CHEM 110 LABORATORY ASSIGNMENT 5

Imine Synthesis

BEFORE COMING TO THE LAB: Read through the assignment carefully and complete
the “110 Pre-Lab Imine Synthesis module” on Canvas. Completion of the pre-lab module
contributes towards your final lab grade.

1. INTRODUCTION
In this experiment you will prepare an imine from an aniline (aromatic amine) and an aromatic
aldehyde. You will then use sodium borohydride to reduce the imine to the corresponding amine.
You will analyse your experiment using tlc and show how 1H NMR can be used to identify the
various components of the reaction. This experiment uses several techniques that you are already
familiar with, including isolation by filtration, recrystallisation, tlc analysis and yield calculations.
You may wish to review your lab assignments for Labs 1 and 3 before attending this laboratory.

The grading criteria for this laboratory are outlined on page 7.

2 CHEMISTRY of IMINES
Imines are a functional group containing a C=N double bond. Imines are important in many
physiological processes and are used in a variety of drugs (e.g., Zetia® for lowering cholesterol,
and Gleevec® and Taxol® for treating cancer). Imines are produced by nucleophilic addition of a
primary amine to an aldehyde or ketone, followed by dehydration. In this experiment an aromatic
aldehyde (salicylaldehyde) will be reacted with an aromatic amine (4-ethoxyaniline) to produce
the corresponding imine.

In this step you will be using ethyl lactate as the solvent. This is a ‘green’ solvent in that it is
biodegradable and available from renewable sources. The development of green alternatives to
traditional organic synthesis is a topical area of research, both at the University of Auckland and
internationally.
Imines undergo many of the same reactions as aldehydes and ketones, including reduction. While
most imines require strong reducing conditions (e.g. LiAlH4) some aromatic imines are sufficiently
nucleophilic to react with a mild reducing reagent, such as sodium borohydride (NaBH4).

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 CHEM 110 Laboratory Assignment 5 [2020] 2
3. TECHNIQUES
3.1 Vacuum Filtration (Assignments 2, 4)
Solid products are often collected by vacuum filtration. The
apparatus used is a heavy walled flask with a side arm (Büchner filter paper

flask) fitted with a plastic or porcelain funnel with a perforated flat clamp here rubber disk

bed (Büchner funnel). heavy-walled

To assemble the apparatus, first clamp the flask to the clamp stand. tubing to
vacuum outlet .
Next, using heavy walled tubing connect the side arm to a vacuum
source, before the funnel is placed through the rubber adaptor which
fits over the neck of the flask. A piece of filter paper is placed in the bed of the funnel and should
cover all holes and be moistened with water (or the solvent being used) before the vacuum is turned
on and the slurry containing solid is poured in.

3.2 Recrystallisation (Assignment 4)

Solids are often purified by recrystallisation from a solvent in which the solid has a high
solubility at high temperature and a low solubility at room temperature. To recrystallise a solid it
must first be dissolved in the minimum amount of heated solvent. Less soluble impurities can be
filtered off at this point. If the solvent has been chosen correctly the solubility of the solid
decreases as the solution cools and the solid crystallises out.

3.3 TLC (Assignment 1)

Look back at your Assignment 1, particularly with respect to thin layer chromatography and how
the various drugs were identified.
Recall the term RF distance travelled by the spot (Measure to centre of spot.)
RF =
distance travelled by solvent (Measure from point of spotting.) )
Remember, if the preparation of the plate and its development have been carried out under strictly
constant conditions, the ratio of the distance travelled by the spot to the distance travelled by the
solvent front from the original spot will be constant for a given compound.
T.l.c is frequently used to determine whether a reaction is finished (has gone to completion) by
comparing the reaction mixture with a sample of the starting material. If the spot corresponding to
the starting material is also present in the reaction mixture, it can be inferred that the reaction has
not gone to completion. In this experiment the positions of ‘spots’ will be identified by placing the
t.l.c plate under a UV light.
3.4 Interpreting NMR (Assignment 3)
In this assignment, you will be asked to match a series of 1H NMR spectra to the corresponding
materials used and/or prepared in this experiment. In preparation for this exercise, you will need
to review your Block 2 notes. You may also wish to attempt some exam-style spectroscopy
questions as preparation.

You do NOT need to fully explain any of the spectra, and some will contain signals outside of what
you may have seen previously.

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 CHEM 110 Laboratory Assignment 5 [2020] 3
4. SAFETY INFORMATION
We are required under the Health and Safety in Employment Act, 1992, to present safety
information concerning the chemicals used in experiments. Read this before beginning the
experiment.
Precautions: Safety glasses, a laboratory coat and appropriate footwear must be worn at all times.
Spillages: If you get a chemical in your eyes, call for help. Use the eye-wash immediately.
Avoid skin contact with chemicals. Wash off any spilled on your skin immediately.
For chemicals spilled in the work area: Dilute water-soluble chemicals with water.
Put on protective gloves and mop up with paper towels. For water-insoluble
chemicals (many organic chemicals), shut off ignition sources. Notify a staff
member.
Disposal: Most materials in this lab will need to be placed in the appropriate organic waste
containers, located in the fume cupboards. Please read the clean-up information
carefully BEFORE starting the experiment. This can be found at the end of the
experimental procedure.

salicylaldehyde 4-ethoxyaniline
Description: Colourless liquid Description: amber liquid.
Risk: Highly flammable. Risk: Harmful in contact with skin.
Harmful by ingestion. Harmful by ingestion.
ethyl lactate methanol
Description: Colourless liquid. Description: Colourless liquid.
Risk: Flammable. Avoid inhalation. Risk: Flammable. Avoid inhalation.
Harmful by ingestion. Harmful by ingestion.

WHEN YOU HAVE READ THIS SECTION ON SAFETY, AND ARE SURE YOU UNDERSTAND THE
INFORMATION, SIGN IN THE BOX AT THE TOP OF THE REPORT SHEETS (PAGE 6).

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 CHEM 110 Laboratory Assignment 5 [2020] 4
5. EXPERIMENT
5.1 Preparation of Imine
1. In a fume cupboard dispense 3.2 mL of ethyl lactate into a clean, dry 50 mL beaker labelled
with your initials.
2. Add 0.7 ml (5.0 mmol) of 4-ethoxyaniline and swirl gently.
3. Finally, add 0.55 mL (5.2 mmol) of salicylaldehyde to the mixture and again, swirl gently.
4. Set the beaker in the fume cupboard (do not bring it back your workbench) and allow to
stand for 10-15 minutes while you prepare your filtration equipment. You will need ice-cold
brine in the next step, so start cooling this now.
5. Add 10 mL of ice-cold brine to the reaction mixture and stir gently with a spatula.
6. Vacuum filter the mixture, using two 5 mL portions of ice-cold deionised water to wash the
beaker and product. Dry for 5 minutes.
7. Transfer your imine into a clean dry reaction vial.
8. Put your filtrate into the waste bottle provided (located in the fume cupboard) and rinse out
the Buchner funnel.
9. Show your product to your group supervisor before proceeding to the next step.

5.2 Reduction of Imine

1. Add your magnetic stirrer bar to the reaction vial.
2. Using the dispenser provided, add 5 mL of methanol. Place your vial on a magnetic hot-plate
in the fume cupboard and set to stir. DO NOT HEAT.
3. Your group supervisor will give you 0.3 g (approx.) of NaBH4. This compound is moisture
sensitive and will react slowly with the water in the air so work efficiently from this point.
4. Add 4-5 grains of the NaBH4 to your vial using a spatula. It will bubble vigorously. Once the
bubbling has slowed, add a second portion (3-4 grains). Continue in this manner until all of
the NaBH4 has been used.
5. Record all of your observations, including any colour changes. If there is still solid on the
sides of the vessel, scrape it down into the mixture with a spatula.
6. Allow the reaction to stir for a further 5-10 minutes.
7. Add 10 mL of 5% sodium bicarbonate solution to your reaction mixture while it is still stirring,
using a measuring cylinder.
8. Vacuum filter the resulting solid using a clean funnel and flask, rinsing the vial and solid with
10 mL of ice cold deionised water. Remove stirrer bar.
9. Transfer the product into a clean 25 mL conical flask (using a solids addition funnel). Add 3
mL of methanol. Stand a boiling stick in the flask and then place the flask on the hot plate
on your bench. Bring the contents of the flask to the boil, stirring occasionally with the boiling
stick to encourage dissolution. If all the solid has not dissolved to give a clear solution once
it has reached boiling, add more methanol in small portions (~1 mL at a time) and continue
heating.
10. As soon as the amine has dissolved take the flask off the hot plate and remove the boiling
stick (we don’t want to ‘overcook’ our solution!). Use a pipette to transfer 1-2 drops of this
solution to a 5 mL vial (transfer it quickly, otherwise it will crystallise inside the pipette); this
will be used for the tlc exercise (5.3). Leave the remaining solution to stand undisturbed
while cooling to room temperature.
11. Complete the t.l.c exercise while waiting for your product to crystallise.
12. Collect the recrystallised product by vacuum filtration, using the same filtration equipment
but with a clean piece of filter paper.
13. Collect a plastic petri dish from the benches near the window and transfer your product to the
petri dish.
*ASSESS YOUR PRODUCT (RECORDING YOUR DECISION ON PAGE 9) BY
COMPARISON WITH THE SAMPLES ON THE SIDE BENCH, BEFORE
GIVING YOUR PRODUCT TO YOUR SUPERVISOR FOR ASSESSMENT.*
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 CHEM 110 Laboratory Assignment 5 [2020] 5
CLEAN-UP INFORMATION
The majority of chemicals used in this experiment CANNOT be washed down the sink. Please
follow the following protocols for cleaning your equipment. Report any spills to your group
supervisor.

• All filtrates should be placed in appropriate waste solvent bottles in the fume cupboard.
• All glassware used in the preparation and purification of the imine and amine should be
returned to the appropriate trays on the window benches (Büchner funnel, Büchner flask,
beaker). Do not wash these yourselves. Technical staff will process appropriately.
• Magnetic stirrer bar should be rinsed clean.
• Excess of the following solutions may be put down the sink: deionised water, brine, sodium
carbonate solution. Glassware used to hold these solutions may be washed and returned to
your drawer in the usual fashion.
5.3 Thin layer chromatography
Preparing the plate:
1. On the tlc plate provided carefully draw a faint pencil line 1 cm up from the short edge.
2. On the line drawn, without damaging the surface of the plate, make three pencil dots,
equidistant from each other and from the sides of the place.
3. Label the three positions starting material (sm) co-spot (co) and reaction mixture (r), from
left to right.
4. Take the small vial containing a sample of your reaction mixture and add 4 drops of
dichloromethane (DCM) to the vial to dissolve any solids.
5. Use a capillary dropper provided to place one small drop of A (imine) on the adsorbent layer
on the left most dot on the pencil line. It is advisable to repeat the spotting procedure at the
same point three to four times, allowing it to dry after each application to make the spot
sufficiently concentrated so that all components can be seen after they have separated.
6. Spot the same sample on the centre dot.
7. Using a different capillary dropper, repeat the procedure using your sample, prepared
above, onto both the right-most and centre dots. The centre dot, where you have spotted both
samples is called the co-spot.

Development: [DO NOT REMOVE THE SOLVENT TANKS FROM THE FUME HOOD]
Let the t.l.c. plate dry for at least 2 minutes. Jars containing a 7:3 mixture of hexane and ethyl
ethanoate (ethyl acetate) as solvent are provided in the fume hood. Lower the thin layer plate
into the solvent. Loosely replace the jar lid, and allow the plate to develop until the solvent is
about 1 cm below the top of the plate.
Remove the plate from the jar and mark with a pencil the position of the solvent front when the
plate was removed from the jar.
Allow the solvent to evaporate before removing the plate from the fume hood.

Viewing the developed plate:

Place the plate under the UV light.
Carefully, with a pencil, circle all spots for the imine and for your reaction mixture.

Staple your plate to page 9.

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 CHEM 110 Laboratory Assignment 5 [2020] 6

CHEM 110 SPECTROSCOPY DATA SHEET

TABLE 1
IMPORTANT INFRARED ABSORPTION REGIONS
(STRETCHING FREQUENCIES)
Bond Present n (cm-1)
O-H (alcohol, phenol) 3700 – 3200
(carboxylic acid) 3600 – 2500 (broad)
N-H 3500 – 3100
C-H 3100 – 2800
C=O 1850 – 1600
C=C (aliphatic) 1680 – 1600
(aromatic) 1620 – 1550
C-O 1250 – 1050 (less definitive)
TABLE 2
1
H NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (1H NMR)
Approximate Chemical Shifts (d) of Hydrogens in Organic Compounds
Hydrogens (H) in various d
chemical environments (ppm downfield from TMS)
(CH3)4Si (TMS) 0
R2CHCR 1.0 (approx.)

R2CHCZ* 1.0 - 2.0

Hydrogens bonded
to sp3 carbon R2CHC=C 1.6 - 1.9

R2CHC=O 2.0 - 2.5

R2CHAr 2.5 - 3.5

R2CHX** 2.0 - 3.7
R2CHO- 3.3 - 4.5

HC=C 4.5 - 7.0

Hydrogens bonded 6.5 - 8.0
H-Ar
to sp2 carbon
HCR 9.5 - 10.0
O
HOR 3.0 - 6.0 (variable)
Hydrogen bonded HOCR 10.5 - 12.0
to oxygen
O

Notes: **X = Cl, Br, I or the N of an amine

*Z = O, C=O, or X (as defined above)
R = H or alkyl group

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 CHEM 110 Laboratory Assignment 5 [2020] 7

TIME MANGAGEMENT PLAN

To complete this lab on time, you need to work effectively. Here is a suggested time management
plan for the lab, but you may wish to develop your own.

1. Welcome and group introduction (15-20 min)

2. Imine Synthesis

- Set up imine synthesis. While waiting for this to reach completion (10-15 min):
o Prepare iced brine and set up filtration equipment.
o Record experimental observations.

- When 10-15 minutes has elapsed, isolate your product (5.1, steps 5-6).
- While this is drying (5 min), collect equipment for the next step.

3. Amine Synthesis (imine reduction)

- Set up imine reduction. After all of the sodium borohydride has been added:
o Collect equipment you will need for work-up and isolation (5.2, steps 7-8)
o Clean up/return all equipment used for the preparation of the imine.

- Isolate amine product. While this is drying (5-10 min):

o Record experimental observations from imine reduction.

- Set up recrystallisation. While this is heating:

o Prepare TLC plate (labels, spot imine etc). However keep careful watch of your
recrystallisation while you do this.
- As soon as the amine has dissolved, remove from heat and remove boiling stick.

- While recrystallisation is cooling:

o Complete TLC exercise, including Rf calculations.
o Set up equipment for final filtration.
o If solution still needs to cool further, start on UV-Vis section of the spectroscopy
exercise.

- Filter product. While it is drying:

o Complete UV-Vis section of the spectroscopy exercise and/or start NMR
exercise.

- Transfer product to petri dish and assess your product.

- Clean up any remaining glassware.

- Complete NMR section of the spectroscopy exercise.

Please remember: cleaning up and handing in on time is one element of the marking rubric
for this lab.

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 CHEM 110 Laboratory Assignment 5 [2020] 8
REPORT SHEETS

Name:________________________________ Stream#:______ Gp:____ Bench#:_______

Supervisor:________________________

I have read and understood the safety

information for this assignment. Signed:_________________________

SUPERVISOR USE ONLY

0 (not achieved) 0.5 (progress) 1 (mastery)

Overall Report Quality More than 2 questions Major errors or 1-2 All questions answered.
unanswered/incomplete missed questions. Minor errors only.
Use recrystallisation to Poor Average and Dry Excellent
make a compound of OR OR AND
high quality Average and Wet Excellent and Wet Dry
Record experimental No observations recorded Limited or unclear Clear and concise
observations recording of recording of
observations observations
- tlc run correctly
- tlc labelled correctly
Missing 3 or more Missing 1-2 criteria for - Rf calculated correctly
Run and analyse a TLC criteria for mastery mastery - Rf correct
experiment - compounds correctly
identified
- appropriate
justification (minor
errors acceptable)

Use spectroscopic data Major issues/errors in Minor errors in structure Structure correctly
to determine the structure identification. identification and/or identified and soundly
structure of a OR major errors/omissions justified, with only
compound No justification provided in justification. minor omissions in
justification.

Issues

Safety non-compliance Incomplete tidy-up Late submission

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 CHEM 110 Laboratory Assignment 5 [2020] 9

6 IMINE SYNTHESIS and REDUCTION

Experimental Observations: (During imine synthesis)

___________________
Supervisor’s signature

Experimental Observations: (During imine reduction)

Recrystallisation:

Volume of methanol used in recrystallisation = ________ mL

Product assessment: STUDENT SUPERVISOR

Quantity:
[Based on samples A B C A B C
available for comparison.]
Crystalline / Powdery Crystalline / Powdery
Quality:
Colourless / Yellow Colourless / Yellow
Dry / Wet Dry / Wet
Supervisor’s signature:

___________________________

6.2 T.l.c Analysis of Reaction Staple plate here

Calculate the Rf for the imine and the amine:

Rf Imine _______________ Rf Amine____________

At the time your sample was taken, had the reaction gone to
completion? YES / NO
Justify your choice.

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 CHEM 110 Laboratory Assignment 5 [2020] 10

7 SPECTROSCOPY EXERCISES
7.1 UV-VIS Spectroscopy
What property of the imine and secondary amine allowed them to be viewed under the UV lamp
in the t.l.c exercise?

After isolation by filtration the imine was _______________ in colour and the amine was
_______________ in colour. UV-Vis spectrometry shows that the imine has a λmax at a much
longer wavelength than the amine. Use the structure of the two compounds to explain these
observations.

1
7.2 H NMR
Your group supervisor will provide you with four 1H NMR spectra, corresponding to
salicylaldehyde, 4-ethoxyaniline, your secondary amine and a fourth compound not used in this
experiment. Match the spectra to the corresponding compounds, giving at least 2 pieces of data
that support your choice.

Salicylaldehyde is spectrum ____________

Justification.

4-ethyoxyaniline is spectrum ____________

Justification.

The secondary amine is spectrum ____________

Justification.

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