Combinatorial Analysis of An Azo Dye Chemistry Lab Report

CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
1
Many plants yield dyestuffs that will dye wool or silk, but there are few of these that will dye
cotton as well. Most will not dye synthetic fibers like polyester or rayon. In addition, the natural
dyes, with a few exceptions, do not cover a wide range of colors, nor do they yield “brilliant”
colors. Even though some people prefer the softness of the “homespun” colors from natural dyes,
the synthetic dyes, which give rise to deep, brilliant colors, are much preferred today. Also,
synthetic dyes that will dye the popular synthetic fibers can now be manufactured. Thus, today
we have available an almost infinite variety of colors, as well as dyes to dye any type of fabric.
Before 1856, all dyes in use were obtained from natural sources. However, an accidental
discovery by W.H. Perkin, an English chemist, started the development of a huge synthetic dye
industry, mostly in England and Germany. Perkin, then only age 18, was trying to synthesize
quinine. Structural organic chemistry was not very well developed at that time, and the chief
guide to the structure of a compound was its molecular formula. Based on formulas, Perkin
thought that it might be possible to synthesize quinine by the oxidation of aniline and toluidine
(methylaniline).
N
He obtained no quinine, but he did recover a
blackish precipitate with properties that could
be extracted with ethanol to give a beautiful
H2N
N
NH
purple solution. This purple solution
subsequently proved to be a good dye for
fabrics. After receiving favorable comments
Mauve
from dyers, Perkin resigned his post at the
Royal College and went on to found the
British coal tar dye industry. He became a
very successful industrialist and retired at age
36 to devote full time to research. The dye he
synthesized became known as mauve. The structure of mauve was not proved until much later.
From the structure it is clear that the aniline which Perkin used was not pure and that it contained
all three toluidine isomers as well.
Mauve was the first synthetic dye, but soon the triphenylmethyl dyes, pararosaniline, malachite
green, and crystal violet were discovered in France. The triphenylmethyl dyes were soon joined
by synthetic alizarin, synthetic indigo, and the azo dyes. The azo dyes, also manufactured from
aromatic amines, revolutionized the dye industry.
The azo dyes are one of the most common types
of dyes still in use today. They are used in dyes
N
for clothing as food dyes and as pigments in
paints. In addition, they are used in printing inks,
N
and in certain color printing processes. Azo dyes
have the following basic structure:
The group containing the nitrogen-nitrogen double bond is called an azo group. To produce an
azo dye, an aromatic amine is treated with nitrous acid to give a diazonium ion intermediate.
This process is called diazotization.
Cl
NH 2
+ NaNO2 + 2HCl
N
N
+ 2H2O + NaCl
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
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The diazonium ion is an electron deficient (electrophilic) intermediate. An aromatic compound,
suitably rich in electrons (nucleophilic) will add to it. The most common used nucleophilic
species are aromatic amines and phenols. The addition of the amine or the phenol to the
diazonium ion is called diazonium coupling reaction.
N
N
N
nucleophile
N
N
N
+H
electrophile
&
N
N
N
+H
Azo dyes are both the largest and most important group of synthetic dyes. In making the azo
linkage, many combinations are possible. These combinations give rise to dyes with a broad
range of colors, encompassing yellows, oranges, reds, browns, and blues.
The azo dyes, the triphenylmethyl dyes, and mauve are all synthesized from the anilines and
other benzene derivatives. All of these substances can be found in coal tar, a crude material that
is obtained by distilling coal. Perkin’s discovery led to the formation of a multimillion dollar
industry based on coal tar, a material that was once widely regarded as a foul smelling nuisance.
Today, these same materials can be recovered from crude oil or from petroleum as by-products
in the refining of gasoline. Although we no longer utilize coal tar, many of the dyes are still in
extensive use.
Azo Dye history at DU:
Year
Aniline derivative(s)
2004
4-aminobenzene sulfonic acid
2005
2, 3, and 4-aminobenzene sulfonic acids
2006
2, 3, and 4-aminobenzene sulfonic acids
2008
2, 3, and 4-aminobenzene sulfonic acids
2009
2, 3, and 4-aminobenzene sulfonic acids
2011
2013
2015
2, 3, and 4-aminobenzene sulfonic acids
2, 3, and 4-nitroanilines
2, 3, and 4-nitroanilines
2015
2017
2017
2018
2, 3, and 4-nitroanilines
2019
2, 3, and 4-aminobenzene sulfonic acids
2020
2, 3, and 4-aminobenzene sulfonic acids
2021
2, 3, and 4-aminobenzene sulfonic acids
2, 3, and 4-aminobenzene sulfonic acids
2, 3, and 4-aminobenzene sulfonic acids
Aromatic coupling reagent(s)
dimethyl aniline
1 & 2-naphthol, salicylic acid
1 & 2-naphthol, salicylic acid
1 & 2-naphthol, 1-bromo-2-naphthol
1 & 2-naphthol, 1,5dihydroxynaphthalene
1 & 2-naphthol, 1-nitroso-2-naphthol
1 & 2-naphthol, salicylic acid
2-naphthol, 2-methoxynaphthalene,
2,3-dihydroxynaphthalene
1-naphthol, 1-methoxynaphthalene,
2,7-dihydroxynaphthalene
1 & 2-naphthol
2,3-dihydroxynaphthalene
2,7-dihydroxynaphthalene
1,5-dihydroxynaphthalene
1.6-dihydroxynaphthalene
1,5-dihydroxynaphthalene
1-amino-5-dihydroxynaphthalene
N,N-dimethylaniline
N,N-dimethyl-1-naphthylamine
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
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From “Introduction to Organic Chemistry Laboratory Techniques: a contemporary approach.”
1976 By D.L. Pavia, G.M. Lampman & G.S. Kriz Jr. WB Saunders Company
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
4
CHEMICAL COMPOSITION OF FIBERS
O
O
H O
O
H
O
H
H
O
O
H
OH
H
HO
H
H
O
O
H
H
OH
O
O
HO
H
H
O
O
O
H
H
H
O
H
O
H
O
Diacetate (cellulose acetate)
O
OH
H OH
H
H
O
O
HO
H
HO
O
HO
H
H
Cotton (cellulose)
O
O
OH
OH
H
H
OH
H
O
H
H
H
H
H
OH
OH
H
H
O
H
N
Nylon (polymamide)
N
H
O
O
O
O
O
O
O
O
Dacron (polyester)
N
N
N
O
O
N
Orlon (polyacrylonitrile)
O
O
H
N
H
N
N
H
N
H
O
O
OH
Wool (protein)
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
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As shown on the previous page, in order for a dye to be effective it must “stick” to the fabric
that is being dyed. There are several ways that dyes interact with fibers including:
1. Physical “trapping” of dye molecules in fiber matrices.
2. Binding by chemical weak forces such as
a. Hydrogen bonding,
b. Van der Waals forces,
c. Ionic forces and
d. Hydrophobic interactions.
3. Dye molecules can also be covalently bound to fibers.
The chemical composition of fibers and the chemical nature of the dye determine their
interaction.
We will be dying fabric swatches containing strips of spun diacetate, bleached cotton, nylon 6.6,
Dacron 54, orlon 75 and worsted wool are available.
Diacetate
(shiny)
Cotton
Nylon
Dacron
Orlon
Wool
(Fuzzy
Off-white)
This gives us not only the ability to test the dye with different fabrics but also creates a sort of
color “fingerprint” for each dye compound.
Arguably, the most notable development in synthetic organic chemistry in the last decade is
probably the so called combinatorial chemistry. The goal of combinatorial chemistry is to
prepare a large number of structurally diversified but related compounds efficiently. A new
journal has emerged that is devoted entirely to combinatorial chemistry. The pharmaceutical
industry has embraced this new development and invested millions of dollars into the area. The
products from a combinatorial synthesis are usually called a library, which must be screened
for a desired activity. This desired activity could range from anti-tumor or anti-HIV properties
to effective catalytic properties.
In this experiment, the principle of combinatorial chemistry is shown through preparing azo
dyes using the combinatorial approach. The coupling reactions involve an aromatic diazo
compound and a nucleophilic, water-soluble aromatic compound as the coupling partners.
Each aromatic ring can be diversified by substitution pattern. Each student is assigned a
unique coupling reaction. The entire class will perform the same basic reaction, but will attain
a variety of products. Assay and the identification of individual compound are a
straightforward process for this parallel experiment. A fabric dying experiment follows the
coupling experiment. The color of the dyed multi-fiber strip serves as the final assay for this
experiment. At the end of this experiment, the colors of the dyed multi-fiber strips from the
entire class should be compared. Any conclusions from the correlation of the product
structures to the colored strips should be discussed in the lab report.
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
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The product designations are arranged on a grid with the aminobenzenesulfonic acids going
across and the nucleophilic benzene derivatives (aromatic coupling reagents) going down. Each
square of the grid is a unique combination of two reactants.
aminobenzene
sulfonic acid →
A
B
C
SO3H
SO3H
SO3H
NH 2
aromatic coupling reagent ↓
NH 2
H2N
4-aminobenzene
sulfonic acid
3- aminobenzene
sulfonic acid
2- aminobenzene
sulfonic acid
1
A1
B1
A2
B2
C1
N,N-dimethyl-m-toluidine
2
C2
N,N-dimethyl-p-toluidine
“Warm red, intensified by a suitable yellow, is orange. This blend brings red almost to the
point of spreading towards the spectator. But the element of red is always sufficiently strong
to keep the color from flippancy. Orange is like a man, convinced of his own powers. Its note
is always that of the angelus, or of an old violin.” V. Kandinksky, Concerning the spiritual in
art.
CHEM254
Procedure:
Combinatorial Synthesis of an Azo Dye
Fall 2022
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I. Preparation of Nucleophile and Diazonium Salts
In a 150 mL beaker place 1.0×10-2 moles of either N,N-dimethyl-p-toluidine or N,N-dimethyl-mtoluidine. Add 8.0 mL of a 2.5 M aqueous solution of sodium hydroxide (measure with a plastic
pipet). Stir to dissolve. Place the beaker in an ice-water bath.
In a small Erlenmeyer place 1.0×10-2 moles of your aminobenzene sulfonic acid and 20 mL of
saturated aqueous sodium carbonate (solution already made). Break up the clumps with a glass rod
if all of the solid does not dissolve. Add of 1.0 g of sodium nitrite and 2 mL of water. Stir to
dissolve.
Weigh out 12 g of ice in a 100 mL beaker. Add 1.0 mL of concentrated HC1 (measure with a
plastic pipet). Add the solution from the small Erlenmeyer dropwise to the 100 mL beaker with a
plastic pipet and transfer any solids with a spatula. Place the mixture in an ice-water bath.
II. Coupling reaction
Add the suspension of the diazonium salts in the 100 mL beaker 1 mL at a time to the 150 mL
beaker with the aromatic nucleophile coupling reagent. Stir the mixture in the 150 mL beaker with
a glass rod after each addition. Let the reaction proceed for at least 10 min with occasional stirring.
Then heat the suspension on a hot plate till it starts to boil. Take the solution off the hotplate. Add
1.0 mL of concentrated HC1 (measure with a plastic pipet). Cool the beaker in an ice-water bath. If
the solution is still basic you may add more HCl until a solid forms. Recover the solid by vacuum
filtration with a Hirsch or Buchner funnel. Wet the filter paper with water before filtering. Rinse
the beaker and wash the solid with 10 mL of saturated NaCl solution and let it dry in the air.
Weigh the solid product when it appears to be dry. Save the filtrate if there is not sufficient solid
retained by the filter paper.
III. Dying a fiber strip
Disposable gloves should be worn in this experiment. Dissolve approximately 0.5 g of the azo
dye prepared in the previous step in about 50 mL of water in a 100 or 150-mL beaker. Put a
multi-fiber strip in the solution of the azo dye and keep it immersed. Boil the solution for about 5
minutes. Remove the multi-fiber strip from the hot solution with a tweezers and rinse the multifiber strip in a large beaker of tap water. Pat dry the dyed multi-fiber strip with a paper towel,
record the fabric colors, and take a photo of the swatch.
IV. Analysis
Take a UV-vis spectrum between 300 and 600 nm of your dye in water.
You may use the previously prepared dye bath.
Enter the dye designation (A1, A2, etc..) and lambda max on the UV-vis notebook before you leave
the lab.
Send the instructor a photo of your dyed swatch. Include the photo on your lab report too.
Hand in your synthetic dye in a properly labeled vial:
Your name, compound designation (A1, A2, etc…) and the date.
V. Clean up: Dispose of filtrate and dye bath in a container in the waste hood.
Wash glassware with soap and water.
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
Checklist for completing the “Prelab” section:
(refer to Laboratory Syllabus for complete directions)
Title and date
Purpose. (4 points)
8
The purpose of this experiment are to learn about synthetic chemistry by preparing the azo dye
compound, and learning about colour of the compounds and knowing the relationship between
the colour of the compound and its structure.
in this experiment we are being provided with a molecule containing diazo group and a
nucleophillic water soluble aromatic compound. Then, we are going to couple them by
adding them in a reaction mixture and providing the required conditions.
We, are going to make a different compounds (azo dyes) by using this coupling reaction
and then compare them based on their colors and also understand why they give that
particular color.
Physical constants.
m.p. oC
b.p. oC
172.18g/mo
le
C6H7NO3S 173.19g/mo
le
C6H7NO3S 173.19
g/mole
Na2CO3 or
105.988
CNa2O3
g/mole
NO2.Na or N 68.99g/mol
aNO2
e
288
XXXX
density
g/mL
XXXX
>300 (SA)
XXXX
XXXX
~300
XXXX
XXXX
~0
~100
~1.0
271
XXXX
XXXX
aqueous hydrochloric
acid
N,N-dimethyl-mtoluidine
HCl
36.46
~0
~100
~1.0
C9H13N
XXXX
215 °C
0.93 g/mL
N,N-dimethyl-p-toluidine
C9H13N or
CH3C6H4N(
CH3)2
135.21g/mo
le
135.21g/mo
le
XXXX
211 °C
0.9366g/
mL
2.5M aqueous
sodium hydroxide
sodium
chloride
saturated aqueous NaCl
NaOH
40.00
~0
~100
~1.0
NaCl
58.44
XXXX
XXXX
XXXX
XXXXX
XXX
−21.12
108.7
1.2
Name
Formula
4-aminobenzenesulfonic
acid
C6H6NO3S-
3-aminobenzenesulfonic
acid
2-aminobenzenesulfonic
acid
aqueous sodium
carbonate
sodium nitrite
Name
aminobenzenes
ulfonic acid
M.W. g/mole
Solubility
Somewhat soluble in water
soluble in alcohol
insoluble in nonpolar solvents
Safety Information
Warning: Corrosive!
Caution: Moderately toxic by various routes.
Caution: Moderate fire and explosion hazard.
CHEM254
aqueous sodium
carbonate
sodium nitrite
aqueous
hydrochloric
acid
glacial acetic
acid
N,N-Dimethylp-toluidine
10% aqueous
sodium
hydroxide
sodium
chloride
Combinatorial Synthesis of an Azo Dye
Soluble in water,
Insoluble in alcohol
Insoluble in nonpolar solvents
Soluble in water
soluble in alcohol
insoluble in nonpolar solvents
Soluble in water
soluble in alcohol
insoluble in nonpolar solvents
miscible with water,
Miscible with alcohol immiscible
with nonpolar solvents
Some soluble in water, soluble in
alcohol and nonpolar solvents
soluble in water
some soluble in alcohol
insoluble in nonpolar solvents
soluble in water
some soluble in alcohol
insoluble in nonpolar
Fall 2022
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Caution: Irritation of eyes, nose and throat.
Caution: Irritation of eyes, nose and throat.
Caution: Flammable
Caution: Caustic
Warning: Caustic, can cause burns
Warning: Vapor irritating to eyes, nose ant
throat.
Caution: Flammable
Moderately toxic. Irritating to eyes, skin and
respiratory system.
warning: Caustic
Not harmful
References:
MI = Merck Index, 11th ed.
Hazardous Chemical Desk Reference, Lewis and Sax, 1987
SA = Sigma-Aldrich catalog online
https://pubchem.ncbi.nlm.nih.gov/compound/4-Aminobenzenesulfonate
https://pubchem.ncbi.nlm.nih.gov/compound/3-Aminobenzenesulfonic-acid
https://pubchem.ncbi.nlm.nih.gov/compound/2-Aminobenzenesulfonic-acid
https://pubchem.ncbi.nlm.nih.gov/compound/Sodium-carbonate
https://pubchem.ncbi.nlm.nih.gov/compound/Sodium-nitrite
https://pubchem.ncbi.nlm.nih.gov/compound/N_N-Dimethyl-m-toluidine
https://pubchem.ncbi.nlm.nih.gov/compound/N_N-Dimethyl-p-toluidine
___ (4 points) Flowchart. Refer to “Procedure.” The following products will be synthesized
ON A Paper
(16 points) Structures and equations.
ON A Paper
Please use your assigned aminobenzenesulfonic acid and aromatic nucleophile coupling reagent
(N,N-Dimethyl-m-toluidine or N,N-Dimethyl-p-toluidine) to complete these questions.
Thursday: CA A1, ZA A2, AmAr B1, SD B2, JH C1, SL C2, ML A1, LM A2, GP B1, IR B2,
LR C1, AZ C2,
Friday: AbAl A1, NB A2, JF B1, KK B2, TL C1, GO C2, ZO A1, MP A2, ER B1, IS B2,
Please use your assigned aminobenzenesulfonic acid and aromatic nucleophile coupling reagent
(N,N-Dimethyl-m-toluidine or N,N-Dimethyl-p-toluidine) to complete these questions.
___ Draw the structure of your diazo intsermediate as a chloride salt.
___ Draw the possible positional isomers that could be produced with your reactants.
CHEM254
Combinatorial Synthesis of an Azo Dye
___ Draw the structure of your azo dye (all atoms neutral).
Fall 2022
10
___ Write the balanced equation for the synthesis with structural formulas:
aminobenzenesulfonic acid + sodium nitrite + HCl + aromatic nucleophile coupling reagent →
azo dye + water + sodium chloride
(12 points) Calculations. Show your calculations, please.
___ Calculate the mass 1.0×10-2 mole of your aminobenzenesulfonic acid.
Mine was aminobenzene sulfonic acid.
1.0*10^-2*173.19g/mole =173g
___ Calculate the number of moles in 1.0 g of sodium nitrite.
1.0g* 1/68.99g/mole= 0.0149 mol3 (0.015 mole)
___ Calculate the mass of 1.0×10-2 mole of your aromatic nucleophile coupling reagent.
___ Calculate the molecular formula, molecular weight of your dye (all atoms neutral).
___ Identify the limiting reagent and calculate the theoretical yield of your dye.
___ Safety Question: (4 points) A Review article in the Indian Journal of Experimental Biology
entitled “Toxicity assessment and microbial degradation of azo dyes” was published in 2006.
The authors say “Toxic effluents containing azo dyes are discharged from various industries and
they adversely affect water resources, soil fertility, aquatic organisms and ecosystem integrity.
They pose toxicity (lethal effect, genotoxicity, mutagenicity and carcinogenicity) to aquatic
organisms (fish, algae, bacteria, etc.) as well as animals,” What evidence do the authors give to
support these claims? Two points please.
1- The author mentioned the “Impacts of aromatic hydrocarbons” supported his
claim with Oxidative metabolism of polynuclear aromatic hydrocarbon (PAH)
is accomplished through highly electrophilic oxidases, some of them may bind
covalently to macromolecules such as DNA, RNA and proteins and induce
mutations. PAH are active inducers of liver enzymes of fish and produce
metabolites that are capable of inducing cancer. Even in small concentration,
naphthalene is capable of inducing hepatic histopathological alterations,
which may cause severe physio-metabolic dysfunction leading to death.
2- The author mentioned the “Impact of dyeing factory effluent on plants” and
he supported his claim, the Intensive irrigation of agricultural lands with water
polluted with various industrial effluents severely affects soil fertility and
plant growth. Plant growth parameters namely germination percentage,
seedling survival and seedling height have been taken as criteria to assess
CHEM254
Combinatorial Synthesis of an Azo Dye
Fall 2022
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plant response to specific pollutants. Some of the dyes used in dyeing
industries are even carcinogenic and mutagenic and the effluents even
reduce the rate of seed germination and growth of crop plants.
CHEM254
Combinatorial Synthesis of an Azo Dye
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CHEM254
Combinatorial Synthesis of an Azo Dye
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CHEM254
Combinatorial Synthesis of an Azo Dye
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Lab Report:
Write a Journal of Organic Chemistry style “Results and Discussion” for this experiment.
(40 points)
Address, at minimum, the following points in your discussion…
1. Report the percent yield of your dye- show your calculations. (Include a chemical equation
with molecular structures) Discuss the yield: why is it greater or less than 100%?
2. Discuss the UV-vis class data. (include at least one table or graph of class data – do not “cut
and paste” raw data)
3. Discuss the fabric strip class data.
a) The general trend observed by changing the amine for the same aromatic nucleophile
coupling reagent (A1, B1, C1 and A2, B2, C2).
b) The general trend observed by changing the aromatic nucleophile coupling reagent for the
same amine (A1,A2 and B1,B2, and C1,C2).
4. Discuss the reasons why the same dye gives a different color with different fabrics.
5. Discuss the reasons why the same fabric gives a different color with different dyes.
Include an image of your UV spectrum
Include an image of your swatch.
Now Israel loved Joseph more than any of his other sons, because he had
been born to him in his old age; and he made him a coat of many colors.
Genesis 37:3
Experimental procedures are based on the lab manual: Daniel R. Palleros, Experimental Organic
Chemistry, John Wiley & Sons, Inc., New York, 2000, pp. 627-628.
A Combinatorial Experiment Suitable for Undergraduate Laboratories
Benjamin W. Gung and Richard T. Taylor
Journal of Chemical Education, Vol 81, No. 11, November 2004
Swatches
CHEM254 lab
Fall 2022
A1
wool
orlon
dacron
nylon
cotton
diacetate
A2
B1
B2
C1
C2
A1
A2
B1
B2
C1
C2