114
Practical Exam
Lab 11. Practical Exam: Synthesis and
Chemiluminescence of Luminol.
11.1. Objective
The goal of this laboratory practical exam will be to assess your ability to prepare for and
conduct an independent two-step organic synthesis of luminol from 3-nitrophthalic acid
using the techniques you have learned this semester in CHEM 233. The structure of
your final product will be confirmed by observing its ability to chemiluminesce when
reacted with caustic hydrogen peroxide and potassium ferricyanide.
NO2 O
OH
OH
O
NO2 O
H2NNH2/H2O
220 °C, 3-4 min
HO
O
O
(triethylene glycol = solvent)
3-nitrophthalic acid
(Does NOT Chemiluminesce.)
NH
NH
OH
O
a.) NaOH/H2O, Na2S2O4
~100 °C, 5 min
b.) CH3COOH
3-nitrophthalhydrazide
(Does NOT Chemiluminesce.)
NH2 O
NH
NH
O
Luminol
(DOES Chemiluminesce.)
Figure 11.1.1
11.2. Assessment
You will be assessed out of 100 points according to the performance elements in the
Grading Rubric, which include a prelab quiz (30 pts), reaction table (20 pts), product
yield (10 pts), general technique (10 pts), positive chemiluminescence of product (20
pts) and drawer check out (10 pts).
The prelab quiz will cover the theory and practical aspects of the synthesis of
luminol as well as the academic article by Creamer, which compares the luminol test for
blood to other forensic techniques. (You will need to find this article according to the
instructions on the following pages.) You should understand the basic functional group
transformations taking place in each step as well as have a working knowledge of the
chemiluminescence exhibited by luminol. Most importantly though, you will be asked
in the prelab quiz to explain and rationalize the importance of the steps taken in the
procedure outlined by the textbook. Some of those answers will require thought and
possibly a little research outside your text. For example, why did the authors choose to
use triethylene glycol as the solvent for the first reaction? I may ask you to list three
reasonable explanations. Or, what is the purpose of adding glacial acetic acid in the
final reaction? Or even, what does glacial mean and what is the structure of acetic acid?
The popularity of this synthesis makes the internet a convenient source, but be careful
to double check your information and scrutinize your web sources for validity. You may
use any notes you take in your lab notebook to aid you during the quiz, but you will only
be given 20 minutes at the beginning of the class period.
© 2009 Chad L. Landrie
115
Preparation is essential for this experiment because you will not be allowed to use
any resources other than your lab notebook, which may contain hand written notes and
procedures for your experiment. No typed or photocopied materials may be used during
the lab time. Your prepared notes will be turned in with the practical to aid TA’s in
assessing your performance, preparedness and general technique. Although you may
use the procedure in the textbook as a guide when preparing your notes, you will be
issued a vial containing a preweighed amount of 3-nitrophthalic acid which is not the
1.0 g recommended by the text. In addition, a slight modification has been made in the
number of equivalents of each reagent used. These two changes will require you to
recalculate the exact quantity of each reagent and solvent needed for your specified
amount of starting material. This will be done on lab day in the Reaction Tables
worksheet, so be sure to understand how to perform this math beforehand.
11.3. Required Reading
1. From your textbook: pages 671 – 675 (Amides and Chemiluminescence).
2.
pages 676 (Miniscale Procedure for Luminol Synthesis).
3.
pages 677 – 678 (Chemiluminescence Procedure).
4. Creamer, Jonathan I., et al. A comparison of the presumptive luminol test for blood
with four non-chemiluminescent forensic techniques. Luminescence 2006; 21(4):
214-220.
Finding and Printing the Journal Article by J.I. Creamer
Fortunately, many academic journals are now online, which makes finding and printing
articles relatively easy, provided that you or your library has an online subscription. The
article by Creamer that you are required to read (item 4 above) is in the journal,
Luminescence. This journal is online and can be accessed by all UIC students, faculty
and staff. Follow the instructions below for finding and printing this article.
1.
2.
3.
4.
5.
Direct your web browser to the UIC library website (www.uic.edu/depts/lib/).
From the Quick Links/Shortcuts menu, choose Electronic Journals.
Using the search bar or the alphabet links, navigate to the journal Luminescence.
Click on the link Wiley Interscience Journals under the title Luminescence.*
Using the year, volume number, issue number and page numbers listed above, find
the article by Creamer.
6. Click on the PDF link to download article. Alternatively, right-click on the PDF link
and save the file to your desktop. You will need a pdf viewer such as Adobe Acrobat
in order to view this pdf file.
*
Note: you may be asked to login to your Bluestem account if you are trying to access
this journal off campus.
11.4. Pre-lab Tasks
1. Read the suggested reading from the textbook and the included journal article. You
may take notes in your lab book to aid you during the quiz on lab day. I am not
expecting you to understand everything you read in the journal article describing the
chemiluminescent test for blood. Look for big concept ideas such as, which test
showed the greatest sensitivity, what materials were the tests performed on, what is
© 2009 Chad L. Landrie
116
the relative toxicity of luminol compared to the other tests, what species is the
catalyst in the test for blood, how might a lawyer discredit the luminol test for blood
to help his client, do the authors address the specificity of the luminol test in their
currently reported research?
2. Consider the exercises on page 678 – 679 as a further review for the quiz.
3. Consider the calculations required to complete the Reaction Tables worksheet
included in this packet. You will receive an exact copy of this worksheet during the
lab practical, but you will not be able to use the worksheet from this packet.
Therefore, I suggest you ensure that you are proficient in these calculations and take
adequate notes in your lab notebook.
4. Review the techniques you will be using to complete the synthesis of luminol.
11.5. Lab Day Tasks
1. Take the quiz, which is on the last page of the packet that will be distributed that day,
during the first 20 minutes of the class period. Remove the quiz from your packet
when finished and turn it in to your TA.
2. Take your Grading Rubric to the stockroom and obtain your vial.
3. Fill out the Reaction Tables worksheet.
4. Perform the synthesis of luminol. There is no need to reweigh your sample. Use the
mass listed on the label at the top of the Grading Rubric.
5. Have your TA weigh a piece of filter paper and record this value on your Weights and
Measures worksheet. Your TA must sign next to each measurement value to receive
credit.
6. Use the above filter paper in your final vacuum filtration of luminol. You may wash
the filter cake with three 10 mL portions of 95% ethanol to remove most of the water.
Aspirate air through the filter cake for at least 10 minutes to ensure dryness. TA’s
will substract 30% from the yield for wet samples.
7. Take the dry luminol, still on your filter paper, to your TA for weighing and record
this value on your Weights and Measures worksheet. Your TA must sign next to
each measurement value to receive credit.
8. Prepare both solutions A and B carefully using the same quantities recommended by
the textbook. Caution: using excess reagents may increase the intensity, but will
result in shorter chemiluminescent times. Your chemiluminescence must persist for
15 seconds.
9. Bring solution A, solution B and an empty vessel to the instrument room. Turn the
lights off and perform the chemiluminescent experiment in front of your TA. (This
should take ~15 seconds per student.) Have your TA indicate the length and
strength of your chemiluminescence on your Weights and Measures worksheet.
Again, your TA must sign next to each measurement value to receive credit.
10. Turn your Grading Rubric, your Reaction Tables worksheet and your Weights and
Measures worksheet into your TA.
© 2009 Chad L. Landrie
117
11.6. Policies
1. No collaboration with classmates will be allowed. Students heard speaking with
other classmates about the lab practical will receive an automatic zero.
2. You may only use your handwritten notes and a calculator during the practical. The
handwritten notes may not contain typed or photocopied materials.
3. All measurements must be entered by the teaching assistant on the Weights and
Measures worksheet. Failing to obtain the TA’s signature on the Weights and
Measures worksheet will result in no credit.
© 2009 Chad L. Landrie
118
11.7. Lab Practical Grading Rubric
The following rubric will be completed by your teaching assistant and used to asses your
performance during the practical exam.
Performance
Element
Excellent
Fair
The prelab
quiz will cover the
theory and practical
lab aspects of the
synthesis of Luminol.
You will have 15
minutes to complete
the quiz beginning
precisely at the class
start.
Late students
will not be allowed
additional time.
Use the questions on
pages 678 – 679 as a
study guide for the
quiz.
Fair questions will also
ask for the justification
for
the
steps
performed
in
the
experiment.
You
should seek additional
sources
when
the
textbook does
not
provide answers.
20 pts:
You will fill
out the reaction table
worksheet for both the
quantities
and
equivalents presented
in the text and for the
amount of starting
material indicated on
your assigned vial.
Each box is worth 1.0
pts.
Values must
contain at least three
significant figures for
credit.
10 pts:
8 pts:
6 pts:
4 pts:
100 - 70%, measured
by TA
Wet solids subtract
30% from yield.
SHOW ALL WORK
69 – 50%, measure by
TA
Wet solids subtract
30% from yield.
NO WORK =
NO
CREDIT
49 – 30%, measured
by TA
Wet solids subtract
30% from yield.
29 – 10%, measured
by TA
10 pts:
Student is
well prepared with
detailed
procedure
notes written in their
lab
book,
works
efficiently
and
demonstrates proper
techniques.
8 pts:
Student is
well prepared with
detailed
procedure
notes written in their
lab
book,
works
efficiently, but has
some difficultly with
one
or
two
procedures.
4 pts:
Student’s
prelab procedure notes
lack detail requiring
frequent guidance and
reminders by the TA.
Student
may
also
show difficulty with
procedures.
2-0 pts
20 pts:
15 pts:
10 pts:
5 pts:
Strong persistent (> 15
sec)
chemiluminescence is
observed.
Moderate or brief (< 15
sec)
chemiluminescence is
observed.
Chemiluminescence is
weak, but visible.
10 pts:
5 pts:
5 pts:
Chemilu
minesce
nce
General
Technique
Yield
Reaction
Table
Prelab
Quiz
30 pts:
Check
Out
Earned
Points
Good
Lab
drawer was restocked
and checked out by
TA. Participated in lab
cleanup.
Checked out
drawer, but did not
participate
in
lab
cleanup.
Poor
0pts:
Example question:
List two reasons why
triethylene glycol was
chosen as a solvent in
the formation of 3nitrophthal- hydrazide.
0 pts:
0 pts: <10%
or not
meas.
Student is
mostly unprepared to
complete
the
lab
independently. Lack of
technical skill impairs
the students ability to
work efficiently within
the lab time.
Chemiluminescence is
only faintly observed.
0 pts: Not observed
or was not completed.
Participated
in lab cleanup, but did
not checkout drawer.
0pts:
Did
not
checkout or participate
in the lab cleanup.
Total Score (100 pts) 
© 2009 Chad L. Landrie
119
11.8. Reaction Tables
On the day of the practical exam, you will receive a page with the exact reaction tables
listed below. Note: we will be using sodium dithionite during the practical exam
(bottom table), not sodium dithionite dihydrate (top table), which is used by the
textbook.
1. (8 pts/1 pt per box) The table below contains the quantities of reactants and solvents
from the textbook (Martin, 676) for the miniscale synthesis of luminol. Fill in the
missing values for mmol and equivalents. Always use 3-nitrophthalic acid as your
reference for determining equivalents. (To save time, you may complete this prior to the
laboratory practical exam and write those calculations in your lab notebook.)
Reactant or Solvent
d (g/mL) or
M (mol/L)
Molecular
Reaction Weight (g)
Weight (g/mol)
or Volume (mL)
3-nitrophthalic acid
Hydrazine (8 wt %
aqueous solution)
NA
1.00 g/mL
8% by weight
211.13
1.00
g
32.05
2.00
mL
mmol
Triethylene Glycol
Sodium Hydroxide
(aqueous solution)
Sodium Dithionite
Dihydrate
1.125 g/mL
1.00 g/mL
3.00 mol/L
150.17
3.00
mL
22.47 mmol
NA
5.00
mL
mmol
NA
192.12
3.00
g
mmol
Acetic Acid
1.049 g/mL
60.05
2.00
mL
mmol
mmol
Equivalents
4.736 mmol
1.00
4.75
2. (12 pts/1 pt per box) A specific mass of 3-nitrophthalic acid has been preweighed for
you in a vial, but is not the 1.00 g suggested by the textbook. Also, I have slightly
adjusted the number of equivalents for reactants and solvents. Using your mass of 3nitrophthalic acid and the new equivalent values, determine what quantities of reactants
and solvents are needed for the specific mass of 3-nitrophthalic acid in your vial. These
are the quantities you will use for your synthesis of luminol. (Complete the table below
immediately after receiving your vial during lab.)
Reactant or Solvent
d (g/mL) or
M (mol/L)
3-nitrophthalic acid
Hydrazine (8 wt %
aqueous solution)
NA
1.00 g/mL
8% by weight
211.13
g
mmol
1.00
32.05
mL
mmol
1.20
Triethylene Glycol
Sodium Hydroxide
(aqueous solution)
1.125 g/mL
1.00 g/mL
3.00 mol/L
150.17
mL
mmol
5.00
NA
mL
mmol
3.50
Sodium Dithionite*
NA
174.10
g
mmol
4.00
1.049 g/mL
60.05
mL
mmol
8.00
Acetic Acid
Molecular
Reaction Weight (g)
Weight (g/mol)
or Volume (mL)
© 2009 Chad L. Landrie
mmol
Equivalents
120
11.9. Weights and Measures
Below is part of the Weights and Measures page that you will receive as part of your
practical exam. Nothing needs to be completed on this sheet prior to the practical. This
is provided as an example only.
1. Pre-weighed Filter Paper (Mass and Signature boxes should be filled out with pen
only by the teaching assistant or instructor.)
0.20 grams
No Signature required. Assume all filter
papers have the same mass.
2. Filter Paper + Dry Luminol (Mass and Signature boxes should be filled out with pen
only by the teaching assistant or instructor only.)
grams
Teaching Assistant/Instructor Signature
3. Percent Yield Calculation (Performed by the student using above values.) Show all
work. No work, no credit.
3. Chemiluminescence (Instructor or TA circle one for each category and sign.)
Intensity:
Duration:
Strong
Moderate
> 15 seconds
Weak
Not Seen
< 15 seconds
Not Seen
Teaching Assistant/Instructor Signature
© 2009 Chad L. Landrie