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Experiment

Synthesis of Fluorescein

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Fluorescein is an organic fluorescent dye used in several areas, ranging from medicine to
research applications. Structurally, fluorescein is similar to phenolphthalein, a well known dye
indicator used in acid-base titrations. The difference can be seen in Figure 1, illustrating oxygen
bridging the aromatic rings on fluorescein. This relatively simple change has a significant impact
on the spectral behavior of these two dyes. Fluorescein is fluorescent whereas phenolphthalein is
not.

Fluorescein
Phenolphthalein
Figure 1 Neutral structures of fluorescein and phenolphthalein

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Fluorescence occurs due to the emission of light from the relaxation of the molecule from the
excited state to the ground state. The electronic transition to the excited state occurs when the
excitation wavelength corresponds to the energy difference between the ground state and the
excited state. In the excited state, some energy is lost to vibrational relaxation. As a result,
fluorescence energy is less than the absorption energy and the emitted light is observed at a
longer wavelength than the excitation wavelength.

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In this experiment, you will synthesize fluorescein by the reaction shown in Figure 2. Visible
absorbance and fluorescence spectroscopy will be used to identify the wavelength of maximum
absorbance ( max) and emission wavelength.

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Phthalic anhydride

Resorcinol
Fluorescein
Figure 2 Synthesis of fluorescein

OBJECTIVES

In this experiment, you will

Synthesize fluorescein.
Identify the wavelength of maximum absorbance ( max).
Determine the purity of the sample based on Beers law.
Identify the emission wavelength.

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Experiment 23

MATERIALS
Part I Synthesis of Fluorescein

50 mL round bottom flask


heating mantle apparatus
ring stand and utility clamp
magnetic stir bar
10 mL graduated cylinder
250 mL beaker
125 mL Erlenmeyer flask
vacuum filtration apparatus
watch glass
disposable Pasteur pipets and bulb

resorcinol
phthalic anhydride
0.5 M sulfuric acid
1.0 M hydrochloric acid
1.0 M sodium hydroxide
0.1 M sodium hydroxide
DI water
250 mL volumetric flask
Wide-Range Temperature Probe or
thermometer

Part II Spectrophotometric Absorbance

LabQuest or computer interface


LabQuest App or Logger Pro
SpectroVis Plus Spectrophotometer
plastic cuvette with lid

fluorescein sodium salt


product from Part I
0.1 M sodium hydroxide
100 mL volumetric flask

Part III Measure Fluorescence (Emission Wavelength)

LabQuest or computer interface


LabQuest App or Logger Pro
SpectroVis Plus Spectrophotometer
plastic cuvette with lid

fluorescein solution from Part I


0.1 M sodium hydroxide
100 mL volumetric flask

PROCEDURE
Part I Synthesis of Fluorescein

1. Obtain and wear goggles. Protect your arms and hands by wearing a long-sleeve lab coat and
gloves. Conduct this reaction in a fume hood.
2. Clamp a 50 mL round bottom flask to a ring stand and lower into a heating mantle.
3. Prepare the reaction mixture.
a. Weigh out 0.3 g of resorcinol and transfer to the round bottom flask. Record the actual
mass to the nearest 0.001 g.
b. Weigh out 0.2 g of phthalic anhydride and transfer to the round bottom flask. Record the
actual mass to the nearest 0.001 g.
c. Slowly add approximately 1 mL of 0.5 M H2SO4 to the mixture of solids in the round
bottom flask. CAUTION: Handle the sulfuric acid with care. Can cause painful burns if
it comes in contact with the skin.
4. Heat the reaction at approximately 160180C for 30 minutes. Monitor the temperature using
a Wide-Range Temperature Probe or thermometer. Note: A red-brown solid will form as the
reaction progresses.
5. After 30 minutes, turn off the heating mantle and let the flask cool for 15 minutes.
6. While waiting, prepare an ice water bath in a 250 mL beaker.
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Synthesis of Fluorescein
7. After the flask has cooled, dissolve the reaction mixture in 25 mL of 1.0 M sodium
hydroxide then transfer the solution to a 125 mL Erlenmeyer flask. CAUTION: Sodium
hydroxide solution is caustic. Avoid spilling it on your skin or clothing.
8. Place the 125 mL Erlenmeyer flask in the ice-water bath and slowly add 25 mL of 1.0 M
hydrochloric acid. CAUTION: Handle the hydrochloric acid with care. Can cause painful
burns if it comes in contact with the skin.
9. Collect the solid using vacuum filtration. Note: Be sure to record the mass of the filter paper
before placing it in the vacuum funnel.
10. Place the filter paper containing the solid on a watch glass and gently direct a stream of air
(low flow) to thoroughly dry the solid. When the solid has dried, weigh the recovered solid
and record the mass to the nearest 0.001 g. Save the solid for analysis in Part II and III.
11. Weigh out approximately 0.010 g of your synthesized fluorescein and transfer to a
250 mL volumetric flask. Record the actual mass to the nearest 0.001 g.
12. Add 0.1 M NaOH to the volumetric flask. Set this solution aside for analysis in Part II.
Part II Spectrophotometric Absorbance

You will analyze several samples to determine the amount of fluorescein in your synthesized
sample. You can use this information to calculate its purity. Follow Steps 1320 to prepare a set
of fluorescein standard solutions and conduct testing to develop your own Beers law plot of the
standards.
13. Quantitatively prepare a 7.5 10-5 mol/L stock fluorescein solution in 0.1 M NaOH.
14. Prepare five dilutions in 0.1 M NaOH of the fluorescein stock solution with the following
concentrations: 7.5 10-6, 6.0 10-6, 4.5 10-6, 3.0 10-6, 1.5 10-6 mol/L. Summarize the
volumes and concentrations for the trials in the data table.
15. Prepare a blank by filling a cuvette 3/4 full with 0.1 M NaOH.
16. Connect the Spectrophotometer to the USB port of LabQuest or a computer. Choose New
from the File menu of the data-collection program.
17. Calibrate the Spectrophotometer with the blank.
18. Determine the optimal wavelength for analysis by collecting a full absorbance spectrum.
Record the wavelength in the data table.
19. Change the mode of data collection to Events with Entry (absorbance vs. concentration).
20. Collect data for the five standard solutions. Record the absorbance and concentration values
in the data table.
21. Perform a linear regression analysis. Record the equation in the data table.
22. Measure and record the absorbance of the fluorescein sample prepared in Step 12 and
determine its concentration. Note: If the absorbance value does not fall within range of your
standard solutions, prepare a more dilute or more concentrated sample, depending on the
absorbance value from your test.
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Experiment 23
Part III Measure Fluorescence (Emission Wavelength)

23. Prepare the fluorescein sample solution for testing by transferring a small amount of the
sample prepared in step 12 to a clean cuvette. Fill the cuvette about 3/4 full with the sample.
24. Prepare the Spectrophotometer for measuring the fluorescence of the solution.
Logger Pro: Choose Change Units Spectrometer: 1 Fluorescence 405 nm from the

Experiment menu.

LabQuest App: Choose Change Units USB: Spectrometer Fluorescence 405 nm from

the Sensors menu.

25. Set an appropriate sampling time for collecting fluorescence data.


Logger Pro: Choose Set Up Sensors from the Experiment menu. Change the Sample Time

to 150 ms, the Wavelength Smoothing to 0, and the Samples to Average to 1.


LabQuest App: Tap the Mode: Full Spectrum box on the Meter screen. Change the Sample
Time to 150 ms, Wavelength Smoothing to 0, and Samples to Average to 1.
26. Place the cuvette containing your solution into the cuvette slot of the Spectrophotometer.
Make sure that you place the cuvette in the Spectrophotometer in the same orientation for all
tests.
27. Start data collection. A full spectrum graph of the sample will be displayed. Stop data
collection.
28. Observe the peak or peaks on the graph. Fluorescence is measured on a relative scale of
01, thus the peak fluorescence needs to be less than 1.0. If the peak intensity is greater than
1, repeat Step 25 and decrease the sample time by 10 ms. If the peak is below 0.5, increase
the sample time by 10 ms. Collect a new fluorescence spectrum. Increase or decrease the
sample time until the peak amplitude is 0.51.0. Record the emission wavelength in your
data table.
29. Discard the solutions as directed by your instructor.

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Synthesis of Fluorescein

DATA TABLE
Part I Synthesis of Fluorescein
Mass of resorcinol (g)
Mass of phthalic anhydride (g)
Mass of filter paper (g)
Mass of filter paper and product (g)
Mass of product (g)
Part II Spectrophotometric Absorbance
Trial
number

Volume of 7.5 10-5


stock (mL)

Volume of 0.1 M
NaOH added (mL)

Concentration
(mol/L)

Absorbance
reading

1
2
3
4
5
Best fit line equation

Absorbance wavelength, max (nm)

Mass of synthesized
fluorescein (g)

Total volume
(mL)

Concentration
(mol/L)

Absorbance
reading

Volume used in
dilution (mL)

Total volume
(mL)

Concentration
(mol/L)

Absorbance
reading

Part III Measure Fluorescence (Emission Wavelength)


Emission wavelength (nm)

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Experiment 23

DATA ANALYSIS
1. What is the theoretical yield of fluorescein in your synthesis? What is the percent yield?
2. Determine your product purity based on your spectroscopy results.
3. Explain the difference between an absorbance and fluorescence (excitation and emission)
spectra.
4. Compare your results using fluorescent spectroscopy to your results using traditional
spectroscopy. Is one method better than the other? Please explain.
5. Draw the three charged species of fluorescein (cation, monoanion, dianion).
6. Draw the three neutral species of fluorescein (quinonoid, lactone, zwitterion).

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Vernier Lab Safety Instructions Disclaimer


THIS IS AN EVALUATION COPY OF THE VERNIER STUDENT LAB.

This copy does not include:

Safety information

Essential instructor background information

Directions for preparing solutions

Important tips for successfully doing these labs

The complete Organic Chemistry with Vernier lab manual includes 26 labs and essential
teacher information. The full lab book is available for purchase at:
http://www.vernier.com/products/books/chem-o/

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