12/6/23, 2:35 PM Chemistry 211 Experiment 10

Experiment 10

Multi-step Synthesis Coenzyme Catalyzed Synthesis of

Benzoin and Derivatives

Objectives
For most of the experiments we have performed this semester, we have set out to convert one
chemical into another with various different methods. For a synthetic organic chemist the product of
one reaction is often used as the reactant in another. This is also the situation that occurs in
biochemistry where different chemical pathways use a newly synthesized product to be a reactant for
the next reaction in that pathway. This is particularly important for the current experiment because
the first chemical transformation occurs using a vitamin as the "co-enzyme" for the synthesis of
benzoin. While enzymes are normally considered to the biological catalysts in cells, it is often a co-
enzyme, derived from a vitamin, that actually performs the biochemical transformation. Thiamine,
vitamin B1, will be used in the current experiment to convert benzaldehyde into benzoin. The
benzoin produced in the first experiment will then be used in the second to produce benzil.
Likewise, the benzil will then be used to synthesize benzilic acid. Hence, a multi-step synthesis of
different compounds. These new compounds can then be used in various different applications, some
of which are for medicinal purposed. The student can research their ultimate uses to observe that no
chemical synthesis is for naught. The last experiment requires the student to devise a method to
convert benzaldehyde into benzoic acid, perhaps employing oxidation methods used previously, or
use a method that might be new to the student, even something not previously discussed during
lecture or lab. Therefore, to provide a variety of methods for producing benzoic acid, students should
work independently, examining general oxidation methods for converting aldehydes into acids
obtained searching the chemical literature.

Procedure
This multi-step experiment is divided into four sections:

Part 1: Synthesis of Benzoin

Part 2: Synthesis of Benzil
Part 3: Synthesis of Benzilic Acid
Part 4: Synthesis of Benzoic acid from Benzaldehyde

Each part will be performed on successive days. The product from the previous day's experiment
will be used as the reactant on the next day's experiment. It is vital that you do the experiments on
the days they are described. Part 4 is a "self-designed" experiment where you devise a protocol to
follow to prepare and purify benzoic acid from benzaldehyde. You must design this experiment, and
get the approval of your instructor. You must then make a list of reagents you will need to fulfill this
experiment.

There are five laboratory periods devoted to this experiment. The first four lab periods are used to
make the chemicals listed for those days. The fifth day can be used to finish up any incomplete
analysis, including melt points, yield, etc.

Background

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

Vitamin B1, thiamine, as its pyrophosphate derivative (shown below), is a coenzyme universally
present in all living systems. It was originally discovered as an essential nutrient required to prevent
the human disease beriberi, which affects the peripheral nervous system. Symptoms include pain and
paralysis of the extremities, emaciation, or swelling of the body. The disease is still common in the
Far East.

In biochemical terminology, thiamine functions as a coenzyme, a biological molecule that assists in

enzymatic reactions. In most cases, coenzymes are directly involved in the biochemical reaction that
the enzyme catalyzes since they usually bind the substrate (reactant) for the reaction. Without the
coenzyme, no reaction will take place. The enzyme, which is the biological catalyst, binds the
substrate, controlling stereochemistry, energetics, and entropic factors. As indicated above, the
vitamin (derived from VITal AMINe) thiamine is required for many enzymatic reactions. In the
current experiment, we will use thiamine to catalyze the reaction of benzaldehyde into benzoin.

Thiamine, functioning as a coenzyme, can be used for (1) the non-oxidative decarboxylations of a-
keto acids, (2) the oxidative decarboxylations of α-keto acids, and (3) the formation of α-hydroxy
ketones. Most biochemical processes are no more than organic chemical reactions carried out under
special conditions. Like most reactions in organic chemistry, many biochemical reactions can now
be explained using familiar reaction mechanisms. To enhance reactivity, and to be stereoselective,
enzymes are used to bind the substrate in a manner that allows only a single reaction, with
stereoselectivity to occur. In addition, enzymatic reactions can be carried out in mild conditions and
at moderate pH values. Reactions which involve hydrophobic (lipid loving or water hating)
conditions that might not otherwise be possible in an aqueous, biological environment.

Part 1 of this experiment is designed to illustrate these types of processes. As a biological reagent,
the coenzyme thiamine in this reaction will be used to carry out an organic reaction chemistry
reaction without using an enzyme. The reaction is an acylion condensation to benzaldehyde (as an
example, if you have 10.00g of benzaldehyde that you start with, seeing that it takes two moles of
benzaldehyde to produce one mole of benzoin, what is the theoretical yield of benzoin?):

From a chemical point of view, many coenzymes have what we call "a business end" to the molecule
and the rest of the molecule. The reactive part of thiamine is the thiazole heterocyclic ring (a 5-
membered ring containing both a sulfur [thio] atom and a nitrogen [azo] atom). This ring is the
reactive portion of the coenzyme. The rest of the molecule is important biochemically for enzyme
associate, etc., but the thiazole ring is the reactive portion. The rest of the molecule is important
biochemically, but it is not required for the reaction described here.
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12/6/23, 2:35 PM Chemistry 211 Experiment 10

Experiments with the model compound 3,4-dimethylthiazolium bromide have explained how
thiamine-catalyzed reactions work. Part of the chemistry of thiamine is the acidic proton located on
the carbon between the sulfur and nitrogen atoms. Using 3,4-dimethylthiazolium bromide, it was
found that there is a rapid exchange of the C-2 proton for deuterium in the D2O solution. At a pD of
7 (No pH here!), this proton was completely exchanged in seconds!

This experiment indicates that the C-2 proton is much more acidic than one would have expected.
This hydrogen, bonded to the imine carbon has a pKa of 12.7, because the carbanion formed when
the proton is removed is stabilized by the adjacent positively charged nitrogen, yielding the highly
stabilized ylide. An ylide is a compound, or reaction intermediate, with positive and negative formal
charges on adjacent atoms. This ylide can react with an aldehyde to produce an enamine:

The enamine which we will produce, using benzaldehyde, can react with a second benzaldehyde
molecule to produce the desired product, following the acyloin condensation pathway. The enamine
functions much like the enolate partner in an acid-catalyzed aldol condensation. It can condense
with a suitable carbonyl-containing acceptor to form a new carbon-carbon bond. Decomposition of
the intermediate to regenerate the thiamine ylide yields the protonated acyloin, benzoin, in this
reaction. The final product depicted below just needs to undergo deprotonation to produce benzoin.

The reaction pathway outlined above describes the pathway we will follow to produce benzoin using
thiamine as catalyst. The benzoin produced will be used for the sequence of reactions which will be
followed to produce benzil and benzilic acid.

Part 1: Synthesis of Benzoin

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

In this, the first step of our multistep reaction sequence (performed during day one), benzaldehyde
will be condensed, using the thiamine as a coenzyme catalyst, to produce benzoin. The same
reaction can be performed using the cyanide ion (you should include a plausible mechanism for
cyanide catalyst and include it with your report). The reaction mechanism for thiamine catalysis is
described above. (It is imperative that the benzaldehyde be from a newly opened bottle because of
the ease of oxidation, producing benzoic acid, which will interfere with the reaction. The thiamine
hydrochloride must also be from a newly opened bottle, although it is not quite as critical as for the
benzaldehyde.)

The overall reaction for conversion of benzaldehyde into benzoin is:

Procedure
Benzoin is made following using the chemicals listed below:

Add 1.75 g of thiamine hydrochloride to a dry 50-mL flask.

Dissolve the thiamine hydrochloride solid in 5.0 mL of water by swirling.
Add 20 mL of 95% ethanol (remember that standard ethanol liquid is 95%) and cool the
solution for a few minutes in an ice bath.
Very carefully and slowly, add 3.33 mL of 3 M NaOH drop-wise. Mix after every 2-3 drops by
gentle swirling, making certain that the temperature of the solution never rises above 20oC.
(The best way to add such a small volume of NaOH is to place about 10 mL of this NaOH
solution into a 10-mL graduated cylinder; then, withdraw using a Pasteur Pipette liquid down
to about the 6.67 mL marking, thus ensuring that you have added the correct amount of
NaOH.)
To the yellow solution, add 10.0 mL of pure benzaldehyde from a newly opened container,
never a previously opened bottle.

Heat the mixture at 60oC (using a constant temperature water bath set to 60-65oC) for about 1.5
hour. However, an easier and more straightforward procedure is to heat the sample at 60oC for 15
minutes after reaching this temperature (allow about 5 min for warming the sample before starting to
record the time, then incubate for the 15 minutes; going long is not bad.)

Caution: The temperature of this reaction cannot go above 65oC. Constant monitoring of
temperature is paramount during this part of the reaction and must be maintained between 60-65oC.

As mentioned above, you can let the reaction procedure for the full 90 minutes. However, the
following alternative incubation should be used, since it only required about 20-30 minutes of total
incubation time. The 1.5-hour reaction described above could still be used, but the shorter time is
preferred. Therefore, incubate your reaction mixture at 60-65oC for at least 15 min (allow five
minutes to equilibrate temperature). After this minimal incubation time, you will store the reaction
mixture until the next lab period (for at least 24-48 hours) at room temperature. The Erlenmeyer
flask containing the reaction mixture will be sealed using a regular cork to seal the flask. At the
beginning of the next lab period, collect all of your solid material using vacuum filtration as normal.
(Remember that the rate of reaction usually doubles for every 10oC increment in temperature.)

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

At the beginning of the next lab, retrieve your reaction flask from the reagent cart. You should be
able to observed crystals, which are a slight yellow in color. If crystals did not form after storage,
withdraw a drop of the solution on a stirring rod and let it dry to produce a solid on the glass rod;
then, rub it against the inside surface of the flask to induce crystallization.

Collect your crystals via vacuum filtration (wash it free of the yellow mother liquor with a 1:1
mixture of 95% ethanol and water; prepare by mixinf 20 mL of 95% ethanol with 20 mL of DI
water). Since crystals will be moist you cannot do a yield and melt temp. However, your sample
should be dry enough to proceed to the next experiment making benzil.

Optional: Remember, only if your instructor instructs you to perform a re-crystalization, then use
the following procedure, but only if your instructor wants you to do this re-crystallization. In most
cases it is not necessary, just proceed with the Part 2 experiment.

Dispose of the liquid filtrates in the liquid waster container.

Part 2: Synthesis of Benzil

Starting with the α-hydroxyketone benzoin (prepared in Part 1), you will prepare an oxidation
product, benzil, which is an α-diketone. Using the still moist product isolated from the solid material
from Part 1, you may desire to re-crystallize this benzoin using hot 95% ethanol (you will need
about 8 mL of ethanol per gram of product; determine an approximate yield from the filtration
performed above. Let air be drawn through the filter before weighing to enhance the drying of your
benzoin. You will use the amount specified below to do your benzil reaction. Your chemical must be
dry before beginning Part 2 (re-crystallized from ethanol). Typical yield should be about 5-6 g,
although some students recover less than 4 grams. Be sure to record the amount of benzoin isolated,
and dry in the drying oven the benzoin not used in the benzil reaction. The product should be nearly
colorless and of sufficient purity (mp 134-135oC) to use in the next reaction.

This oxidation can easily be done with a variety of mild oxidizing agents, including Fehling's
solution (an alkaline cupric tartrate complex) or copper(II) sulfate in pyridine. In addition, benzoin
could be oxidized by sodium dichromate, but the yield of benzil is lower because some of the
benzoin is converted back into benzaldehyde following cleavage of the bond between the two
oxidized carbon atoms, which is activated by the phenyl rings, producing benzoic acid as the final
product. In this experiment, due to ease of use and consistent results, we will use nitric acid as the
oxidizing agent.

Caution: Concentrated nitric acid is highly corrosive and causes severe burns if spilled onto your
skin. Nitrogen dioxide (NO2) fumes are highly toxic and can damage the lungs due to inflammation.
Do not breathe NO2 fumes, and perform this part of the experiment in the hood.

Procedure
Benzil will be synthesized as follows, and must be performed in a hood:

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

Weigh out 4.8 g of benzoin (prepared in Part 1; be sure to save enough for analysis, but if
using less than the desired 4.8 grams of benzoin, simply proportion the other reagents as
needed) and add to a 100-mL round-bottom flask.
Carefully add (with a funnel) 16.8 mL (but it may be easier to measure 17.0 mL in graduated
cylinder) of concentrated nitric acid.
Add a clam-shell-shaped stirring bar and attach a condenser to the top of the flask for reflux.

Set up this reflux in the hood to vent NO2 gas which is produced during the reaction. With stirring,
heat the reaction mixture. Begin timing the reaction when NO2 (red-brownish colored gas) are
visible above the reaction mixture and gas bubbles are present on the stir bar. Reflux for at least 30
minutes, or until no more NO2 gas is being produced. Do not stop the reaction until the reaction is
complete (there might still be a slight brownish color inside the flask, but as long as no more gas is
being produced, the reaction can be stopped). Stop the reaction by removing the heating mantle, and
letting the reaction mixture cool for about 5 minutes. Add about 75 mL of cold water to the reaction
mixture, cool to room temperature, and swirl for a minute or two to coagulate the precipitated
product. Collect and wash the yellow solid using vacuum filtration. Continue drawing air through
the crystals on the funnel by suction for about 5 minutes to assist in drying the crystals.. The crude
product (dry weight close to 4.0 grams) need not be dried but can be crystallized at once from
ethanol. Dissolve the product in 10 mL of hot ethanol. To enhance precipitation of solid product
from the cooled mixture, you can add some DI water drop wise until you reach "the cloud point" (the
mixture goes from clear to slightly cloudy and solids start to appear), and set aside to allow crystals
to form, and placing your flask on ice will enhance recovery. Record the yield, describe the
crystalline form, color, and melting point of the purified, and recrystallized benzil.

Optional (at discretion of your Instructor): Test for the presence of unoxidized benzoin (only if
requested by your instructor): Dissolve about 0.5 mg of crude or purified benzil in 0.5 mL of 95%
ethanol or methanol and add one drop of 10% NaOH. If benzoin is present the solution soon
acquires a purplish color owing to a complex of benzil with a product of autoxidation of benzoin. If
no color develops in 2-3 min, and indication that the sample is free from benzoin, add a small
amount of benzoin, observe the color that develops, and not that if the test tube is stoppered and
shaken vigorously the color momentarily disappears; when the solution is then let stand, the color
reappears.

Cleaning up: The aqueous filtrate (containing HNO3) should be neutralized with sodium carbonate,
diluted with water, and flushed down the drain. Ethanol used in crystallization should be placed in
the organic solvents container.

QUESTIONS

Show the correctly balanced oxidation-reduction reaction for this reaction (the "N" gets reduced, and
the "C" gets oxidized: determine oxidation state for each, on the reactant side and on the product
side to properly balance).

HNO3 → NO2
Benzoin → Benzil (pick only the single carbon which gets oxidized to determine oxidation
state)

Overall reaction would be as follows.

Benzoin + HNO3 → Benzil + NO2

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

Remember to

balance electrons gained verses electrons lost

balance oxygen atoms by adding H2O
balance hydrogen atoms by using protons (H+)

Part 3: Synthesis of Benzilic Acid

In this experiment, benzilic acid will be prepared by causing a rearrangement of the α-diketone
benzil. (Preparation of benzil is described in Part 2 of this experiment.) The rearrangement of benzil
proceeds as follows:

The driving force for he reaction is provided by the formation of a stable carboxylate salt (potassium
benzilate). Once this salt is produced, acidification yields benzilic acid. The reaction can generally
be used to convert aromatic α-hydroxyacids. Other compounds, however, also will undergo benzilic
acid type of rearrangement.

Procedure
Add 3.0 g of benzil and 9.0 mL of 95% ethanol to a 100-mL flask. Place a stirring bar in the flask
and attach a reflux condenser. Heat the mixture with stirring until the benzil is dissolved. Add drop
wise 7.5 mL of an aqueous potassium hydroxide solution1 downward through the condenser into the
flask. Gently reflux the mixture for 15 minutes with stirring. After the mixture has dissolved and
heated for a few minutes, the mixture will turn blue-black in color. As the reaction proceeds, the
reaction product will turn brown and the solid may, or may not, be completely dissolved. At the end
of the reaction, remove the flask and let it cool.

When the apparatus is cool enough to handle, remove the condenser and transfer the contents,
including any solids, into a 150-mL beaker. Allow the mixture to cool to room temperature (do not
rush!). When the mixture is cooled, continue the cooling in an ice-water bath for an additional 15
minutes, when crystallization should be complete. Crystallization is complete when it appears that
virtually the entire mixture is solidified. If this does not occur in 15 minutes, allow the mixture to set
overnight, or until complete (it is possible to store in an ice bucket or in a refrigerator, if necessary).
Collect the crystals using vacuum filtration and wash the crystals thoroughly with three 15-mL
portions of ice-cold 95% ethanol. The solvent should remove most of the color from the crystals.

Transfer the solid, which is mostly the potassium benzilate salt, to a 125-mL Erlenmeyer flask
containing 30 mL of hot water. Stir the mixture until all the solid has dissolved or until it appears
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12/6/23, 2:35 PM Chemistry 211 Experiment 10

that the remaining solid will not dissolve. If solid still remains in the flask, filter the mixture through
a Hirsch funnel to remove any particulate material. (If all the solid dissolved, then filtration is not
required.)

With stirring, add drop wise 15 mL of 1 M HCl to the solution of potassium benzilate. The pH
should be about 2; if it is higher than this add a few more drops of acid and check the pH again.
Allow the mixture to cool to room temperature and then complete the cooling in an ice bath. Let the
solid form in the ice bath for at least 30 min, up to about 60 min. If solid has not formed after an
hour, you can store your sample until the next lab period.

Collect the benzilic acid by vacuum filtration. Wash the crystals with 30-40 mL of water to remove
salts. Dry the product thoroughly in a desiccator until the next laboratory period.

Weigh the dry product and determine percent yield. Determine the melting point. Pure benzilic acid
melts at 150oC. At the option of the instructor, determine the IR spectrum of the benzilic acid in
KBr. Turn in the entire sample to the instructor in a labeled vial. Your grade will be based on the
purity of your final product, and the amount of benzilic recovered.

___________
1The aqueous potassium hydroxide solution should be prepared for the class by dissolving 27.5 g of KOH in 60.0 mL
of water. This will provide enough solution for 8 student groups, assuming little solution is wasted.

QUESTIONS

1. Show how to prepare the following compounds, starting from the appropriate aldehyde,
following the same procedure performed in this experiment.
2. Give the mechanisms for the following transformations
3. Interpret the infrared spectrum of benzilic acid

Part 4: Synthesis of Benzoic Acid from Benzaldehyde

In this experiment, you will design and perform an experiment to prepare benzoic acid from
benzaldehyde.

You will devise a procedure to perform this experiment and to fully characterize your product. The
maximum amount of benzaldehyde you can use is 2.0 mL (2.09 g). Prior to doing this part of the
experiment, you must compose an experimental protocol in complete detail, which must be
approved by your instructor. Your experimental procedure but be complete, to carry out an oxidation
reaction. The only things that must be done are:

Start with no more than 2.0 mL (2.09 g) of benzaldehyde. The amount of the oxidizing agent (e.g.,
KMnO4 or chromic acid, etc.) you add will be based on a properly balanced oxidation-reduction
equation. For example, if your balanced redox equation had 3 mol benzaldehyde and 2 mol KMnO4,

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12/6/23, 2:35 PM Chemistry 211 Experiment 10

then determine the correct masses of each chemical based on the mass of benzaldehyde to determine
the mass of KMnO4.

Use a procedure that will oxidize the benzaldehyde to benzoic acid. You must use a 250-mL round
bottom flask for this oxidation reaction.

Perform a recrystallization on the benzoic acid you have prepared. Remember, recrystallization is
when you take a solid chemical, and without any chemical modification, dissolve it in a solvent to
supersaturate the solution, and then cool to let crystals form. Do not confuse recrystallization with
the formation of a solid (e.g., treating benzoate ion with HCl to produce benzoic acid is NOT
recrystallization, it is just forming an insoluble form of benzoic acid).

Authorization to perform this experiment will be given only after your protocol has been approved
by your instructor (his initials on the Chemical Request Form are required). You are responsible for
requesting the appropriate chemicals, and for performing the experiment safely and for
characterizing your product fully. (You might have a little reprieve, since other students will likely
be requesting chemicals, and if you forgot, you can still use the chemicals on the cart, even if
requested by another group.)

The chemical request sheet, that must be turned in during the third day of this experiment, is
available online: Chem211 Experiment 10, Part 4 Chemical Request Form

Chemicals, Reagents, and Supplies

Compound MW Amount mmol mp bp Density ηD msds
benzaldehyde 106.1238 10.0 mL (for benzoin) 98.5 -26 179 1.045 1.5454 msds
benzoin 212.2476 4.8 g (for part 2) 22.6 137 344 msds
346 -
benzil 210.2318 3.0 g (for part 3) 14.2 95
348 msds

150 -
benzilic acid 228.247
153 msds

ethanol (95%) 46.0 15.0 mL -114.1 78.5 0.8 msds

benzoic acid 122.1232 122.4 249 1.08 msds
KMnO4 158.0256 240 2.703 msds
2.0 g (for benzoic acid
benzaldehyde 106.1238 18.8 -26 179 1.045 1.5454 msds
synthesis)

Compound g/mol grams or mL 10-3 oC oC g/mL ηD msds

mol

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Copyright Donald L. Robertson (Date last modified: 11/20/2012)

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