CENTRAL SCIENCE LABORATORIES - METHODS

Pharmacy Chemical prep for Cycle 3 Experiment 1

— Synthesis of acetylated caffeic acid phenethyl ester
— LJ180 prep: CAPE 1

1 Scope (mandatory)
Preparation of acid chloride of acetylated caffeic acid using oxalyl chloride and reaction. Followed by
preparation of acetylated caffeic acid phenethyl ester by the reaction of the acid chloride with
phenylethanol.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments)
applies.

Quality manual CSL Quality Manual needs reference number here

COSHH – Health and Safety Executive, “Control of substances hazardous to health (Sixth
edition)”, Health and Safety Executive, Stationery Office, Norwich 2013.

EH40/2005 – “Workplace exposure limits - (Fourth Edition)”, Health and Safety Executive,
Stationery Office, Norwich, 2020

HSG65 – “Managing for health and safety (Third edition)”, Health and Safety Executive,
Stationery Office, Norwich 2013.

G201 “Fume cupboards - COSHH essentials - Control approach 2 Engineering control”,

Health and Safety Executive, Stationery Office, Norwich 2013.

EN 13150:2005 “Workbenches for laboratories - Dimensions, safety requirements and test

methods”
GMP Good Manufacturing Practice (GMP) guidelines; Volume 4 of "The rules governing
medicinal products in the European Union" contains guidance for the
interpretation of the principles and guidelines of good manufacturing practices for
medicinal products for human and veterinary use laid down in Commission
Directives 91/356/EEC, as amended by Directive 2003/94/EC, and 91/412/EEC
respectively.

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3 For the purposes of this document, the terms and

definitions apply.
CAS Number Chemical Abstracts Service Registry Number as published by “Chemical
Abstracts Service (United States)”
Fume cupboard A fume cupboard, also known as a fume hood, fume cabinet or chemical cabinet
is a type of local ventilation device that is typically a large piece of equipment
enclosing five sides of a work area with a moveable sash window, the bottom of
which is most commonly located at a standing work height.
air is drawn in from the front (open) side of the cupboard, and either expelled
outside the building via ducting.

A fume cupboard, functions by maintaining a relatively negative pressure in the

interior of the fume cupboard to prevent any contaminant from escaping while
drawing air in through the hood opening at a consistent rate.
Balance table The balance table is used for sensitive measuring equipment. Balance tables
usually contain a heavy slab of concrete mounted on a supporting construction.
The supporting construction for the slab is vibration isolated.
Lab Cup LabCup is a chemical inventory software, that assists in risk assessment/COSHH,
equipment booking and asset inventory.
Auto Pipette ACCUMAX A Variable Volume Pipette 100-1000 µl
hot plate IKA™ RCT Basic Hot Plate Stirrer, Ambient to 310°C, Aluminum
stirrers
Student COSHH The documents that matches this particular laboratory session for students
Assessment rather than for the preparation for the lab. This is titled
“COSHH Cycle_3 Exp_1 CAPE Synthesis of acetylated caffeic acid phenethyl ester
NG 2019-20”
Student solvent Shall be glass bottles 250 mL sized supplied by: DWK Life Sciences item code
bottles 218603607. The bottles shall be labelled in accordance with “Chemical
Labelling” — “Student chemical bottles”. For purpose of this method only these
substances supplied by CSL chemical stores shall be regarded as solvents, the list
of solvents is:
Solvent CAS number
Chloroform 67-66-3
Dichloromethane 75-09-2
Petroleum ether 8032-32-4
Toluene 108-88-3
Tetrahydrofuran 109-99-9
Diethyl ether 60-29-7
Hexane 110-54-3
Cyclohexane 110-82-7
Acetonitrile 75-05-8
Acetone 67-64-1
Ethyl acetate 141-78-6
Methanol 67-56-1
Ethanol 64-17-5

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4 Checks before starting session

Follow the method document; “LJ180 Checks before Lab session”

5 Expectations for student working (with COVID

restrictions)
Students are expected to work individually one per fume cupboard. During the session it is expected
that a fume cupboard is used to hold 2 rotary evaporators. The maximum capacity will be 16
students for the LJ 1.80 lab.

6 Preparing dry solvent bottles

6.1Preparation of molecular sieve
The 500 g of molecular sieve type 4A (see 13 Materials to be purchased) needs to be dried at a
temperature of 200 to 335 °C for 16 hours (the vacuum oven is ideal for this). Ideally a glass screw
top glass bottle should be used to contain the molecular sieve. After 16 hours the air in the oven
should be pumped out and clean dry air introduced via a Schlenk line, with liquid N 2 used to dry the
input air. The bottle cap should be placed on the bottle once the oven has cooled down sufficiently.
The bottle should then be kept in the “Technicians drying oven” shown in Figure 3.

6.2Preparation of bottles & vials

The bottles are specified in section 13 Materials to be purchased. Any new/replacement bottle
labels should be printed from “LabCup” using the “solvent” template. Prior to filling the bottles,
they should be held overnight (for 16 hours) in the “Technicians drying oven”.

Before any solvent is added to any bottle, pour the molecular sieve type 4A into each bottle to be
used to a depth of 10 mm.

6.3Filling the bottles

Careful consideration was given to using airtight syringes to move solvent from purchased bottles to
the student bottles. Unfortunately, it would require many syringe transfers for each student bottle,
that each pose a risk to the technician (handling needles repeatedly). The solvents;
dichloromethane and toluene do not readily absorb water vapour from the air and can be poured
from one bottle to another.

The solvent N,N-dimethylformamide readily absorbs water and along with pyridine should be
transferred via air tight syringe and needle, with nitrogen filled balloon. The bottle used are shown
in Figure 1.

The types of bottles to be used and solvent type are shown in Table 1 and further details for
purchasing are set out in Table 8.

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Solvent Volume (mL) Bottle type
Dichloromethane 100 Soda Lime Glass Bottle with 28mm Fitted
Closure, Amber
Toluene 100 Soda Lime Glass Bottle with 28mm Fitted
Closure, Amber
Pyridine 25 Type 1 Diagnostic Clear Glass Vials 25 mL
25 Type 1 Diagnostic Clear Glass Vials 25 mL
Table 1 Solvent and bottle type

Dry dichloromethane

Dry toluene

Dry pyridine

Dry N,N-dimethyformide

Figure 1 Chemicals to in to student fume cupboards

Each fume cupboard to be used by students will need one bottle of each of the follow:

 Dry dichloromethane
 Dry toluene
 Dry N,N-dimethyformide
 Dry pyridine

To reduce moisture ingress, it is ideal that once solvents have been used in the lab session, they are
collected in by demonstrators and their lids checked.

7 Labels specific for pharmacy

To conform to GMP standards all raw materials must be traceable. The raw material packs should
have “dates received “and “dates opened” written on to them, (see Table 2). It is reasonable to
write on supplier the outside of a supplier’s bottle with marker pen.

Date received: Date at which the pack was received from the supplier
Date opened: Date at which the seal on the pack was broken
Table 2 Additional details

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Materials that prepared specifically for pharmacy students, require additional details for students
filling in batch record sheets, (see Table 3). These details can be printed on to a small self-adhesive
label that can be replaced after the bottle has been used.

Date of fill: Date when the bottle was filled from the supplier's pack.
Technician: The person carrying out the filling.
Supplier: The name of the supplier of the raw material.
Supplier ID: The catalogue ID used to purchase the raw material from the supplier.
Supplier lot: / The unique serial number used by the supplier to distinguish production runs.
batch number:
Table 3 Additional labelling requirements for pharmacy student bottles

8 Drying tubes
Drying tubes are made up of an angled “Quickfit” drying tube, MF23/1 with desiccant and held in
place with glass wool. All the components required can be purchased from fisher scientific and are
listed in Table 8. The items are assembled as shown in Figure 2. The drying tube are kept in the
“Technicians Drying Oven”, shown in Figure 3.

Figure 2 Example of a drying tube, with a B14 connector, with desiccant trapped by glass wool

Figure 3 Location of drying tubes

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9 Preparation of acetylated caffeic acid

Place 50 g of caffeic acid in a 500 mL round-bottom flask, followed by 100 mL of DI water. Place the
flask on a hotplate with no heat but stirring. Add 30 g of sodium hydroxide pellets and wait for the
caffeic acid and pellets to dissolve. Once dissolved, clamp the flask in an ice bath on top of a
stirrer/hotplate, add 100 mL of acetic anhydride to the flask and stir for 30 minutes. Continue to stir
the mixture until the reaction is complete (check by TLC, using 50:50 ethyl acetate: petroleum ether
as the eluent).

Once the reaction has run to completion, recover the solid material by vacuum filtration using a
Buchner funnel and flask. Wash the solid on the filter paper with a small amount of ice-cold
Deionised water. Dry the material using the Buchner funnel and then record the crude yield and the
appearance of your product. Whilst crude product is drying on the Buchner apparatus, heat 500 mL
of ethanol to boiling.

The filtrate still contains acetic anhydride and needs another 13 g of sodium hydroxide pellets to
neutralise it and make safe for disposal to the sink.

Re-crystallise the crude material using the boiling ethanol. After the solid has dissolved, remove it
from the heat and allow the solution to cool to room temperature. The crystals should form over a
period of 20 minutes. The crystallisation process is completed in an ice bath. Isolate the purified
product by Buchner filtration. Dry the product in the technician's drying oven shown in Figure 3.
Dispose of the ethanol filtrate to the non-halogenated waste.

Once the acetylated product is dry, run IR, melting point and nmr to confirm synthesis.Figure 3
Location of drying tubes

10 Delivery of oxalyl chloride to students

Oxalyl chloride is very hazardous and must not be inhaled. The lab script say that the students must
calculate the amount of oxalyl chloride to be added to their reaction and that the addition must be
carried out by a demonstrator. This is to restrict access to the oxalyl chloride.

In addition, the safety, requiring students to tell the demonstrator the volume of oxalyl chloride they
need, “builds quality” in the multi-step synthesis. Using the mass of acetylated caffeic acid recorded
by the students the amount of oxalyl chloride can be read off from a simple “ready reckoner” to
check student calculations, (see section 11 Quantities per student).

During the lab a fresh bottle of oxalyl chloride should be placed in a large Gartrell's tray in a fume
cupboard. The bottle should only be opened while stood in the tray, this is to prevent any spillage
going direct to drain. Once the bottle has been opened and seal broken the top should be screwed
back on. The bottle should then be placed in a large 1 L beaker within the tray. To prevent the
bottle moving within the beaker it can be surrounded by tissue. The tissue also helps to clean up any
odd drops that fall when pipetting.

Normally transferring a liquid direct from a bottle is considered poor practice, but in the case of
oxalyl chloride the hazards outstrip this, so transfer direct from the bottle to student flask is carried
out. The tray should be used to transport oxalyl chloride from fume cupboard to fume cupboard.
The students should be asked to clear a space in their fume cupboard to allow the tray to be set

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down. This way all transfer of oxalyl chloride is carried out in fume cupboards. The students should
also be asked to supply a 100 ml beaker containing 80 mL of tap water (do not place this in the tray).

A 1000 microlitre autopipette with disposable tips are used to transfer the oxalyl chloride. A clean
pipette tip should be used for each transfer to minimise the risk of contamination of the oxalyl
chloride. After transfer the pipette tip should be ejected in to the 100 ml beaker containing 80 mL of
tap water.

Preparation for lab Expectations on students Expectations on demonstrators

 100GR Oxalyl chloride, 98%  To calculate the volume of oxalyl  To check student calculations
 1 L beaker chloride required  To keep the cap on oxalyl chloride
 Deep Gratnells tray  Clear a space in their fume while outside the fume cupboard
 1000 microlitre autopipette cupboard for the tray  Not to take used pipette tips out of
 1000 microlitre pipette tips  Supply a 100 mL beaker with 80 the fume cupboard
mL of tap water
Table 4 Expectations for demonstrators to deliver oxalyl chloride to students

11 Delivery of phenethyl alcohol to students

Phenethyl alcohol is much less hazardous than oxalyl chloride, however students are attempting a
multi-step synthesis and error reduction in the early steps is essential. This is again achieved by
asking students to calculate the volume of phenethyl alcohol and again ask demonstrators to deliver
this quantity. Once again, a simple “ready reckoner” can be used to check student calculations,
provided students state the mass of acid chloride they produced, (see section 12 Quantities per
student). Students that fail this check can be asked to re-do their calculations, and if this still results
in a problem referred on to another demonstrator the check their calculations.

Just as with oxalyl chloride, a 1000 microlitre autopipette with disposable tips are used to transfer
the phenethyl alcohol. A clean pipette tip should be used for each transfer. There is no need to
dispose of the tip in to water, simple disposal in the used “pipette tips and Pasteur pipettes” beaker
is fine.

A summary of expectations for the delivery of phenethyl alcohol is set out in

Preparation for lab Expectations on students Expectations on demonstrators

 Phenethyl alcohol  To calculate the volume of  To check student calculations
 1 L beaker phenylethanol required  To keep the cap on phenyl alcohol
 Deep Gartrell's tray  Clear a space in their fume while outside the fume cupboard
 1000 microlitre autopipette cupboard for the tray  Not to take used pipette tips out
 1000 microlitre pipette tips of the fume cupboard
Table 5 Expectations for demonstrators to deliver phenethyl alcohol to students

12 Quantities per student

The lab script asks students to use 2.00 g of acetylated caffeic acid. Given that there are 16 fume
cupboards available for students, but 2 fume cupboards need to be kept free for the rotary
evaporators, a maximum of 28 student pairs can carry out the lab. This means that a minimum of 56
g of acetylated caffeic acid is required. To ensure that spare material is available to cover spillages,
each of the six bottles of acetylated caffeic acid should be filled with about 20 g for the start of each
lab.

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Name, element content, Mr Structure
Acetic anhydride
Formula: C13H12O6
Element amu Atoms
C 12.01 9 108.09
H 1.01 8 8.08
O 16.00 4 64.00
Mr: 180.16

Acetic anhydride
Formula: C13H12O6
Element amu Atoms
C 12.01 4 48.04
H 1.01 6 6.06
O 16.00 3 48.00
Mr: 102.09

Acetylated caffeic acid

Formula: C13H12O6
Element amu Atoms
C 12.01 13 156.14
H 1.01 12 12.10
O 16.00 6 96.00
Mr: 264.23

Oxalyl chloride
Formula: C2Cl2O2
Element amu Atoms
C 12.01 2 24.02
Cl 35.45 2 70.91
O 16.00 2 32.00
Mr: 126.93 Density = 1.48 g/mL

Diacetyl caffeic acid chloride

Formula: C13H11ClO5
Element amu Atoms
C 12.01 13 156.13
H 1.01 11 12.10
Cl 35.45 1 35.45
O 16.00 5 80.00
Mr: 282.69

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Phenylethanol
Formula: C8H10O
Element amu Atoms
C 12.01 8 96.08
H 1.01 10 10.08
O 16.00 1 16.00
Mr: 122.16

Table 6 chemicals used for student calculation along with

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An example calculation is set out below:

Mass of c acid used ¿ 2.0000 g

mass 2.0000
Number of mols of diacetyl caffeic acid ¿ =mols= =0.007569 mols
Mr 264.23
Convert mols to mmols ¿ 0.007569 ×1000=7.569 mmols

mmols of two equivalents of oxalyl chloride ¿ 7.569 ×2=15.14 mmols

15.14
Convert mmols to mols ¿ =0.01514 mols
1000
Mass of oxalyl chloride ¿ 0.01514 × 126.93=1.9215 g

mass 1.9215
Volume of oxalyl chloride required ¿ = =1.298 mL
density 1.48
Auto pipettes deliver volume in units of 5 microlitres

Converting mL to microliters and rounding ¿ 1.298 ×1000=1300 μL

If 2.0000g of diacetyl caffeic acid were used as the starting material, then the mass of diacetyl caffeic
acid chloride would be:

mols of diacetyl caffeic acid chloride = mols of diacetyl caffeic acid

So, mols of diacetyl caffeic acid chloride ¿ 0.007569 mols

mass of diacetyl caffeic acid chloride ¿ 0.007569 ×282.69=2.1397 g

The lab script asks students to weigh the amount of diacetyl caffeic acid chloride produced and
calculate the addition of phenylethanol assuming 0.9 equivalence.

mass 2.1397
mols of diacetyl caffeic acid chloride ¿ =mols= =0.007569 mols
Mr 282.69
Convert mols to mmols ¿ 0.007569 ×1000=7.569 mmols

mmols of 0.9 equivalents of Phenylethanol ¿ 7.569 ×0.9=6.812 mmols

6.812
Convert mmols to mols ¿ =0.006812 mols
1000
Mass of Phenylethanol ¿ 0.006812 ×122.16=0.8321 g

mass 0.8321
Volume of Phenylethanol required ¿ = =0.8159 mL
density 1.02
Auto pipettes deliver volume in units of 5 microlitres
Converting mL to microliters and rounding ¿ 1.298 ×1000=815 μL

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Diacetylated Oxychloride Acid Chloride 2-phenylethanol

caffeic acid (g) addition (mL) (g) addition (mL)
0.1 0.065 0.1 0.038
0.2 0.130 0.2 0.076
0.3 0.195 0.3 0.114
0.4 0.260 0.4 0.153
0.5 0.325 0.5 0.191
0.6 0.390 0.6 0.229
0.7 0.455 0.7 0.267
0.8 0.520 0.8 0.305
0.9 0.585 0.9 0.343
1 0.650 1 0.381
1.1 0.715 1.1 0.419
1.2 0.780 1.2 0.458
1.3 0.845 1.3 0.496
1.4 0.910 1.4 0.534
1.5 0.975 1.5 0.572
1.6 1.040 1.6 0.610
1.7 1.105 1.7 0.648
1.8 1.170 1.8 0.686
1.9 1.235 1.9 0.725
2 1.300 2 0.763
2.1 1.365 2.1 0.801
2.2 1.430 2.2 0.839
2.3 1.495 2.3 0.877
2.4 1.560 2.4 0.915
2.5 1.624 2.5 0.953
2.6 1.689 2.6 0.991
2.7 1.754 2.7 1.030
2.8 1.819 2.8 1.068
2.9 1.884 2.9 1.106
3 1.949 3 1.144
3.1 2.014 3.1 1.182
Table 7 Ready reckoner for student additions

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13 Materials to be purchased
All the items listed in Table 8 are taken from the Fisher scientific catalogue, selected as suitable for
this particular lab.

ID Supplier Description CAS

10246563 Acros Organics 100ML Phenethyl alcohol, 99% 60-12-8

10542351 Acros Organics 2.5LT Acetic anhydride, 99+%, pure 108-24-7

100GR 3,4-Dihydroxycinnamic acid, 99+%,

11955091 Acros Organics 331-39-5
predominantly trans isomer

100ML N,N-Dimethylformamide, 99.8%,

10045421 Acros Organics 68-12-2
Extra Dry over Molecular Sieve, AcroSeal

2.5LT Dichloromethane, 99.8%, Extra Dry

10295661 Acros Organics 75-09-2
over Molecular Sieve, Stabilized, AcroSeal

2.5LT Toluene, 99.85%, Extra Dry over

10150662 Acros Organics 108-88-3
Molecular Sieve, AcroSeal

10497900 Acros Organics 100GR Oxalyl chloride, 98% 79-37-8

100ML Pyridine, 99.5%, Extra Dry over

10700693 Acros Organics 110-86-1
Molecular Sieve, AcroSeal

500GR Molecular sieve type 4A, technical,

10336430 Fisher Chemical 70955-01-0
2.5-5.0mm beads (4-8 mesh)

14843582 Fisherbrand Type 1 Diagnostic Clear Glass Vials 25 mL Non

Screw cap; I-LOC Closures, Thread Size 20-

DWK Life Sciences
11788709 400, Material Polypropylene, Liner Non
Wheaton™
Material Bromobutyl.

Soda Lime Glass Bottle with 28mm Fitted

12880944 Fisherbrand Non
Closure, Amber

10276750 Acros Organics Drierite, with indicator, 8 mesh Non

12373866 Edge Azpack™ Quartz Wool Non

Glass Drying Tubes With Standard Ground

10614182 Duran Non
Cone NS 14/23
Table 8 Materials to be purchased

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