Squaraine Handout - URI Department of Chemistry

STUDENT HANDOUT
PURPOSE OF THE EXPERIMENT
Synthesize a near-infrared emitting organic fluorophore via multi-step organic synthesis; purify the
product by recrystallization, and characterize the photophysical properties of this unique compound.
BACKGROUND REQUIRED
You should be familiar with the set-up of standard organic reactions using a reflux condenser,
recrystallization and extraction techniques, and analysis of organic compounds by 1 H NMR and FT-IR
spectroscopy. You will learn how to use a Dean-Stark trap to remove water from a reaction, and how to
perform UV-visible and fluorescence analysis.
BACKGROUND INFORMATION
Organic dyes are important for a number of applications, including the detection of explosive molecules
and the imaging of cancer cells and other biological analytes. Some examples of commercially available
organic dyes are shown below (Figure S1).
Figure S1: Examples of common organic dyes
Researchers are continually working to synthesize new dyes with better properties, such as: (1) a higher
fluorescence quantum yield; (2) better stability in biological environments; and (3) absorption and
emission maxima that are in the red to near-infrared region (because this region will have little
interference from other molecules).
Note: Fluorescence quantum yield is defined as the number of photons emitted divided by the number of
photons absorbed. When a molecule absorbs a photon of light, that photon can undergo a variety of
procedures, one of which is emission to cause fluorescence. High quantum yields (near 1) mean that a
high proportion of the photons that are absorbed are also emitted.
Squaraines are a class of organic dyes that possess a high fluorescence quantum yield, and red to nearinfrared
absorption and emission maxima (2 of the 3 properties mentioned above). The general structure
of one type of squaraines is shown in Figure S2. These compounds possess an electron-deficient
cyclobutenone central ring substituted with two electron-rich aromatic rings. One stable structure of
squaraine compounds is the zwitterionic form shown, where the cyclobutene has a +2 charge, and both
oxygens are negatively charged.
Figure S2: General structure of squaraine compounds
Most red-emitting dyes have large, planar conjugated structures (Figure S3), because the extended
conjugation often leads to red-shifted absorption and emission maxima. In contrast, squaraines have a
relatively small size, yet still absorb and emit in the red to near-infrared spectral region. This small size
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enables them to be encapsulated easily in macrocycles, such as cyclodextrins or synthetic rotaxanes, and
allows them to be used in biological detection schemes without interfering with natural cellular pathways.
Figure S3: Examples of near-infrared emitting organic dyes
The synthesis of squaraines is usually accomplished by reacting two equivalents of an electron-rich
aromatic compound with squaric acid (3,4-dihydroxycyclobut-3-ene-1,2-dione). The mechanism of this
reaction is shown below:
Scheme S1: Mechanism for the synthesis of squaraine compounds
Squaraines are also interesting because of their unique absorption and emission spectra. The particular
squaraine you will synthesize in this experiment is shown in Figure S4 (compound 1a) with its
absorbance and emission spectra (measured in chloroform).
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Figure S4: Absorption and emission spectra of the target squaraine compound
The narrow absorption band around 630 nm and emission band around 650 nm are typical of squaraine
compounds. The squaraine compound is also brightly colored (green-blue solid, bright blue dilute
solution, reddish-blue concentrated solution) (Figure S5).
Figure S5: Colors of the squaraine product 1a under various conditions (measured by the authors)
OBJECTIVES
The purpose of this experiment is to synthesize squaraine compound 1a in a two-step procedure, and
characterize that compound using UV-Vis and fluorescence spectroscopy, as well as 1 H NMR analysis.
PROCEDURE
Laboratory Period 1:
Using a graduated cylinder, measure 20 mL of water and add to a 50 mL round-bottom flask. Add 20 mg
of sodium dodecyl sulfate and 920 mg of sodium bicarbonate. Then use a syringe to add 4.6 mL of aniline
to the aqueous solution (CAUTION: This step should be done in a fume hood). Add a stir bar and stir the
mixture vigorously at room temperature for 5 minutes, then add 1.3 mL of benzyl bromide via syringe.
(You can do this using a 1 mL disposable syringe by dispensing 1 mL first, then 0.3 mL. Alternatively,
your instructor may dispense this compound for you.) Add a reflux condenser to the top of the round-
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ottom flask and make sure that the water or other coolant is flowing appropriately through the
condenser. Heat the reaction mixture to 80 o C using an oil or sand bath for approximately 90 minutes.
Monitor the progress of your reaction using TLC. Use a solvent mixture of 95:5 hexanes: ethyl acetate as
the mobile phase. Both of your reactants and your desired product are visible under a hand-held UV lamp,
or can be stained with a potassium permanganate stain.
After 90 minutes (or when the TLC shows that the reaction is complete), cool the reaction to room
temperature and transfer it to a separatory funnel. Add 20 mL of ethyl acetate to the funnel using a
graduated cylinder. Separate the layers and collect the ethyl acetate layer. Do another extraction by
adding an additional 20 mL of ethyl acetate to the funnel with your aqueous layer, then separating the
layers. Combine the ethyl acetate layers in a round-bottom flask. Remove the ethyl acetate on the rotary
evaporator to yield a crude oil.
Add a small amount of n-hexanes via a Pasteur pipette to the oil and cool the mixture to 0 o C. Crystals
will form, and these crystals should be filtered using a Buchner funnel. Transfer the filtrate to a roundbottom
flask and remove the solvent on the rotary evaporator. This will yield more crude oil that has not
yet crystallized. Do a second round of recrystallization by adding more n-hexanes to that oil, cooling it to
0 o C, and filtering the crystals using a Buchner funnel.
Obtain a mass for your crystals and calculate your percent yield. Dissolve approximately 10 mg of your
product in 1.0 mL of CDCl 3 and transfer it to an NMR tube. Obtain a 1 H NMR spectrum. Obtain an IR
spectrum as well. Save your crystals for the second reaction which will occur during the next lab period.
Safety: Aniline and benzyl bromide are volatile chemicals and should be used only in the fume hood.
Make sure to dispose of all syringes and chemical waste properly. Hexanes and ethyl acetate are volatile
and flammable and should only be used in a fume hood. Deuterated chloroform is a potential carcinogen
and should be used in the fume hood.
Laboratory Period 2:
Use the crystals obtained during the previous laboratory period as the starting material for this second
reaction. Add 270 mg of dibenzylaniline (your product from the first reaction) and 57 mg of squaric acid
to a round-bottom flask, followed by 2.0 mL of toluene and 2.0 mL of n-butanol, which you can measure
with a graduated cylinder or syringe. (CAUTION: These solvents should be measured and transferred in a
fume hood. They are volatile and have potentially irritating odors).
Add a Dean-Stark trap to the top of the flask, followed by a reflux condenser. Make sure all of the joints
are properly secured and greased. Then add a sufficient amount of a 1:1 mixture of toluene and n-butanol
from the top of the condenser to fill the Dean-Stark trap completely. Heat the reaction to reflux for two
hours. (NOTE: The two hours do not start until the reaction mixture starts to boil).
As the reaction proceeds, you should observe an intense green color developing. After two hours, slowly
cool the reaction mixture to room temperature. Green crystals should spontaneously form. Filter the
crystals using a Buchner funnel and wash them with a small amount of isopropanol. The filtrate will be a
dull green color, and the crystals will be bright green. Obtain a mass of these crystals and calculate the
percent yield. Save your crystals in a vial for the next laboratory period, where you will analyze and
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characterize your product. If you do not obtain crystals, then remove your solvent on the rotary evaporator
to obtain a green solid, which should be sufficiently pure for UV-visible and fluorescence analysis.
Safety: Toluene and n-butanol are volatile solvents with potentially irritating odors. Isopropanol is a
volatile solvent. Make sure to use them only in a fume hood and dispose of them properly. Dispose of all
syringes and chemical waste properly.
Laboratory Period 3:
In this lab session, you will thoroughly analyze the crystals that you formed in the previous session.
1. 1 H NMR analysis: Dissolve a small amount of your crystals in CDCl 3 . Transfer the solution to an NMR
tube, and collect the spectrum.
2. IR analysis: Take a small amount of your crystals and obtain an IR spectrum. Look for the presence of
key functional groups (i.e. C=O, OH, etc).
3. UV-Vis analysis: Dissolve a small amount of your crystals in chloroform to obtain a concentration of
10 mg/L (solution 1). Transfer some of the solution to a cuvette and obtain a UV-Vis absorbance
spectrum. Then dilute some of your solution 1 to a new final concentration of 0.50 mg/L (solution 2).
Transfer some of the solution to a cuvette and obtain a UV-Vis absorbance spectrum of solution 2. Look
for any differences in the spectra of the two solutions.
Note 1: You need approximately 3.0 mL of each solution to obtain a good absorption and emission
spectrum.
Note 2: You cannot record these spectra using plastic/disposable cuvettes. The organic solvent will erode
the plastic.
4. Fluorescence analysis: Using the same solutions that you used for UV-visible analysis, obtain a
fluorescence spectrum of both solution 1 and solution 2. Excite the solutions at 600 nm and record the
fluorescence from 610-800 nm. Look for any differences in the spectra of the two solutions.
Safety: Make sure to dispose of all your solutions using the proper waste containers. Chloroform is a
potential carcinogen and should be used in a fume hood.
POST-LAB QUESTIONS
1. What is the theoretical yield for each step of the reaction
2. What is your percent yield for each step
3. What are the key peaks that you can identify in the IR and 1 H NMR spectra for step 1 of the reaction
How does this help you figure out the structure of your product
4. Why is the first reaction considered to be an example of “green chemistry” What is the role of SDS in
the reaction mixture
5. What is another potential product that you could have formed from step 1, and how would you
distinguish that product using 1 H NMR and IR spectroscopy
6. What are the key peaks that you can identify in the IR and NMR for step 2 of the reaction How does
this help you figure out the structure of your product
7. Analyze your UV-vis spectra for the two solutions. What are the key differences between the two
How can you explain these differences
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