CHEM 2020 3.

0
Introductory Organic Chemistry I
Lab Manual

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 Table of Contents
Page
Introduction 4
Experiment 1: Chromatography – Separation of a Dye Mixture 13
Experiment 2: Isolation of Cholesterol from Gallstones 19
Experiment 3: Resolution of (±)-α-phenylethylamine Part B 24
Experiment 4: Separation of Acids and Neutrals 32
Experiment 5: The Grignard Reaction 34
Pre-labs 39

Lab Performance Rubric

1/1 0.75/1 0.5/1 0/1
Adherence to Safety No violations Not wearing personal Repeated notices Refuses to comply
Guidelines protective equipment about not wearing with lab safety
(PPE) when the lab starts PPE or removes guidelines (The
or removes goggles twice goggles more than student should be
during the lab twice asked to leave the lab)

1/1 0.75/1 0.5/1 0/1

Effort and On time and fully 5 minutes late and/or 5 minutes late and/or 15 minutes late
Preparedness focused, efficient, somewhat focused, distracted, somewhat and/or irresponsible,
vigilant, and adequate knowledge of unprepared, and entirely unprepared,
shows great team the experiment, and overly relies on lab- relies too heavily on
work works effectively with mate lab-mate and/or the
lab-mate TA

2/2 1.5/2 1/2 0/2

Pre-lab Assignment All questions Most questions answered Some questions Pre-lab incomplete,
answered correctly with some answered correctly, major segments
completely and minor errors many errors present missing, questions
correctly consistently incorrect

For labs 1, 2 & 4. Total of 4 performance marks. 1/0.75/0.5/0 & 2/1.5/1/0 (per rubric above)
For labs 3, multiply each performance component by 2. Total of 8 performance marks. 2/1.5/1/0 & 4/3/1.5/0
For lab 5, multiply each performance component by 4. Total of 16 performance marks. 4/3/1.5/0 & 8/6/3/0
Detailed marks breakdown on page 5.

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INTRODUCTION TO THE LAB PORTION OF CHEM 2020 3.0

The experiments in this laboratory manual are designed to more or less accompany the lecture material in CHEM 2020
3.0; however, it is only a minor function of the experiments to illustrate the lecture material. The main purpose is to
expand and extend the lecture material, and, above all, through the teaching of the techniques, skills and philosophies
involved in carrying out experimental work, to make organic chemistry more real and concrete than can the lectures
alone. These objectives should be kept in mind as the experiments are performed. Some of the theoretical bases for
some of the experiments are not specifically covered in lecture, so some of that is provided but you will rely on your
own ability to find knowledge.
Since thoughtless performance of the experiments rapidly degenerates into routine following of a prescription, as from a
cookbook, with the chemical receiving more exercise than the student, the study of the experiment prior to its
performance is of prime importance. Further, an explanatory and discussion period before each experiment will take
place in the laboratory in order to emphasize and clarify the principles involved in the experiments, demonstrate any
new techniques and/or apparatus and point out any physical hazards that may be encountered. For obvious reasons,
attendance at this portion of the session is absolutely mandatory.
The laboratory experience is designed to acquaint you with fundamental laboratory practices and operations (use and
assembly of laboratory glassware, properties and use of organic solvents) and the illustration of certain techniques
commonly used in the laboratory such as extraction, crystallization and chromatography.

Who’s in Charge?
The Course Director is responsible for all aspects of the course and works with the Lab Coordinator to ensure that the
laboratory component runs smoothly. The TAs work with both.
You answer directly to your TA. He or she is there to assist you and will determine your lab grades, including
performance and penalties (see below). Appeals must be directed to the Course Director, and not to the Lab
Coordinator, as the Lab Coordinator is much too busy for this. The TAs are paid agents of the university, with
professional obligations and responsibilities for the safety of students. They are not paid overtime. They have the
authority to penalize you, to refuse you entry into the lab room and to expel you from the lab room (for conduct or
safety violations or for not finishing an experiment at the end of a session). The Lab Coordinator will oversee the
experiments as they run, making sure things run smoothly. She also has the authority to refuse entry to students or to
expel them for cause, but is not concerned with marking issues and cannot overrule a TA’s mark. The Course Director
will not overrule a TA’s professional assessments but will consider methodological and fairness concerns.

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Your Obligations and Our Expectations
The laboratory is a required portion of the course and consists of a three-hour session weekly according to the lab
schedule. You are expected to show up for your experiments at the appropriate session and on time.
The laboratory portion of your final grade will be worth 20% in total and this will be proportionally weighted between
the five experiments, as shown in the table below.

Table I: The detailed marks breakdown of the lab component of the course.

Marks Lab
Component
Laboratories
Performance & Report Total Marks
Percentage
Pre-lab

Lab 1 Chromatography 4 16 20 2%

Lab 2 Isolation of Cholesterol 4 16 20 2%

Lab 3 Resolution of α-PE 8 32 40 4%

Lab 4 Separation of Acids/Bases 4 16 20 2%

Lab 5 The Grignard Reaction 20 80 100 10%

200 20%

Note that each lab will be evaluated on TWO components: a performance (and pre-lab) grade and a report grade.

The performance grade consists of both work completed before the lab session (your prelab assignments) as well as
your actual in-lab performance, based on your TA’s assessment of your skill, technique and general efficiency while
working in the labs. At the end of the course, the course directors may adjust laboratory grades slightly to account for
the differences in subjective marking between all the TAs on this component. A detailed rubric for performance grades
is on page 2 of this manual you are advised to read it over.

The report grade consists of your writeups after the experiment is complete. For labs 1, 2, 4 these will be in-lab reports,
meaning you will complete them on a handout you will be given once you have completed the experiment and they are

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due at the end of the lab sessions (there is no post-lab work to be completed at home). The report for lab 3 will be
longer and more challenging, hence its greater weighting. Lab 3 will be a semi-formal report, meaning you will answer
questions at home but not in a formal style. Lab 5 will be formal lab report that must be completed at home following
guidelines we will be posting to eClass. Reports for labs 3 and 5 and are nominally due one week after the lab is
performed. More details on writing formal lab reports will be posted to eClass throughout the semester.

In order to successfully pass the laboratory component, you must obtain a final laboratory mark of 67% or higher. The
laboratory component of the course must be passed independently of the lecture component, as detailed in the
course syllabus.

No exceptions will be made. Any missed1 or failed experiments might result in failing the lab portion of the course, no
matter how well you are doing with the lecture material. If, by the drop deadline, you find that you are not in a position
to pass the laboratory component, you would be well advised to drop the course and avoid a failing grade on your
transcript.2 You can later be made exempt for any and all ‘passed’ experiments in any new attempt at the course.

You will need to bring with you to each lab session as proper equipment:
(a) safety goggles (only indirectly vented chemical splash goggles are allowed – NOT safety glasses),
(b) a lab coat,
(c) clothing that covers your legs and feet,
(d) a hardcover lab notebook with numbered pages, and
(e) a pen.

A calculator is a useful tool, though not required. If you forget your safety glasses, you can rent a pair, but we do not
provide any of the other required items. If you forget one or more of these, you may be asked to leave the lab and you
would then need to proceed as if you missed the lab for other reasons.1

You will be expected to come on time to each lab session equipped and prepared to work. You will be expected to be
focussed on the tasks at hand and on nothing else, and to finish on time. This means no idle chit-chat with others, no
horseplay, no leaving the lab room and no cell-phone usage. Indeed, any cell-phone use during the lab session, whether

1 See below concerning missed experiments.

2 Please be reminded that you are allowed three attempts at any course at York University. If more than one grade is
earned, the most recent grade will count.
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in the lab room or out in the hallway) will result in expulsion from the lab, unless justified by a verifiable personal
emergency. Inefficient progress (because of ignorance or disregard of the lab manual instructions) will not be without
consequence. You will also need to submit something as you leave or as directed on the Lab Report requirements.

For each experiment, you will be marked on the five Ps: punctuality, preparedness, performance, production and
professionalism.
Punctuality means arriving in the lab room on time and being ready and attentive when the pre-lab session starts. Those
arriving later WILL NOT be allowed to continue3 and must therefore proceed as if the session was missed.1 Students
attending CHEM 2020 lectures and labs on Monday and Friday afternoons will be given more leniency in their arrival
times. Punctuality also means being on time when submitting reports and data sheets.
Preparedness means arriving ready to proceed. To be ready at your next lab session, you must (a) read the lab manual
description of the experiment for that session, (b) answer the pre-lab questions before entering the lab, and (c) bring
with you a lab coat, safety goggles or safety glasses and a hard-cover lab notebook. After being allowed into the lab
room, you go to your assigned bench to deposit what you will need, you put on your lab coat and goggles/glasses and
you hang your jacket/coat and/or backpack near the entrance (backpacks can also be stowed in the cupboards under
the benches), then you need to be attentive when your TA starts the pre-lab presentation. Being attentive means being
silent, pen and paper in hand and looking to the TA. The TA may also wish to check your pre-lab questions before
allowing you to proceed.
Performance means many things which can be summarized as ‘safe and careful effort’, that is following instructions
(written and verbal) without undue delays and in respect of all safety requirements.4 It is neither necessary to carry out
tasks perfectly well nor to get perfect results, but it is necessary to do so intelligently, carefully, neatly, safely and in
coordination with your lab partner. Following the instructions should get you very good results. A poor performance
mark can result from inappropriate behaviour (wasting time, socializing, cellphone use, leaving the lab), disorganization
and/or lack of neatness, carelessness (poor set-ups, poorly acquired measurements, poor notebook entries, violations of
safety requirements, poor clean-up, etc.) and insufficient contribution to team effort. Finishing the experiment on time
is also a sign of good performance. While some extra time may be available, depending on circumstances, none can be
expected.

3 If you arrive late, you will be asked to leave the laboratory. Security will be called to escort you should you resist.
4 The initial meeting will introduce safety issues and requirements. A later section of this Introduction summarizes the
safety issues.
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Production refers to what you are expected to produce and deliver (the deliverables). This includes measurements and
observations, lab notebook entries, samples of products (when required), data sheets and lab reports (when required)
and their content. Not completing an experiment on time may mean not being able to deliver on all deliverables and this
cannot be expected to NOT affect your lab grade.
Professionalism refers to what is expected of a professional. Professionalism includes all of the other Ps but also includes
focus, ethical behaviour, honesty, civility, courteousness and cooperation. No rude or disorderly conduct will ever be
tolerated.
All five Ps obviously contribute together to success in an experiment, and neglect of one or the other can seriously
jeopardize the outcome.

Lab Reports
A laboratory report is required for some experiments and must be handed in as described on a due day set by the
Course Director or the Lab Coordinator. There will be severe penalties for lateness. All lab reports must be
independently produced pieces of work. Precise instructions and expectations for the deliverables on each experiment
are to be found on the Moodle course website.

Laboratory reports, when required, must be typed and made available in both printed and electronic formats. BOTH
must be submitted on time, as specified for each experiment, for the maximum grade to be earned. No mark will be
recorded without both submissions. Lateness in either submission will incur penalties or may be rejected entirely. In the
unlikely event that you do not know how to use a word processor such as Microsoft Word, now is the time to learn.
Computers are available for student use throughout the university. You are also required to generate your own chemical
structure diagrams and reaction schemes. Instructions on how to do this will be available on the Moodle course website.
The qualities of a good lab report include thoroughness, quality of presentation, appropriate language, truthfulness and
thoughtfulness. Notice that the results you obtain are not part of the lab report expectations. The quality of your results
are assessed as part of your performance (data sheet and notebook notations) and/or as separate deliverables
(samples). Hence, do not be tempted to bend the facts in order to fit them to some preconceived notion of what ought
to have happened, but always record what you actually observe. A data sheet describing your experimental results is to
be included in the hard-copy submission of each lab report, in which case it must be initialled by the demonstrator
before leaving the lab. For experiments 4 the data sheet will be provided on the Moodle website and you will be
required to have it with you at the lab session. Remember, observations must always be entered using a pen (no red,
please) and pencilled entries will be considered blank.

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Attendance
You are expected to attend all five lab sessions so long as you remain enrolled in the course. People dropping the course
cannot continue in the lab for insurance reasons. Unless justified with appropriate documentation, an absence will
result in a grade of zero for that session. While this may not prevent you from passing the lab, it will lower your overall
lab grade (worth 20% of your final course grade).
In the event of unavoidable problems, such as severe illness, please arrange a make-up session through the Lab
Coordinator (Olga Girina) by contacting her in her office in 308 CB. If a make-up session is not possible, then, in order to
become exempted from that session so that your overall lab grade will not suffer, procure documentary evidence
justifying your absence and provide it to the Course Director as soon as is feasible (without delay). Only the Course
Director can exempt you from a lab session. Remember that if you plan to retake the course, you can only be eligible for
lab 99 if you have completed EVERY single lab.

Pre-Lab Assignments
Prior to beginning an experiment, you must complete a pre-lab assignment, which are found on perforated pages in the
lab manual. These are due to your lab TA ONCE YOU ENTER THE LAB ROOM. You will not be allowed to hastily complete
a pre-lab assignment once you enter.

Lab Exemptions
If you repeat the course, you may be exempted from lab sessions for which you have already received a passing grade,
so long as they were passed in-person in the past three years. Contact Ms. Michelle Barton, at [email protected]
before labs begin to request lab exemptions. No credit will be given for any lab work done after dropping the course in
previous years.

LABORATORY SAFETY
The chemistry lab can be a dangerous environment in which to work. Good lab practice and sensible precautions go a
long way to minimize those dangers. The experiments in this course have been chosen to exclude or restrict the use of
exceptionally hazardous materials, however most chemicals are considered harmful in some respect and the particular
hazards associated with each are described in Material Safety Data Sheets (MSDS), located in the lab. During each pre-
lab discussion, listen carefully to your demonstrator’s remarks about specific hazards associated with the particular
experiment to be conducted that day.

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Lab safety ultimately lies with the individual; you are responsible for carrying out the experiment in a safe manner
without endangering yourself or others, and it is your responsibility to observe the essential safety rules of the chemistry
lab listed below. The TAs and the Lab Coordinator will not hesitate to remove unsafe individuals from the lab.
Our obligations toward ensuring your safety and the safety of others are limited toward providing you with the needed
information and instruction to proceed safely and to providing the non-personal equipment that you need to proceed
safely. We will, of course, exert due diligence and step in when a hazard becomes apparent, but we cannot prevent all
possible hazards that you can personally create or exacerbate. In other words, after you have had the opportunity to be
informed of the hazards and how to deal with them, you become responsible for your own well-being and for the well-
being of those around you.

ESSENTIAL SAFETY RULES

1. ALWAYS:

● Wear Goggles - You will NOT be permitted to work in the lab without them. Safety goggles must be worn even
over prescription glasses.
● Lab coats are mandatory and can be purchased from bookstore.
● Dress sensibly; open-toed shoes and uncovered legs are not allowed.
● Know the location of emergency exits, showers, eye wash fountains and the First-Aid kit.
● Know how to operate emergency showers and eye wash fountains.
● Read experiment instructions carefully before the laboratory.
● Pay attention to TA’s remarks about potential hazards with any experiment.
● Check that the apparatus is assembled correctly.
● Handle chemicals with care, wear gloves and obey Safe Handling Instructions on labels.
● Know the location of Material Safety Data Sheets (MSDS) in the lab.
● Keep your work area tidy and attend to spills immediately.
● Dispose of all waste in the appropriate waste bottles provided.
● Unplug hotplates and melting point apparatus before leaving the lab.
● Wash your hands before leaving the lab.
● ASK THE TA IF IN DOUBT.

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2. NEVER:
● Heat flammable solvents on an electric hotplate; use a hot water bath or a steam bath instead.
● Eat, drink or smoke in a lab.
● Sniff or taste chemicals or solvents.
● Work alone.
● Wear open-toed shoes or contact lenses in the lab. Shorts and skirts can be worn with leggings but not with
uncovered legs. Lab coats do not adequately protect the lower legs.
● Wear gloves outside the lab.
● Run, fool around or distract your neighbours.
● Put glass in the regular garbage (grey bins); this is very dangerous to our cleaning staff. Dispose of waste
pipettes in the blue bins provided in the lab.
● Throw broken glassware (flasks, beakers, etc.) in the blue bins without first consulting your TA, a technician or
the Lab Coordinator.
● Put waste chemicals in the sink.
(Adapted from Laboratory Safety Handbook, by Anne Alper et al, The Chemical Institute of Canada, 1987.)

In case of fire, either in the laboratory or as indicated by the sounding of the alarm in the building, University policy
stipulates that students must leave the building immediately, taking all personal effects. Appropriate action will be taken
by others. STUDENTS MUST LEAVE – that is the law.
In the event of an accident or spill, notify the TA or technician immediately. If any material enters the eye while in the
laboratory, wash the eye with copious amounts of water. Eye wash stations are located in every lab. Chemical spills on
oneself require the removal of clothes from the affected area and washing with large amounts of water for 15 minutes.
Safety is everyone’s responsibility. The cooperation of everyone working in the lab is essential in maintaining safe
conditions. Students must dress and behave appropriately, be aware of emergency procedures and observe all rules of
lab safety. Violators will be penalized and/or barred from continuing in the lab.

Care of Glassware and Equipment

Good lab practice requires that reactions be carried out in clean, dry glassware, which is in good condition, free of
scratches or chips. Each student is responsible for leaving his or her equipment, glassware and bench space in a clean
and safe condition for other students. Most experiments will require the cleaning of glassware with an initial rinse of

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95% ethanol followed by soap and water. Bottles for waste will be provided. TAs will deduct lab performance marks for
unclean glassware or bench space left behind.
Any damage to equipment or glassware must be reported to the TA. Consult the TA, technician or Lab Coordinator
before disposing of chipped glassware; York’s glassblower can repair most apparatus.

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 EXPERIMENT 1: CHROMATOGRAPHY: SEPARATION OF A DYE MIXTURE
and EXPERIMENT 3: RESOLUTION OF (±)-α-PHENYLETHYLAMINE (PART A)

During this session, you will complete experiment 1 and prepare the crystals for experiment 3, which is to be completed in a future
lab session.

i. CHROMATOGRAPHY - SEPARATION OF A DYE MIXTURE

Introduction
Chromatography (from the Greek chroma, meaning colour, and graphein, meaning to write) is a technique frequently
employed by chemists and biochemists to separate the components of a mixture. In its original application the method
was used for the separation of coloured substances, but colour is not required for compounds to be separated by this
method. Colourless compounds may be rendered visible either by the use of ultra-violet (UV) light or by chemical
means. A number of separation techniques using the principles of chromatography have been developed, some of which
are specific to a class of material (e.g. proteins, polysaccharides, lipids, organic polymers, etc.).
All techniques depend upon the differential distribution of various components (analytes) of a mixture between two
phases, a mobile phase (liquid, gas or supercritical fluid) and a stationary phase (solid or liquid adsorbent). Usually (and
in this experiment), the stationary phase is a high-surface-area solid and the mobile phase is a liquid solvent. In that
context, the differential distribution usually (but not always) depends on the relative strengths of the intermolecular
interactions in a three-way competition between analyte, solvent and adsorbent: If the interactions of the analyte with
the stationary phase are much stronger than with the solvent and stronger than those between the stationary phase
and the solvent, then the analyte will tend to move slowly up the plate. If the interactions of the analyte with the
stationary phase are much weaker than they are with the solvent or weaker than those between the stationary phase
and the solvent, then the analyte will tend to move quickly up the plate. The differences in mobility between analytes in
a sample mixture then depend on how differently the analytes interact with both solvent and stationary phase. The
choice of solvent is critical: Some solvents will out-compete both analytes for the surface interactions, and both analytes
will travel quickly, with little net separation. Some solvents will fail to compete with either analyte for the surface, and
neither analyte will move much, with again little net separation.
The stationary phase may be polar or apolar. In this experiment, the surfaces of the stationary phases will be polar, with
both charged groups and neutral ones, and with hydrogen-bonding and other dipolar interactions at play. Hence, both
charged and neutral polar analytes will interact strongly with such surfaces, and solvents that will adequately compete
will need to be polar if the most polar analyte is strongly polar. Solvents that have too little polarity will not compete and
afford little mobility, while those that are too polar will compete too successfully and afford too much mobility.

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Similarly, the solvent will need to be less polar if the most polar analyte is less polar. An ideal separation occurs when
the solvent polarity is such that it outcompetes one analyte but not the other. In practice, however, it is difficult to
predict with any exactness just how polar the mobile phase needs to be, and both experience and trial-and-error will
help determine the appropriate solvent for a given analyte.
Thin layer chromatography, frequently referred to as TLC, is largely an analytical technique to separate nanomole to
micromole quantities of analytes. It is used for determining the purity of materials, for preliminary identification
purposes and for determining the best solvent system to use for later separations of mixtures on a larger, preparative
scale for instance by column chromatography. There is also a preparative version of TLC that uses much thicker and
wider layers to separate larger (milligram-scale) quantities of material. Paper chromatography is a kind of thin-layer
chromatography appropriate for analytes that can interact with cellulose.
Analytical TLC plates are prepared as follows: A film of approximately 0.3 mm thickness of alumina, silica gel, or other
surface-active adsorbent material containing a small amount of binding material (usually calcium sulphate) and
optionally a fluorescent dye is applied as a slurry to one side of a clean glass plate, aluminum sheet or plastic film. The
slurry is then dried either at room temperature or by heating. In this laboratory, we will use pre-prepared TLC plates
available commercially. Microliter-sized samples of mixtures to be analyzed are applied as solutions, placing a small spot
near the bottom of the plate or film using a glass capillary. The solvent is allowed to air-dry, then the TLC plate is placed
in an upright position in a chamber containing a small volume of developing solvent (eluent). As the solvent ascends the
thin layer by capillary action, components of the mixture become separated and appear as distinct coloured spots (if the
mixture contains coloured substances). This is called a chromatogram. If the analytes are not coloured, UV light can be
used to visualize the migrated spots so long as the adsorbent contains a fluorescent dye and the analytes can quench
the fluorescence. Otherwise, a number of reagents can be used to reveal spots by inducing a chemical reaction that
produces a coloured product. Unlike in paper chromatography, the thin material layer on the surface of a TLC plate can
be treated with even very corrosive reagents.
By marking the solvent front and measuring the distance travelled by each component, the retention factors (Rf values)
for individual compounds may be determined. In addition to being rapid (from start to finish is usually a matter of
minutes), the method is very sensitive using very little material.
Column chromatography is a preparative technique used for separating mixtures of two or more components, once an
appropriate solvent has been determined using TLC. This technique uses gravity or pressure (instead of capillary action)
to move analytes downward along a vertical column packed with a finely divided adsorbent already wet with a solvent
(and thus free of air pockets). The solvent used to prepare the column is often less polar than the one(s) used to elute
the analytes. An analyte mixture is introduced as a solution at the top of the column, allowed to penetrate the
adsorbent, then fresh solvent is added continuously to the top of the column to carry the analyte down and through the

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adsorbent. Each component of the mixture, in descending through the column packing, partitions itself repeatedly
between the adsorbent (the stationary phase) and the solvent (the mobile phase). If the solvent is well chosen, the
distribution coefficient with respect to these two phases is different for each compound comprising the mixture, and,
therefore, each travels through the adsorbent at a different rate. Components of the mixture thus become separated
within a short time. If the analytes are coloured substances, the compounds appear in the column packing as distinct,
coloured bands. Continued addition of solvent to the column finally elutes each compound as a coloured solution out
the lower end and into a receiving flask. A more polar solvent may be required to elute the more polar analyte(s) into
separate receiving flasks. The recovery of each compound as a pure substance than is possible by simply evaporating the
solvent. Columns are usually prepared just before use, as in this experiment.

Experimental Procedure

1. Thin Layer Chromatography

The mixture to be analyzed by TLC is a solution containing 0.2% sodium fluorescein dye and 0.2% methylene blue in 95%
ethanol.

1. Obtain two pre-coated thin-layer alumina plates. Use a pencil and draw a line across the chalky side of the plate about
¾ cm from the shorter edge of the plate. DO NOT draw with such force as to remove the alumina from the plate (this
prevents the dyes from flowing upwards).

2. Draw a small quantity of the dye mixture into a capillary tube and lightly touch it to the plate on the line previously
drawn. Keep the spot sizes to less than 1 mm in diameter. Repeat, spotting solutions of pure methylene blue solution
and pure fluorescein dye solution on the left and right sides of the dye mixture, parallel with the original spot as shown
below. These additional spots on the side will serve as controls.

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3. Carefully place the TLC plate in the ‘developing chamber’ (a 150 mL beaker) and allow the back of the plate to lean
against the side of the beaker at a slight angle from the vertical. Add 95% ethanol to the beaker such that the solvent
level is just below the level of the spots when the plate is immersed in the chamber.

4. Cover the beaker with a watch glass to prevent solvent evaporation from the plate. It is important that the level of the
solvent not reach the level of the sample spots, otherwise the sample will simply be dissolved off the plate instead of
being carried up by the rising solvent.

5. Once the solvent front has reached about 90% of the height of the plate, remove it from the chamber and
immediately mark the solvent front by drawing a straight line using pencil.

6. After letting the solvent dry, measure the Rf values for methylene blue and fluorescein (note the figure below for
help). Note that the methylene blue may contain a small amount of a pale purple impurity. If this additional purple spot
appears on your TLC plate, calculate its Rf value as well. Sketch the finished TLC plate to include with your results on the
report sheet.

𝑏 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑎𝑛𝑎𝑙𝑦𝑡𝑒

𝑅𝑓 = =
𝑎 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝑓𝑟𝑜𝑛𝑡

7. Repeat the entire procedure (steps 1-6) using 65% ethanol as the eluting solvent (mobile phase).

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2. Column Chromatography
The system to be separated is the same dye mixture used for the TLC exercise.

1. To prepare the column, clamp it in a vertical position with its Teflon stopcock closed. Insert a plug of cotton into the
bottom of the column with a long glass rod, using enough cotton to form a mat 1 cm thick.

2. With the aid of a plastic funnel, pour a layer of clean sand (about 1 cm in depth) on top of the cotton. In a 50 mL
beaker, prepare a “slurry” of alumina (6 g) in ethanol (15 mL) and add to column. This aids in the formation of an evenly
packed column.

3. Wash down the column walls with ethanol and add a 1 cm layer of sand to the top of the alumina. The figure below
shows what your prepared column should look like before proceeding to the next step. If unsure, ask your TA.

4. Open the stopcock and allow the solvent to drain just to the top of the upper layer of sand. At no time should the
solvent level be allowed to drain below this level; as this will produce entrapped air bubbles, which forms channels that
hinder separation.

5. With the level of the eluent just above the sand, carefully add ~0.5 mL of the dye mixture using a pipette. In order to
maintain the proper flow rate of the liquid at 1 drop per second from the column, attach one end of a rubber tube to the
air pump and the other end to the top of the column. Your TA may demonstrate this procedure.

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6. Slowly open the stopcock. Introduce a steady stream of air through the column. Run the mobile phase down and then
carefully add fresh 95% ethanol at the top of the column using the squeeze bottle. Proceed to collect the blue dye
eluent. This is repeated until the eluent appears colourless.

7. Wash the column with 0.1 M aqueous sodium hydroxide solution until no more yellow dye remains in the tube and
collect the eluent in another beaker. Record your observations on your report sheet.

ii. RESOLUTION OF (±)-α-PHENYLETHYLAMINE, PART A

Note: The purpose of these steps is to prepare crystals which will be analyzed in lab 3. It is not necessary at this point to fully
understand what is happening as it will be covered later in lecture.

1. Dissolve 12.0 g of (+)-tartaric acid in 165 mL of methanol using a 250 mL Erlenmeyer flask by heating the unstoppered
mixture on a steam bath.

2. Remove the flask from the steam bath and slowly add 10.0 mL of racemic (±)-α-phenylethylamine (also called (±)-α-
methylbenzylamine) to the warm solution (~1 mL each addition) while swirling the flask to mix well. CAUTION: This
reaction is exothermic and rapid addition of the amine will cause the solution to boil over and splash upwards, possibly
onto your face and hands.

3. The desired salt will crystallize slowly as large clear prisms. The first crystals obtained are often fine white needles
instead of prisms; these needles do not provide optically pure amine and must be avoided. To ensure that the desired
prisms form, add a few ‘seed’ crystals of the correct composition. Cork and label the flask and set it aside until your
scheduled day to complete Experiment 3, Part B.

18
 EXPERIMENT 2: THE ISOLATION OF CHOLESTEROL FROM GALLSTONES

* Pre-lab assignments are found at the end of the lab manual *

Introduction
This experiment illustrates two very common techniques used in the purification of organic materials: extraction and
recrystallization. It goes without saying that recrystallization applies to crystalline solids only. The technique of liquid-
liquid extraction will be learned in experiment 3. This experiment will include a solid-liquid extraction.
Extraction is one of the oldest of all chemical operations, and one which is used in everyday life. The simple act of
making a cup of tea or coffee involves the extraction of various components responsible for flavour, odour, and colour
from tea leaves or ground coffee beans by hot water. Extraction in the chemical sense means ‘pulling out’ a compound
from one phase to another, usually from a liquid or solid to another liquid.
Crystallization is the deposition of crystals from a solution or melt of a given material. During the process of crystal
formation, a molecule will tend to become attached to a growing crystal composed of the same type of molecules
because it can fit well into a crystal lattice of the same molecules, and tending to exclude other molecules. If the
crystallization process is allowed to occur under near-equilibrium conditions, the preference of molecules to deposit on
surfaces composed of like molecules will lead to an increase in the purity of the crystalline material. Thus, the process of
recrystallization is one of the most important methods available to the chemist for the purification of solids. Additional
procedures can be incorporated into the recrystallization process to remove impurities. These include filtration to
remove undissolved solids, and adsorption to remove highly polar impurities. Activated charcoal and Celite
(diatomaceous earth) are both useful clarifying and decolorizing agents.
Recrystallization usually takes advantage of the difference in solubility of a substance in hot versus cold solvent. It is
desirable that the solubility of the substance be high in the hot solvent and low in the cold solvent to enable a more
thorough recovery of the starting material.5 The choice of solvent is therefore critical and, for a new or unknown
substance, this may require several trials to discover, as it is difficult to predict. If the molecular structure of the
compound to be recrystallized is known, the adage "like dissolves like" provides a useful starting point. Solvents used in
recrystallizations should be relatively low-boiling so that any solvent adhering to the crystal surfaces can be readily
removed by evaporation. Some common solvents used in recrystallization are listed in the following table.

5 The supernatant solution remaining is known as the ‘mother liquor’.

19
Table 1. Common solvents for recrystallization listed in order of decreasing polarity

Solvent boiling Useful for Common co-solvents

point
(oC)

Water 100 Salts, amides, some Acetone, methanol, ethanol

carboxylic acids
Methanol 65 General use Water, diethyl ether, benzene
Ethanol 78 General use Water, diethyl ether, benzene
Acetone 56 General use Water, hexane
Ethyl acetate 77 General use Hexane
Dichloromethane 40 General use, low melting Ethanol, hexane
compounds
Diethyl ether 35 General use, low melting Methanol, ethanol, hexane
compounds
Chloroform 62 General use Ethanol, hexane
Toluene 111 Aromatic compounds
Benzene 80 Aromatic compounds Ethanol, diethyl ether, ethyl acetate
Hexane 69 Hydrocarbons All solvents listed except water and
methanol
Petroleum Ether 35-60 Hydrocarbons All solvents listed except water and
methanol

Cholesterol (C27H46O) is a normal and abundant component of blood lipids but certain physiological abnormalities lead to
the deposition of lumps of almost pure cholesterol in the gall bladder. Cholesterol was first isolated from gallstones in
1769; this experiment therefore has a long history.

20
The purpose of this experiment is to learn the techniques of solvent extraction, recrystallization and determination of a
melting point. The melting point of a solid substance gives an indication of its purity and can also assist greatly in its
identification. A sharp melting point range (< 2oC) between the first appearance of liquid droplets within a solid sample
to the disappearance of the last trace of solid constitutes good evidence high purity. Unless the solid decomposes as it
melts, a broad melting point range is strong evidence for impurity.

EXPERIMENTAL

1. Weigh (to 0.1 g accuracy) about 1.5 g of crushed gallstones on a piece of weighing paper. Transfer it to a 50-mL
Erlenmeyer flask.

2. Add 15 mL of 2-butanone (ethyl methyl ketone) and about a half-spatula of Celite. Heat and swirl the mixture on the
steam bath for about 5 min. There will remain an undissolved amorphous solid impurity.

3. Use a conical plastic funnel with a loose plug of cotton (1-1.5 cm in diameter) placed over the funnel hole. Use a
spatula to gently push the cotton into the stem of the funnel and place the funnel on a clean 125-mL Erlenmeyer flask.

4. Holding a glass rod in one hand in such a way as to lead the flow of liquid down onto the cotton, use your other hand
to pour the hot butanone solution down the glass rod and into the funnel as quickly as possible and without pausing (to
prevent the solution from dribbling along the outside of the flask).

5. Add about 5 mL more of butanone into the first flask, heat for 1 min on the steam bath, and tip it rapidly into the
funnel as a rinse. Next heat about 5 mL of the solvent in a small beaker on the steam bath, and use this hot solvent to
rinse down the walls of the funnel.

6. The filtrate should now be nearly colourless, and if you carried out the operations without spillage, it should contain
virtually all of the cholesterol that was originally in the gallstones.

7. Heat the filtrate on the steam bath and add 20 mL of 3:1 ethanol-water mix. Swirl briefly over the steam bath, and
then set in an ice-water bath to cool. Swirl from time to time to make sure the contents are well cooled.

21
8. Suction filter the resulting precipitate on a Büchner funnel (with a filter paper disk in it, your TA will demonstrate how
to use the vacuum aspirator). Use a little cold ethanol as a rinse.

9. Turn off the suction and press the crystals down well onto the paper with a flat spatula blade so as remove as much of
the mother liquor as possible. Spread the crystals out on a watch glass to air-dry.

10. Weigh the “crude cholesterol” obtained and measure its melting point range. Only a few crystals are needed, and it
is recommended that one partner take the melting point, and the other partner begin the recrystallization with the
remaining crystals. Determine the % yield of your pure cholesterol product based on the initial gallstone mass you
started with (don’t expect a yield of 100% since the gallstones are not pure cholesterol!)

11. Recrystallize the crude cholesterol from 95% ethanol. To do so, transfer the cholesterol to a 100 mL round bottom
flask and add about 10 mL of 95% ethanol and 1-2 boiling stones (anti-bumping granules) to ensure smooth boiling.

12. Clamp the neck of the flask securely to a support stand (see diagram) so that the flask is immersed about 2 cm into
the water bath. Do not begin heating until the apparatus is completely assembled as shown in the diagram below. Attach
a condenser to the flask, and use a second clamp to secure the condenser to the support stand. Using amber tubing,
connect the water-inlet arm of the condenser (the lower arm) to a cold water tap. Connect a second tube to the water-
outlet arm of the condenser (the upper arm) and run this tube into a drain. Slowly open the tap so that a gentle stream
of cold water circulates through the condenser. See the figure below:

22
13. Heat the suspension for 10 min over a steaming water bath while cold water circulates through the condenser. This
process is called heating under reflux. The solvent repeatedly vaporizes then condenses back into the reaction flask. If
the cholesterol does not completely dissolve, add 5 mL more of 95% ethanol from the top of the condenser and resume
heating.

14. Once a clear solution is obtained, raise it out of the water bath (turn the heat off), allow to cool and, when cool
enough to handle, place in an ice-water bath.

15. Suction filter and air-dry the pure crystals as before using a Buchner funnel with fresh filter paper.

16. Weigh your pure product and determine its melting point range. Show your product to your TA before you clean up
and leave the lab. Determine the % yield of your pure cholesterol product based on the initial gallstone mass you started
with.

23
 EXPERIMENT 3: RESOLUTION OF (±)-α-PHENYLETHYLAMINE, PART B

* Pre-lab assignments are found at the end of the lab manual *

Introduction
Chirality, enantiomers (optical isomers), diastereomers, optical activity and optical purity are discussed in Chapter 5 of
the textbook. Be sure to review this material and be familiar with the terminology.

One key point to retain from Chapter 5 is that enantiomers are identical with respect to chemical properties (reactivity
with achiral reagents) and almost every physical property such as melting/boiling point or solubility, the exception being
their behaviour in a beam of polarized light. On the other hand, diastereomers can have very different physical and
chemical properties.

Resolution is the isolation of one enantiomer from an optically impure mixture of enantiomers. In this experiment,
resolution is accomplished by transforming a racemic mixture into a mixture of diastereomers, isolating one by
exploiting a difference in physical properties, then converting it back to optically pure material. In particular, we will
exploit the different solubilities of diastereomeric salts in order to effect a resolution of a racemic chiral amine after
reaction with an optically pure acid. This is only one of a number of possible ways of resolving racemic (or optically
impure) mixtures. It affords a modest yield of one optically pure enantiomer (maximum 50%) and the balance is optically
enriched in the other enantiomer (but almost never optically pure). Other methods of resolution include:
● performing an enantioselective chemical transformation of one or the other enantiomer with a chiral reagent or
chiral catalyst (including enzymes, which are chiral), leaving the other enantiomer untouched, then separating the inert
enantiomer from the transformed one using some difference in a physical property; this is called kinetic resolution since
it involves reactivity differences, one example being the enzymatic resolution of alcohols by highly selective acetylation
of one enantiomer; this affords a maximum 50% yield of one enantiomer;
● chiral chromatography, whereby the stationary phase is modified to be chiral and optically pure such that the
elution of racemic analytes generates diastereomeric interactions and therefore exploits different chromatographic
mobilities for each analyte enantiomer; this can afford high yields of both enantiomers in optically pure form;
● dynamic resolution, in which one enantiomer is converted to the other through an achiral intermediate (for
instance, converting an (R)-alcohol to a ketone, and then to an (S)-alcohol) using special catalysts, for a maximum 100%
yield of a single enantiomer.

24
When a racemic (±) amine is treated with one enantiomer of a chiral acid, the resulting salts are diastereomeric. If the
two enantiomers of the amine are designated (+) and (-), according to their optical rotations, 6 and if the acid is
designated (+), the salts are (+)(+) and (-)(+) combinations, as shown in the schematic reaction below:

(±)-RNH2 + (+)-RCOOH → (+)-RNH3+ (+)-RCOO- and (-)-RNH3+ (+)-RCOO-

These salts no longer have a mirror image relationship; they are diastereomeric and should have different physical
properties. In this experiment, a solvent is used in which one salt is more soluble than the other, and the other can then
be separated by fractional crystallization. This is the case with a 1:1 mixture of α-phenylethylamine and tartaric acid,
forming α-phenylethylammonium hydrogen tartrate. The crystallization, however, is a slow process, and that is why you
set up the crystallization after experiment 1. In this session, the crystals will be isolated, transformed back to the amine
and the yield, optical identity and optical purity of the product will be assessed.

Tartaric acid is a diacid with two chiral centres. It is thus available in three forms, as (+) and (−) enantiomers and in an
achiral meso diastereomer. Tartaric acid was the first compound ever resolved, by Louis Pasteur in the 19th century,7 at a
time when organic structures were much less well known or understood, including the tetrahedral nature of saturated
carbon. This feat was seminal in establishing the modern-day concepts of chirality and stereochemistry. The technique
that Pasteur used was fortuitous: Using a magnifying glass or microscope, he noticed the presence of mirror-image
crystals, which he was able to carefully segregate, and each crystal orientation corresponded to a single enantiomer.
This technique is rarely useful, since many (if not most) racemic materials crystallize in a single crystal form. Fortunately,
optically pure tartaric acid forms as a byproduct of winemaking.

Extraction: A method for the separation of mixtures

The separation of organic mixtures is accomplished by various methods such as chromatography, distillation,
crystallization and extraction. Extraction uses a two-phase liquid solvent system and a separatory funnel. By taking
advantage of the differential solubilities of organic compounds in the two phases, a means of separation can be
accomplished.

6 Note that the (+) descriptor is equivalent to d and that the descriptor (−) is equivalent to l. Racemic material is identified
with () or rac descriptors.
7 This is the same Pasteur who gave us vaccines and pasteurization.
25
The figure below illustrates a separatory funnel containing two immiscible liquids once the phases have separated on
standing.

When a substance A is partitioned between two liquid phases, the ratio of the concentrations in each phase is a
constant defined as the distribution ratio Kdist = [A]1/[A]2. If the compound has finite solubility in both solvents, Kdist can
be calculated from the ratio of solubilities, each of which can be measured separately. Because solubility will vary with
solvent and temperature, Kdist is a function of the solvent pair and the temperature. For any solvent pair at a given
temperature, calculations will show that, as a result of the constancy of Kdist, regardless of the relative volumes, the
extraction of a substance from one phase into another is more efficient if several small portions of the extracting solvent
are used than if the same total volume is employed in one portion.

The most commonly used solvent systems in extraction are ether-water and dichloromethane-water (but any water-
immiscible solvent can be used). Because of the usually large differences in solubility of organic and ionic compounds in
water and ether or dichloromethane, neutral compounds (e.g. amines at high pH) may thus be separated from salts (e.g.
carboxylates) by extraction. This technique will be used in later experiments as well.

The Measurement of Optical Activity

The apparatus used to measure optical activity is called a polarimeter. A schematic of a polarimeter and a description of
its operation appear in the textbook. A sodium vapour lamp is most often used as the light source, with measurement of

26
the intense yellow light given off by excited Na atoms at 589 nm, the so-called sodium D line.8,9 By convention, optical
rotations are reported at 589 nm. Occasionally, however, rotations at that particular wavelength are too weak, and
other wavelengths are sometimes used, using for instance a Hg vapour lamp. For the sake of reproducibility, therefore,
the wavelength at which optical rotation measurements are made is always specified. A related and informative
technique that scans optical rotations over a whole spectrum of wavelengths is called circular dichroism, but this is
beyond the scope of this experiment.

In a polarimeter, a polarized light beam is generated by a combination of a monochromator (a wavelength selector), slits
(forming a beam) and a polarizer (often a calcite prism) setting the plane of polarization relative to some set of
coordinates (for instance, relative to the surface of the Earth). A sample of an optically active substance placed in the
beam can rotate the plane of polarization of that beam, and the resulting angle can then be measured by rotating a
second polarizer to counteract the rotation produced by the sample and then carefully measuring the angle between
polarizers.10 The direction of rotation is defined by convention as being positive (+) or dextrorotatory (d) if clockwise
from the detector’s point of view, or negative (−) or levorotatory (l) if counter-clockwise. Together with the number of
degrees of rotation, the measurement of direction and amount of rotation is called the optical rotation. Calibration of
the instrument is performed using an optically inactive sample (such as air), with a rotation setting of 0°. The best
precisions in angle of rotation can reach 0.001° but usually one decimal place suffices.

The sample needs to be in liquid form to transmit light (solids and suspensions cause too much dispersion by diffraction
and reflection). The molecular origin of the optical rotation phenomenon is complex, and it is very difficult to predict
from a chiral substance’s structure in which direction and to what extent it will rotate plane-polarized light at this or that
wavelength. There is no necessary relationship to the R or S designations used to categorize chiral molecules. Suffice it
to say that is has something to do with molecular dipoles, keeping in mind that molecules in liquid states are randomly

8 Street lamps often are Na vapour lamps and give this yellow light. Mercury vapour lamps give a brighter, whiter light.
9 The unique series of wavelengths emitted by excited atoms and ions have long ago been measured and quantified. The
observation of particular wavelengths allows the identification of elements in a technique called atomic absorption
spectroscopy. It also allows the identification of the elements present in stars of various ages, interstellar dust clouds and
so on.
10 No polarized light is transmitted through a polarizer oriented 90 to the plane of polarization (polarized sunglasses
exploit this to reduce the glare from reflected and partially polarized sunlight). A polarizer placed parallel to the incident
plane of polarization transmits the maximum amount of the polarized light. The angle of rotation by an optically active
sample is then measured by optimizing the amount of transmitted light through rotation of the second polarizer.
27
oriented and that the net rotation of the light plane is therefore the result of rotations by all possible molecular
orientations. It is also important to keep in mind that a molecule’s structure can vary in time (through bond rotations,
for instance) and be influenced by more or less strong interactions with the solvent, both of which also depend on the
temperature. In other words, the end result will depend on both the nature of the solvent and the temperature.
Therefore, the solvent used and the temperature of the measurement also need to be reported with any measurement
of optical rotation (by convention, measurements are made at room temperature, approximated as 20°C, but more
precise control of the temperature can be had with a jacketed polarimeter cell through which water from a circulating
water bath can be pumped; temperature variations can also be studied with such a cell).

A more important consideration is that if N molecules between the two polarizers rotate the plane of polarized light to
an extent represented by α, then xN molecules will rotate that plane to an extent xα. Hence, the net rotation is
determined by the number of molecules encountered by the light beam. That number will then depend on the thickness
of the sample (the light’s path length L) and by the concentration c of the molecules in the sample. In practice, the
linearity xN → xα holds best in dilute solutions, but if the sample substance is itself a liquid (and enough is available),
then the neat liquid can be used to give reproducible measurements, in which case the equivalent of concentration is
the liquid’s density.11 Otherwise, the sample needs to be dissolved in some solvent to a known concentration c. Hence, a
reproducible measurement takes into account both L and c at a conventional temperature (usually 20°C) and at a
conventional wavelength (usually the sodium D line) in a stated solvent. Dividing the observed rotation αλT (at
conventional temperature T in °C and wavelength λ) by c and L then annuls the effect of c and L and the result is solely
due to the molecule itself. This is called the specific rotation [α], as shown below, with L conventionally measured in
dm12 and c measured in g/mL, for convenience.
𝛼λ
[𝛼]λ =
𝑐𝐿

For instance, the specific rotation of (+)-tartaric acid is [α]D20 = +12.0o (c = 20 g/100 mL in H2O). The subscript D indicates
that the sodium D line was used as the monitored wavelength at 20°C. The concentration (in conventional units) and the
solvent are also reported.

11 So long as the optical rotation is not too strong, then a single measurement can be used. If the anticipated rotation is
unknown, then it must be kept in mind that a +10 rotation cannot be distinguished from a +370 rotation nor from a −350
rotation. These three situations can be distinguished from each other by a variation of the sample concentration or the
light’s pathlength.
12 The typical polarimeter cell has l = 10 cm = 1 dm.
28
When using pure liquids, the conventional formula is shown below, where ρ is the liquid’s density at temperature T. For
instance, the reported specific rotation of pure (−)-α-phenylethylamine is [α]D20 = −40.4o ± 0.2o (neat).

𝛼λ
[𝛼]λ =
𝜌𝐿

EXPERIMENTAL

1. Obtain the corked flask set aside after experiment 1. Carefully decant (pour off) the liquid into the appropriately
labelled waste container, keeping the crystals in the flask. Add 10 mL of methanol to the crystals and suspend and break
the mass up into individual crystals with a stirring rod.

2. Using a small Büchner funnel and filter flask, clamped properly to the retort stand, collect the crystals by vacuum
filtration. Wash any remaining crystals out of the flask with a small amount of the filtrate and rinse the crystals on the
funnel with 10 mL of fresh methanol. Spread the crystals on paper to air-dry, then weigh the product and calculate the
percentage yield of the (-)-α-phenylethylammonium hydrogen (+)-tartrate salt.

3. Place the crystalline salt product in a 125 mL Erlenmeyer flask. Add 30 mL of 2 M aqueous NaOH solution. Swirl the
mixture until all of the crystals have dissolved. You may notice the water turning cloudy or oily; this is normal (what is
this due to?)

Note: The next steps require a separatory funnel. Its use will be demonstrated by your TA.

4. Transfer the aqueous NaOH-salt mixture to the separatory funnel using a plastic funnel (to minimize spillage). Rinse
the flask with 10 mL of dichloromethane. Add this rinse to the separatory funnel. Allow sufficient air space in the funnel
to mix the liquids (as a general rule, never fill a separatory funnel more than two-thirds to three-quarters full).

5. Stopper the funnel. Lift the separatory funnel out of its ring support and hold it with both hands to prevent the
stopper from falling out. Hold the rounded body in one hand, near the stopcock so that you can open and close the
stopcock quickly to release pressure, but keep a finger of the other hand over the stopper at all times.

29
6. Invert the funnel (so that the stem points upwards and the airspace is at the stopcock end). With the stem aimed well
away from yourself and others, immediately open the stopcock to release any pressure arising from the mixing of the
two solvents. Most organic solvents are fairly volatile and their vapour pressure causes buildup of pressure in the funnel.
When venting a funnel in this way, never point the stem towards yourself or your neighbours. Be sure to always wear
your safety glasses in a lab full of separatory funnels.

7. After the first venting, close the stopcock, swirl the inverted funnel for a few seconds, and then vent it again. If
excessive pressure build-up is evident, repeat the swirling-venting process until the pressure build-up is negligible. Then,
and only then, the funnel may be shaken vigorously for about 2 minutes with venting every 20 seconds or when
pressure builds. At the end of the shaking, vent the funnel again, return it to the ring support, remove the stopper, and
allow the aqueous and organic layers to separate.

8. To withdraw the dichloromethane phase (and hence your amine), you must first know which layer is the organic layer.
As a general rule, the denser liquid is the layer on the bottom. Most organic solvents are less dense than water, with the
exception of solvents containing halogen atoms. Withdraw the dichloromethane layer into a clean and dry 50 mL
Erlenmeyer flask, leaving the aqueous layer in the separatory funnel.

9. Extract this aqueous phase twice more with 5 mL portions of dichloromethane and combine these extracts with the
first in the Erlenmeyer (repeat steps 5-8 with fresh solvent each time). Once all the extractions are finished, the aqueous
solution in the separatory funnel can be discarded into the appropriate waste container.

10. To dry the dichloromethane extract, Add about 1 gram of anhydrous potassium carbonate (don’t measure this mass
exactly) to the Erlenmeyer flask and swirl, then let it stand for a few minutes. When the dichloromethane is dry, it will
appear clear (perhaps with a yellow colour) and the potassium carbonate will be easily settled on the bottom of the
flask. If this is not the case, you may need to add more potassium carbonate.

11. Weigh a clean and dry 50 mL beaker containing 1 or 2 boiling stones.

12. Filtering the dichloromethane solution into the pre-weighed beaker using a small cotton plug fitted at the
constriction of a plastic funnel. Rinse the cotton plug with 5 mL of fresh dichloromethane.

30
13. Place the beaker onto a steam bath to remove the dichloromethane. It will be completely removed when the
solution stops boiling, leaving behind the free liquid amine.

14. Remove the beaker from the steam bath, let it cool to room temperature then weigh the beaker again. The weight
thus gained is the weight of the amine that it contains. Calculate the percent yield of the isolated amine and proceed
with the polarimeter measurement.

15. The laboratory technicians will assist you in using the polarimeter. Record the observed optical rotation from your
amine sample and convert that into a standard optical rotation on your report sheet. This will allow you to calculate the
optical purity of your amine product.

31
 EXPERIMENT 4: THE SEPARATION OF ACIDS FROM NEUTRALS BY EXTRACTION

* Pre-lab assignments are found at the end of the lab manual *

Introduction
Carboxylic are much stronger acids than alcohols or phenols. This is clear from a comparison of acid dissociation
constants (the pKa values for CH3COOH, PhOH and CH3CH2OH are approximately 5, 10 and 16, repsectively). Because of
their relatively strong acid character, carboxylic acids can be converted to ionic salts using relatively weak bases such as
bicarbonate, HCO3− (pKa of H2CO3 ~6-7). For this reason, most carboxylic acids are soluble in dilute (e.g. 5%) aqueous
NaHCO3. On the other hand, phenols are less acidic and the corresponding equilibrium lies predominantly on the side of
undissociated phenol. However the use of a stronger base such as hydroxide OH− (pKa of H2O near 16) or carbonate
CO32− (pKa of HCO3− near 12) will convert phenols to water-soluble salts. Much stronger bases (e.g. NaH, NaNH2) are
needed to deprotonate alcohols; such strong bases will also deprotonate H2O and so alkoxides cannot be prepared in
aqueous media. Since most organic compounds are insoluble or only partially soluble in water but are completely
soluble in water-immiscible organic solvents, the conversion of carboxylic acids and phenols in water to their ionic salts
allows for their separation from mixtures with non-acidic organic materials. After the separation, they can be
regenerated by the addition of a strong mineral acid to reprotonate the ionized group(s).
This experiment will exploit acid-base properties to effect a separation of a carboxylic acid from a neutral organic
compound.

Safety Information
Handle concentrated HCl with gloves, dispense in a fumehood.
Ether is very flammable and should only be evaporated on a steam bath.

Experimental
A stock solution in ethanol containing benzoic acid and biphenyl (both at 0.05 g/mL) will be provided. Transfer 20 mL of
water to a large separatory funnel using a plastic funnel and, using a graduated cylinder, measure 5 mL of the stock
solution and pour it into the separatory funnel. Rinse the graduated cylinder into the funnel with a small amount of
ether from a wash bottle, then add 15 mL more ether to the funnel (the volumes do not have the be exact). Shake well,
and let the layers separate, then discard the aqueous phase. Use the proper technique for liquid extractions as you
learned them in CHEM 2020. If you are rusty with your technique, your TA can provide a quick demonstration. Every
time you remove the stopper from the separatory funnel, remember to rinse it and the mouth of the funnel with fresh

32
ether into the separatory funnel since evaporating ether leaves behind a white residue containing your compounds of
interest.

Add ~15 mL of 5% NaHCO3 solution to the separatory funnel and shake thoroughly. Let the layers separate and drain the
aqueous layer into a 125 mL Erlenmeyer flask (flask A). Repeat the process with an additional ~15 mL portion of 5%
NaHCO3 and add the aqueous phase to flask A. Repeat a third time but, before adding the extract into Flask A, draw off a
few mL of the aqueous phase into a test tube. Add to this a few drops of concentrated HCl to make it acidic. If there is no
appreciable precipitate of benzoic acid in the test tube, the third aqueous layer can be discarded. If there is a
precipitate, add the third extract to Flask A. Then, heat Flask A on a steam bath for at least 10 min to drive off any
dissolved ether.

While flask A is heating, shake the ether phase in the separatory funnel with 10-15 mL of saturated NaCl solution and
discard the aqueous layer. Drain the ether layer into a 50 mL Erlenmeyer flask containing about 5 g of anhydrous Na2SO4
(does not have to be measured accurately) and swirl the contents of the flask for 3-5 min.

Prepare a dry, 50 mL Erlenmeyer flask (flask B) containing a boiling chip and weigh them together. Filter the ether
solution through a small plug of cotton in a plastic funnel into flask B. Rinse the flask containing the Na2SO4 with one
small portion of ether (5 mL) through the filter funnel and into flask B. Place flask B on a steam bath in the fumehood to
evaporate the ether until the boiling stops. The molten product should solidify readily upon cooling. Weigh flask B and
calculate the yield of crude product. Redissolve the crude product in a minimum volume of 95% ethanol with heating,
and boil for several minutes. Upon cooling, the product should crystallize to white plates, which should be suction-
filtered and washed with a few drops of chilled 95% ethanol. Let the crystalline product air-dry, remove the boiling chip
and weigh the pure product. Measure its melting point and identify.

After the aqueous solution in flask A has been freed of ether on the steam bath, let it cool and add concentrated HCl
dropwise until the solution is acidic and a product precipitates from solution. The crude product is recrystallized by
heating the suspension to boiling on a hotplate (if necessary, add sufficient additional water so that the benzoic acid
dissolves in the boiling solution). Let the solution stand and cool to room temperature. Collect the crystals by suction
filtration and wash with a little cold water. Let it air dry, weigh the pure product, measure its melting point range and
identify it. Show both products to your TA before disposing into the waste container.

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 EXPERIMENT 5: THE GRIGNARD REACTION

* Pre-lab assignments are found at the end of the lab manual *

Introduction
Review the sections in Chapter 10 in the textbook dealing with organometallic reagents. As will be evident in this and
later chapters, Grignard reagents play a commanding role in organic synthesis, adaptable to the preparation of a large
variety of functional systems, and comprise one of the major uses of alkyl halides. This experiment involves a synthesis
diphenylmethanol using phenylmagnesium bromide and benzaldehyde.
Reaction of a halide and magnesium occurs on the metallic surface and is formally an oxidation of the metal. The
reaction is usually carried out in ether solution. The ether solvent functions as a Lewis base, solvating the Grignard
reagent and permitting it to diffuse away from the metal. As stated in Chapter 10, the major pitfall in preparing a
Grignard compound is the presence of alcohol or any other active hydrogen (protic) compound, as this consumes and
wastes the reagent, and may also seriously complicate its formation, since the magnesium salt that forms from reaction
with a proton source is often much less soluble and can coat the surface of the metal, preventing reaction of the alkyl
halide.
Experimental
The glassware shown in this picture has been dried in an oven and should be
taken directly from there.

1. Assemble the apparatus as shown in the picture: fit the round-bottom

flask with a Claisen adapter; fit the Claisen adapter with a condenser and a
glass stopper; and fit the condenser with a drying tube filled with blue
desiccant13. Ensure that all joints are fitted well and all clamps are secured
tightly. Verify with your TA that your set-up is correct before adding any
chemicals. Run a gentle stream of tap water through the condenser.

2. In the clean and dry 50 ml Erlenmeyer flask (from the oven), make up a
solution of 1.8 ml of bromobenzene in 9 ml of dry (anhydrous) ether using
oven dried 10 ml graduated cylinders. Be sure to gently mix the solution
before proceeding.

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3. Stopper the flask with a dry rubber stopper and set aside. Add 0.4 g of dry magnesium turnings to the 50 mL round-
bottom flask14. Add some of the bromobenzene solution to the magnesium, just enough to cover magnesium turnings.
The formation of the Grignard reagent is indicated by the appearance of cloudiness in the solution and the production of
heat.

4. If no change is observed, carefully scratch the magnesium pieces using a clean and dry glass rod. Once the solution is
cloudy, add the dry stirring bar15 to the flask, then return the flask to your setup and turn stirring on. Allow the mixture
to reflux for 10 - 15 min while the ether boils gently.

5. When the initial reaction subsides (not before) add the remainder of the bromobenzene solution in small portions
over a 3 - 5 min period through the side arm of the Claisen adapter.16

6. Rinse out the 50 ml Erlenmeyer flask with dry ether (2 – 3 ml) and add the rinsings to the reaction mixture.

7. Leave the stirring mixture to reflux for another 5 – 10 min until reaction is complete17 and use this Grignard reagent
for the next step.

8. Make a solution of 1.5 mL of benzaldehyde in 4 mL dry ether in another oven dried flask. Whilst keeping the setup still
assembled, add this solution DROPWISE (using the long Pasteur pipette) through the Claisen adapter to the reaction
mixture, where vigorous fizzing should be observed, then leave the mixture to stir for 5 min.

9. Allow the reaction mixture to cool, then add 5 ml of cold water to the flask through the Claisen adapter. Then add 6
ml of 20% HCl DROPWISE through the Claisen adapter to your reaction mixture. After the addition is complete, stop the
stirbar and you should observe two homogeneous layers.

14 The magnesium turnings have been pre-weighed prior to the lab and will be provided to you in a vial ready for use.
15 Ask your TA for a stirring bar. Make sure to return it to your TA at the end of the lab. Students who lose the stirring bar
will get 25% deduction from the performance mark.
16 Make sure to re-stopper the Claisen adapter with a glass stopper after each addition of the bromobenzene solution.
17 The reaction is complete when mixture stops boiling (no more bubbles appear) and is cooling down.
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10. Turn off the condenser water and dismantle the reflux apparatus. Transfer the entire contents of the flask to a
separatory funnel and allow the aqueous and ether layers to separate. Try not to lose the stir bar in the process!

11. Remove the aqueous layer from the separatory funnel (which one is it?) and discard into the appropriate waste
container.

12. Wash the ether layer with fresh water18, then with saturated sodium bisulphite solution, and again with fresh water,
each time making sure to drain off the aqueous wash.

13. Collect and dry the ether layer by swirling it over a few grams of anhydrous magnesium sulfate in a 50 mL
Erlenmeyer flask for 3-4 min. Remove the magnesium sulfate by filtering through a plastic funnel plugged with a small
piece of cotton into a clean and dry flask. Rinse the cotton plug with additional ether.

14. Concentrate the ether solution by heating the flask on a steam bath to remove most of the ether. Then, blow air
slowly into the flask to remove the remaining ether.

15. Immediately recrystallize using petroleum ether. Suction-filter, dry and weigh the product, then measure its melting
point range and calculate the percentage yield.

16. Clean your used glassware immediately and return the drying tubes to the dessicator before leaving the lab.

18 “Washing” means shaking the mixture with water.

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Pre-Lab Assignment 1 (detach and submit to your TA at the beginning of your lab session)
Name: Date: TA:

Given the structures of benzophenone and biphenyl (shown below), answer the following questions.

1. Which of the two structures is more polar and explain why?

2. Which structure would be expected to have a higher Rf value on a polar TLC plate (ex: silica, alumina) and why?

3. Why is it recommended to spot the analyte on the TLC plate in a position that it will not be immersed in eluent
solvent (Figure A)? What is wrong with figure B?

Figure A Figure B
4. Why is it recommended to use pencil to mark your TLC plate instead of pen?

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Pre-Lab Assignment 2 (detach and submit to your TA at the beginning of your lab session)
Name: Date: TA:

1. Please complete the following table, ignoring the shaded areas.

Molecular weight mp, °C bp, °C

Cholesterol

2-butanone

Ethanol

2. What is the benefit to performing a recrystallization to isolate a product as opposed to simply evaporating the solvent
and recovering the product?

3. Why should melting points always be reported as a range rather than as a single temperature?

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Pre-Lab Assignment 3 (detach and submit to your TA at the beginning of your lab session)
Name: Date: TA:

1. Complete the table, excluding the shaded areas. Refer to experiment 1 for some values.
Molecular Density Mass Volume mmol
Name of Compound weight (g/mL) (g) (mL)

(±)-α-phenylethylamine
(+)-tartaric acid
(−)-amine-(+)-tartrate salt
(+)-amine-(+)-tartrate salt

2. During the extraction step, two distinct layers will form (namely, aqueous and organic), which solvent will be used to
form the organic layer? Will the organic layer be formed on the top or bottom layer? Explain why.

3. What is the role of adding the 2 M NaOH to the amine tartrate salt prior to the extraction step with dichloromethane?

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Pre-Lab Assignment 4 (submit to your TA at the beginning of your lab session)

Name: Date: TA:

1. Draw proper chemical structures of benzoic acid and biphenyl. Look up and report the acidity constant (pKa) of benzoic
acid, as well as the melting points of both compounds.

2. What is the purpose of the anhydrous sodium sulfate (Na2SO4) in this experiment?

3. The following organic compound is very insoluble in water. Propose a chemical reagent that can transform it into a water
soluble ion, and draw the product you would expect from such a reaction.

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Pre-Lab Assignment 5 (detach and submit to your TA at the beginning of your lab session)
Name: Date: TA:

1. Complete the Table, excluding the shaded areas:

Molecular Bp, (°C) Mp, (°C) density Mass Volume mmol
Weight (g/ml) (g) (mL)
bromobenzene

benzaldehyde

magnesium

diphenylmethanol

diethyl ether

2. Why should all of the glassware and the magnesium used for the preparation of the Grignard reagent be dry?

3. What is the role of the anhydrous calcium chloride used in this experiment?

4. Calculate the theoretical yield (in g) of diphenylmethanol in this experiment. Show your work.

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