Jacob Lytle

CHEM 2020-PA
April 6, 2010
Synthesis of an Azo Dye

Introduction

Most azo dyes are synthesized through the process of diazotization. The process involves using
nitrous acid, HNO2, to create the nitrosonium ion, N=O+. The nitrosonium ion is eventually turned into a
diazonium salt by reacting it with an aromatic amine. Since the diazonium ion is a weak electrophile, it
reacts with electron-rich, aromatic coupling agents to produce an azo dye. The products are mainly
ortho and para due to the reaction being an electrophilic aromatic substitution.

In this experiment, p-nitroaniline was reacted with HCl and NaNO2 to produce the diazonium
ion, which was then reacted with 2-naphthol to create the azo dye, Para Red. The diazo component,
p-nitroaniline reacted with hydrochloric acid to produce a salt. The salt then reacts with the sodium
nitrite. Losing water, after two proton transfers, produces the diazonium ion. Sodium 2-naphthoxide
reacts with the diazonium ion to produce the azo dye.
Procedure

Chemical Amount
p-nitroaniline 1.381 g
2-naphthol 1.442 g
hydrochloric acid 8 mL
sodium nitrite 10 mL
sodium hydroxide 20 mL
sulfuric acid 5 mL

Part A:
Dissolve 10 mmol of the phenol coupling component in 20 mL of 1 M NaOH in a 125 mL
Erlenmeyer flask. Cool the solution in an ice/salt bath 0 C.
Part B: Diazotization of an Aromatic Amine
Wear gloves and work under the hood when performing this procedure. Dissolve 10 mmol of
the assigned diazo component in 8 mL of 3 M HCl. Heat the solution gently. If necessary add up to 10 mL
of water in order to dissolve the solid. After the solid has dissolved cool the solution to 0 C in an ice/salt
bath with stirring. While stirring add 10mL of freshly prepared 1.0 M NaNO2. Adjust the rate of addition
so that the temperature does not rise above 10 C. Test the solution with starch iodide paper. If the
paper does not immediately turn blue-violet, add 1.0 M NaNO2 dropwise until the paper turns blue-
violet. The diazonium salt must be used immediately. While stirring add the diazonium salt solution to
the solution of the coupling component from Part A. Allow the solution to remain in the ice bath for 15
minutes until the crystallization is complete. Collect the diazo dye by vacuum filtration. Dry the crystals
in a vacuum oven at 80 C before weighing.
Part C: Direct Dyeing with the Azo Dye
Suspend 0.5 g of the dye in 100 mL of hot water. Acidify the solution with a few drops of H2SO4.
Obtain two pieces of special cloth containing strips of different fabrics. Submerge the cloth in the dye
solution for 10 minutes. Remove the cloth, rinse with cold water and allow the fabric to air dry. If the
fabric does not appear to pick up the dye it may be necessary to adjust the pH with dilute HCl or NaOH.
To test for fade resistance wash one of the dyed pieces of cloth with detergent five times. Compare the
colors of the fabrics after drying.
Part D: Recording the UV-Visible Spectrum of the Dye.
Dissolve 100 mg of the dye in 100mL of ethanol using a 100mL volumetric flask. Record the
spectrum from 800-350 nm in 25 nm intervals. It may be necessary to dilute the dye solution to keep the
spectrum on scale.
Part E: Determining the pH Range of the Dye
Transfer 1mL of the solution from part D to a 10mL volumetric flask using a volumetric pipette.
Fill the flask to the mark with ethanol. Add two drops of the dye solution to 20 mL of deionized water in
a small beaker. Swirl the solution to mix thoroughly. Divide the solution into 3 test tubes. Record the
color of the solution and the pH using pHydrion paper for test tube #1. To test tube #2 add dropwise 0.1
M HCL, swirling well after each addition, until a color change occurs. Record the color and pH of test
tube #2. To test tube #3, add 0.1 M NaOH dropwise, swirling well after each addition, until a color
change occurs. Record the color and pH of test tube #3. Determine whether or not the dye prepared
would be a good acid-base indicator. If so indicate the pH range of the indicator activity.
Part F: Determining the Antibacterial Properties of Dye
Obtain two Hinton Mueller agar plates, a broth culture of Staphylococcus aureus and Escherichia
coli, 2 sterile swabs, a pair of forceps and a Petri dish containing sterile blank disks. Swab the first agar
plate with Staphylococcus aureus and the second agar plate with Escherichia coli. Cover every square
mm of the plate. Label the plates. Dissolve 30 mg of the dye in 10 mL of deionized water. Dip a sterile
disk into the dye solution using a pair of forceps. Place three disk equally space on each plate. Incubate
the plates for 48 hours. Measure the zone of inhibition around each disk. The larger the zone of
inhibition the more effective the dye is at controlling bacterial growth. Dispose of all plates and broth as
biohazard waste.

Results Data

Actual Yield Theoretical Yield Percent Yield

2.147 g 2.932 g 73.23%

Theoretical Yield

1.381 g p-nitroaniline mol 293.277 g azo dye = 2.932 g azo dye

138.124 g mol

Percent Yield

% Yield = (Actual/Theoretical) x 100

2.147 g x 100 = 73.23%

2.932 g

Direct Dyeing with the Azo Dye

Fabric Acid - Unwashed Acid - Washed Base - Unwashed Base - Washed

Spun Diacetate yellow bright yellow bright yellow yellow
Bleached Cotton pink white white purple
Spun Polyamide yellow yellow yellow yellow
Spun Polyester pink pink pink purple
Spun Polyacrylic pink pink pink purple
Worsted Wool red light yellow light yellow dark yellow
Determination of the pH Range of the Dye

pH Range Test Color pH

Initial Solution pale orange 6
0.1M HCl pale orange 1
0.1M NaOH purple 10

UV-visible Spectrum Absorbance

nm Absorbance nm Absorbance
800 0.003 575 0.011
775 0.004 550 0.034
750 0.003 525 0.295
725 0.003 500 0.692
700 0.003 475 0.706
675 0.004 450 0.529
650 0.005 425 0.661
625 0.006 400 1.533
600 0.007 375 2.188
350 1.891

Discussion

Para Red is an example of an azo dye and was the first azo dye used. The vivid colors of azo dyes
are due to the delocalization of electrons in the aromatic rings and the nitrogen-nitrogen double bond
(the azo bond).
Several types of fabric were tested for colorfastness of the azo dye before and after being
washed. Spun diacetate is a cellulose fabric that has been modified to remove many OH-groups found
on the surface of the threads. Cotton is natural cellulose found in plant material. Nylon is a synthetic
material made of many linked amide groups, somewhat similar in structure to wool or silk. Polyester
(Dacron) is a synthetic material made from many linked ester groups. Acrylic (Orlon) is another synthetic
fabric made from many acrylonitrile (CH2=CHCN) units linked together. After dyeing all previous fabrics
mentioned with the azo dye, only the spun polyamide remained colorfast when dyed with both acidic
and basic solution. The spun polyester and polyacrylic remained colorfast when dyed in the acidic
solution, and the remaining fabrics remained colorfast in neither acidic nor basic solution.
The visisble spectrum absorbance of the azo dye was tested using spectroscopy. When a
compound absorbs a portion of the visible spectrum, it appears colored. If no visible light is absorbed,
the compound appears colorless or white. The apparent color of the compound is due to the portion of
the visible spectrum that is not absorbed. Different azo dyes absorb light of different wavelengths due
to their different structures. For example, more highly conjugated systems will absorb light with longer
wavelengths (lower frequency, lower energy). Also, having an electron-donating and an electron-
withdrawing group para to each other on an aromatic ring increases both the wavelength of absorption
and the intensity of the absorption. However, Para Red has two electron-withdrawing groups para to
each other on the aromatic ring, meaning it should absorb shorter wavelengths of light (higher
frequency, higher energy). When tested, Para Red absorbed violet light, around 400 nm-1.
This azo dye can act as an acid-base indicator because the structures of its acid and conjugate
base forms absorbed light at different wavelengths. The conjugate base form was a purple/violet color,
while the acid form was pale organge. However, the neutral solution was also pale orange. This means,
as an indicator, Para Red would show no color change when added to an acidic solution. The azo dye
would not be the most desirable indicator, but can partially function as one.
The anti-bacterial properties of the dye were tested using agar dishes and two bacteria.
Staphylococcus aureus and Escherichia coli were swabbed on the dish and the dye was place on the dish
in several spots. The azo dye was shown to be ineffective at preventing the growth of the bacteria on
the plate.