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Experiment #5: Post Lab Assignment Luminol and Chemiluminescence

Below are the assigned questions for this weeks post lab assignment, which you will submit in lieu of a laboratory
report. Each question must be answered in five sentences or fewer. Use the same heading, font, and spacing as in the
report template. Figures may also be used in lieu of words. DO NOT INCLUDE THE QUESTIONS IN YOUR
POSTLAB. If you do, the report will not be acceptable for submission to turnitin.com and will therefore count as a zero
grade.

1. The two carboxylic acid groups in 3-nitrophthalic
acid have different pK
a
values. The pK
a
value of
the carboxylic acid at the 1-position is about 3, and
the pK
a
value of the acid at the 2-position is about
2. Which of these carboxylic acid functionalities is
more acidic and explain why?

2. Given the approximate pK
a
s provided, compute K
eq

for the reaction below.



3. In the first step of the luminol synthesis (shown
below), explain why 3-nitrophthalhydrazide (1)
was formed as the major product instead of
compound 2, as shown below. (This happens even
if 5 equivalents of hydrazine are used relative to 3-
nitrophthalic acid.)

4. Hydrazine (H
2
N-NH
2
) and 3-nitrophthalic acid
undergo a double condensation reaction to form
compound 1 (shown above). If propane-1,3-
diamine (H
2
N-CH
2
-CH
2
-CH
2
-NH
2
) were used
(instead of using hydrazine) to form an analogous
product, we expect that (A) the rate of reaction
would be slower, and (B) product would be less
stable than compound 1. Explain why you would
expect BOTH of these results to be true (provide
two separate/different answers, i.e., one for each).
Note that the answer is not deeply embedded in the
mechanism. Although variability in nucleophilicity
may play a role, exclude this argument from your
answer for this problem. This may be the most
challenging question for some, so perhaps you will
want to come back to it.

5. After luminol is synthesized/isolated, it was re-
dissolved in an aqueous NaOH solution. Explain
why amide protons are removed under the given
basic conditions, whereas the amine protons are not
removed from the molecule.

6. Upon treatment of luminol with
K
3
Fe(CN)
6
/H
2
O
2
you should have
observed an emission of light (see
image on right if your sample did not
yield chemiluminescence). There was
a range of wavelengths emitted; select
one wavelength that you would
expect to be toward the middle of the
range of emission wavelengths. Think back to what
you learned in general chemistry. Briefly explain
why you selected the wavelength.

7. Explain why you think luminol would be a good or
bad choice as a light source for a large blue sign in
Times Square (the sign would need to be blue and
to run 24 hours a day).

8. Imagine that you travel to one of Puerto Ricos two
Bioluminescent bays (very interesting, look them
up), collect a large quantity of dinoflagellates
(unicellular species that exhibit bioluminescence
upon agitation), return to your laboratory, and
extract (isolate) the compound responsible for the
luminescence. Your colleague is convinced that the
compound responsible for the luminescence is
luminol, while you suspect luciferin (the same
molecule responsible for fireflies luminescence)
may be involved. In order to identify the unknown
compound you will employ several techniques
such as gas chromatography/mass spectrometry
(GC/MS), infrared (IR) spectroscopy, and nuclear
magnetic resonance (
1
H-NMR and
13
C-NMR)
spectroscopies.




(See the next page for the rest of this question!)
NO
2
OH
O
OH
O
NO
2
O
O
OH
O
H
2
NNH
2
H
3
NNH
2
+ +
pK
a
~2 pK
a
~8
NH
NH
O
O
NH
2
Luminol
O
O
O
O
NH
2
1) NaOH
2) K
3
Fe(CN)
6
+ Light
S
N
S
N
OH
O
HO
Luciferin
Luciferase/O
2
S
N
S
N
O
HO
+ Light
OH
OH
O
O
NO
2
H
2
N NH
2
hydrazine
3-nitrophthalic acid
NH
NH
O
O
NO
2
1
N
N
O
O
NO
2
NH
2
NH
2
H
H
2

2
Assuming you develop a method to isolate and
study the unknown the luminescent compound
without oxidation taking place. For EACH possible
compound (luminol AND luciferin), list the
following data that you would expect to obtain
from:
(A) GC/MS: including the exact mass (to the
hundredths place) and the mass of the two most
abundant isotopes,
(B) IR Spectroscopy: indicate one unique
differentiating signal and clearly state which bond
it is associated with,
(C)
13
C-NMR Spectroscopy: indicate the number
of signals with different chemical shifts, and
(D)
1
H-NMR Spectroscopy: indicate the number of
signals with different chemical shifts.

9. List all resources (websites, books, journal articles,
etc.) you used to answer these questions. Make
sure that if you used five or more words directly
from any reference, the words are in quotes with a
reference crediting the source. Failing to do so is
plagiarism.