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Experiment 8
A Biomimetic O2 Carrier: Co(Salen) Synthesis and Kinetics
Week 1: Biomimesis of Hemes: Co(Salen) Synthesis
Week 2: Oxygen Binding to a Heme Model Compound: Co(Salen) Kinetics

Naturally occurring oxygen carriers and storage proteins contain transition metal ions to
which O2 can reversibly bind. Typically these are iron (in the form of ferrous heme in proteins
such as myoglobin and hemoglobin) or copper (hemocyanin). In this experiment a simple cobalt
complex will be prepared which also reversibly binds dioxygen. Many complexes of this type
have been used as models to aid in the understanding of how the proteins function. The complex
Co(salen) (where salen = N,N-bis(salicylaldehyde)ethylenediamine, see Figure 1) reversibly
binds O2, thereby acting as a functional model for myoglobin. As one might imagine, most
spectroscopic and measurement techniques are easier to perform and interpret on a molecule
which has two orders of magnitude fewer atoms than a protein.
HO

HO

N
Fe

2+

Co

N
O

Co(salen)
Ferrous Heme

Figure 1. Ferrous heme is the cofactor which binds O2 reversibly in hemoglobin and myoglobin. Co(salen) is a
coordination compound that can also bind O2 reversibly.

When Co(salen) was first prepared, it was observed that the red-brown crystals darkened
on exposure to air. However, it was not until five years later that it was established that the color
change was due to reversible uptake of O2. SalenH2 (figure 2) is a Schiff-base ligand formed by
the condensation of two molecules of salicylaldehyde (sal) with ethylenediamine (en).

NH2 + 2

H2N
en

OH

OH HO

salenH2

sal

Co(II)

N
O

Co

N
O

Co(salen)

Figure 2. Synthesis of Co(salen).

Later, it was found that different crystalline forms existed depending on the solvent used
in the preparation, and that these crystalline forms had varying capacity for oxygenation in the
solid state. This variation in oxygenation has been related to the presence of voids in the crystal
lattice, sufficient to allow the passage of molecular oxygen. This suggestion is supported by the
x-ray crystal structure determination of the so-called "inactive" form which shows that the
structure consists of dimeric units [Co(salen)]2. The active forms of Co(salen) are presumed to
contain dimeric units with a more-open lattice packing relative to the inactive form.
N
N
Co
O
O
O
O
Co
N
N
Active structure

N
N
Co
O
O
O
O
Co
N
N
Inactive structure

Figure 3. Structures of active and inactive Co(salen) in the solid state.

In anaerobic solution, it has been found that the cobalt(II) may be four, five or six
coordinate, depending on the solvent. For example, in a strongly coordinating solvent such as
pyridine (py, C5H5N), both [Co(salen)(py)] and [Co(salen)(py)2] exist, while in chloroform, the
only species is Co(salen). When 5- or 6-coordinate species can exist, O2 will form a complex
with Co(salen). The oxygenated product may be a 1:1 (Co:O2) or a 2:1 (2Co:O2) complex. It is
thought that O2 adds as the sixth ligand to a 5-coordinate complex, or replaces one of the
coordinating solvent molecules. The final structure may be either a 6-coordinate 1:1 complex, or
a 6-coordinate 2:1 complex. It is worth noting that the conventional way to view the formation

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of an O2-adduct is as a redox process, in which O2 accepts one electron from each Co2+. This
forms superoxide, O2-, bound to Co3+, in the 1:1 complex. O2 accepts one electron from both
Co2+ ions, forming peroxide, O2-2, bound to two Co3+, in the 2:1 complex.
Sol
N
N
Co
O
O
O
O
O
O
Co
N
N

Sol
N
N
Co
O
O
O
O

Sol
2:1 structure

1:1 structure

Figure 4. Two possible structures for the O2 adduct of Co(salen), in which Sol represents a coordinating solvent
molecule.

In this experiment, the inactive form of Co(salen) is syntheszied. The uptake of dioxygen
is then investigated for the complex dissolved in dimethylsulfoxide (DMSO), a stronglycoordinating solvent that generates the active form of the complex. You will use the kinetic data
of oxygen uptake to establish whether a 1:1 or a 2:1 complex is formed under these conditions.

Week 1: Synthesis
SAFETY NOTES: H2salen is a skin irritant; if exposed, wash thoroughly with water.
H2salen
Synthesize the salen ligand by placing 6 mL of 95% ethanol in a test tube that also has a
small magnetic stirring bar. Heat the ethanol to boiling on a sand bath while stirring.
Immediately, with continued heating and stirring, add 0.60 mL (5.6 mmol) of salicylaldehyde
and then 0.20 mL (2.8 mmol) of ethylenediamine (98%). Stir the solution for three to four
minutes and then cool in an ice-water bath to precipitate the yellow salen. Filter your reaction
mixture with a Hirsch funnel and wash your crystals with several drops of cold ethanol. Dry the
crystals as much as possible on the filter using vacuum, and then collect them and spread them
on a piece of filter paper to air dry.

Co(salen)
NOTE: This preparation is sensitive to air, so it MUST be performed under a nitrogen-filled
balloon. The TAs will degass the ethanol solvent. This involves bubbling nitrogen through the
solvent until all dissolved oxygen is replaced with dissolved nitrogen.

Add H2salen (0.46 g, 1.72 mmol) to a 50 mL round-bottom flask containing a large stir
bar. Apply a thin layer of stopcock grease to the male ground glass joint of a reflux condenser,
and place it on the flask. Attach the flask-and-condenser apparatus to a ringstand.
To a separate round-bottomed flask, add cobalt(II) acetate tetrahydrate (0.4 g) to 3.2 mL
of water and swirl to dissolve the solid. All the solid must dissolve; add a few more drops of
water, or warm the solution slightly as you swirl it, or add a stir bar and stir the reaction mixture
to dissolve. When all the cobalt acetate has dissolved, seal the flask with a rubber septum. Place
this flask in a beaker to hold it.

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The TAs will help you flush the cobalt acetate flask with nitrogen, using purge and vent
needles, to remove all air and create an anaerobic nitrogen atmosphere in the flasks and
condenser. Five minutes of flushing is sufficient to accomplish this.
Add 24 ml of previously degassed ethanol to the H2salen flask, then warm the flask with
stirring to dissolve all the salen. Then remove the sand bath, stop heating, and continue stirring.
Now seal the top of the condenser with a red rubber septum while the reaction mixture is still
warm. and flush the apparatus with nitrogen while it is still warm.
Add the cobalt acetate solution to the ethanol solution of H2salen via a syringe. While
flushing the cobalt acetate flask with nitrogen, puncture its rubber septum with a needle-andsyringe, and draw the cobalt acetate solution up into the syringe. Then change the purge and
vent needles to the septum on top of the reflux condenser to flush the ethanol/salen solution with
nitrogen.
Puncture the septum on top of the reflux condenser while you are still flushing the
flask/condenser with nitrogen. Now add the Co acetate solution by pressing the syringe plunger.
As the stir bar mixes the two solutions a brown gelatinous precipitate should form. You should
now remove all the nitrogen-flushing purge and vent needles (remove the silver purge needle
first), and cease flushing the apparatus with nitrogen.
(If, while you are adding the cobalt acetate solution to the solution of H2salen, the
pressure is too great and the stoppers pop out, remove the nitrogen purge needle, and slowly add
your cobalt acetate solution with just the pink vent needle in place.)
Replace the septum on top of the reflux condenser with a nitrogen-filled balloon. This
requires that a TA assist you. One of you should hold the nitrogen-filled balloon close to the top
of the reflux condenser, while the other holds and unfolds the rubber septum, ready to remove it.
At the count of three, one removes the septum and the other quickly places the balloon over the
top of the condenser. If there is a problem, and the condenser remains open to the air too long,

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re-attach the purge and vent needles to the septum, and flush the condenser/flask apparatus with
nitrogen for a few more minutes. Then try to place the balloon again.

Once the nitrogen-filled balloon is in place, set the round-bottomed flask on a sandbath
on top of a magnetic stirring plate, and heat at a reflux for at least 1 hour while stirring
vigorously. During this period, the initially formed brown "active" complex should slowly
change to the brick-red "inactive" complex. Inefficient stirring during the reflux period leads to a
blue compound (probably a Co+3 complex) forming at the bottom of the flask.

At the end of the reflux period, cool the solution to room temperature (in air--you may
remove the reflux condenser at this point), and then in an ice bath. Filter to collect the solid, and
wash it with 2 ml of ice-cold ethanol. Dry the solid as much as possible on the filter using the
vacuum, and then collect it and spread it on a piece of filter paper to air dry.

Place your Co(salen) in a vial and record your name or initials on the vial. Then try to
break up any large chunks of the solid into finer pieces (to facilitate drying of the solid) using a
spatula. Place the open vial into the dessicator provided, to dry the sample for the next week. A
dessicator is a container which is partly filled with a drying agent. A rack for holding sample
vials is placed above the agent. The open sample vials are placed in the rack and the dessicator is
then sealed with an air-tight lid. Over time, the drying agent absorbs ethanol from the samples
and ethanol vapor in the interior of the dessicator, thus helping to dry the samples placed within.

Record your yield of Co(salen) the following week.

Week 2: Kinetics of O2 Absorption by Co(salen) in Dimethylsulfoxide

SAFETY NOTES: DMSO is not itself poisonous, but it is readily absorbed by the skin and
will carry other compounds through the skin with it. If exposed, wash thoroughly with water.

Grind your Co(salen) to a fine powder with an agate mortar and pestle. CLEAN THE
MORTAR AND PESTLE with acetone immediately when you have finished. Accurately weigh
out 50-75 mg of the fine Co(salen) powder and immediately record in your notebook how much
you have weighed. Place the solid in a filter flask with a stir bar. Larger amounts of Co(salen)
make for more accurate results: a good strategy might be to use less than half of your material for
the first run (as long as this is not greater than 75 mg), and the remaining material for a second
run, when you will be more aware of experimental challenges, and are more likely to get good
data.
Connect tubing and a graduated pipette to the sidearm of the filter flask, as shown in
Figure 1 of this EP, and place the filter flask on a magnetic stirrer. Now clamp the filter flask and
the graduated pipette to a ringstand, and fill the tubing and pipette with water. The flask will
need to be clamped low on the ringstand to sit on the magnetic stirrer; the tubing of water will
probably have to drape beneath the bench level.
If bubbles of air appear in the tubing, squeeze them out by squeezing the tube. Or, place
your finger over the top of the pipette and turn it vertically, below the level of the tubing, to let
the bubbles rise out.
Adjust the movable tubing to make the water levels equal in both the tubing and in the
graduated pipette: this ensures that the pressure within the apparatus is atmospheric. The water
level in the tubing must not be so high that it might be sucked or splashed into the filter
flask. But, during the experiment, as the Co(salen) takes up oxygen, the water level in the pipette
might drop, and the level in the tubing might rise, anywhere from two (more likely) to as much
as twenty milliliters, so be sure that the water level in the graduated cylinder is at a reasonable
level for 1) it not to be sucked into the flask, and 2) you to read if it drops this much. Add or

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remove water as necessary, and finally re-adjust the tube so that the water level on both sides is
equal. Record the water level in the graduated tube.
Place a black rubber stopper on top of the filter flask, cocked just enough to one side to
allow a purge needle to slip through into the flask, and bring your ringstand and apparatus (not
the magnetic stirrer) over to the oxygen flushing stations. With a long purge needle that almost

Figure 1. Experimental apparatus for measuring the kinetics of oxygen uptake by Co(salen).
reaches the Co(salen), flush the flask thoroughly with oxygen: five minutes should be necessary.
(If the oxygen purge blows the Co(salen) all around the flask, raise the purge needle to a higher
level.)
After purging with oxygen, seal the flask tightly with the stopper, take your apparatus
back to your hood, and place your flask on the magnetic stirring plate. Record the time and the
water level.

Then, fill a needle and syringe with 20 mL of previously degassed DMSO, and again
cock the stopper to one side just enough for a needle to pass through. Add the DMSO to your
flask, immediately seal the flask tightly with the stopper, and stir the solution. Then
immediately record the time and the water level.
Continue to record the water level in the pipette at thirty second time intervals until the
water level stops dropping, or for about twenty minutes if the water level is still dropping at that
time. If your timer does not record seconds, use one-minute time intervals.
You should perform this experiment a second time, if possible, to try to acquire two good
data sets. However, some exceptions given below may make it better for you to perform only one
run:
Some samples of Co(salen) are not pure enough, or not dry enough, to convert to the
active form upon dissolution in DMSO, and therefore will not take up oxygen or show any
decrease in the water level of the graduated pipette. In this case, ask someone who does have
Co(salen) that shows activity to give some of their sample to you, and perform the experiment
using that sample (Record in your notebook and lab report whose sample you used). Those
students who share their samples may then have only enough Co(salen) to perform one good run,
and you also may only be able to obtain enough Co(salen) to perform the experiment once.

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Data Analysis
1. (3 points) Write the balanced overall reaction that was used for the of synthesis of Co(salen).
2. (1 point) Record your per cent yield of Co(salen).
3. (6 points) Make a table of your data in the form of total time passed and total volume
change in the water.
So, if at the 30-second reading, the volume change is one mL, and at the 60-second
reading the volume change is an additional 4 mL, you would list as the second entry in your table
that the "total time change" is 90 seconds, and the "total volume change" is 5 mL.
4. (6 points) Plot the above data in three ways: as volume change vs time, as log(volume change)
vs. time, and as volume change vs 1/time.
5. (8 points) Extrapolate on all three plots to estimate the time that the volume change ended (or
would have ended had we extended the experiment longer). Use the three different plots to try to
determine which is the most accurate extrapolation, or combine several reasonable extrapolations
and average them. Record the final total water volume change (in mL) at the extrapolated total
time passed as the overall total oxygen uptake in mL.
4. (7 points) Using the total number of mL of oxygen absorbed, calculate at room temperature
and atmospheric pressure the number of moles of oxygen that were absorbed. Then calculate the
ratio of the number of moles of Co that you used to the number of moles of oxygen that was
absorbed. Show all your calculations.

Discussion
1. (2 points) Discuss whether you got a reasonable yield of Co(salen), and, if not, what problems
in its synthesis might have lowered the yield.

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2. (6 points) Discuss whether your Co(salen) showed oxygen uptake activity. If it did not, discuss
what factors may have prevented this. Give specifics on how problems during its synthesis,
possible oxidation of the Co(II), or Co(salen) that was not dry enough, might have resulted in
giving the inactive form of the complex. How could any ethanol that remained in the Co(salen)
inhibit its oxygen-uptake activity?.
3. (3 points) Discuss what chemical differences between chloroform and pyridine result in
chloroform being unable to coordinate to Co(salen), while pyridine can readily be coordinated.
4. (6 points) Explain the changes that occur and the molecular processes that are important when
DMSO is added, that allow oxygen uptake to begin.
5. (7 points) Using your best extrapolation(s), how many moles of dioxygen were actually
absorbed per mole of Co(salen)? Discuss whether the bound O2 exists in the peroxide or
superoxide form.
If your calculated stoichiometric ratio of Co to O2 is larger than expected (ex., 4 Co: 1
O2), was the Co(salen) more active in absorbing O2, or less active, than expected? Discuss
possible reasons for this greater or lesser activity.
(10 points) Conclusions: Discuss how your results on the form and stoichiometry of oxygen
uptake in the biomimetic Co(salen) could be used to give information on biological oxygen
uptake and release in metal-containing heme groups.