EDB workshop
The Hong Kong University of Science and Technology
Department of Chemistry
MICROSCALE ORGANIC EXPERIMENT
Microscale Separation of Components in a Mixture
Dr TSUI, W. M.
Basic Safety & Waste Disposal Procedures
Personal Safety
1. Safety glasses must be worn at all times in the labs. Contact lenses should not be worn
in the lab because chemicals and particulates can get caught behind them, causing severe
eye damage.
2. Lab gown must be worn in the labs where an unexpected chemical spill may expose you
to the risk of injury. The following clothing is not permitted in the labs unless covered
by protective clothing:
 Open-toed shoes, sandals or other uncovered footwear; clothes that expose above the
ankles;
 Eating, chewing gum, and drinking in the lab.
 Untied long hair, dangling jewelry, loose clothing, and anything else that may get
caught in equipment, or dipped in chemicals.
3. Never work alone or unsupervised in the labs. Work only during the scheduled
laboratory periods and perform only authorized experiments.
 Wash your hands, arms, and then face, with soap and water as soon as possible after
leaving the lab.
 If you are uncertain about any safety aspect of an experiment, please ask your TA.
Lab Safety
 Make sure you know the exact locations of the safety features of the lab; e.g.,
eyewash fountains, safety showers, chemical spill kits, fire extinguishers, fire alarms,
fire blankets.
 Whenever possible, do not deal with incidents on your own. Your TA, the lab
instructor and the technician are all trained to respond to the sort of incidents that
may occur in this lab; e.g. chemical spills, cuts, burns, fires, medical emergencies, etc.
 Keep your work area clean and organized to reduce the possibility of accidents.
Know what you are doing and don't be careless.
 Avoid unnecessary exposure to chemicals. Never pipette by mouth. Never taste or
inhale a chemical on purpose. Wear gloves when directly working with hazardous
chemicals. Use hoods when appropriate.
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


Take appropriate precautions. Keep flammables away from hot plates and open
flames. Wear gloves when using toxic, carcinogenic, or other hazardous chemicals.
Take care with corrosive acids and bases. Always pour concentrated acid slowly into
water (never water into acid). Read the Safety Issues section at the beginning of each
experiment.
Be informed. Material Safety Data Sheets (MSDS) summarize known hazards
associated with every chemical are available from the lab.
Chemicals & equipment may not be removed from the lab without permission from
the Lab Instructor.
Disposal of Chemical Waste
It is very important to properly dispose the chemical waste you generate. Follow these
guidelines and dispose of your waste properly, to avoid adding to the contamination of our
environment.
• Generate as little waste as possible. It is expensive to have hazardous waste removed and
disposed. Don't prepare more of a chemical than you expect to use.
• Never return unused portions of chemicals to the reagent bottle. At the end of your
experiment, unused reagent must be disposed of as waste, so don't pour out more than
you need.
• Don't discard chemicals down the sink or in the wastebasket, unless you are explicitly
told that it's okay to do so. Most of your chemicals will pose a threat to the environment
if disposed improperly.
• Place chemical waste only in the appropriate container. Often, more than one waste
container is provided to separate certain chemicals for safety or easier disposal. Pay
attention to the Waste Disposal information for each experiment in this lab manual, and
use the waste containers indicated. If you cannot find a waste bottle labeled with your
particular chemical, ask your TA where to dispose it.
• Fill in the appropriate waste inventory sheet. There is separate inventory sheet for each
waste container. Use it to record the chemical(s), concentrations and volume you dispose.
• Do not over-fill a waste container. Tell the technician the bottle is getting full and they
will replace it.
• Use the clearly marked GLASS containers to dispose of broken glass and Pasteur
pipettes. Do not place broken glass in the sink or wastebasket, to avoid serious injury to
an unsuspecting person.
• Use the clearly marked WASTE SOLIDS wide-mouth bottles to dispose of waste solids.
Waste solids include solid chemicals, filter paper, and weighing paper.
• If you realize that you disposed of a chemical in the wrong container, use the waste
inventory list provided by the waste container to let us know.
• If you have waste whose identity you can’t recall, you can often test your waste, e.g.,
with litmus paper, to deduce its identity. Do not add unidentified waste to the waste
bottles. You will force us to categorize the entire waste container as “unknown” which
becomes extremely expensive to identify and dispose of.
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DEPARTMENT OF CHEMISTRY
Hazard Identification Sheet
Name: ________________________
Experiment Title:
MICROSCALE ORGANIC EXPERIMENT – Microscale Separation of Components in a
Mixture
Date of Experiment: ________________________________
 I have read and understand the instructions stated in ‘Basic Safety & Waste Disposal
Procedures’ concerning the proper use of the chemicals.
 I shall work in compliance with the aforementioned instructions made by the ‘Basic
Safety & Waste Disposal Procedures’.
SECTION I:
General Hazards Identified for the Experiment

I shall handle hazardous inorganic chemicals during the experiment. I shall follow
the instructions specified in ‘Basic Safety & Waste Disposal Procedures’.

I shall handle organic solvents and chemicals during the experiment. I shall follow
the instructions specified in ‘Basic Safety & Waste Disposal Procedures’.

I shall dispose hazardous waste chemicals during the experiment. I shall follow the
instructions specified in ‘Basic Safety & Waste Disposal Procedures’.
[please tick  where it is appropriate]
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SECTION II: Specific Chemical Hazards
For classification of hazards, please check the Material Safety Data Sheet (MSDS)
established
by
The
Sigma-Aldrich
Company
at:
http://www.sigmaaldrich.com/chemistry.html
Hazard Flammable Corrosive
Toxic
Irritant
Harmful
Oxidizing Explosive
Others
Chemical
Acetaminophen
Caffeine
Dichloromethane
Diethyl ether
1,4Dimethoxybenzene
Hexane
Hydrochloric acid
(6M)
Sodium chloride
(Brine)
Sodium hydroxide
(2M)
Sodium Sulphate
[please tick  where it is appropriate]
* Others: Carcinogenic, Allergic, Lachrymatory, Narcotic, Teratogenic, Reproductive
hazard, Central nervous system depression, Dangerous to the environment, Asphyxiant,
Poison
SECTION III: Declaration
This form to be completed by participant:
I am well aware of the hazard(s) of the experiment(s) to be carried out in the Chemistry
Laboratory.
Name:
Signature:
Date:
(in block letters)
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EDB workshop
The Hong Kong University of Science and Technology
Department of Chemistry
MICROSCALE ORGANIC EXPERIMENT
Microscale Separation of Components in a Mixture
Dr TSUI, W. M.
Chemical hazard notes
Dichloromethane is harmful, irritant and carcinogenic; it is an asphyxiant, can cause
central nervous system depression and reproductive hazard.
Disposal of wastes
Organic reagents and solvent must be disposed to appropriate waste solvent bottle.
Introduction
Separation and isolation of target compounds from a mixture is important in
organic chemistry experiments, knowing their physical and chemical properties is
essential for the choice of separation methods. For example, filtration is the simplest and
direct technique in separating solid from liquid. Sublimation is ideal for isolating volatile
compound from non-volatile solid impurities.
This experiment involves the separation of caffeine, acetaminophen and pdimethoxybenzene in a powder mixture. Find out the structure of these compounds and
you will realize that the mixture consists of neutral compound and compounds with
acidic or basic property. Their separation can be done easily based on the different
solubility, principles of acid/base chemistry and the physical properties associated with
each of the functional group. One of these components having different solubility in a
given solvent can be easily separated by simple solid-liquid extraction in the first step. To
test the solubility of a compound in a solvent, just simply take out a small amount of the
single component and try to dissolve in different kinds of solvent. This is so called
“solubility test”. Liquid-liquid extraction is another key method emphasized in this
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experiment, the component is isolated from the mixture by reaction with an appropriate
acid/base to form either conjugated acid or base which is ionic, and thus have vastly
different solubility from other compounds in a given solvent that makes separation
possible.
OCH3
CH3
N
N
O
N
N
N
H3C
H
CH3
O
Caffeine
O
OCH3
HO
p-Dimethoxybenzene
Acetaminophen
Read through the following experimental procedures and prepare a flow chart
for the separation in advance can help you to have a clearer picture on the steps and make
understanding more easily. Do not commence the experiment until you have familiarized
yourself with this flow chart and understand the principles behind it.
Procedure
Part I: Isolation of Compound 1: Solid-liquid extraction
(a) Solubility test:
1. Take 3 small test tubes, put to each test tube approximately 0.01g of the given pure
sample of caffeine. Label the test tubes C1 – C3.
2. Take another 3 small test tubes, put to each test tube approximately 0.01g of the given
pure sample of acetaminophen. Label the test tubes A1 – A3.
3. Take another 3 small test tubes, put to each test tube approximately 0.01g of the given
pure sample of 1,4-dimethoxybenzene. Label the test tubes D1 – D3.
4. Add approximately 1 mL of water to test tube C1, A1 and D1. Shake well.
5. Determine the solubility of each compound in the solvent and record your results in the
data sheet as “soluble”, “slightly soluble” and “insoluble”.
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6. Repeat step 4 – 5 with 1 mL of dichloromethane (C2, A2 and D2) and 1 mL of hexane
(C3, A3 and D3).
(b) TLC analysis:
7. Spot the pure sample of caffeine, acetaminophen and 1,4-dimethoxybenzene on a TLC
plate and run a TLC experiment using acetone : dichloromethane = 5 : 3 as eluting
solvent.
(c) Solid-liquid extraction:
8. According to the results obtained in solubility test, add 3 mL of the most suitable
solvent for solid-liquid extraction to the sample mixture.
9. Stir the mixture well with a glass rod.
10.Suction filter and suction dry the solid. Rinse the flask with about 1 mL of
dichloromethane and suction dry the solid. This is Compound 1.
11. Record the weight of the isolated compound.
12. Keep the filtrate for isolation of Compounds 2 and Compound 3.
Part II: Isolation of Compound 2: Liquid-liquid extraction
13. Evaporate the filtrate obtained from step 12 with a stream of air to dryness.
14. Dissolve the residue in 2 mL of diethyl ether and 2 mL of 6M HCl in a screw capped
test tube.
15. Shake well and vent the tube by loosening the screw cap. Repeat the shake and vent
step for two more times.
16. With the screw cap opened, wait for the separation of the two layers.
17. Separate the organic layer with a dropper.
18. Wash the aqueous layer with 2 mL of diethyl ether.
19. Collect and combine the organic layers, keep this organic layer for the separation
of Compound 3.
20. Transfer the aqueous layer to a larger test tube. Add 2M NaOH dropwise to the
solution until the pH of the solution is higher than 10.
21. Extract the solution twice with 2 mL portions of dichloromethane.
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22. Collect and combine the organic layers to a 10-mL conical flask.
23. Dry the organic layers with Na2SO4, pipette filter the solution to a pre-weighted small
vial.
24. Evaporate the solvent with a stream of air to obtain Compound 2.
25. Record the weight of the isolated compound.
Part III: Isolation of Compound 3: Evaporation
26. Wash the organic layer obtained from Step 19 with 2 mL of distilled water followed
by 2 mL of brine solution.
27. Dry the layer with Na2SO4, pipette filter the solution to a pre-weighted small vial.
28. Evaporate the solvent with a stream of air to obtain Compound 3.
29. Record the weight of the isolated compound
30. Run a TLC experiment for Compound 1 – 3 with the solvent system stated above.
Data
Solubility test: Water
Sample
Caffeine
Acetaminophen
p-Dimethoxybenzene
Degree of solubility
Solubility test: Dichloromethane
Sample
Degree of solubility
Caffeine
Acetaminophen
p-Dimethoxybenzene
Solubility test: Hexane
Sample
Caffeine
Acetaminophen
p-Dimethoxybenzene
Degree of solubility
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TLC anlaysis (Step 7): Sketch the TLC plates in 1:1 scale below
Rf values:
Isolated weight of Compound 1 = ____________
Isolated weight of Compound 2 = ____________
Isolated weight of Compound 3 = ____________
TLC results of Compound 1 – 3: Sketch the TLC plates in 1:1 scale
Rf values:
Questions
1. Match Compound 1-3 with caffeine, acetaminophen and p-dimethoxybenzene
respectively.
2. Draw a flow chart for the separation of the 3 compounds in the powder mixture.
3. Briefly explain the separation of the above 3 compounds. Chemical equations can be
used for the explanation.
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