothermic,
Hair strand B -> B R -> R
colorless solution clear oily solution
Insoluble, heterogenous and
colorless solution with Forms gas, exothermic,
Alanine B -> B B -> R
reddish brown oily insoluble clear oily solution
particles, bubble suspensions
Insoluble, homogenous and
Forms gas, exothermic,
Casein colorless solution with white B -> B R -> R
clear oily solution
precipitates
Insoluble, turbid white Forms gas, exothermic, very
Albumin solution with gel like B -> B R -> R fine white precipitate,
particles soluble

Table 2. Preparation and Reactions of Diazonium Salts

Preparation of Replacement Reaction Coupling reaction of diazonium salt
diazonium salt of diazonium salt
Pale yellow Upon shaking,the 12 drops of HCl were used to eliminate the amine but
solution with solution slightly turned there are still little brown emulsions. Addition of
settled white golden brown to red diazonium salt turned the solution into dark red orange
precipitate orange solution solution. Addition of sodium acetate turned the
solution into darker red orange with dark red
precipitate
Table 3. Color Reaction of Amino Acids and Proteins

Ninhydrin test
Alanine Violet (+)
Aniline Pale orange inside the spot and yellow on the surface(-)
Casein Pale violet (+)

Biuret test
Blank Pale blue, homogeneous solution
Alanine Pale blue, homogeneous solution(-)
Casein Pale turbid violet solution with white precipitates(+)
Pale turbid violet solution with gel-like white particles and blue
Albumin
precipitates (+)

Reaction with Lead Acetate

The strands dissolved and turned the solution into black with black
Hair strands
precipitates.

II. Discussion

Amines are compounds and functional groups that contain a basic nitrogen atom with a
lone pair. Amines are formally derivatives of ammonia, wherein one or more hydrogen atoms
have been replaced by a substituent such as an alkyl or aryl group. Organic compounds
containing amine (-NH2) and carboxyl (-COOH) are called amino acids. These amino acids are
the building blocks of proteins.

In this experiment, the differences in the chemical reactivity of amines, amino acids and
proteins were examined through various chemical tests.

Water Solubility

The first part of the experiment involves water solubility. The samples used were
methylamine, alanine, dimethylamine, casein, aniline, albumin, and hair strands. Based on the
experimental results, methylamine, dimethylamine, and aniline are soluble in water while the rest
of the samples are insoluble.

The small amines of all types such as methylamine and dimethylamine are very soluble in
water. Aniline, prototypical aromatic amine is moderately soluble in water. All of the amines
can form hydrogen bonds with water, even the tertiary ones. Although tertiary amines lack
hydrogen atom attaching to the nitrogen, they cannot form hydrogen bonds with themselves
instead, they form hydrogen bonds with water molecules by using the lone pair on the nitrogen.
Solubility decreases as the hydrocarbon chain gets longer, noticeably after 6 carbon atoms. In
testing the acidity and basicity of the amine samples, it should be concluded that amines are
basic in nature.
 Amino acids are generally soluble in water and insoluble in non-polar organic solvents
such as hydrocarbons. Alanine is the only amino acid sample in the experiment. In water, the
ionic attractions between the ions in the solid amino acid are replaced by strong attractions
between polar water molecules and the zwitter ions. A zwitter ion is a molecule or ion having
separate positively and negatively charged groups. This is much the same as any other ionic
substance dissolving in water. The extent of the solubility in water varies depending on the size
and nature of the "R" group. Alanine is neither basic nor acidic, so it is neutral.

Proteins samples in the experiment include hair stands, casein and albumin. Casein
contains a high number of proline residues, which do not interact. There are also no disulfide
bridges. As a result, it has relatively little tertiary structure. It is relatively hydrophobic, making
it poorly soluble in water. Albumin, a type of protein that is soluble in water and in water half
saturated with a salt such as ammonium sulfate. Hair strands are made up of a tough protein
called keratin and is insoluble in water. All the protein samples were neutral in nature.

Aqueous Acid Solubility

In this part of the experiment, the solubility of aqueous mixtures of amines were tested
using concentrated HCl. Based on experimental results, all amine compounds were found to be
soluble in the aqueous acid.

Reaction of amines with concentrated HCl would end up with a solution containing
ammonium chloride - the chloride ions, of course, coming from the hydrochloric acid.

The chemistry of amines is dominated by the lone pair of electrons on nitrogen which
accounts for their alkalinity. Most amines are Bronsted and Lewis bases like ammonia, 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. The solubility of amino acids and proteins depends on the pH of the
solution. In acidic solutions, both amino and carboxylic groups are protonated while in basic
solutions, both groups are unprotonated. From the N-containing group, the lone pairs were
attracted due to the ionizing power of concentrated HCl which made the Aniline soluble.

Preparation and Reactions of Diazonium Salts

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.
 After the preparation, 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. In the replacement
reaction of diazonium salt, the diazonium group being a very good leaving group is lost as N 2. It
is a withdrawing group of substituent of the aromatic ring due to the positive charge in N=N. The
attachment of some other atom or group to the aromatic ring system is done by electrophilic
aromatic substitution. Moereover, the diazonium group leaves the ring and is replaced by SO3H
(Sulfonic Acid) in the presence of sulfuric acid. At temperatures higher than 5C, diazonim salts
in aqueous solution are unstable and can decompose giving 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
preparation, they are immediately used in further reactions. A fast decomposition proceeds on
standing even when cold. Therefore, to avoid immediate decomposition, the salt was kept in an
ice bath until its usage in the next parts of the experiment. The purpose of cooling the system
thoroughly is that in aqueous solutions, the diazonium salts are unstable at temperatures higher
than 5 C. The N+N group tends to be lost as N2 so cooling the system is important.

In the coupling reaction of diazonium salts, the reaction between diazonium salt and
dimethylaniline produces an azo compound. Coupling reaction 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. Para postion is favored in the
substitution process. Since no reaction will take place when the pH is low, a mildlyacidic or
neutral solution is required. The reaction occurs by electrophilic aromatic substitution where
diazonium salt is an electrophile and dimethylaniline is a nucleophile. 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, done in the presence of a mineral acid (HCl) making the
addition of some HCl necessary until the N,Ndimethylamine dissolves. 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 dark red solution . 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.

Color Reaction of Amino Acids and Proteins

This part of the experiment examines the color reaction of amino acids and protein done
through different chemical tests such as ninhydrin test, Biuret test and reaction with lead acetate.

In ninhydrin test, the samples used were aniline, alanine, and casein. A small amount of
sample was spotted in different strips of filter paper and dried. It was dipped in 2% ninhydrin
solution in acetone. After 30 minutes, the color development was observed. This test shows a
positive result on alpha amino acids and proteins that contain free amino acid groups indicated
by the formation of red, blue or purple color. In the experiment, alanine and casein showed a
postive result while aniline was negative. The reaction mechanisms are shown below.
 In Biuret test the samples used were alanine, casein and albumin. The biuret test is a
chemical test used for detecting the presence of peptide bonds. The Biuret reagent is made of
sodium hydroxide (NaOH) and hydrated copper (II) sulfate, together with potassium sodium
tartrate. In the presence of peptides, a copper (II) ion forms violet-colored coordination
complexes in an alkaline solution. A positive result is denoted by a deep blue-violet color. In the
experiment, there were positive results for casein and albumin while negative for alanine. Biuret
test cannot determine the presence of proteins or polypeptides. Using other tests is applicable to
indicate if the substance is either a polypeptide or a protein.

The last part of the experiment involves reaction with lead acetate. This was done using
two strands of hair in a test tube. It was added with 1ml of 20% NaOH together with 2 drops of
10% lead acetate. The mixture was then boiled for 5 minutes and was examined. The formation
of a black precipitate indicates a positive result for this test. 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. It is the principal active
ingredient in progressive types of hair coloring dyes when it is at low concentrations. 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 forms long, stiff fibers that comprise the
cytoskeleton due to polymerization. This allows the cell to maintain its shape and size. Lead
acetate reacted with the protein indicated by the hair integration shown in the reaction. 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 black
color.
 III. Conclusion

The small amines of all types such as methylamine and dimethylamine are very soluble in
water. Aniline, prototypical aromatic amine is moderately soluble in water. Solubility decreases
as the hydrocarbon chain gets longer, noticeably after six carbon atoms. Amino acids like alanine
are generally soluble in water and insoluble in non-polar organic solvents such as hydrocarbons.
Solubility of protein samples such as casein, albumin and keratin (hair strands) vary. Casein
contains a high number of proline residues, which do not interact with water molocules. Albumin
is soluble in water and in water half saturated with a salt such as ammonium sulfate. Hair strands
are made up of a tough protein called keratin, is insoluble in water.

In amines, the higher the pKa is the stronger the base. The solubility of amino acids and
proteins depends on the pH of the solution. In acidic solutions, both amino and carboxylic groups
are protonated while in basic solutions, both groups are unprotonated. From the N-containing
group, the lone pairs were attracted due to the ionizing power of concentrated HCl which made
the Aniline soluble.

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 NN 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 5C since this can decompose to give phenol and gaseous N2.
Therefore, cooling is required.

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. Answers to Questions

1. Explain why milk and egg white are 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 Hg2+, Pb2+, Ag+, Tl+, Cd2+ 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.
2. In diazotization, what is the purpose of cooling the system thoroughly?
The purpose of cooling the system thoroughly is that in aqueous solutions, the diazonium
salts are unstable at temperatures higher than 5 C. The N+N group tends to be lost as
N2 so it is important to cool the system.
3. Explain the difference in pH (if any) of the different amines.
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.

V. References

An Introduction to Amines. Retrieved April 29 form http://www.chemguide.co.uk/organicprops/

amines/background.html

An Introduction to Amino Acids. Retrieved April 29 form http://www.chemguide.co.uk/

organicprops/aminoacids/background.html

Amines as Bases. Retrieved April 29 form http://www.chemguide.co.uk/organicprops/

amines/base.html

The Ninhydrin Test. Retrieved April 29 form http://www.harpercollege.edu/tm-ps/chm/100/

dgodambe/thedisk/food/ninhy/ninhy.htm