Weeks 4 & 5

Indigo Synthesis
I believe that the great Creator has put ores and oil on this earth to give us a breathing spell. As we
exhaust them, we must be prepared to fall back on our farms, which is Gods true storehouse and can
never be exhausted. We can learn to synthesize material for every human need from things that grow.
George Washington Carver
Scientist

An understanding of the transformation of matter is fundamental to the study of chemistry. The synthesis
of new molecules and new materials is the basis of the chemical industry. The synthesis of new chemicals
involves a number of different linked processes. First, reactions are performed that generate new
chemicals. Then the products are isolated from the other materials present, purified and characterized.
This last procedure involves measurement of their chemical and physical properties to confirm their
identity and purity.
This two-week experiment is designed to introduce you to some of the issues involved in transforming
matter. You will investigate the synthesis of the dye indigo. This common dye will be made, isolated and
evaluated.
Educational Objectives: A student who has successfully completed this experiment will be able to

synthesize indigo,

determine percent yield for a reaction,

make leuco-indigo from indigo,

dye cloth using vat dying,

perform a sulfonation reaction, and

use absorption spectroscopy to determine yield.

Experimental Objectives: A student who performs this experiment is asked to

synthesize indigo from smaller precursors,

isolate the indigo using filtration and determine a yield,

prepare leucoindigo and use it to dye a cloth test strip,

synthesize indigo carmine from indigo, and

quantify the indigo carmine produced using absorption spectroscopy.

Background
Indigo is the name given to an organic molecule with an intense blue color and to the plant (a member of
the pea family) from which it is extracted. Indigo has been used to dye cotton and wool fabric for
millennia. It is the source of the color in blue jeans. The molecule was first made synthetically in 1856.
By the turn of the 20th century a commercial process had been developed and synthetic production quickly
replaced agricultural production.
Synthesizing Indigo
Many different ways are now known to make indigo. The one presented here is quick and produces indigo
in very high yield. The reaction is between 2 nitrobenzaldehyde (a.k.a. o-nitrobenzaldehyde or orthonitrobenzaldehyde) and acetone in the presence of a strong base (NaOH).
2 C7H5NO3
+
2-nitrobenzaldehyde

2 C3H6O
acetone

C16H10N2O2 +
indigo

2 CH3CO2H + 2 H2O
acetic acid

(2-1)

The organic molecules are dissolved in water. A NaOH solution is added to start the reaction. Since
indigo is virtually insoluble in water, it precipitates as fast as it forms. The product can be readily
collected by filtration and quantified.
Dying Cloth
Indigos insolubility in water not only makes it easily to isolate, but also makes it a good dye. Once
impregnated in the cloth, washing with water will not easily dislodge the dye molecules. Getting the dye
into the cloth, however, is a problem. Fortunately, the chemistry of indigo provides a solution. Indigo can
be reduced to form a compound known as leucoindigo. Equation 2 shows half of the reaction for this
reduction. It can be coupled with any suitable reaction that provides the two electrons.
C16H10N2O2
indigo

2 H+ +

2e

C16H12N2O2
leucoindigo

(2-2)

Leucoindigo is a colorless molecule that is quite soluble in water. To dye cloth, indigo is reduced to
leucoindigo with an appropriate reagent and dissolved in water. The cloth to be dyed is then dipped into
the solution. When the cloth is removed from the solution and allowed to dry, the oxygen in the air will
oxidize the leucoindigo to indigo. Simultaneously, the dye becomes fixed in the cloth and the color
appears.
A suitable reaction to provide the needed electrons is the oxidation of the stannite ion to stannate. This is
the reaction that will be available for you to use.
SnO22- +
stannite ion

2 H2O

SnO32- +
stannate ion

2 H+ +

2e

(2-3)

Making Indigo Carmine

Although indigo itself is quite insoluble in water, it can be reacted with sulfuric acid to produce watersoluble forms that retain the characteristic intense blue color. When placed in a concentrated solution of
sulfuric acid, the indigo will add two, three or four sulfonic acid (HSO3) units. The actual number will
depend on the strength of the acid, the temperature and the reaction time.
C16H10N2O2 +
2 H2SO4
Indigo
sulfuric acid

C16H8N2O8S22
+
indigo disulfonic acid

2 H2O

C16H10N2O2 +

3 H2SO4

C16H7N2O11S33 +
indigo trisulfonic acid

3 H2O

3 H+

(2-5)

4 H2SO4

C16H6N2O14S44 +
indigo tetrasulfonic acid

4 H2O

4 H+

(2-6)

C16H10N2O2

2 H+

(2-4)

The disulfonic acid form is extensively used in industry. As a dye in the food and drug industry, it is
known as FD&C Blue #2. It is also used as a functional kidney test and to color nylon surgical sutures.
One gram of indigo carmine will dissolve in about 100 mL of water.

The Problem
The problem put to your group is to investigate the properties of the organic dye indigo. The process you
will use can be summarized as follows.
1.
Make some indigo.
2.
Isolate the indigo and determine a yield.
3.
Convert some of the indigo into leucoindigo and use that to dye a cloth sample.
4.
Convert the remaining indigo into indigo carmine and determine the yield using absorption
spectroscopy.
The first three activities will be performed the first week. Your charge is to determine the efficiency of
this synthesis method in preparing indigo.
Indigo carmine will be synthesized the second week. But indigo carmine is only one of three possible
products. In fact, the extent of the reaction and the mixture of products will be affected by the conditions
under which the reaction is performed. Your group is asked to evaluate the effect of one of the following
conditions on the extent of reaction and the mixture of products: strength of the acid, the temperature and
the reaction time.

Procedures
Making Indigo
The quantities given here are intentionally
small. Larger quantities of the indigo can be
made by proportionately increasing the
amounts.

Caution!
Indigo is a highly colored dye. Very
small amounts can impart lots of
color to all kinds of things
(clothing, hands, books, etc.). Be
careful to avoid coloring things best
left uncolored.

You will need to write procedures in your lab

notebook based on the descriptions given here.
Parts of these procedures will be used as
answers to the TA-graded pre-lab questions. Dont worry about having correct procedures. You will
have the chance to modify them based on what you learn during the pre-lab discussion.
Heres a short video of indigo being made.
http://youtu.be/S8JsdLly-sk
Write a procedure for making indigo. Incorporate the following features.

Use approximately 0.4 g of 2-nitrobenzaldehyde. This is a solid reagent.

Use about 5 mL of acetone. This is a liquid reagent.
Dissolve the nitrobenzaldehyde in the acetone and then add the NaOH. You should use about 6
mL of 1.0M NaOH. You will need to add the NaOH slowly the reaction generates a lot of heat.
Dont add the NaOH so fast that the acetone evaporates.
After thoroughly mixing these chemicals you will need to allow the mixture to sit for at least 5
minutes to ensure the reaction is complete.
Isolate the indigo using vacuum filtration as described in chapter 5-2 of the lab book.
Allow the indigo to dry. When its dry you can determine the mass.

Characterizing Indigo: Dyeing Cloth

Indigo is used to dye cloth in a process known as vat dyeing. The indigo is first converted to the watersoluble leucoindigo (2-2). The cloth is then dipped into a solution of the leucoindigo, allowed to soak,
removed, and allowed to dry. As the cloth dries, oxygen from the air oxidizes the leucoindigo back to
indigo, and in the process, also fixes the indigo in the cloth.
Preparation of leucoindigo is a two-step process. First, the reductant must be made, then the leucoindigo
prepared. The reductant provided for your use is the sodium stannite reagent. Because it is a strong
reductant, it easily reacts with atmospheric oxygen. Thus, it must be prepared fresh every time it is used.
Heres how.
Sodium Stannite Reagent
Put one gram of stannous chloride dihydrate in a small beaker (150 mL).
Add 10 mL of 1 M HCl and swirl to dissolve. This should take a few minutes. Dont worry if the
solution is cloudy.
Add 20 mL of 3 M NaOH. Swirl to mix. At first a white to off-white precipitate will form. It
should re-dissolve with mixing. Again, this should take a few minutes. Dont worry if the final
solution is slightly cloudy. This solution is the stannite reagent.
Preparation of Leucoindigo
Place half of the indigo you made earlier in a small
beaker. Save the other half for next week.
Add 3 mL of a 3 M NaOH solution. Heat to boiling in a
hot water bath.
Slowly add stannite reducing reagent, with constant
stirring. The indigo dissolves rather slowly. The
stannite reagent should be added a mL or so at a time,
with mixing, until a total of 25 mL has been added.
Continue to heat and stir for another 10 minutes.
Take a clean small beaker (150 mL) and add 25 mL of
distilled water. Gravity filter the leucoindigo solution,
collecting the filtrate in this beaker. The dilute filtrate is
what you will use to dye the cloth.
Work a small piece of cloth into the dye solution and
allow it to soak. When saturated, remove and allow it to
drain and dry. Note that the leucoindigo cannot be reoxidized until the cloth has dried out. Once the cloth is
dry, the color will slowly develop.

Figure 2-1. Cloth swatch for testing

indigo.
Making Indigo Carmine
Performing the Reaction
In addition to reacting with
concentrated sulfuric acid, indigo is
also soluble in this reagent. Thus, the
reaction system is rather simple. Solid

Caution!
Remember that concentrated sulfuric
acid, like all concentrated acids, is
strongly corrosive and will cause the
immediate destruction of clothing and
skin. Should you suspect youve gotten
some on you, begin washing with
water immediately and consult your
instructor. Make sure all spills are
neutralized and cleaned up
immediately.

indigo is dissolved in 18 M H2SO4 and the mixture heated to promote the reaction. After a sufficient
period of time, the reaction can be stopped by the addition of water which will also dilute the sulfuric acid
and the un-reacted indigo will precipitate out. The following is an outline of a procedure for evaluating
this reaction.
You should use your remaining indigo for this procedure. It should be at least 0.05 g. If you
dont have enough consult your instructor.
Dissolve the indigo in concentrated sulfuric acid (H2SO4). You should use 20 drops (1 mL) for
every 0.1 g of indigo you have with a minimum amount of 1 mL.
Swirl to dissolve the indigo in the acid.
Heat in a simmering water bath. How long depends on what you and your group decide on but it
should be for at least 10 minutes and not longer than 30.
When it is time to stop, pour the reaction
Caution!
mixture into distilled water. But first,
Do
not
add
water to acid. You
cool the solution. Use 5 mL of water for
can
get
a
steam
explosion.
every mL of acid you started with. The
Pour
the
acid
into
water. And
unreacted indigo will precipiatate
do not pour hot acid into water.
leaving the sulfonated products
Again, a potential explosion.
dissolved in the water.
Set up a clean vacuum filtrate apparatus.
Remember that you will be keeping the filtrate. Filter the indigo solution. Use a small amount of
distilled water to wash all solution through the funnel. You may discard the solid, un-reacted
indigo residue.
Determine the volume of your filtrate. A graduated cylinder is sufficient for this measurement.
Spectrophotometric Determination of Product Yield
You will be evaluating your product using absorption spectroscopy. Since you want to know the total
moles you will need to know both the molarity and the total volume. You will need to develop a
procedure to determine the max and the absorbance. Remember that if the absorbance is more than 1.0
you will need to dilute the solution. Finally, you will need to know the molar absorptivity for all the
appropriate indigo sulfonate known solutions.

Data Analysis and Report Considerations

Data Analysis

For the synthesis of indigo you will need to determine your percent yield which is discussed in
chapter 2-4 of the lab book. This will require figuring out the experimental yield and the yield theoretical
yield which is best done while still in lab in collaboration with your fellow group members.

For the synthesis of indigo carmine, determine your percent yield. Again you will need to know
the experimental yield and the theoretical yield. And again, this is best done while still in the lab in
collaboration with your fellow group members.

For the synthesis of indigo carmine you will also have to evaluate which of the three sulfonated
indigo carmines you made. This will be done by evaluating the spectrum of your product solution.