Techniques for Characterizing and

Purifying Organic Compounds

CHML 2210-Organic Chemistry Lab
Eastern Florida State College
Palm Bay Campus

Fall 2014 EDITION

TABLE OF CONTENTS
Page
Laboratory Safety

Laboratory Notebook

A Sample Preparative Laboratory Notebook example

13

Sample Organic Laboratory Report

20

Characterization and Purification

23

Washing Dishes

23

EXPERIMENT 1: Melting Point

24

EXPERIMENT 2: Column Chromatography

27

EXPERIMENT 3: Thin-Layer Chromatography

32

EXPERIMENT 4: Extraction

48

EXPERIMENT 5: Distillation

53

EXPERIMENT 6: Acid-Base Extractions

57

EXPERIMENT 7: Identification of Organic Compounds by Qualitative

Analysis

62

EXPERIMENT 8A: Effect of Structure on the Free-radical Bromination

of Hydrocarbons

69

EXPERIMENT 8B: Relative Rates of Nucleophilic Substitution of

Reactions of Halides

72

EXPERIMENT 9: Medicinal Chemistry

75

EXPERIMENT 10: Nylon Experiment

78

Appendix A: Gas Chromatography

80

Appendix B: Infrared Spectroscopy

85

Appendix C: Nuclear Magnetic Resonance Spectroscopy

92

LABORATORY SAFETY RULES

Chemistry laboratories are places with unusual hazards, which for the most part, are associated
with the chemicals in them. Chemicals are unforgiving: if you mishandle them, you can be
seriously hurt. TREAT THE LABORATORY AND EVERYTHING IN IT WITH RESPECT. The
following rules will minimize risks in the lab:
1. Safety goggles MUST be worn. When anyone is working, EVERYONE must wear
goggles. There are NO exceptions. Eyes are priceless and irreplaceable. When
goggles fog, step outside to mop them. Goggles on top of the head do not protect the
eyes.
2. An apron or lab coat MUST be worn when working. This adds an extra layer of
protection between chemicals and your body as well as protects your clothes from
discoloration or mutilation.
3. Shoes MUST cover the entire foot. NO exposed toes. NO flip flops, sandals or
extreme ballet flats that expose the entire top of the foot. You may carry your shoes in
a pack to lab.
4. Contact lenses tend to complicate eye injuries. The use of contact lenses during lab
periods is strongly discouraged.
5. Wearing long pants to lab is required. Tie back long hair to keep it out of reactions.
6. Absolutely no smoking, eating or drinking in the lab.
7. Locate the fire extinguishers, eye washes and safety showers BEFORE you begin any
experimental work.
8. Do not run. Do not push. Do not work and socialize. IF youre sure your sample can be
left safely you can talk. If you are not sure about the safety of your sample or reaction
give it your attention and save the conversation for later.
9. Do not alter the procedure for the experiment you are doing. Follow all directions
carefully.
10. Unauthorized experiments will guarantee an F for CHML 1045 for any student involved.
(Note on academic honesty: failure to perform analyses that you report, will result in a
zero for the experiment.)
11. Never taste or smell any chemical (to detect an odor, use your hand to waft the chemical
towards your nose). Do not touch chemicals with your hands. At the least, you will get a
skin rash. Many chemicals are absorbed through the skin and some can cause serious
burns.
12. WARNING: All ethanol in this laboratory is denatured. Denatured means that it is toxic
as it contains poisons, which cannot be removed by distillation. If you take any ethanol
from the laboratory for drinking purposes, you will kill or seriously injure yourself or
whomever you give it to.
13. Never heat a sealed apparatusit WILL explode. The flying glass will cause serious
injuries.
14. Never heat a sample or reaction rapidly, always gradually. NEVER leave a reaction
unattended.
15. Your instructor will clean up broken glass (after you inform the instructor); dont pick it up
yourself. Please treat glassware with care.

16. Wipe any spilled chemicals up immediately. Note where acidic and caustic spill kits are
in the laboratory.
17. Reset balances to zero when you finish. Do not leave weights on a balance.
18. Wash your hands before leaving the laboratory.
19. NO chemicals are to be flushed down a drain unless specifically instructed to do so by
the lab procedure.
20. Wastes are to be poured into the appropriately labeled waster container.
21. DO NOT mix wastes from different categories.
22. Report all accidents to the instructor. Clean up solid and liquid spills immediately, but
only after checking with your instructor for possible safety hazards.
23. Take containers to the stock of chemicals. Do not bring stock containers to your lab
table.
24. Read the label on chemical bottles carefully. Insure that you have the correct chemical.
25. Do not insert a pipette or medicine dropper into a stock bottle. Avoid contamination by
pouring a small quantity into a flask or beaker.
26. Use special care when handling stoppers or tops of bottles so as not to pick up
contamination.
27. Take no more of a chemical than an experiment requires. Do not pour extra back into
the stock container.
28. Set up your glassware and apparatus away from the front edge of your laboratory bench.
29. When an experiment is completed, clean all glassware and other materials appropriately
and return them to the drying racks by the sink. Laboratory equipment, glassware and
benches are to be returned or left in equal or better shape than when they were found.
30. Always add acid to water, never the reverse. Improper addition can lead to violent
reaction and splattering.
Follow any other housekeeping, safety or disposal rules given by your instructor.
Federal and State regulations require compliance with strict guidelines for handling, storage and
disposal of hazardous chemical wastes. Because of the wide variety of chemicals used in a
teaching laboratory, it is imperative that all students follow proper disposal procedures so as to
not pollute our environments and groundwater supply.
Willful violations of these rules can result in a zero being given for that lab. Repeated willful
violations can result in an F being given for the course.
Every effort has been made to minimize the hazardous chemicals used in the lab. All chemicals
provided can be used safely by following correct procedures. However, any chemical can be
dangerous if used improperly. Material Safety Data Sheets (MSDS) are provided for each
chemical having possible hazardous effects. MSDSs are available for review upon request.
Students with Disabilities: Eastern Florida State College is committed to the success of all
students. A person with a disability may qualify for reasonable accommodations. Contact the
Office for Students with Disabilities, 433-5172, for eligibility criteria and more information; we
recommend you do this within the first two weeks of class or preferably, before classes begin.
Your expectation for confidentiality will be respected and maintained in accordance with the law.

LABORATORY SAFETY PROCEDURES

Safety is one of the major concerns in any lab. Safety for humans is always a primary
concern. The location of safety equipment should be common knowledge to anyone working in
the lab. In order to become familiar with the safety equipment in this lab, make a map on the
reverse side of this sheet showing the location of the following safety items. Indicate the fire
escape with an arrow.
1. Emergency shower and eye wash station
2. Fire extinguisher
3. Fire blanket
4. Exits from room
5. Fire escape route
6. Fire alarm boxes
7. Container for broken glass
8. Electrical power cut off switch
9. First aid box

I have read and agree to follow ALL of the listed safety procedures and precautions.

_________________________________________
Signature

________________
Date

_________________________________________
Instructor

________________
Section

Note: The next few pages are a general description of how to prepare for lab, write up your
experiments, write lab reports, etc. Each lab instructor has personal preferences concerning
these topics. These preferences may differ somewhat from what is presented here, but your
instructor will tell you what their preferences are. You should use the following information as a
guideline, but follow any specific directions your instructor gives you.
THE LABORATORY NOTEBOOK
Keeping organized, accurate records of procedures, observations and data in a lab notebook is
the cornerstone of all kinds of scientific work. This includes organic chemistry lab. A lab
notebook is your record of procedures, observations, amounts of chemicals and other
information as you complete experiments. Your lab notebook should give a step-by-step record
of what you do and observe and measure. Writing your lab reports is easier for you to do with a
well-organized lab notebook. It also makes studying for the final exam easier.
Why bother with a lab notebook if I'm not majoring in chemistry?
In most scientific careers, graduate work, clinical studies, or forensic analysis, etc., it is vital to
properly record procedures, data and results in a laboratory notebook. Improperly documented
procedures and data have resulted in patents being denied, research findings being rejected or
forensic tests being thrown out of court. Maintaining a lab notebook takes some work, but many
instructors in upper-division science courses (and most supervisors in the real world) will
assume that you can properly use and maintain a laboratory notebook.
What should be recorded in the lab notebook?
Everything relating to carrying out the experiment (title, objective(s), reaction, reagent table,
procedures, observations, raw data, etc.) should be written directly into the notebook. At a
minimum, all steps that must be done in the lab (procedure, measuring mass, obtaining spectra,
etc.) must be recorded directly into the notebook.
Notebook guidelines
Write all observations and data in ink directly into your notebook as you carry out the
experiment. (Yes, you have to take your notebook to the balance or laboratory instrument to
record masses and/or measurements!) If you make an error in your notebook, cross it out with a
single line and re-enter your data nearby. Do not obliterate your error with a large blob
because if the first entry was right, it is now unreadable. Do not write the correction directly on
top of the error; some notebooks use carbon paper, and if the original copy is hard to read
then carbon copy is usually impossible to read!) Write data only in your notebook. Never write
data on scratch paper or in the margins of the lab manual! This very poor lab practice can (and
does) lead to lost or garbled data. The instructor reserves the right to confiscate materials used
to improperly record data and/or deduct points from an offender. Please find below a summary
of some guidelines for keeping a laboratory notebook for organic chemistry lab courses. These
are taken from the UCL Journal of Technology (Jan 2000).
1. DO use bound books. Permanently bound books should be used. They should be
consecutively numbered and each page should be dated, signed and witnessed.
2. Do use ink. Notebook entries should be made in ink and in chronological order. Entries
should not be erased or whited out. If an entry contains an error, a line should be
drawn through the error and new text should continue in the next available space.
3. Dont leave blank spaces. Blank gaps between entries should be avoided. If a blank
space is left on a page, a line or cross should be drawn through the blank space and the
pages dated to prevent subsequent entries.

4. Dont modify. Prior entries should not be modified at a later date. If data were omitted,
the new data can be entered under a new date and cross-referenced to the previous
entry. Record experiments when they are performed.
5. Do use past tense. Use the past tense (e.g., was heated) to describe the experiments
that were actually performed.
6. Do explain abbreviations and special terms. Explain all abbreviations and terms that
are nonstandard. Explain in context, in table of abbreviations or in glossary.
7. Do staple attachments. Attachments such as graphs or computer printouts should be
permanently affixed in the notebook (by stapling) and both the attachment and the
notebook page signed and dated.
8. Dont remove originals. No original pages should be removed from the notebook.
9. Do outline new experiments. When a new project or experiment is started, the
objective and rationale should be briefly outlined e.g., in a short paragraph or by
providing a flowchart.
10. Do provide detail. Record experimental descriptions, including operating conditions,
test results, and all explanation of the results, and thin layer chromatography sketches of
the results. Any conclusions should be short and supported by factual data. DO NOT
COPY THE DIRECTIONS IN THIS MANUAL. DO NOT wait until the end of the
experiment to write down observations write them down as you make them. Also, write
directly into your notebook. NEVER take notes on a paper towel or other loose sheet of
paper.
If carbon copies are used, remember that your instructor will grade the carbon copies so
make sure they are readable!
Whatever you write in your notebook must give legible carbon copies. Make it easy for the
instructor to read what you write. Print in your notebook; cursive writing often gives carbon
copies that are very difficult to read. Make sure that you press hard enough to produce clear
copies, and make sure the plastic shield is in place (for a carbon copy notebook). Remember
that tapping your pen, writing on another paper resting on your notebook, or forgetting to put the
shield behind your carbon page will cause marks on the carbons that will obscure what you
write. Periodically check your carbons to confirm that your writing is clear and legible. Use
several paragraphs instead of large blocks of text because small paragraphs are easier to read.
Keep solvents and chemicals away from your notebook because they may dissolve the ink or
destroy the pages.
Your notebook is not expected to be flawless, but it should be easy to follow. Cross-outs and
corrections are expected. Do not skip pages, and do not tear original pages out of your
notebook, even if they are spoiled. If you run out of room on a page, do not try to cram your
writing into the available space, because this typically leads to unreadable carbon copies.
Instead, use Continued on page... and Continued from page to help the instructor follow
your report. Useful tip: Leave a few blank lines between steps of your procedure, so you can
insert any changes or forgotten steps. Also, leave space between sections of your report in case
you need to add something you forgot to include or if you need to correct or better explain
something you previously mentioned.
GOAL: You are keeping a good laboratory notebook if a student who has taken a similar organic
lab course at another university could carry out the experiment based on your notes. You should
work toward this goal.

These sections should be prepared and in your notebook before you come to lab:
Title, lab meeting time, date of experiment: This is self-explanatory.
Objective: Write one to three sentences about the goal(s) of the experiment. This could be the
identification of an unknown compound, the preparation of a compound, isolating a pure
compound from an impure sample, etc.
Main reaction (if applicable): All preparative experiments should include the balanced
reaction. Include reaction conditions, catalysts, etc.
Table of reagents, solvents and products: Prepare a table listing the chemicals used in the
experiment. List headings such as compound names, molecular weights, densities, melting and
boiling points, refractive index, etc. Include the relevant properties in the table. Irrelevant
information such as the boiling point of a solid or the density of a gas can be excluded. You can
find information in your textbook, online (chemfinder.com is a good place to start), in chemical
catalogs (Aldrich, ACROS, Lancaster, etc.) or the CRC Handbook.
Procedure: Do not just copy out of the manual. Summarize the steps you will use. Example: A
miniscale fractional distillation apparatus was set up as in Figure X in the manual page X. Glass
beads were used as packing, as in Figure Y in the manual. You do not have to describe how
you clamped each piece of glassware. (Recipes in a cookbook do not explain how to turn on an
oven or boil water.) Similarly, you do not have to explain each step of obtaining a refractive
index or setting up a filtration because this information is freely available in your manual. Leave
one or two blank lines between steps to give you room to insert any steps you left out or to
include any changes announced by the instructor. Avoid using first person (I/me/my) in your
report. Record the exact amounts of chemicals that you use in the Observations section.
A very convenient and effective way to organize your Procedure and Observation sections is the
two column method shown in the Sample Laboratory Notebook on pages 14-20. You may
also want to create a flow chart if the procedure is complicated or has multiple steps with
several fractions.
These two sections must be also entered in your notebook as you carry out the
experiment:
Changes to the procedure: If you deviate from the written procedure (even if the instructor
announces the changes), you need to write the changes in your notebook. Any changes such
as using different reagents, different amounts, using an additional portion of solvent to wash a
solution, heating for a different time, etc. must be recorded in your notes. Even if you have to
redo a step because of a mistake, you still have to write the changes down. (This is why leaving
a few blank lines between steps is a good idea!)
Observations and Data: Record the exact amounts of chemicals actually used in the
experiment. (The procedure may say to use 0.0100 moles of cyclohexanol. This amount is 10.0
g of cyclohexanol, but you need to record the amount you used (10.03 g, 9.86 g or whatever).
Remember to base your yield calculations on the amounts of reagents you actually used, not on
the amounts called for in the procedure. You should describe the colors and appearances of the
reagents (before and after mixing!), color changes of solutions, temperature changes as a
chemical is added to a solution and so forth. Make as many observations as you can. A
surprisingly large number of students forget to record the appearance of their products.

Sometimes, the cause of an unsuccessful experiment can be pinpointed using observations.

(True story: When half of the students failed to get a reaction to occur, checking the notebooks
showed they used one bottle (with grey powder) and the others used another bottle containing
white powder. It became clear that the first bottle had been mislabeled, so the students
unknowingly used the wrong material.
REMEMBER: Unwritten observations and data never happened!
Data Table: This is for raw numbers such as masses or volumes of reagents, uncorrected
melting points and uncorrected refractive index values or masses of products. Important! All of
this information should have been previously recorded in the Observations section. Remember
to use significant figures appropriately.
The following sections can be entered in your notebook outside of lab time:
Interpretation of Spectra: Report the information (peak values, intensity, interpretation, etc.) in
tables in your notebook. See the Sample report for examples. Do not write on the spectrum
itself except for your name and a title (e.g. John Doe, IR Spectrum of product of Exp. 13, thin
film). Be sure to note the presence (or absence) of significant peaks. For example, if you are
preparing an alcohol from a ketone, you should look for peaks in your spectrum that indicate
that could show the presence of the alcohol product as well as any unreacted ketone.
Calculations: This is where numerical data is processed. Thermometer readings are corrected,
measurements on a TLC plate are converted into R f values, masses of reactants and products
are converted to moles and so forth. Use significant figures (sig figs.) properly in your
calculations! NOTE: In any preparative lab, you need to determine the limiting reagent and
calculate the theoretical and actual yieldeven if you obtain no product at all! If you do not
know or do not remember how to calculate yields, consult your instructor.
Do not create or discard significant figures. If you measured to only hundredths of grams,
your calculations must reflect this. Thus, if your product weighs 3.13 g, do not add a trailing zero
(3.130 g). On the other hand, if a beaker weighed 45.217 g full of reagent and 41.137 g after
adding the reagent to the flask, you added 4.080 g to the flask, not 4.08 grams. The zero in the
thousandths place is a significant figure here.
Discussion of Results: This is the most important section of the laboratory report. Here is
where you explain and interpret the results of your experiment. The length and specific content
will depend on the experiment, but the Discussion section is usually at least 10-15 sentences in
length. The section will be somewhat longer in preparative experiments. You should, of course,
always use correct grammar, spelling and terminology in the lab report, but doing so is
especially important in the Discussion section. Why? Because the quality of your discussion
indicates how clearly you understand the objectives of the experiment and how you interpreted
your results. If you make inaccurate or poorly supported claims, use illogical reasoning, omit
relevant information, or use the wrong terminology, it gives the impressionwhether true or
notthat you do not understand the experiment. This impression will impact your score for that
report. Likewise, unclear wording, misspelled words and poor grammar give the impression of
sloppiness or that you did not proofread your work. Even if an experiment goes poorly, a wellwritten, well-reasoned Discussion will often earn more points than a badly written Discussion
describing a successful experiment. More information concerning what should be included in
the Discussion section is given on the next page. In contrast to the Procedure, using first-person

pronouns (I/me/my) is more acceptable in the Discussion section. See the following page for
more information.
Summary of Experiment: The key word is summary. This section should be brief (typically 35 sentences). The summary should state the outcome of the experiment and a couple of
important details. You are expected to include specific information such as vial numbers,
measured properties, yields, etc. Do not introduce any new information in this section. This
section should only include information that is mentioned elsewhere in the report. The key topics
from the Discussion section will probably be part of your Summary, but remember that the
Discussion and Summary are separate sections of the report.
Example of a poorly-written Summary (The reader has no idea what the result was or what the
data means.):
I successfully accomplished the objective of this experiment. My data was very close to the
expected values, and there were no significant problems in carrying out the experiment.
Example of a much better summary (A reader can clearly see what was done and what the
results meant.):
My unknown liquid (code B-23) was identified as tetralin. The measured boiling point was
about 5 C lower than the listed value for tetralin, but the measured refractive index was very
close to the value in the manual. Tetralin was the only compound with a boiling point and
refractive index that were both fairly close to the measured values, so I am confident that my
identification is correct.
Example of a summary for an experiment involving the preparation of a compound :
Benzhydrol was prepared by the reduction of benzophenone by NaBH 4. The yield of purified
product was 65.8%. The IR spectrum closely matches that of a pure sample and the melting
point is only 1-2 C below the reported value, indicating that the product is probably quite
pure. The loss of mass during recrystallization shows that this is the step where the major
loss of product occurred.
REMEMBER! Focus on the highlights. Think: What in terms of What I did in lab in 50 words or
less.
Answers to follow-up questions: Your instructor may assign some or all of these questions.

Additional Guidelines for Writing Your Discussion Section

The most important rule is: do not make claims in unless they are backed up with specific
information! For example, if you state that the sample you obtained was pure, you must support
this by giving specific evidence that you have recorded such as a narrow melting range, a
melting range or appearance that that matches an authentic sample, etc. Use specific data, IR
absorptions, etc. to support your claims. See these three examples.
Good: My sample melted at 113-114 C, which is close to the m.p of pure acetanilide (114115 C).
So-so: My sample melted at 113-114 C, which is close to the m.p of acetanilide.

10

Poor: My sample had the same melting point as acetanilide. (The reader must then look up
the values.)
Consider all relevant information, even some seems contradictory. For example, if a product
looks identical to a known pure sample, but the melting range data suggests the sample is
impure, the Discussion must address both pieces of evidence. Remember that contradictory
data may be due to misreading an instrument, incorrect calculations, misinterpreting the
instructions or many other sources. If you find contradictions, double-check all of your readings,
calculations, etc. (Having a friend look at your data can be helpful here.) If you cannot find a
problem, but you still have contradictions, then you must judge the importance or reliability of
each piece of data and the weight to give it as you draw conclusions about the experiment.
When you have contradictory data, it is a good idea to consult your instructor before you begin
to write your Discussion.
Examples of appropriate topics for Discussion:
Example #1. In Experiment Y, you will purify a solid by recrystallization, and identify it by melting
point determination. The Discussion section should address issues such as:
1) What is the identity of your solid, and how do you know that the identification is correct?
2) Is the recrystallized material pure? What evidence supports your claim that the product is
pure or not?
3) At which steps was product lost? If possible, point out where major loss of product
occurred.
Example #2. In Experiment Z, you will prepare and purify an alkyl halide and analyze it using
refractometry and infrared (IR) spectroscopy. Good topics for your Discussion include the
following:
1) Did you successfully prepare and isolate the desired product? Was the product pure?
2) What does the IR spectrum indicate about the identity of the product and/or the presence
of impurities?
3) What is the percent yield of isolated product? At which steps were significant amounts of
product lost?
Three things you should not do in your Discussion
1. Do not introduce new data, or observations. Never refer to anything concerning the
experiment that is not recorded in your notebook. For example, your Discussion should not refer
to the color of your product unless it is recorded in your observations. Likewise, if you spill a
third of your product at the end of the experiment, but you fail to document it in your
observations, it is not appropriate to cite spillage as a source of product loss in your Discussion.
Note: it is appropriate to refer to sources such as the Merck Index or the CRC handbook for
information about your product, but you must properly cite the references you use.
Remember: Any unwritten observations or data never really happened!
Therefore, you cannot discuss something that never happened!
2. Do not re-state the procedure as part of the Discussion. Use the Discussion section to
explain and interpret your data and results.
NOTE: It is okay to mention steps that affect the yield or purity of your product. For example: I
started the lab late, so I was only able to heat the mixture for 40 minutes, instead of 90. The

11

reaction probably did not go to completion. In short, discussing how the procedure affects the
results is fine; just restating the procedure is not.
3. Do not combine your Discussion and Summary sections. The Discussion section is where
you evaluate and explain your results. The Summary section is where you wrap up the
experiment.
Keep this in mind when you are sick of preparing your notebook and writing lab reports:
Clear communication of information through writing is the backbone of science. Strive to make
your writing as clear, complete, concise and correct as possible. Employers out in the Real
World place a high value on sound writing skills in their employees. Also, many employers will
expect new employees to have experience in properly maintaining a laboratory notebook and
writing clear, concise lab reports.

Twelve Key Points to Remember as You Prepare for Lab and Write Your Notebook
1. Preparing for lab is absolutely essential. Know what you are supposed to do, what
chemicals you will use, what techniques you will employ and so forth. Being prepared when
you begin the experiment makes it much easier to keep your observations, data, etc.
organized. This, in turn, makes it easier to find the information you need when you write
your Discussion section.
2. Summarize procedural steps when you write them. This saves time for you and the reader
as well. You may assume that the reader is familiar with basic lab techniques, so you do not
have to fully explain them. Always include any additions or changes to the procedure
including things like re-doing steps, performing additional extraction, re-distilling your
product or even restarting the whole experiment.
3. The amounts of chemicals you list in the Procedure are approximately what you will need.
Therefore you will need to record the exact masses and volumes in your Observations.
Always record the appearances of your solvents, reagents, the colors of solutions, changes
in appearance or texture during heating or cooling, colors of the layers when performing
extractions, colors and appearance of products, etc.
4. When performing calculations, use significant figures correctly.
5. Present data from IR and NMR spectra in tables. Try to interpret all relevant signals.
Relevant is loosely defined as a signal or peak that is probably from either the product,
starting material or a solvent. You must develop your skills in interpreting spectra so you can
determine whether a given peak or signal is from the product, starting material,
contaminants, etc. Feel free to consult your instructor if you need some help.
6. The Discussion section should focus on the results. (What did you make/isolate? What is the
purity? What is the % yield/recovery? Where did losses occur? What problems did you
have?) Support all your claims.
7. Some material usually remains behind when pouring or transferring chemicals. For example,
scraping solid out of flasks or off of a piece of filter paper usually fails to remove all of it.
Unavoidable losses caused by transferring materials from one container to another are often

12

referred to as mechanical losses. Spills and similar mishaps are not mechanical losses,
and they should be addressed in the Discussion section.
8. Use correct scientific terms. For example, The plural of spectrum is spectra, not
spectrums. Use percent yield and percent recovery appropriately.
9. Carefully check your spelling and grammar to make sure what you say makes sense. You
may want to have a friend or classmate read your report to help locate mistakes and parts
that should be revised. Terms that are in Mohrig or the manual should always be correctly
spelled.
10. Keep your Summary section brief, but remember to include specifics such as % yield, code
numbers of unknowns, etc.
11. Use common sense in your Discussion. For example, if your yield of crude product was
52.4% and after recrystallization your purified yield was 39.3%, you should not claim that the
major source of product loss was recrystallization. It is true that you lost a quarter of the
crude products mass during recrystallization, but more remember that nearly half of the
theoretical yield was lost even before you isolated the crude product! Other sources of loss
are important and should be considered. Similarly, if some product is spilled or left as
residue on glassware, and if the amount lost is small compared to the amount you actually
isolated, then the transfer losses are not a major source of loss of product. Critically analyze
your Discussion and your reasoning to avoid making contradictory or nonsensical claims.
Reading it aloudor even betterhaving someone else read it aloud can help spot
problems such as flawed reasoning, contradictory sentences, poor grammar and so forth.
Hint: A clearly written report with sound reasoning and analysis shows that the writer really
understands the experiment. Similarly, a report with contradictory reasoning, unreasonable
(or unsupported) claims or that ignores important data or results gives the impression that
the writer did not really understand what they did.
12. Remember to make sure that your carbon copies are legible!
A Sample Preparative Laboratory Notebook
The following pages show an example of how a preparative experiment should be written up in
a notebook. Remember that if your notebook is organized, your carbon copies can serve as the
majority of your report.
Ben Zeen

Sunday, Feb. 30

Experiment 6: Preparation of Ethyl 4-Aminobenzoate (Benzocaine)

Purpose: The goal of this experiment is to prepare benzocaine from p-aminobenzoic acid. The
product will be purified by recrystallization and the purity checked by m.p., IR, and NMR.
Reaction:

H2N

O
OH

OH

H2SO4
reflux

H2N

O
O

+ H2O

13

Table of Reagents, Solvents, Products, etc.

Compound

p-Aminobenzoic acid (PABA)

Ethanol (EtOH)
Sulfuric acid, conc. (18 M)
Ethyl p-aminobenzoate
Ether
Other materials used:
water
10% Na2CO3 solution

MW
137.136
46.068

m.p.,
C
187-9

165.189

b.p.,
C

Density,
g/mL

78

0.789

35

0.71

92

comments
white crystals*
flammable
corrosive
Off-white crystals*
very flammable

*11th Ed. Merck Index

saturated NaCl solution

Procedure

anhydrous MgSO4

Observations

1.

Add 0.020 mol of p-aminobenzoic acid to

a 100-mL round-bottom flask Add 40 mL
of 95% EtOH and swirl to mix. Not all
solid will dissolve.

PABA is white fine crystals.

0.020 mol ~2.7 g. 2.67 g was actually used.
EtOH is clear liquid. Used 41 mL Swirling
dissolves most solid to give pale yellow soln.

2.

Cool flask in ice-water bath, then slowly

add 2.5 mL of concentrated sulfuric acid.
Lots of precipitate will form, but it will
dissolve during reflux.

Acid was thick clear liquid. Adding acid

caused some spattering. Lots of white-yellow
precipitate formed and sank to the bottom of
the flask. Solution turned pale yellow

3.

Add a small magnetic stir bar as boiling

stone. Assemble simple reflux set-up with
flask and reflux for 75-90 minutes. Stop
the reflux every 15 minutes to vigorously
swirl the flask to help solid to dissolve.

4.

After reflux period, remove heating

mantle and let mixture cool for 10
minutes, then cool with cold-water bath.

As solution boiled, it turned yellowish and

solid began to dissolve. Some solid was crust
just above liquid. Crust dissolved when flask
was swirled after 15 minutes of reflux. All
solid dissolved after about 25 min. of reflux.
Soln stayed yellow. Reflux stopped at 75 min.
No changes visible after initial cooling.
No changes after cooling in ice bath.

5. Pour contents of flask into 250-mL

beaker. Rinse flask with 2-3 mL of ethanol

Had to use about 5 mL of ethanol to wash out

the flask. Solution in beaker is yellow.

6. Slowly add 10% Na2CO3 solution (with lots

of stirring) until gas evolution ceases and the
solution is strongly alkaline (pH of 9-10 as
shown by test strips) About 35 mL of base
should be needed. Ppt. should form.

Na2CO3 solution is water-like. Lots of foaming

as added to the beaker. Some solid particles
deposited on sides of beaker. Fizzing stopped
after about 30 mL of solution added. Lots of
ppt formed then.

7. Pour mixture into separatory funnel. Add

40 mL of ether to dissolve as much of the
solid benzocaine as possible. Stopper and
shake funnel. VENT! Separate layers.

Ether is clear liquid. Ether solution after

shaking is slightly yellow. Aqueous layer
(bottom) is sort of yellow, but cloudy, too.

14

8. Pour aqueous layer back into sep. funnel

and extract with 20 mL of ether in same way
as previous step. Separate layers and add
ether layer to earlier ether solution.

Ether solution after second extraction is

nearly colorless. Combined ether solution is
pale yellow.

9. Wash ether layer with 10 mL of satd

NaCl solution, then dry using MgSO4 in
Erlenmeyer.

No change in appearance of organic solution.

MgSO4 made the snow globe when swirled.

10. Gravity filter solution into 24/40 joint

round-bottom flask. Rinse flask and filter
paper with a little ether.

Used 5 mL of ether to rinse flask and filter

paper.

11. Add a glass bead as boiling stone and

remove ether and ethanol using rotary
evaporator. Scrape solid from flask. Weigh
and obtain crude melting point.

Removal of solvent gave pale yellow residue

stuck to sides of flask. Easy to scrape from
sides but some left. Yellowish powder. 2.55 g
2.05 g crude product (damp). m.p = 86-90 C.

12. Purify solid by mixed-solvent

recrystallization using just enough boilinghot ethanol to dissolve product. (10-13 mL)

Used 11 mL of hot ethanol. Gave yellowish

solution.

13. After solid dissolves, add warm water

drop-wise with stirring until the cloudiness
does not disappear. Cool on wood block 10
min. Then chill in ice bath for 15 min.

Cloudiness formed after adding about 1 mL of

water. More solid formed as solution cooled.
Solid was whiter in color than the crude solid.

14. Vacuum filter, rinsing w/ ethanol. Obtain

final mass and m.p. Obtain IR, NMR, and
submit sample in labeled vial.

Solid was creamy white. Filtrate was

yellowish. 1.39 g of dry pdt. obtained. m.p. was
90-91 C. IR, NMR spectra obtained using
CDCl3 solvent and attached to report.

Expect yield of purified product of 40-60%

The IR was obtained by allowing a CH2Cl2

solution to evaporate on a salt plate.

15

Ben Zeen

IR Spectrum of Benzocaine

(CH2Cl2 solution evaporated on a salt plate)

Analysis of the IR spectrum of the product gave the following information:

(The absorptions that were intense or which were easily identified are listed.)
Absorption, cm-1

intensity / shape

Interpretation

3424
3346 + 3225
3047
2986-2900
1687
1634
1601
1518
1476
1450
1367
1312, 1174
849

medium / fairly sharp

2 med-strong bands
sharp / weak
several weak bands
very strong / sharp
very strong
strong / sharp
strong / sharp
weak / sharp
weak / sharp
weak / sharp
very strong
medium / sharp

Overtone of C=O
NH stretches of NH2
aromatic CH stretch
C-H stretches in alkyl groups
C=O stretch of ester
NH bending (probably)
C=C stretch of aromatic ring
C=C stretch of arom. ring (probably)
(unsure)
C-H bend of alkyl groups
C-H bend of CH3 group
CO of ester
o.o.p. C-H bends in 1,4-disub arom. ring

Summary of IR interpretation
I expected benzocaine to show two bands from the NH2 group at around 3300 cm-1, plus
absorptions from the aromatic ring at near 3050, 1600, 1500 and around 835 cm -1. This is basically
what was seen. The absorptions at 3000 and at 1450 and 1380 cm -1 were from the alkyl groups. The
carbonyl group was not at 1735 cm-1, but instead it was at 1687 cm-1. Two strong absorptions at 1312
and 1174 cm-1 are probably the CO stretches from the ester. There also is an unexpected, intense
absorption at 3424 cm-1. I did not observe any strong, broad OH near 3400 that would have been
visible if water or residual alcohol had been present. The 3424 band is fairly sharp, so I believe it is
from something else. It is almost exactly twice the frequency of the C=O band, so I think it is an
overtone of the C=O stretch, but this is far more intense than any I have seen one before. Maybe
the amine group affects the C=O group somehow.

16

I believe that the C=O is the band at 1687 cm -1, but I do not know why it is below 1700. Wade,
chapter 21.4 says the C=O for conjugated esters is around 1710-1725 cm-1, and the book shows C=O
of methyl benzoate at 1723 cm-1. For now, I am assuming that the band at 1687 is the C=O, because
there is nothing else that seems to fit. Maybe the amine group lowers the stretching below 1700. I
was unable to find any information in Wade or Mohrig that explained the result.

NMR SPECTRUM
1
Ben Zeen
H NMR spectrum of my Benzocaine. Solvent is CDCl 3.

3H, triplet

2 H, doublet
2 H, quartet
2 H, doublet
2H, broad singlet

Table of NMR Data

Chemical
Integration
Shift, ppm
7.8
2H
6.6
2H
4.3
2H
4.1
2H
1.3
3H

Splitting
doublet
doublet
quartet
br. singlet
doublet

Assignment
A
B
C
D
E

H2N
D

O
O CH2 CH3
C

Data Table
Mass of 4-aminobenzoic acid (PABA):

2.67 g

Volume of ethanol:

41 mL

Mass of crude product:

2.05 g

Mass of recrystallized product:

1.39 g

Calculation of amounts of reagents used and determining the limiting reagent:

17

Moles of PABA:

2.67 g of PABA x

1 mole PABA
137.136 g PABA

0.0195 moles PABA

Moles of EtOH
41 mL of EtOH

0.95

0.789 g EtOH

1 mL of EtOH

1 mole EtOH

46.068 g EtOH

0.70 moles EtOH

The 0.95 corrects for the fact that the ethanol used contained 95% EtOH, 5% H 2O.
PABA is the limiting reagent.
Calculation of the theoretical yield of Benzocaine:
0.0195 moles PABA

= 0.0195 moles benzocaine

1 mol of Benzocaine = 0.0195 moles benzocaine

1 mol of PABA
x 165.189 g benzocaine = 3.22 g benzocaine
1 mole benzocaine

Calculation of crude yield of benzocaine (assuming it is pure, which it isnt):

2.05 g crude yield
x 100% = 63.7 % crude yield
3.22 g theoretical yield

Calculation of the actual yield of purified Benzocaine:

1.39 g actual yield
x 100% = 43.1 % yield of purified product
3.22 g theoretical yield

Discussion of results:
I believe that I successfully prepared a sample of fairly pure benzocaine. The melting
range of my product was 90-1 C, which is narrow and close to the accepted value for pure
benzocaine (92 C). This is one indication that my product is rather pure. The physical
appearance of my product also fits the description in the Merck Index.
The IR spectrum fits what would be expected for benzocaine. The NH2 group is visible as
two absorptions at 3346 and 3225 cm-1. The ester group is shown by the strong C=O band
at 1687 cm-1 and the CO bands at 1312 and 1174 cm-1. The aromatic ring is shown by the
C-H stretching band at 3047, the C=C stretches at 1601 and (probably) 1518 and the o.o.p
C-H bend at 849 cm-1. The o.o.p bend also showed that the compound was 1,4disubstituted, as would be expected. There were no broad O-H stretches near 3300 cm-1
that would be present if any unreacted PABA, residual ethanol or water were present, so I
know that these are not in my product.

18

I do not know why the C=O stretching frequency is below 1700 cm-1, but my spectrum does
closely match the one the IR spectrum of benzocaine found at the Sigma-Aldrich website
(www.sigmaaldrich.com/spectra/ftir/FTIR000308.PDF). The online IR spectrum also has
the band at 1690 cm-1, and nearly matches my IR spectrum. Therefore, I believe that the
IR suggests that my product is quite pure.
The NMR spectrum is what would be expected for benzocaine. The ethyl group is shown by
the 3H triplet and 2H quartet. The broad singlet at 4.1 ppm is the amine hydrogens. The
aromatic Hs are the two 2H doublets at 6.6 and 7.8 ppm. The pattern indicates a 1,4disubstituted aromatic ring. The carbonyl group deshields the Hs ortho to it, so they are
the ones that are further downfield. There were no unexplained peaks present, so the
NMR spectrum also indicates that my product is pure.
My 43.1 % yield of purified product is within the 40-60% range suggested by the lab
manual, but this means that over half of the potential amount of product was lost. The
most obvious source of loss was the recrystallization step. Some benzocaine remained
dissolved in the ethanol, so it was lost in this step. About 0.66 g of mass was lost, but the
crude product was still damp (probably ether and/or ethanol), so the amount of solid lost
here is less than 0.66 g, but it is not possible to know how much it was.
Also, the reaction was probably incomplete. I stopped the reflux at 75 minutes, instead of
letting it go 90 minutes. The Introduction in the Lab Manual says the reaction was a
Fischer esterification reaction, and these reactions are equilibria that do not go to
completion. If I stopped too soon, then the reaction may not even have reached
equilibrium. The crude yield was less than 64 %, so about a third of the potential yield of
product was lost even before isolating the crude product. However, there is no way to
calculate how much of the product never was formed by stopping the reflux so soon.
Some product was probably lost in the extractions, drying and filtering, but I took care to
rinse my glassware to try to keep this to a minimum. Mechanical losses, such as not being
able to scrape all of the product from the flask and traces left on the glassware and filter
and filter paper, probably added up to a few percent of the loss. I saw no large deposits of
residue on any glassware that would suggest greater losses.
Summary:
Benzocaine was successfully prepared in 43.1 % yield. The product appeared to be quite
pure, as judged by the narrow melting point range, appearance, as well as the IR and NMR
spectra. Recrystallization was a major source of product loss, but it was clear that the
reaction did not proceed to completion.

[Note: Any additional spectra (such as those used for comparison or provided by the instructor),
along with any assigned homework problems, are attached to the lab and turned in as well.]

19

WRITING LABORATORY REPORTS

The cornerstone of science is communicating experimental results. For each experiment, you
will submit a written report that describes the reaction you carried out, the procedures you
followed, the observations you made and the data you obtained and your interpretation of the
results. Lab reports are made up of several sections as described below. Your instructor will
provide you specific information about what is to be included for particular experiments. If you
are unsure what to include something in your report, just ask your instructor.
The following is an example of an excellent organic lab report. Use it as a guideline for your
own reports, but dont plagiarize!
Abstract
Observing the separation made by the spinach extract on the thin layer chromatography plates, a solution
of 7:3 toluene:acetone was identified as the best eluent for calculating retardation factors. Compounds
and Rf values were experimentally calculated as follows. The following compounds separated; Bcarotenes, pheophytin a, pheophytin b, chlorophyll a, and chlorophyll b, along with their respective Rf
values; .98, .90. .68, .61, and .53. Two fractions were collected from the column chromatography
experiment, along with a sample of the original spinach extract. Fraction one resulted in a single
compound with Rf value of .98. Fraction two resulted in two compounds with Rf values of .45 and .10.
The original spinach extract resulted in four compounds with Rf values of .98, .63, .45 and .22. TLC was
used for the identification of compounds in an unknown pain reliever based on Rf values for Tylenol and
Bayer. The unknown resulted in three compounds with Rf values of .64, .46, and .24. Tylenol was
experimentally found to have a Rf value of .45. Bayer was experimentally found to have an Rf of .5.
Introduction
Thin-layer chromatography is a technique used to separate compounds with in mixtures. This is useful
for evaluating the purity of compounds and monitoring the progress of a reaction. TLC consists of two
phases, a mobile phase and a stationary phase. TLC functions by the differences in polarity of the
compounds with the solvent used, result in varying migration in well defined and separated spots.
Reagents and Equipment
Reagents used in this experiment include methanol, hexanes, water, sodium sulfate, toluene, acetone,
spinach, spinach extract from column chromatography experiment, and pain relievers. The equipment
used includes a mortar and pestle, beakers with glass watches, glass funnel, cotton plug, 125 mL
separatory funnel, Erlenmeyer flask, Pasteur pipets, and test tubes.
Procedure and Observations
Ten grams of spinach was cut, dried and drained before weighing. The spinach was added to the mortar
with twelve milliliters of methanol and crushed for three minutes resulting in a bright green mixture. The
methanol was then discarded. Fifteen milliliters of hexanes and five milliliters of methanol was added to
the mortar with the leaves and crushed for five minutes resulting in a dark green mixture. The liquid was
then transferred to a beaker and filtered with a cotton plug into the separatory funnel. Three layers
formed. A dark green layer formed on top followed, by a lighter green layer followed, by an almost clear
layer. Five milliliters of water was added to the separatory funnel. Funnel was inverted and periodically
vented. The dark green organic layer stayed on top. The lower aqueous layer was extracted and
discarded. The organic layer in the separatory funnel was then rinsed three more times, resulting in the
aqueous layer becoming more clear with each rinse. The organic layer was then separated and transferred
to an Erlenmeyer flask. Anhydrous sodium sulfate was added to absorb remaining water. Solution was
then filtered with a cotton plug and funnel into a test tube. The test tube was sealed for one week. With
the test tube concentrated down to one to three milliliters the experiment was continued one week later.

20

Five development chambers were prepared in beakers. Solvents used included hexanes, toluene, 9:1
toulene-acetone, 7:3 toulene-acetone, and acetone. TLC plates were prepared for each development
chamber. The 9:1 toulene-acetone was used to determine the correct size of compound to be used. The
7:3 toulene-acetone was found to have the most separation. The spinach extract from the column
chromatography experiment was then dissolved in a small amount of the solvent used to collect the
fraction. Each fraction and remaining extract was placed on a TLC plate in the 7:3 toulene-acetone
developing chamber. Tylenol, Bayer, and an unknown pain reliever was respectively crushed to a fine
powder and placed in separate test tubes. Each compound was dissolved with a 1:1 mixture of methylene
chloride and ethanol solution. Using .5% acetic acid in ethyl acetate as the solvent, TLC plates were
labeled and ran. The unknown was paired with each comparative pain reliever on a separate TLC plate.
The Unknown resulted in three separate compounds. The Tylenol and Bayer each resulted in one
compound. Ultraviolet light was used to visually see the compounds and mark the TLC plate
appropriately.
Data
The following is a table of the spinach extract with 7:3 v/v toluene:acetone used as the eluent.
TABLE 1
Compound
Rf
B-carotenes
.98
Pheophytin a
.90
Pheophytin b
.68
Chlorophyll a
.61
Chlorophyll b
.53
The following is a table of the fractions and extract from column chromatography experiment with 7:3 v/v
toluene:acetone used as the eluent.
TABLE 2
Compound
Fraction 1
Compound
Fraction 2

Rf
.98
Rf
.45
.10

Compound
Spinach Extract

Rf
.98
.63
.45
.22
The following is a table representing the unknown pain reliever, Tylenol, and Bayer with .5% acetic acid
in ethyl acetate used as the eluent. Ultraviolet light was used to visually see the compounds.
TABLE 3
Compound
Rf
Unknown
.64
.46
.24
Tylenol
.45
Bayer
.50

21

Results
Based on the separation of the spinach extract using hexanes, toluene, 9:1 toluene-acetone, 7:3 toluene
acetone and acetone as the eluents, 7:3 toluene acetone was found to have the best separation of
compounds. Acetone was found to not be a good solvent to separate the pigments due low polarity.
Taking the Rf values of each compound and comparing to the polarity of each pigment, the correlation is
a direct relation. As the polarity of the compound decreases so does the Rf value of the compound.
Using the spinach extract from part one as a basis of comparison for the spinach extract from the column
chromatography experiment, fraction 1 was found to contain B-carotene. Based on the polarity of the
carotenes and the pigment separations of the other eluents tested, a mixture of hexanes and toluene would
provide the best separation. Hexanes provided a minor movement of the carotenes with little movement
of the rest of the pigments due to the differences in polarity. Toluene provided much more movement up
the TLC plate of the carotenes and remaining pigments but not enough separation. To achieve an
acceptable Rf value, close to but not greater than .75, a mixture of the two solvents is necessary. Fraction
2 was found to contain chlorophyll b. Chlorophyll a has a methyl group in the position chlorophyll b has
an aldehyde group, making chlorophyll b more polar. Based on this assessment, the original appearance
of the fraction and the proximity of Rf values, chlorophyll b was determined as the compound. The
additional Rf compound found in fraction 2 is too far from the range of experimental Rf from any data
collected in table 1 to be determined. The column chromatography spinach extract was found to contain
B-carotenes, chlorophyll a chlorophyll b and an additional compound that was too far from the range of
experimental Rf values in table 1 to be determined. The unknown pain reliever was found to have three
separate compounds with similar components of both Tylenol and Bayer. The Rf values for Tylenol and
Bayer were very close in proximity, along with a component in the unknown. This indicates that the
unknown contains a component of both the Tylenol and the Bayer. The overall TLC plates of the pain
relievers were not as clean in separation as the spinach plates. The compounds came out as large smears
indicating overloading of the TLC plate. For future experiments, a technique of using less compound on
the TLC plate will prove to provide better data.
Follow up questions
(deleted so that you can come up with your own answers)
References
Techniques for Characterizing and Purifying Organic Compounds. CHML 2210-70B Brevard
Community, Palm Bay Campus. Fall 2013 Edition.

22

Characterization and Purification

There are four problems that confront organic chemists on a daily basis:
1. is the sample I have pure?
2. if it is not pure, what can I do to remove the contaminants?
3. if it is pure, what properties does the sample have which will enable me to recognize the
same compound if I obtain it again from a different reaction source?
4. what is this stuff anyway?
Virtually any method, which can be used to characterize a compound, will also serve as another
check of the purity of the sample in question. No single means of characterization is sufficient
to establish the identity of a previously known compound and no single means for indicating
purity is sufficient to establish purity. Good experimental work in the characterization and
purification area systematically includes several indicators of good purity and an array of sample
properties to characterize the compound in question. Multiple redundancies in information
make characterization possible.
In this course, the first half of organic chemistry, you will learn many basic techniques. All of
these techniques have application beyond the chemistry laboratory (biology uses
chromatography extensively).

Washing Dishes
In order to characterize and purify compounds successfully and to carry out actual reactions,
one must start each experiment with clean glassware. The easiest way to obtain this is to do
your dishes at the end of the previous experiment since many procedures need to be done
under dry conditions. This isnt trivial because many organic compounds are not very soluble in
water.
Suggested steps are as follows:
1. Remove grease (if any) from ground-glass joints. If stopcock grease has been used, it
must be removed before washing is attempted, otherwise grease distributes itself over
everything. To de-grease joints, moisten a paper towel with ether or dichloromethane
and wipe the grease off the joint. Ether is marginally better but is flammable.
2. Scrub the glassware with soap and water. Use a brush.
3. Rinse the soap off with water.
4. Visually inspect the glassware to see if its clean. If so, youre done. If not, a little more
work is needed.
5. If the glass isnt clean, get it soapy again; skirt acetone onto the stain and scrub again.
Use a brush. Wet, soapy acetone is a pretty good general solvent for slime.
6. Continue steps 3-5 until glass is clean.

23

Experiment 1: Melting Points

Reference: Williamson, K.L. Macroscale and Microscale Organic Experiments. 1989.
C. Heath and Co. Lexington, MA.

COOH

napthalene

cinnamic acid

Introduction: The melting point and boiling points of a pure solid organic compound are
characteristic physical properties, along with molecular weight, refractive index and density.
Both melting point and boiling point are affected by the forces that attract one molecule to
another: ionic attraction, van der Waals forces, dipole-dipole interactions and hydrogen bonding.
A pure solid will melt reproductively over a narrow range of temperatures, less than 1 0C. Using
less than 1 mg of material, a melting point can be determined. The melting point apparatus is
very simple, consisting of a thermometer, a capillary tube to hold the sample and a heating bath.
Melting points are determined for three reasons: 1) if the compound is known, the melting points
will help characterize the sample in hand 2) if the compound is new then the recording of the
melting point is done to allow for further characterization by others 3) The range of the melting
point is indicative of the purity of the compound; an impure compound will melt over a wide
range of temperatures.
Boiling point is used to characterize a new organic liquid and knowledge of the boiling point
helps to compare one organic liquid with another. Comparison of boiling points with melting
points is instructive but process for determining boiling points is more complex. Boiling point
determination requires more material and is affected less by impurities, it is not as good an
indicator of purity as melting point.
Procedure:
Part I: Calibration of the thermometer: Determination of the melting point of standard
substances over the range of interest gives the difference between the values found and those
expected. This difference constitutes the correction that must be applied to future temperature
readings.
IMPORTANT! The liquid in the thermometers is very thin and can collect in the top part of the
thermometer, resulting in inaccurate readings. Always store your thermometer with the bulb end
lowermost. Also, check your thermometer before use to make sure that the thread of liquid is continuous
and that the top is free of liquid.
Thermometer Calibration (Your instructor may tell you to skip this part; check your syllabus.)
The thermometers used in this course are fairly accurate (1 C) and usually do not need to be calibrated,
so your instructor may have you skip this step. If you are instructed to calibrate your thermometer, follow
the instructions given below.
Why carry out a thermometer calibration at all?

24

When measuring temperatures, the upper part of the thermometer is cooler than the bulb, and the liquid in
the upper part of the thermometer contracts slightly. The resulting reading can be 1-3 degrees below the
temperature of the bulb. Many thermometers are accurate enough to need no correction, but a calibration
curve corrects for less-accurate thermometers.
To prepare a calibration curve, record the identities and true melting ranges of three or four compounds
provided by the instructor. Obtain reliable melting ranges for these solids and record them in your
notebook. Determine the average values for both the observed and the true melting ranges for each
compound..
To prepare your calibration curve, label the X-axis as Observed Temperature and the Y-axis as the
Corrected or True Temperature. Start numbering your graph at 30 or 40, using 1 per division on the
paper. Next, plot the melting points of your known compounds on the graph. Use small dots for the data
points. Draw a straight line which best fits these points (use pencil first). Your line will probably not pass
through the origin. Show your calibration curve to the instructor for checking. Save your approved
calibration curve for use throughout the semester, and label your thermometer with your name and locker
number. If you break or lose your thermometer, you will need to prepare a new calibration curve for your
new thermometer.
Converting measured temperatures to corrected values is simple: The corrected temperature (Y-axis) is
where the measured temperature (X-axis) intersects with the calibration line. You need to do this with
both the lower and upper values of a temperature range. Note: Do not use averages of temperatures in Part
B or any future experiments; average temperatures are only used when constructing a calibration curve!

Take a 10 mg sample of trans-cinnamic acid and prepare a melting point tube by

pushing a melting point capillary into the powder and force the powder down in the
capillary by tapping the capillary or by dropping it through a long glass tube held
vertically and resting on a hard surface. The column of solid should be no more than 2-3
mm in height and should be tightly packed.
Take a melting point range of this compound using the Mel-Temp apparatus in the lab.
The temperature can be raised rapidly until the temperature is about 20 0C below this
point. Then slow down heating considerably so that rate of increase is no more than 1 0C
per minute while the sample is melting. Compare the melting point range with the
published melting point of t-cinnamic acid. Record the difference between published MP
and the MP you obtained.
Repeat the same procedure using urea. Record the differences.
If the differences recorded for t-cinnamic acid and urea agree within 1 0C, use this
correction for every melting point you perform in this laboratory, provided that the same
thermometer is always used. Write this correction in your lab report.
Part II: Melting Points of urea and cinnamic acid mixtures.
Make mixtures of urea and cinnamic acid in the approximate proportions 1:4, 1:1, 4:1 by
putting side by side the correct number of equal-sized small piles of the two and then
mixing them.
Grind the mixtures thoroughly for at least a minute with a mortar and pestle.
Not the ranges of the three mixtures and use the temperatures of complete liquefaction
to construct a rough diagram of mp versus composition.

25

Part III: Determination of Unknowns.

Record this information in your notebook:

Unknown Number: __________
Appearance of Unknown: ___________________
m.p. of Unknown (uncorrected):__________C
(If applicable): Corrected m.p. value: __________C
Determine the melting point of the unknown given by your instructor and on the basis
of that melting point, identify that substance.
Run two capillaries of the unknown. Run a very fast determination on the first to
obtain approximate melting point and then use the second to do a slow, careful
determination.
Record your results and identify the compound you tested in your lab report.

Table 1.1 Melting Points of Unknowns

Compound
Benzophenone
Naphthalene
Benzoic Acid
Urea
Salicylic Acid

Melting Point (0C)

49-51
80-82
121.5-122
132.5-133
158.5-159

Clean Up: Place all leftover solids in the solid waste bottles provided. Capillary tubes can be
placed in the broken glass box in the laboratory.
Suggested topics for Discussion
What is the identity of your unknown? What is your rationale for your identification and how
confident are you in your identification of your unknown? What problems (if any) did you have
during the experiment? Feel free to include additional topics as appropriate.
Follow-up Questions (It is usually helpful to consult your lecture text when answering these.)
1. A student prepared a mixture of two compounds. The mixture showed a narrow melting
range, but the value is considerably lower than the melting range of either component in the
mixture. Explain.
2. A student attempts to obtain a melting range, but he filled the capillary with five times as
much sample as necessary. How might this affect the observed melting point? Explain.
3. A student was trying to obtain the melting point of borneol. While heating the sample, he
stepped away from the apparatus for a few minutes. He saw that the capillary tube was
empty, even though the temperature was well below the listed boiling point of borneol. What
probably happened? What should the student do to obtain the melting range of his solid?

26

Expt 2: Column Chromatography

The leaves of plants contain a number of colored pigments generally falling into two categories,
chlorophylls and carotenoids. Chlorophylls a and b are the pigments that make plants look
green. These highly conjugated compounds capture the (nongreen) light energy used in
photosynthesis.

Carotenoids are part of a larger collection of plant derived compounds called terpenes. These
naturally occurring compounds contain 10, 15, 20, 25, 30 and 40 carbon atoms which suggest
that there is a compound with five carbon atoms that serves as their building block. Their
structures are consistent with the assumption that they were made by joining together isoprene
units, usually in a "head to tail" fashion. Isoprene is the common name for 2-methyl-1,3butadiene. The branched end is the "head" and the unbranched is the "tail". That isoprene units
are linked in a head to tail fashion to form terpenes is known as the isoprene rule. Carotenoids
are tetraterpenes (eight isoprene units). Lycopene, the compound responsible for the red
coloring of tomatoes and watermelon, and -carotene, the compound that causes carrots and
apricots to be orange, are examples of carotenoids.
-Carotene is also the coloring agent used in margarine. When ingested -Carotene is cleaved
to form two molecules of vitamin A and is the major dietary source of the vitamin. Vitamin A,
also called retinol, plays an important role in vision.
Spinach leaves contain chlorophyll a and b and -carotene as major pigments as well as
smaller amounts of other pigments such as xanthophylls which are oxidized versions of
carotenes and pheophytins which look like chlorophyll except that the magnesium ion Mg 2+ has
been replaced by two hydrogen ions 2H+. In this experiment we will isolate and separate the
spinach pigments using differences in polarity to effect the separation. Since the different
components are colored differently, we can follow this separation visually. The structures of the
major components are given below.
Notice that since -carotene is a hydrocarbon it is very nonpolar. Both chlorophylls contain C-O
and C-N bonds which are polar and also contain magnesium bonded to nitrogen which is such a
polar bond it is almost ionic. Both chlorophylls are much more polar than -carotene.
If you look carefully you can see that the two chlorophylls differ only in one spot. Chlorophyll a
has a methyl group (-CH3) in a position where chlorophyll b has an aldehyde (-CHO). This
makes chlorophyll b slightly more polar than chlorophyll a. After we isolate the pigment mixture
from the leaves in a hexane solution we will use the difference in polarity to separate the various
pigments using column chromatography. We will analyze the original extract and the pigment
fractions using thin layer chromatography, which also separates based on polarity.

27

Chlorophyll a
Blue-green, polar
C55H72MgN4O5
M.W. 893.5026

Chorophyll b
Green, polar
C55H70MgN4O6
M.W. 907.4862

-carotene
Yellow, nonpolar
C40H56
M.W. 536.8824
CH3
CH3

CH3

CH3

CH3

CH3
R

CH3

CH3

CH3

CH3

-carotene (R H)
xanthophyll (R OH)
Objective(s): To separate the color pigments in spinach by column chromatography.

28

Technique(s) used: Isolation of a natural product; extraction; preparation and use of column
chromatography.
Zubrick 7th Ed. Column Chromatography and TLC page 221 and page 239
Microscale column chromatography Zubrick 7 th page 243

CAUTIONS: Avoid inhaling the solvent fumes; keep the stoppers in the test tubes.

Procedure:
At the beginning of the lab set up a hot water bath on a hot plate and watch the water
level so it doesnt get too low in the beaker (hot water bath).

Extraction of the pigments:

Weigh out 5 - 6 grams of spinach.
Remove as many of the stems as possible, then put the leaves in a mortar with a scoop of
sand and 5 mL of methanol.
Grind the mixture with a pestle for 10 minutes. Pour the ground mixture into a large test
tube and add about 10 mL of hexane. With a spatula or stirring rod, mix the hexane with the
ground spinach.
Decant the hexane into another test tube. Be sure to press the spinach to get out as much
of the hexane as possible. To the hexane in the second tube, add about 10 mL of DI water,
stopper the tube and shake, but not too hard. Remove the lower aqueous layer with a pipet
and repeat the extraction. This will remove most of the methanol from the hexane.
Dry the hexane by doing the following: set up a vacuum filtration with a Buchner funnel and
filter paper. Place a scoop of drying agent, the anhydrous sodium sulfate, on the filter paper.
Turn on the water to the aspirator to do a vacuum filtration. Carefully pour the green hexane
solution into the Buchner funnel over the drying agent and filter it. Alternate procedure:
Put a scoop of anhydrous sodium sulfate drying agent in the flask. Swirl and let sit 5
minutes. If the drying agent is still clumping, add more until it moves like sand in
bottle. Using a small funnel, put a small piece of a cotton ball loosely over the hole to
the stem. Gravity filter the solution through the cotton ball to separate the extract
from the drying agent. Save the green hexane spinach extract solution in a clean test
tube.
Add a boiling chip to the test tube and evaporate the solution to about 0.5-mL in a hot water
bath on a hot plate. This is the green hexane spinach extract that you will use with your
column.

29

Preparation of the column:

Figure 1. Pasteur pipet wet-column chromatography

Set up a microscale column as shown in Figure 1.

Obtain a chromatography tube such as a Pasteur pipet.
Shorten the tip of the Pasteur pipet; if it is not already shorten.
Gently press a small glass wool plug into the tip of the tube - being careful not to pack it too
tight.
Add about 50 mg of sand on top of the glass wool.
Weigh about 500 mg of alumina and break up any clumps with the stirring rod (if necessary,
use a mortar and pestle to make a fine powder of alumina). Slowly add the alumina to the
column. Every so often, thump the column with your finger to aid in settling the alumina.
After all the alumina has been added, place about 50 mg of sand on top of the alumina.
The chromatography separation:

Clamp the column on a ring stand.

In four large test tubes add the following elution solvents:

1st test tube 10 mL of hexane
2nd test tube 7 mL hexane + 3 mL toluene (7 / 3 hexane/toluene)
3rd test tube 3 mL toluene + 7 mL ethyl acetate (3/7 toluene/ethyl acetate)
4th test tube 10 mL of methanol.

Take two pipets; one for the green hexane spinach extract solution and one for solvents.

Prepare to run the column. The microscale column (Pasteur pipets) doesnt have
a stopcock. Once it starts running, it goes until it runs out. You cant let the column dry
out. Have all your elution solvents and the green hexane spinach extract solution ready and be
prepared to run the column once you start it.

Now slowly add some hexane into the column and let it wet the entire column.

From this point on, it is extremely important that the liquid level never be allowed
to get below the top of the sand.

30

DO NOT EVER LET THE COLUMN RUN DRY! NEVER LET THE SOLVENT LEVEL
FALL BELOW THE LEVEL OF THE ALUMINA!

Carefully pipet the green hexane spinach extract (from the extraction of the
pigments section above) to the top of your column. Dont fill the column with liquid; drip
the solution onto the sand. Leave a small amount for the TLC experiment.
When the green hexane spinach extract is all on the column, just below the sand,
add a small amount of hexane, the first elution solvent.
Watch your column! Do not ever let the column run dry.
Collect the drops off the end of the column in a small beaker.
As long as the first color band is seen moving down the column, continue with hexane.
As the color band reaches the glass wool plug, place a small test tube in place of the
beaker to catch the colored solution in the smallest possible volume. Note in your lab
book, the color, solvent, and approximate volume.
When no color bands are seen moving with your present elution solvent, change to the
next more polar elution solvent.
Continue the process until all the solvents have been used.
Collect each color band in small test tubes as a series of fractions and label all test
tubes.
Any color left at the top of the column cannot be removed.

Store the reserved extract and fractions collected off the column in OPEN test tubes to
be used next week in the TLC experiment.
Notebook Report:
Keep a detailed record of all your observations during the extraction of the pigment and the
chromatographic separation. In tabular format record the number of the fractions being
collected, the time over which the fractions were collected, the concentration of the solvent (10
mL hexane; 7/3 hexane to toluene..) on the column, the appearance of colors on the column,
and the color of the solution in the fraction being collected. Based upon color and polarity,
identify each fraction either as -carotene, chlorophyll a, chlorophyll b, or xanthophyll.

Number
of
Fraction
1.
2.
3.
4.
5.
6.

Collection Time
of Fractions

Concentration of
Elution Solvent

Band Color on
the Column

Color of Fractions in
Test Tube

Under optimum separation, the fractions collected show the following pigments in spinach:
-Carotenes (yellow-orange) nonpolar
Pheophytin a (gray, may be nearly as intense as chlorophyll b)
Pheophytin b ( gray, may not be visible)
Chlorophyll a (blue-green, more intense than chlorophyll b)
Chlorophyll b ( green) more polar than chlorophyll a
Xanthophylls (yellow)

31

Experiment 3: Thin-Layer Chromatography

A characterization method and criterion for purity
Thin layer chromatography (TLC) uses glass, aluminum or plastic plates covered with a layer of
adsorbent. Common adsorbents are silica gel, alumina and cellulose. The sample is applied as
a dilute solution to a position near the bottom of the plate. The plate is stood on end in a
shallow pool of solvent (called the eluant), which will run up the plate.
As the eluant passes over the applied sample, the molecules, which are adsorbed on (stuck to)
the adsorbent, will tend to dissolve in the eluant, move ahead in solution, and re-adsorb further
up the plate. The sample will constantly re-dissolve and re-adsorb while the plate is running
(being developed). The more strongly the sample adsorbs on the adsorbent, the less it will tend
to dissolve, and the more slowly it will move up the plate. Different compounds will, in general,
tend to dissolve, and the more slowly it will move up the plate. Different compounds will, in
general, adsorb with different strengths, so that different compounds usually move different
distances up a give plate.
Spotting and Developing TLC Plates: To apply a sample to a TLC plate, it is dissolved in a
suitable solvent (try CH2Cl2 first) at a concentration of 100 mg per mL of solvent. The solution is
applied to the plate with a double-open ended capillary tube, ~3/8 from the bottom of the plate
so that a spot of ~1/8 diameter is formed (Figure 1).

Figure 1. TLC plate with starting material product and a mixture of both spotted on the plate.
The appropriate solvent (eluant) for development is poured into a TLC chamber (either a special
chamber or a beaker). The solvent should be 1/8 deep, so that the spot will wash the sample
off the chamber (otherwise, solvent will wash the sample off the plate). The plates are then
stood in the chamber (or beaker) and the chamber is covered (with a watch glass). Once the
eluant reaches the top of the plate, the plate is removed from the chamber. Upon removal,
mark, in pencil, where the solvent stopped on the plate. Lay the plate flat to dry.
Silica gel is very polar. Therefore, polar compounds tend to stay near the origin, while non-polar
compounds tend to move much faster up the plate. A suitable eluant is chosen by trial and
error, and often differs from the solvent used to spot the sample. If a compound runs too fast

32

(too far up the plate) your next plate should be run with a less polar eluant. If the compound
runs too slowly, your next plate should be run with a more polar eluant. See Table 1.

Table I. Comparative Polarities of Solvents

LEAST POLAR
hexane *
cyclcohexane
carbon disulfide
carbon tetrachloride
trichloroethene
toluene
benzene
dichloromethane
chloroform*
diethyl ether
tetrahydrofuran
ethyl actetate*
pyridine
acetone
1-propanol
ethanol
methanol*
water
MOST POLAR
*indicates the big four solvents which can be used in mixtures to separate most
organic compounds.
Visualizing Spots. A TLC plate, which has just been developed, looks (unless the sample is
colored) just like an undeveloped plate: a plain white surface. To locate the spots of
compounds, they must be visualized. Visualization can be done either destructively or nondestructively.
a) Non-destructive Visualization. The most common non-destructive technique requires
that the TLC plate be prepared with an adsorbent, which contains a fluorescent indicator
(a compound that fluoresces when you shine UV light on it. The TLC plates that we will
be using have this indicator, which fluoresces green under 254-nm light. If such a plate
is spotted and developed, UV light lets you see the spot(s). The beauty of this kind of
visualization is that you can wash your sample back off the plate and recover it
unchanged; for the closely related techniques thick-layer chromatography, this
advantage is very important.
b) Destructive Visualization. The usual techniques for destructive visualization involve
spraying the plate with a reagent which immediately reacts with adsorbed sample to give
a product which either I) differs in color from the background color of the sprayed plates
or II) has low volatility, so that vigorous heating will lead to charring of the sample, giving
black spots on a white background. A wide variety of general-use and special-purpose
sprays exist. Spray visualization is destructive because the sample is irreversibly
converted into another compound. Every method is unable to detect some samples (for
example, 254-nm UV visualization cant detect a sample that doesnt absorb 254-nm

33

light, such as most chloralkanes). To avoid surprises, use two methods to visualize
each plate.
Spots: Shape and Tailing. The spots on the developed plate should be round or slightly
elliptical. Sometimes the spot has a long tail going back toward the origin (Figure 2); such a
result is useless. The most common cause of tailing is that the plate is overloaded; dilute the
spotting solution and try again. Occasionally, a sample wont stop tailing no matter how dilute it
is; this is uncommon but not unknown. In such a case try a different eluant of similar polarity.
Characterization by TLC. To use TLC in characterizing a compound, one determines the R f
value, defined as (Figure 3).

Rf = distance traveled by the compound (cm)

distance traveled by the eluant (cm)
A compounds Rf value under a given set of conditions should be reproducible to one significant
figure, if distances are measured carefully. However, an R f value depends on several
parameters, and hence lacks meaning unless theyre reported: (i) adsorbent; (ii) thickness of the
adsorbent layer; (iii) eluant. It is also good practice to write down the visualization method(s).
The final picture in your notebook should look like that in Figure 4.
An Rf of zero doesnt tell one anything; it states only that a sample isnt moved by a given
eluant, while a nonzero Rf says both the sample moves and how such it moves. Likewise, R f
values greater than about 0.75 mean only that the compound travels with the solvent front.
Very low or very high Rf values should be used only for comparing two compounds; when using
TLC to help characterize a single compound, try to find at least one eluant in which 0 < R f <
0.75.
Double spotting and identity proofs. TLC evidence can be very useful when you think you
know a compounds identity. Suppose you suspect a sample to be benzaldehyde. Prepare a
dilute solution and a dilute solution of authentic benzaldehyde. On a single plate, spot the
solution of your sample on one side, both solutions in the middle (on top of each other) and the
authentic benzaldehyde solution on the other side. Develop and visualize. If the middle spot is

34

still clean, you can have good evidence, but not proof, that the two compounds are the same
(Figure 5). It the middle spot splits in two, you have proved that the compounds are different.
A Measure of Impurity. If your sample is a mixture, and if the components have different R f
values under your TLC conditions, you will see several spots on the plate after visualization.
TLC alone never tells you how many components a mixture has---it tells you the MINIMUM
number of compounds, which must be present. It is a real possibility that your visualization
method may not show all the spots present, or that one spot may represent a mixture of
compounds, which have the same Rf value (co-elution).
TLC Procedure (http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html)
1. Prepare the developing container.
The developing container for TLC can be a specially designed chamber, a jar with a lid, or a
beaker with a watch glass on the top:

In the teaching labs, we use a beaker with a watch glass on top.

Pour solvent into the

beaker to a depth of
just less than 0.5 cm.

35

To aid in the saturation of the

TLC chamber with solvent vapors,
line part of the inside of the
beaker with filter paper.

Cover the beaker with a watch glass, swirl

it gently, and allow it to stand while you
prepare your TLC plate.

2. Prepare the TLC plate.

TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets.
Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more
samples you plan to run on a plate, the wider it needs to be.

36

Shown in the photo to the left is a box of

TLC plates, a large un-cut TLC sheet,
and a small TLC plate which has been cut
to a convenient size.

Plates will usually be cut and ready

for you when you come to lab.
Handle the plates carefully so that
you do not disturb the coating of
adsorbent or get them dirty.

Measure 0.5 cm from the bottom of the

plate. Take care not to press so hard
with the pencil that you disturb the
adsorbent.

Using a pencil, draw a line across the plate

at the 0.5 cm mark. This is the origin: the
line on which you will "spot" the plate.

It's kind of hard to see the pencil line

in the above photos, so here is a
close-up of how the plate looks after
the line has been drawn.

37

Under the line, mark lightly the name of the

samples you will spot on the plate, or mark
numbers for time points. Leave enough space
between the samples so that they do not run
together, about 4 samples on a 5 cm wide plate is
advised.
Use a pencil and do not press down so hard that
you disturb the surface of the plate. A close-up of a
plate labeled "1 2 3" is shown to the right.
3. Spot the TLC plate
The sample to be analyzed is added to the plate in a process called "spotting". If the sample is
not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes,
ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in
100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a
smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of
thumb" above is usually a good estimate, however, sometimes only a process trial and error (as
in, do it over) will result in well-sized, easy to read spots.

add a few drops of

solvent . . .

. . . swirl until
dissolved

38

The solution is applied to the TLC plate with a capillary tube or a microcap, as seen in the
photos.

Take a tube and dip it into the solution of the sample to be spotted. Then, touch the end of the
tube gently to the adsorbent on the origin in the place which you have marked for the sample.
Let all of the contents of the tube run onto the plate. Be careful not to disturb the coating of
adsorbent.

dip the microcap into

solution - the arrow points to
the microcap, it is tiny and
hard to see

make sure it is filled hold it up to the light if

necessary

touch the filled microcap

to TLC plate to spot it make sure you watch to
see that all the liquid has
drained from the microcap

do this
rinse
process
3 times!

rinse the microcap with

clean solvent by first
filling it . . .

. . . and then draining it by

touching it to a paper
towel

39

If the microcapillary tube breaks or clogs, you may obtain a new one.

here's the TLC plate,

spotted and ready to be
developed
4. Develop the plate.
Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass,
and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter
below the top of the plate (see photos below).

place the TLC plate in the developing container make sure the solvent is not too deep

The solvent will rise up the TLC

plate by capillary action. In this
photo, it is not quite halfway up the
plate.

40

In this photo, it is about

3/4 of the way up the plate.

The solvent front is about

half a cm below the top of the
plate - it is now ready to be
removed.

quickly mark a line across

the plate at the solvent front
with a pencil

Remove the plate from the

beaker.

Allow the solvent to

evaporate completely from
the plate. If the spots are
colored, simply mark them
with a pencil.

5. Visualize the spots

If your samples are colored, mark them before they fade by circling them lightly with a pencil
(see photo above).
Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over
the plate and mark any spots which you see lightly with a pencil.

Beware! UV light is damaging both to your eyes and to your skin! Make
sure you are wearing your goggles and do not look directly into the lamp.
Protect your skin by wearing gloves.
If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run
together. If this happens, you will have to start over with a more dilute sample to spot and run on
a TLC plate.

41

this is a UV lamp

here are two proper sized spots,

viewed under a UV lamp

(you would circle these while

viewing them)
The plate to the left shows three compounds
run at three different concentrations. The
middle and right plate show reasonable
spots; the left plate is run too concentrated
and the spots are running together, making
it difficult to get a good and accurate Rf
reading.

Here's what overloaded plates look like compared to

well-spotted plates. The plate on the left has a large
yellow smear; this smear contains the same two
compounds which are nicely resolved on the plate next
to it. The plate to the far right is a UV visualization of
the same overloaded plate.
NOTE: Your instructor will indicate which parts of the experiment are to be
performed during the laboratory period.

PART ONE: Identification of Components in Spinach by

Thin-layer Chromatography (Week 1-follow Instructors directions)
PROCEDURE:
Reference: Palleros, D. A. Experimental Organic Chemistry. 2001, J. Wiley and Sons, New
York, pgs. 193-195.

42

Vegetable Extract
Weigh out 10 g of spinach leaves and chop them up using scissors. If they were previously
frozen, drain as much water out of the leaves as possible before weighing.
Place the spinach in a large mortar, add 12 mL of methanol, and crush the leaves with a pestle
for about 3 minutes. With the aid of the pestle or spatula, squeeze the spinach against the side
wall of the mortar to remove as much methanol as possible. Transfer the liquid to a 250-mL
Erlenmeyer flask, labeled methanol-water and set aside. The contents of the flask will
eventually be discarded.
Extract the remains of the leaves with a mixture of 15 mL hexanes and 5 mL methanol, crushing
the tissue with the pestle for about 5 minutes. The extract should be deep green. Leaving
behind as much solid as possible, transfer the liquid (a mixture of hexanes and methanol) to a
100-mL beaker. Filter it using a glass funnel (4-6 cm diameter) with a cotton plug. Collect the
filtrate directly into a 125-mL separatory funnel supported on a ring stand (make sure the
stopcock is closed).
Add 5 mL of water to the separatory funnel, shake, vent and allow the layers to separate.
Which layer is the aqueous layer? Collect the aqueous layer in the Erlenmeyer flask labeled
methanol-water. Extract the remaining hexane layer with 5 mL of water in the separatory
funnel. Collect the aqueous layer along with any emulsion present in the methanol-water
flask.
Transfer the organic layer to a clean and dry 50-mL Erlenmeyer flask; add a small spatulaful of
granular sodium sulfate (Na 2SO4) to dry the organic layer. Cap the flask with a stopper and
swirl occasionally. After about 5 minutes, filter the suspension by using a clean and dry microfunnel (about 2.5 cm in diameter) and a cotton plug. Collect the filtrate in a dry 50-mL roundbottom flask. Using the rota-vap, evaporate the solvent until the volume is approximately 2-3
mL. The color of the final extract should be deep green. With the aid of a Pasteur pipet,
transfer the liquid to a labeled test tube.
TLC Analysis
Clean and thoroughly dry five 150-mL beakers and five watch glass covers. Label them with the
names of the solvents to be used: 1) hexanes 2) toluene 3) toluene-acetone (9:1) 4) tolueneacetone (7:3) 5) acetone.
Determining the Optimum Number of Applications. Take the plate labeled tolueneacetone (9:1). This plate will be used to determine the optimum number of applications needed
to visualize the separation of pigments. On this plate, the sample will be applied on three
different spots, each with a different number of applications (1, 4 and 7). Immerse a double
open-ended capillary tube in the extract; the liquid will rise through capillary action. Apply the
liquid to the plate by touching the plate with the tip of the capillary tube and applying slight
pressure. If necessary, gently wiggle the capillary to make the liquid flow. The spot should be
about 0.5 cm from the side edge of the plate. Raise the capillary tube to stop the flow of liquid
when the diameter of the spot is about 3 mm. On the same plate, apply a second and a third
spot at a distance of about 0.5 cm from each other. The number of applications should be 4
and for the third spot, 7. Allow the solvent to evaporate between applications. Failure to do so
will result in very large spots.

43

With the aid of tweezers, place the TLC plate in the chamber with toluene-acetone (9:1) and
allow it to run until the solvent has risen to a height of about 6 cm (0.5-1 cm from the top of the
plate). Remove the plate from the chamber (use tweezers); immediately mark the solvent front
with a pencil and allow the solvent to evaporate in a well-ventilated place. Once the solvent has
evaporated, circle the spots with pencil. Draw the results in your lab notebook, paying special
attention to the colors of the spots (yellow, pale green, deep green, gray or orange). Notice any
spot at the origin. This analysis should be done without delay because the colors of the spots
fade very quickly (within hours). Determine how many applications gave you the best results, or
in other words, clear and well-delineated spots without streaking. If the spots are too faint to be
seen, the extract should be concentrated by further evaporation of the solvent. If the spots run
with tails, too much sample has been applied. Either reduce the number of applications or dilute
your extract with hexanes.
Analyzing your Extract. Spot the extract on the remaining plates, doing as many applications
as you have already determined to be optimum. Develop all the plates simultaneously.
Calculate the Rf values for each pigment in each solvent. Decide which solvent gives the best
separation. Be sure to write down in your notebook all the data that are necessary to reproduce
your Rf values (stationary phase, solvent, number of applications). Draw the plates in your
notebook and indicate colors. Pay attention to the differences between plates.
Under optimum separation conditions, these pigments usually show in the following order (in
order of increasing Rf): chlorophylls (green), followed by xanthophylls(yellow), pheophytins
(green-gray), and then carotenes (yellow-orange) closer to the solvent front.
Clean Up
Let the spinach leaves dry in the hood and dispose of them in the trash.
Dispose of the contents of methanol-water flask and other solvents in the container labeled
non-halogenated solvents.
Dispose of the sodium sulfate and cotton plug in the solid waste bottle.
In the lab report answer the following questions as part of the conclusion:
1. On the plate, where are the carotenes? Where are the pheophytins? Where are the
xanthophylls? Where are the chlorophylls?
2. Which is the best solvent to separate all the pigments?
3. Is acetone a good solvent to separate the pigments?
4. Which solvent would you choose if you have to separate the carotenes from the other
pigments?
5. Calculate the Rf values. Show your work. Using a table format, report R f values for the
identified pigments.
6. Is toluene a good solvent to separate the different chlorophyll types? Which solvent would
you choose to carry out such a separation?

PART TWO: Identification of Spinach Fractions after Column

Chromatography (Week 2follow Instructors directions)
Using the optimal conditions determined in Part One, analyze the spinach extracts from
Experiment 2.
Sample Preparation: Dissolve the oils in a small amount of the solvent that each fraction was
extracted from the column.

44

Run each fraction and identify the major component. When writing the report, discuss whether
these fractions can be considered pure.

PART THREE: Identification of Components in Pain Relievers by

Thin-layer Chromatography
Procedure
You will probably use TLC plates consisting of
silica gel plus a fluorescent indicator fused to a
flexible plastic sheet, but you should check with
your instructor to be sure. Remember to handle the
plates only by the edges to avoid damaging the
coating or leaving fingerprints on the plate.
Using a pencil, carefully mark your plate with a
horizontal line at the bottom of the plate as shown
in the Figure. Do not scratch through the coating, or
you may have to start with another plate! Use small
marks to indicate starting places for each of the
known compounds plus one for your unknown .
Space out the marks so you can keep easily track
of the spots, but do not put marks too close to the
edges. Put your initials at the top of the plate.

Spotting a TLC Plate

10-15 mm

Asp. Caf. Sal. Acet. Dip h . Unk Z

Crush the pain relievers provided using pestle and motor into fine powder and dissolve in 1:1
mixture of methylene chloride and ethanol. Use the small capillaries provided to spot each
known and your unknown on its own mark. When you touch the capillary to the plate, the wet
spot should be fairly small; about 2-3 mm across is ideal. Avoid making very large spots
because these give results that are harder to interpret. When you are ready, spot each known
and your unknown. Let the spots to air-dry for about a minute (do not blow on them).
IMPORTANT: Before developing your plate, check your plate with the UV lamp to make sure
that all of the spots are clearly visible. Re-spot any samples that are very faint or cannot be
seen under the UV light. The spots should not be larger than about 0.5 cm because spots that
are large or contain too much sample usually tend to develop as smears or comets rather than
round or oval spots, making it difficult to calculate a reliable R f value. The spots should not be
closer than about 0.5 cm apart, or they may merge as the plate develops. If stray spots are
visible, you will have to spot a fresh plate.
Carefully place the TLC plate in the developing chamber containing 0.5% acetic acid in ethyl
acetate as developing solvent. The instructor may do this for you. The sides of your plate
should not touch the wall or other plates. Take care not to disturb other students plates.
Replace the top of the developing tank. The plates take about 5-15 minutes to develop. When
the solvent front has risen to within 1 cm of the top of the plate, remove the plate and
immediately mark the solvent front with a pencil. Remember to replace the cover of the tank. Let
the plate dry for a minute, then shine the UV lamp on your plate. Circle any visible spots with a
pencil. Note the colors of the spots. You should make a quick sketch of the plate in your

45

notebook. If so instructed, place the plate in an iodine chamber and wait 5 minutes for spots to
appear, then circle them. Estimate where the center of each spot is and mark it in pencil.
Measure and record the distances that each spot and the solvent moved from the starting line.
Calculate your Rf values and use them to identify the components in your unknown. Show your
calculations for Rf values, rounding your answers to the appropriate numbers of significant
figures. Note: Some compounds have similar Rf values, and if these are in the your unknown,
you may see only one spot or a broadened spot.
Table 1. Possible compounds in the samples (Your instructor may use other compounds.)
Component
Acetaminophen
Aspirin
Salicylamide
Caffeine
Phenacetin

UV Lighta
++
++b
Blue glow
bggww;lkadsj;l
++
kfsadj;lk9glowg
++
lowfluorescence
++

I2 Vapor
++
+
++
+
+
+

++ easily detected

+ detectable

Visible as dark spot on the glowing

background.
Aspirin sometimes shows a second faint spot
caused
by salicylic acid (from decomposition of
aspirin).
b

Prepare a table in your notebook for recording your data. The headings of your table may look
like this:
Substance spotted on
plate

Color under
UV Light

Color
w/ iodine

Distance solvent
moved

Distance
spot moved

Rf

Possible topics for Discussion:

Which compounds are present in your unknown? How certain are you of your identification? If
other possibilities are likely, describe them and address how likely the possibilities are. Discuss
any problems you had in preparing or developing your TLC plate and/or identifying your
unknowns. What could you doif anythingto avoid these problems if you used TLC later in
the semester?
Follow-up Questions
1. A student calculated the Rf value for a spot to be 1.05. Why is
value wrong?

this

2. Thin-layer chromatography can be used to follow the progress

reaction. To do this, a TLC plate is spotted with the starting
material(s) and the product(s), as well as with the reaction
As the reaction proceeds, spots in the reaction mixture will
in intensitybut not Rfas starting material is consumed and
forms.

of

mixture.
change
product

A B C
46

For example, a student is converting benzhydrol to benzophenone. As the reaction

proceeds, he prepares TLC plates that are spotted with the following samples: an authentic
sample of benzhydrol (Dot A in the diagram shown), an authentic sample of benzophenone
(Dot B), and the reaction mixture (Dot C). When ready for development, the plates look like
the one shown at the right. If the Rf for benzhydrol is 0.20 and the Rf for benzophenone is
0.60. Sketch what the developed TLC plate would look like if reaction mixture is from:
a. at the very start of the reaction
b. the reaction mixture when the reaction is roughly 30% complete
c. the reaction mixture when the reaction is roughly 80% complete
How would the student know when to stop the reaction?

47

Experiment 4: Separation of Acidic and Neutral Compounds

by Extraction
Background
Extraction is a useful technique for separating two compounds that differ in their solubility
properties. For example, one component of the mixture may be soluble in acid while the other
component is insoluble.
Consider a mixture of the solids: biphenyl (an organic hydrocarbon) and phenylacetic acid (an
organic carboxylic acid). Neither compound is water-soluble, and both compounds dissolve in
ether, so they cannot be separated by selectively dissolving one component in water or in ether.
However, carboxylic acids phenylacetic acid react with bases such as NaOH to form
water-soluble carboxylate saltsin this case sodium phenylacetate. Biphenyl does not react
with NaOH. This difference in reactivity allows the separation of these compounds.
Suppose one has an ether solution of phenylacetic acid and biphenyl in a separatory funnel. If
an aqueous solution of NaOH is added to the funnel, the phenylacetic acid will react with the
NaOH to form the salt. sodium phenylacetate, This salt is water-soluble, so it will dissolve in the
aqueous solution. The biphenyl (and any unreacted phenylacetic acid) are insoluble in water,
but will remain in the ether solution. Ether and water are largely insoluble in each other, and this
means that two layers will form in the funnel, with the less dense liquid (ether) on top. The
aqueous layer containing the sodium phenylacetate can be drained. Repeating these steps one
or more times converts nearly all of the phenylacetic acid into the water-soluble salt, leaving
nearly pure biphenyl in the ether layer. The biphenyl can be recovered by simply evaporating
the ether. The phenylacetic acid can be recovered by adding enough of a strong acid (such as
HCl) to the NaOH solution. The HCl converts the phenylacetate salt back into phenylacetic acid,
which is insoluble in water. The solid can be isolated by vacuum filtration.
The name for the overall process is reactive extraction because a chemical reaction (acidbase in this case) is involved in separation of the components. NOTE: Several smaller portions
of NaOH solution were used because this removes more acid than using one large portion. It is
difficult to completely extract the phenylacetic acid, but the amount remaining in the organic
layer left after three extractions is negligible.
Introduction
In this experiment, you will separate and identify the components of a mixture containing a
neutral organic compound and an organic acid. Both components are soluble in organic
solvents, but are either insoluble or only slightly soluble in water.
Important: Be sure that you have several clean flasks and/or beakers handy. You must keep
track of which solutions are in which containers. Confusing one solution with another usually
leads to loss of your product. To help avoid confusion, label your containers or make a placemat
with labeled spots for the beakers and flasks containing the solutions you are working with.

48

Separatory Funnel Extraction Setup

Procedure
Select your mixture from one of the jars provided by the instructor. Weigh out about 4 g of solid,
recording the code number and the appearance of the solid. About half of the mixture is an
organic acid; the rest is a neutral organic compound. Neither compound is soluble in water, but
both will dissolve in ether or methylene chloride. (You will use methylene chloride because ether
is highly flammable.). Set up a ring stand with a separatory funnel. Obtain about 45 mL of
methylene chloride. Add 1-2 mL to the funnel to confirm that the stopcock fits properly, and if it
leaks, consult the instructor. Add the solid to the separatory funnel, followed by the remaining
methylene chloride. Swirl the funnel to dissolve the solid. Any remaining solid will dissolve
during the next step. (If the residue is undissolved acid, it will dissolve in the NaOH solution you
will be adding in the next step. If the solid is undissolved neutral compound, it will dissolve into
the organic layer after the organic acid is extracted into the aqueous layer.)
Obtain 45 mL of 3 M NaOH. (You have to prepare your own solution by diluting 6 M NaOH
solution.) Pour about 15-mL of the 3 M NaOH solution into the separatory funnel, replace the
stopper and repeatedly invert the funnel to mix the layers. Avoid shaking the vessel too
vigorously or an emulsion may form. Vent the funnel periodically to relieve built-up
pressure. Replace the funnel in the ring, remove the stopper, and allow the vessel to stand so
that the layers will separate. Drain off the lower (methylene chloride or organic) layer into a

49

container labeled Extracted Organic Layer. Empty the remaining (aqueous) layer into a
container labeled Extracted NaOH layer. You will probably see colored droplets of the
methylene chloride solution in the aqueous layer, but do not worry about it at this point, because
you will recover this material later in the experiment.
Close the stopcock and pour the methylene chloride layer back into the funnel. Repeat the
extraction process with a second 15-mL portion of NaOH solution. After the layers have
separated, drain off the organic layer into the appropriate container. Drain the aqueous layer
into the Extracted NaOH layer container. You should now have 30 mL of NaOH solution in this
flask. Repeat the extraction using the organic layer and the remaining NaOH solution. Again,
drain the lower layer into the CH2Cl2 container. The NaOH solution should be delivered into
the container with the other extracted NaOH solution. When you have completed the extraction
and separation, you should have one flask containing about 40 mL of methylene chloride and
one containing about 45 mL of NaOH solution.
At this point, the NaOH solution contains the organic acid (as its sodium salt), but the solution is
probably contaminated by some of the methylene chloride solution. The presence of colored
droplets on the bottom of the Extracted NaOH layer flask is due to the CH2Cl2 solution
containing some of the neutral compound. This solution is removed by a so-called back
extraction. Pour the aqueous layer into the separatory funnel and add about 5-10 mL of
methylene chloride. Gently invert the funnel a few times. Allow the layers to separate and then
carefully drain the methylene chloride layer into the vessel containing the other methylene
chloride solution. Your NaOH solution should now be free of color. If it is not, repeat the
extraction with a fresh 5-mL portion of methylene chloride. Set aside your purified NaOH
solution for later recovery of the organic acid.
Your organic layer will contain a little of the NaOH solution. Wash (extract) the organic layer with
20-mL of water to remove residual NaOH. The rinse water should be put in a labeled container.
Repeat the rinsing of the organic layer with 20 mL of fresh water. The organic layer should now
contain only the neutral compound plus a little water. Wash the organic layer with 15 mL of
saturated NaCl solution to remove most of the water. Transfer the organic layer into a clean,
dry, 125-mL Erlenmeyer flask, followed by a marble-sized portion of anhydrous magnesium
sulfate (MgSO4). Magnesium sulfate is a drying agent that absorbs the remaining water. Swirl
the flask for a few seconds and then watch as the MgSO 4 settles. If all of the MgSO4 is clumped
or pasty at the bottom of the container or if the solution is cloudy, then the solution still contains
water, and you need to add a little more drying agent. However, if a substantial portion of the
drying agent settles slowly to the bottom of the container (like a snow globe), and the solution
is not cloudy, then the traces of water have been removed. Cover or stopper the flask and set it
aside for 15 minutes, swirling it occasionally. If you are unsure whether to add additional drying
agent, consult your instructor.
While your organic solution is drying, you should turn your attention to your aqueous solution.
Calculate the volume of 6 M HCl needed to neutralize the NaOH solution, and obtain this
amount of acid. (Note: the concentrations of the stock acid and base solutions are not precise,
and you may need a bit more or less acid than you calculate.) Slowly add the acid to your NaOH
solution with stirring. As you add the acid, a precipitate will appear, but it will disappear as the
solution is stirred. The precipitate will remain visible longer as you approach the equivalence
point, and once the solution is acidic, the precipitate will no longer dissolve. Stir the solution
thoroughly and confirm that the solution is acidic to litmus. If the solution is still basic after
adding all of your acid, add 2-3 mL of additional HCl and repeat the litmus test. If no precipitate
is visible at this point, consult the instructor. Once the solution is definitely acidic, cool the

50

solution in an ice-water bath for about 15 minutes. Collect the solid by vacuum filtration. Wash
the solid with a small amount of ice-cold water, and allow air to be pulled through your sample
for a few minutes, then remove the filter cake from the funnel. Allow the crystals (and the filter
paper if some solid is sticking to it) to dry in an open beaker or dish in your locker until the next
lab period. You will obtain the mass and melting point of this solid then.
After the organic layer has time to dry, filter the organic solution into a round-bottom flask. You
may be provided with wide-neck flasks for this step. Rinse the filter paper and drying agent with
about 5 mL of methylene chloride. The drying agent may remain slightly colored, even after
rinsing. Use the rotary evaporator to remove the solvent. Your instructor will operate it or will
show you how to use it. When the solvent is removed, scrape out as much solid as you can.
You may need to bend the spatula to reach some parts of the flask. If there are not enough
flasks to go around, be sure to do this step in a timely fashion so others do not have to wait too
long. Rinse your flask with acetone and return it to your instructor for others to use.
Analysis of your products
After your samples are dry, determine their masses and melting ranges. Be sure to remove the
boiling stones from the solid you scraped from the flask before you calculate masses! Identify
your unknowns from the list provided by the instructor. Compare your starting and final masses,
and calculate the percent recovery of each component. Remember that your sample is
approximately a 1:1 mixture of the two components. Your particular sample could have a quite
different composition than your neighbor because the samples are not uniform, so you cannot
assume that each component made up 50% of the starting mass. Besides, some components
are easier to recover than others.
Remember to submit your samples in properly labeled vials. Each label should include your
name, the name of the compound, the mass and the melting range that you measured.
Suggested topics for Discussion
What is the identity of each component?
How pure is each component and how do you know?
Comment on the percent recovery of each component and suggest likely places in the
procedure where product was lost. (Remember that your sources of loss should be reasonable!)
Waste disposal and cleanup
The aqueous solutions including the NaOH and HCl solutions may be disposed of by flushing
them down the sink with water (watch splashing!). Any remaining methylene chloride should be
disposed of in the Halogenated Waste container provided in the hood. Any residue from the
organic solids can be removed from glassware using acetone.

COMMON ERRORS
1. Insufficient mixing of the NaOH and CH2Cl2 layers during extraction.
2. Emulsion formation by shaking the funnel too vigorously.
3. Leaving the used NaOH solution in the separatory funnel, and then adding new NaOH
solution into the separatory funnel.
4. Leaving the stopcock open, so solution added to the funnel spills onto the desktop.
5. Not performing a back-extraction, causing the aqueous product to be contaminated.
6. Mixing up the layers and adding drying agent to the aqueous solution.

51

7. Adding insufficient drying agent.

8. Using insufficient CH2Cl2 to rinse the drying agent out just before rotary evaporation.
9. Allowing water to get into the dried CH2Cl2 solution, making re-drying necessary.
10. Rotary evaporation with a flask that is too full, causing the solution to bump up into the
apparatus.
Follow-up Questions
1. Why is it important to cool the acidified aqueous layer before vacuum filtration?
2. If insufficient acid were added to the aqueous layer, what effect, if any, would this have on
the percentage of the acid compound isolated?
3. Instead of using a slight excess of HCl to precipitate the acidic compound, a student used a
five-fold excess of HCl solution. Why will this reduce the mass of acidic compound that is
isolated?
4. This experiment deals with the separation of a neutral compound from an acidic compound.
Could you use the same reagents and procedure to separate an organic acid and an
organic base? If not, how would you alter the procedure? (Hint: consider the acid/base
properties of the components and whether the components would react with the extraction
solvents.)
5.

Outline a brief (6-10 sentence) procedure for separating solid mixture of a neutral
compound and an organic amine (base).

52

Experiment 5: Distillation: Isolation of Anise Oil

Ref: Palleros, D.R. Experimental Organic Chemistry, 2001, John Wiley & Sons, New York, pgs.
113-142.
Introduction: Distillation is one of the oldest methods ever developed for the purification of
liquids. It has been used to concentrate dilute alcoholic solutions such as wine and beer and to
obtain perfumes from fruits and flowers. Distillation consists of heating a liquid to its boiling
point and separating the condensed vapors. There are different types of distillation used in the
organic laboratory: simple, fractional, vacuum and steam.
At any given temperature, a liquid is in equilibrium with its vapor. As the temperature increases,
the vapor pressure of the liquid also increases. When the vapor pressure equals the applied
pressure, the liquid boils. Like melting point, the boiling point is an important physical property
used to characterize organic materials. Pure compounds normally boil within a range of 1 oC.
Boiling points are not very affected by changes in external pressure that makes them less
reliable to assess purity and identify compounds. Boiling points should always be reported with
the external pressure at which they were determined.
The boiling point of a liquid depends on its polarity, its molecular mass and the overall size and
shape of the molecule. For a liquid to boil, the intermolecular interactions that keep the
molecules together should be disrupted. These interactions include hydrogen bonds and
dipole-dipole interactions. No covalent bonds are broken during boiling.
The distillation apparatus for simple distillation is shown in Fig 4.1 below. It consists of a round
bottom flask called a distillation flask, where the liquid to be distilled is placed; a distillation
head with an attached thermometer connects the flask to the condenser, and a distillate
take-off adaptor connects the condenser to the receiving flask. The condenser consists of a
glass tube with a jacket through which tap water circulates against gravity; Water enters at the
lower end and exits at the upper end; circulation of water in the opposite direction results in an
incomplete filling of the water jacket. When liquids with boiling points higher than 150 oC are
distilled, breakage of the water condenser may occur due to thermal shock caused by the
difference in temperature between the vapors and the cooling water. For such distillations, an
empty condenser without circulating water is called an air condenser, is recommended.

Before the distillation is started, a few pieces of a porous material called boiling chips are
added to the liquid. They produce smooth bubbling and prevent bumping of the liquid during
distillation. To avoid boil-over, the liquid should fill no more than to 2/3 of the total capacity of
the distillation flask. Depending on the boiling point to be reached, the system is heated with
the aid of a heating mantle, a sand bath or a water bath on a hot plate. Vapors ascend through
the distillation head and condense around the thermometer and the condenser, which prevents
vapors from escaping. The distillate is collected in the receiving flask. The distillation apparatus
should always have an opening to the atmosphere at the end where the distillate is collected.
NEVER HEAT A CLOSED SYSTEM OF AN EXPLOSION WILL OCCUR. Figure 4.1 gives the
distillation setup that will be used in this lab.

Figure 4.1 Simple distillation apparatus

53

Isolation of Anise Oil

Overview
Anise is one of the oldest flavorings ingredients of which we have on record. The anise plant
probably originated in the Middle East, from where, it expanded to the entire Mediterranean
region. The sweet-smelling oil is obtained by steam distillation of the dried ripe fruits (anise
seeds). The three main components of anise oil are trans-anethole, estragole and panisaldehyde. Anise seeds are used in French and Italian pastries. The oil with its distinctive
licorice flavor is employed in the confection of candies and beverages. In this experiment you
will isolate anise oil by steam distillation of the anise seeds and analyze and characterize the oil
by physical and chromatographic methods identify the main component in the oil as either
estragole, trans-anethole, and p-anisaldehyde by gas chromatography.

54

H3 C

H3 C
H
H

CH3
OMe

trans-anethole

C C CH2
H2 H

estragole

p-anisaldehyde

Procedure:
Crush 25 g of anise seeds in a mortar and pestle.
Transfer the crushed material into a 250-mL round-bottom flask.
Add 100 mL of distilled water to the flask and assemble the simple distillation apparatus
as in Fig 1.
In another 100-mL round-bottom flask, pour 30 mL of water and mark the level of the
liquid with a marker.
Pour the water out of the flask and use this flask as the receiving flask for the distillate.
Heat the distillation flask using a heating mantle. Adjust the heat supply so the liquid
distills at an approximate rate of one drop per second.
Collect approximately 30 mL of distillate. Record the temperature in the distillation head
during the distillation.
Add 0.5 g of sodium chloride to the distillate, stir with a glass rod to mix, and transfer the
liquid to a 125-mL separatory funnel.
Extract the aqueous later with 15 mL of t-butyl methyl ether.
Transfer the organic layer in a 125-mL Erlenmeyer flask (Hint. Check density; the denser
solvent will be at the bottom).
Extract the remaining aqueous layer with another 15-mL portion of t-butyl methyl ether.
Collect the organic later in the same 125-mL Erlenmeyer flask.
Combine the organic layers and add enough magnesium sulfate until the drying agent
runs freely in solution (a spatulaful is usually enough).
Cap the Erlenmeyer flask with a cork and let the suspension stand for about five minutes
on the bench top with occasional swirling.
Filter the suspension by gravity filtration using a piece of fluted filter paper. Collect the
liquid in a dry and pre-tared 100-mL round-bottom flask.
Evaporate the solvent in the rotary evaporator. Weigh the flask with the oil and calculate
the mass of oil by difference.
Clean-Up

55

Dispose of pot residue in the garbage.

Aqueous extraction waste should be placed in the aqueous waste bottle.
Magnesium sulfate and filter paper should be placed in the solid waste bottle labeled
magnesium sulfate waste.
Be sure to rinse the syringes with acetone between and after uses.

In your lab report address the following questions:

1. Calculate the % yield of anise oil from anise seeds % yield = mass oil/mass
seeds x 100.
2. Report and discuss the boiling point of the mixture
3. Why does the distillate look milky?
4. What sources of error during isolation may have resulted in a lower yield?
5. Why do you add sodium chloride before performing the extraction with tert-butyl
methyl ether?
6. In the extraction with tert-butyl methyl ether, is the organic layer the top or the
bottom?

56

Experiment 6: Acid-Base Extraction: Separation of Eugenol

From Cloves
Reference: Palleros, D.R. Experimental Organic Chemistry, 2001, J. Wiley and Sons, New
York, pgs 307-312.
In this experiment, you will isolate the essential oil from cloves by co-distillation with water.
Cloves are flower buds from a tree native to the Molucca Islands of Indonesia. Today,
Madagascar and Tanzania, off the east coast of Africa, are the major producers of cloves. The
essential oil content of cloves averages 17% by weight; with only half of this isolated by water
co-distillation; the amount of oil in the aqueous distillate ranges between 0.6 and 0.8% (by
weight). Clove oil is a food flavoring agent as well as a dental anesthetic.
The oil of cloves has only three major components: eugenol, acetyleugenol or eugenol acetate,
and -carophyllene. Eugenol accounts for 70-90% of the oil, acetyleugenol ranges from 2-17%
and -carophyllene comprises 5-12%. Part of the fruity fragrance is due to a 1% component of
2-heptanone. The structures of the components are given below:

O-CO-CH3
OMe

OH
OMe

CH2 CH=CH2

eugenol

CH2 CH=CH2

H3C H
H3C

CH3

H
H2C

acetyleugenol

b-caryophyllene

Eugenol is acidic because of the phenol group. Eugenol acetate is an ester and carophyllene is
a hydrocarbon of the family sesquiterpenes. Eugenol acetate and carophyllene are neutral and
can be successfully separated from eugenol by acid-base extraction. Analysis will be done via
gas chromatography and infrared spectroscopy.
Gas chromatography is a method of separation similar to column chromatography. In gas
chromatography the mobile phase is a gas and the stationary phase can be a solid or a liquid.
A solution of the sample to be analyzed is introduced into the gas chromatograph, where it is
vaporized and carried by the mobile phase, or carrier gas, through a narrow column filled with
the stationary phase. Separation of the mixture into its components is achieved in the column
by partition or adsorption mechanisms. Single components come out of the column at different
times and pass through the detection device, which sends a signal to the recorder. This
separation in time is based on the properties of boiling point, vapor pressure and molecular
polarity. The result is a gas chromatogram or GC trace. When compounds are clearly
separated, we say that they are resolved. The position of the peak in time is called the
retention time and is the characteristic used to identify the compound. The size or integration of
the peak area is used to prepare a calibration curve for quantitative analysis of the material
(How much?)

57

Infrared (IR) Spectroscopy was one of the most powerful tools for elucidation of chemical
structures in the 1950s and 1960s. With the explosive development of nuclear magnetic
resonance (NMR) in the last 30 years, IR has been forced to a less prominent position. IR
however is still routinely run because it is inexpensive and it provides direct information about
the presence or absence of certain functional groups in a way that no other spectroscopic
technique can.
The interaction between infrared radiation and matter causes vibrational excitation through the
absorption of energy quanta (photons). Most organic molecules absorb IR radiation in the
wavenumber region of 700-4000 cm-1. A record of transmittance or absorbance as a function of
wavenumber constitutes an IR spectrum. Characteristic IR frequencies are given in Table 6.1
below.

TABLE 6.1
58

CHARACTERISTIC INFRARED ABSORPTION FREQUENCIES

Compound Type
Frequency range, cm-1

Bond
C-H

Alkanes
CH3 Umbrella Deformation

C-H Alkenes
Aromatic Rings
C-H Phenyl Ring Substitution Bands
Phenyl Ring Substitution Overtones
C-H Alkynes

2960-2850(s) stretch
1470-1350(v) scissoring and bending
1380(m-w) - Doublet - isopropyl, t-butyl
3080-3020(m) stretch
1000-675(s) bend
3100-3000(m) stretch
870-675(s) bend
2000-1600(w) - fingerprint region
3333-3267(s) stretch
700-610(b) bend

C=C Alkenes

1680-1640(m,w)) stretch

C C Alkynes

2260-2100(w,sh) stretch

C=C Aromatic Rings

1600, 1500(w) stretch

C-O Alcohols, Ethers, Carboxylic acids, Esters

1260-1000(s) stretch

C=O Aldehydes, Ketones, Carboxylic acids, Esters 1760-1670(s) stretch

Monomeric -- Alcohols, Phenols
O-H Hydrogen-bonded -- Alcohols, Phenols
Carboxylic acids
N-H Amines

3640-3160(s, br) stretch

3600-3200(b) stretch
3000-2500(b) stretch
3500-3300(m) stretch
1650-1580 (m) bend

C-N Amines

1340-1020(m) stretch

C N Nitriles

2260-2220(v) stretch

NO2 Nitro Compounds

1660-1500(s) asymmetrical stretch

1390-1260(s) symmetrical stretch

v - variable, m - medium, s - strong, br - broad, w - weak

Procedure:
WEEK I

59

Weigh about 10 g of fresh cloves and grind them using a mortar and pestle.
Transfer the powder to a 500-ml round bottomed flask and add 200 mL of distilled water.
Assemble a simple distillation apparatus.
In a 100-mL round bottomed flask, pour 60 mL of water and mark the level of the liquid
with a marker or tape. Pour the water out and use this flask as the receiving flask for the
distillate.
Heat the distillation flask using a heating mantle. Adjust the heat supply so that the
liquid distills at a rate of 1 drop per second.
Distill the mixture until about 60 mL of distillate has been collected. Read the
temperature in the distillation head during the co-distillation.
Transfer the distillate to a 125-mL separatory funnel and extract it with 25-mL portion of
tert-butyl methyl ether (t-BME; density 0.7404g/cm3) by shaking well and venting
occasionally.
Separate the layers and transfer the organic layer in an Erlenmeyer flask or beaker
Extract the aqueous layer again with another 25-mL portion of t-BME.
Collect the aqueous layer in a beaker labeled Aqueous 1. This layer will be kept until
the end of the experiment and then it will be discarded.
Collect the organic layers in a 125-mL Erlenmeyer flask.
Transfer the combined organic layer back to the separatory funnel and extract it with a
25-ml portion of a 3% NaOH solution. Separate the layers and collect the aqueous layer
in a 500-ml Erlenmeyer flask.
Extract the remaining organic layer two more times with 25 mL of the 3% NaOH
solution. Collect all the aqueous layers and set them aside for the next step.
Transfer the organic layer to a 125-mL Erlenmeyer flask labeled neutral compounds.
Add enough anhydrous magnesium sulfate until the solid runs freely in suspension; let it
dry for 5 min.
Filter the suspension using a glass funnel with a cotton plug, and collect the liquid in a
dry and pre-tared 100-mL round bottom flask.
Evaporate the solvent using the rota-vap. Record the weight of the neutral compounds
left in the flask. Set aside for later analysis.
Acidify the combined aqueous layer, in the 500-mL Erlenmeyer flask, by slowly adding
with a Pasteur pipette a 6M HCl aqueous solution until the liquid turns permanently
cloudy; the final pH should be around 1.
Transfer the acidified aqueous layer to a 250-mL separatory funnel and extract it with a
15-mL portion of t-BME each time.
Separate the layers and repeat the extraction of the aqueous layer two more times with
15 mL t-BME each time.
Combine all the organic layers in a 125-mL flask labeled eugenol. Transfer the
aqueous layer to a beaker labeled aqueous 2.

WEEK II
Transfer the combined organic layer labeled eugenol to a 125-mL separatory funnel
and extract it with 10 mL of water. Separate the layers and extract the organic layer
again with 25 mL of saturated sodium chloride solution in water.

60

Transfer the organic layer to the 125-mL Erlenmyeter flask labeled eugenol (that has
been rinsed and dried with paper towels.
Dry the solution by adding anhydrous magnesium sulfate.
After 5 minutes filter the suspension using a glass funnel with a cotton plug.
Collect the liquid in a pre-tared, 100-mL round bottom flask and evaporate the solvent.
Determine the weight of eugenol by difference. With the aid of 0.5-1 mL of t-BME,
transfer the residue to a screw capped vial and label it eugenol.
Obtain the IR spectrum of eugenol using 1-2 drops of 10-20% eugenol solution in t-BME
on the sodium chloride IR plates. Allow solvent to evaporate before obtaining IR
spectrum.
In your lab report, include answers to the following:
1. Outline the learning objectives of this experiment.
2. Calculate the percentage yield of eugenol and neutral compounds from whole cloves.
3. Report the boiling point during co-distillation and compare with the expected boiling
point.
4. Why does the aqueous layer turn cloudy when 6 M HCl is added? What compound
forms?
5. Assign as many bands as you can to the IR spectrum of eugenol. Present your data as
a table showing observed band frequency, expected frequency and assignment
columns.

61

Experiment 7: Identification of Organic Compounds by

Qualitative Analysis
Introduction
In this experiment, you will receive two or three unknown compounds, each containing one of
these functional groups: carboxylic acid, phenol, amine, alcohol, aldehyde, ketone, alkyl halide
or hydrocarbon. In addition, the compounds may or may not contain an aromatic ring. Using
chemical tests, you will determine which functional groups are present in each unknown.
Classify your compounds as specifically as possible. Use the flow chart below to organize your
analysis. Some functional groups have additional classification tests that can help you make
your identification more specific. For example, instead of just identifying a compound as a
ketone, you should be able to determine whether or not it is a methyl ketone.
Some important notes and helpful hints
1. Test results do not always give a clear positive or negative result. If you have iffy results
for a test, redo the test with slightly more or less unknown. Also, if you are unsure of one
result, be wary of the results of subsequent tests in case you went down the wrong path of
the flow chart. Of course, all observations, whether clear or otherwise, should be carefully
recorded in your notebook.
2. Running tests using a known compound (one that you know will give a positive or negative
result for a particular test) is sometimes useful because comparison to the results for your
unknown can help determine whether your unknown gave a positive result or gave a
negative result.
3. Regardless of the results of your other tests, be sure you carry out the flame test
(Test 16) to determine if aromatic rings are present in your unknown.
4. Most of the tests call for specific volumes of materials. You can save yourself time by
preparing several pre-marked test tubes and pipets. Add measured volumes of water (1 mL,
2 mL, etc.) to test tubes, then mark them. Mark several pipets in similar fashion. This saves
time and reduces chances of contamination.
Test 1: Water solubility (Indication of low molecular weight)
Compounds with low molecular weights (1-4 carbons) or those with multiple -OH or -NH groups
(low C : O ratio) are usually quite soluble in water. Compounds with increasing numbers of carbons or a higher C : O ratio are only slightly soluble in water. Compounds with C : O ratios
greater than > 6 : 1 are generally insoluble in water. Note: Because solubility is not a yes/no
phenomenon, some of a sample may dissolve while some remains insoluble. When in doubt,
treat the unknown as insoluble, You can re-check water solubility later.
Add about 6 drops of your liquid unknown (about 0.1 g if it is a solid) to a test tube containing
about 2-3 mL of water. Look for evidence of dissolving such as the formation of schlieren (wavy
lines) in the liquid as the unknown dissolves. If the droplets or crystals appear largely
unchanged after considerable mixing, the compound may be classified as insoluble in water. If it
is soluble, run the litmus test (Test 2) on the aqueous solution.
Test 2: Litmus test (Indication if compound is acidic or basic)
If your unknown is water-soluble, test the resulting aqueous solution with litmus paper. A
strongly basic response suggests that compound is an amine. If no change is observed, your
compound can be classified as neutral and you should go to Test 7. A strongly acidic response
to the litmus paper suggests that your compound is probably a carboxylic acid. You should
confirm your results by performing Test 5. Note: Some phenols are sufficiently water-soluble

62

and acidic to turn litmus red or give bubbling with NaHCO 3. If Tests 2 and 5 give positive results,
you should perform Test 15 in case your compound is a phenol.
Test 3: Solubility in HCl (Indicates organic amines)
Add 5 drops of your sample (about 0.1 g of a solid unknown) to a test tube containing about 2
mL (about 20 drops) of 5% hydrochloric acid (HC1). If the unknown is soluble, then it is an
amine. Heat is often evolved during a positive test. Perform Test 16 to further classify your
amine. If the result is negative, go to Test 4.
Test 4: Solubility in NaOH (Indicates phenols and carboxylic acids)
Add about 5 drops or 0.1 g of your known to a test tube containing about 2 mL of 5% sodium
hydroxide (NaOH) solution. If the unknown is soluble in 5% NaOH, (positive result) then it is
either a carboxylic acid or a phenol. Perform Test 5 to distinguish between these two groups. If
the result is negative, then go to Test 6.
Test 5: Solubility in NaHCO3 (Distinguishes between most phenols and carboxylic acids)
Add 5 drops or 0.1 g of your unknown to a test tube containing about 2 mL of 5% sodium
bicarbonate (NaHCO3). If the unknown dissolves in the sodium bicarbonate solution, especially
if fizzing or bubbles are visible, then it is probably a carboxylic acid. If the unknown is insoluble
(negative result), then it is probably a phenol, and you should perform Test 15. Note: some
phenols give positive results with NaHCO 3, so you should also perform Test 15. If Test 5 is
positive, perform Test 16 to further classify your unknown.
Test 6: Solubility in conc. H2SO4 (Detects alcohols, aldehydes and ketones)
CAUTION: Concentrated sulfuric acid can cause severe burns and eats through paper!
Add about 1-1.5 mL (10-15 drops) of concentrated sulfuric acid (H2SO4) to a dry test tube, and
then carefully add 5 drops (or about 0.1 g) of your unknown. Carefully mix the contents with a
stirring rod. A positive (soluble) response is indicated by substantial color changes, usually with
the evolution of heat. A positive test may also cause formation of a tarry residue from
polymerization of the unknown. A negative (insoluble) response produces two clearly defined
phases, but there may be slight darkening of either phase (due to trace impurities).
A positive result indicates that the unknown is an alcohol, aldehyde or ketone (Almost any group
with oxygen or nitrogen atoms present will give a positive result, but most other groups have
been screened out by the time you get to this test.) If Test 6 gives a positive result, perform Test
7. If Test 6 is negative, your compound is either a hydrocarbon or alkyl halide. Go to Test 12
(the Beilstein test).
Test 7: DNP test (Distinguishes alcohols from aldehydes or ketones)
Add 1 mL of 2,4-dinitrophenylhydrazine (DNP) reagent to a dry test tube, followed by add 4-6
drops of your unknown. (If your unknown is a solid, dissolve about 0.1 g in about 0.5 mL of nondenatured ethanol, then add the solution to the DNP reagent.) Formation of a yellow, orange, or
red precipitate is a positive result, indicating that your unknown is either an aldehyde or a
ketone. If the test is positive, then perform Test 8 to distinguish between aldehydes and
ketones. If Test 7 is negative, then you have an alcohol, and should perform Test 10 to
determine more information about your alcohol.
Test 8: Chromic acid test (Distinguishes between aldehydes and ketones)
CAUTION! Chromic acid is highly corrosive and a suspected carcinogen.
Carefully add two drops of chromic acid to a test tube containing 2 mL of acetone. Next, add 2
drops of your unknown and mix thoroughly. Formation of a blue-ish to green-ish precipitate

63

within 5 seconds shows that an aldehyde group is present, and you should perform Test 16 to
further classify your aldehyde. A negative result means that your compound is a ketone.
Comparing your results to those using an authentic aldehyde is recommended. Perform Test 9
to further classify the ketone. Dispose of the chromium wastes in the indicated container in the
hood.
Test 9: Iodoform test (Distinguishes between methyl ketones and other ketones)
Be sure your test tube is cleaned and does not contain any traces of acetone. If your compound
is water soluble, mix 1 drop or 0.15 g of your unknown with 1 mL of water in a test tube. If the
unknown is insoluble in water, dissolve your sample in 0.5 mL of 1,2-dimethoxyethane in a test
tube. Add 0.5 mL of 3 M NaOH solution to your unknown. Add 0.75 M KI/Iodine solution in a
drop by drop manner until the iodine color persists after shaking. Place the tube in a warm water
(60 C) bath for 10 minutes. A positive test is shown by the formation of a yellow precipitate
(iodoform, CHI3). You should compare your results with results from a known methyl ketone
such as acetone, and a known non-methyl ketone such as cyclohexanone. Perform Test 16 to
further classify your ketone.
Test 10: Chromic acid test (Confirmatory test for 1 and 2 alcohols. This is the same
basic test as #8)
CAUTION! Chromic acid is highly corrosive and a suspected carcinogen.
Carefully add two drops of chromic acid to a test tube containing 2 mL of acetone. Cautiously
add 2-3 drops of your unknown and mix the contents thoroughly. A positive result (appearance
of a blue-green precipitate within two seconds) indicates a primary or secondary alcohol.
Tertiary alcohols give a negative result. You should compare your results to known primary,
secondary and tertiary alcohols. If the result is positive, perform Test 11 to further classify your
alcohol. Dispose of the chromium wastes in the indicated container in the hood.
Test 11: Iodoform test (Detects the methyl carbinol [R-CH(OH)-CH3] group)
Be sure your test tube is cleaned and does not contain any traces of acetone. If your compound
is water soluble, mix 1 drop or 0.15 g of your unknown with 1 mL of water, then add 0.5 mL of 3
M NaOH solution. (If it insoluble in water, dissolve your sample in 0.5 mL of 1,2dimethoxyethane (ethylene glycol dimethyl ether) before adding the NaOH solution) Add 0.75 M
KI/Iodine solution in a drop by drop manner until the iodine color persists after shaking. Place
the tube in a warm water (60 C) bath for 10 minutes. A positive test is shown by the formation
of a yellow precipitate (iodoform, CHI3). A positive test indicates that your alcohol contains the
methyl carbinol group [R-CH(OH)CH3].You may want to compare your results with results with a
known methyl carbinol such as 2-butanol. Perform Test 16 (Flame Test) to further classify your
alcohol.
Test 12: Beilstein Test (Indicates halogenated compounds)
NOTE: When you carry out this test, you are also performing Test 16 (Flame Test), so you can
interpret your observations for that test at the same time as you do this test.
Clean a copper loop by holding it in a burner flame until the flame no longer has any greenish
color. Do not overheat the loop or you will melt the wire. Put a few drops or crystals of your
unknown into a watch glass, let your loop cool, and dip it into your sample. (If your sample boils
or smokes, clean the loop again, then let it cool for a bit longer before dipping it in the sample.)
Hold the loop in the upper part of burner flame.
A positive test (halogens present) is shown by a greenish flash or glow, often as a glow after the
flame subsides. If your results are positive, perform Tests 13 and 14 to further classify your

64

halide (primary, secondary, tertiary or aromatic). A negative result (no green flame) means that
your compound is a hydrocarbon. (See Test 16 for more information about classifying
hydrocarbons.)
Classification of halides
Note: These tests are fairly sensitive to contamination, so your glassware must be clean and dry
or false results may occur. You may want to run these tests on authentic samples to compare to
your results.
TEST 13: Silver Nitrate Test (Test for secondary and tertiary halides)
To a test tube containing 2 mL of 1% silver nitrate in ethanol, add 5 drops (0.1 g) of your
unknown. If no precipitate forms at once, heat the mixture for 20 minutes in a hot-water bath. A
light-colored precipitate is a positive result for a secondary or tertiary alkyl halide. Primary and
aromatic halides give negative results. Perform Test 14.
TEST 14: (Sodium Iodide Test (Test for primary and secondary halides)
To a test tube containing 2 mL of 15% sodium iodide in acetone, add 2-3 drops (0.1 g) of your
unknown. If no precipitate forms, heat the mixture for 20 minutes in a hot-water bath. A white or
yellow precipitate indicates a primary or secondary alkyl halide. Tertiary and aromatic halides
give negative results.
Test 15: Ferric Chloride Test (Confirmatory test for phenols)
To a test tube containing 2 mL of 0.6 M ferric chloride (FeCl 3) solution, add 2 drops or 0.1 g of
your unknown and mix very thoroughly. A green, red, blue, or purple color is a positive test for
phenols. If a negative result is obtained, your compound is a carboxylic acid. Perform Test 16.
All phenols give a positive result for Test 16.
Test 16: Flame Test (Indicates aromatic compounds)
Clean a copper loop by holding it in a burner flame until the flame no longer has any greenish
color. Do not overheat the loop or you will melt the wire. Put a few drops or crystals of your
unknown into a watch glass, let your loop cool, and dip it into your sample. (If your sample boils
or smokes, clean the loop again, then let it cool for a bit longer before dipping it in the sample.)
Hold the loop in the upper part of burner flame.
A sooty flame (positive test) means that one or more aromatic rings are present the unknown. A
negative result (no soot) means that no aromatic rings are present.
NOTE: Some halogenated compounds (Test 12) also give a somewhat sooty flame, but the
sooty flame from aromatic compounds is much more pronounced. You may wish to compare
your results to a known aromatic compound, non-halogenated compound and to a non-aromatic
halogenated compound.

65

66

Positive

Positive

Aldehyde

Amine

Basic

Negative

Soluble

2SO

Soluble
3

Insoluble

Non-methyl ketone

to
oneis,orcarry
compound
your
compound
forcontains
16)your
compound
class
your
(Test
rings.of
Test
what
whether
Flame
aromatic
theREMEMBER!
more
determine
out
Regardless
Negative

(Try Test 10,

Positive
Negative
then Test 11
all other
tests
(ifnegative)
Hydrocarbon
Halide
if needed)
using
13 + 14)
Tests
(Classify

Test)
(Beilstein
Test 12

Insoluble

5
NaHCO
(5%
Test

Soluble

Test 4 (5% NaOH)

)
Carboxylic
Phenol
Acid with
Test 15)
(Confirm

Insoluble

Test 3 (5% HCl)

Insoluble

Insoluble

Test 6 (Conc. H

Negative
4
)
Alcohol

Ketone
Test 9 (Iodoform Test)

Methyl ketone

Positive

Soluble

Amine

Test 7 (2,4-DNP)

Alcohol, Aldehyde
or Ketone

Neutral

Test 8 (Chromic Acid)

Aldehyde or Ketone

15)
phenol?
Do(orTest

Carboxylic Acid

Acidic

Test 2 (Litmus)

Soluble

Test 1 (Water)

Unknown Compound

Outline for Qualitative Analysis

Sample layout for data tables (Record data directly in your notebook!)
Unknown #:___________
Color and appearance of unknown:
Test

Observations

Interpretation

1
2
3

Use the results of your tests to classify your unknowns. Your classification should address the
functional groups, molecular weight and whether any aromatic rings are present. Be as detailed
as you can, given the results of your tests. Provide detailed reasoning for your classifications,
explaining how each positive or negative result supports your classification. How sure are you of
your classification? Explain any other possibilities for your unknown if there is some doubt to
your classification. If there are any conflicting results, attempt to explain them.
Note: A compound may contain an aromatic ring and a halogen, and yet not be an aromatic
halide. Aromatic halides can be defined as compounds with a halogen attached directly to an
aromatic ring. If the halogen is attached elsewhere in the molecule, then the compound will
behave as a primary, secondary, or tertiary halide.
Follow-up Questions
1. Most ketones react readily with 2,4-DNP, but benzophenone (diphenyl ketone, Ph-CO-Ph)
reacts very slowly with 2,4-DNP.
a. Give the structure of the product of the reaction of 2,4-DNP with benzophenone.
b. Suggest an explanation why it reacts so slowly. Hints: Look up the structures of
benzophenone and
2,4-DNP and consult Chapter 18 of Wade.
2. One possible problem with qualitative analysis is that impurities can
lead to inaccurate conclusions. Consider the following scenario: Longterm exposure to oxygen (such as sitting in a half-empty bottle) can
cause slow oxidation of alcohol 1 to give ketone 2.
a. Mary analyzes a pure sample of alcohol 1, following the chart on p.
40. List the tests she would perform and the observations she
should obtain in each test. Based on these results would she
identify her sample as an aromatic alcohol? (Hint: 1 is insoluble in
water.)

OH

1
O

2
b. John analyzes a pure sample of ketone 2, following the chart on p.
40. List the tests he would perform and the observations he should obtain in each test.
Based on these results would he identify his sample as an aromatic ketone? (Hint: 2 is
insoluble in water.)

67

c. Steve analyzes a sample that he believes it is a pure compound. However, the sample
contains roughly equal amounts of alcohol 1 and ketone 2, which means any tests that
would be positive for 1 will give positive results, and any tests that would be positive for
2 will also give positive results. Assuming that Steve follows the sequence of tests on
page 40, list the tests he would perform and the observations he should obtain in each
test. Based on these results, what functional groups would he think are present in the
sample? (Remember, Steve has no idea what is in the sample, let alone that the sample
is impure; he thinks that the sample contains only one compound.)

68

Experiment 8A: The Effect of Structure on the Free-radical

Bromination of Hydrocarbons
Background Information
Free-radical bromination is believed to proceed by a free-radical chain reaction. In the first step,
called initiation, the Br-Br bond breaks, giving a pair of bromine radicals (Br). In the second step,
propagation, bromine radicals abstract (pluck off) hydrogen atoms from hydrocarbon molecules,
giving alkyl radicals and HBr. When the alkyl radical collides with a bromine molecule, it abstracts a
halogen atom, forming the product and another bromine radical. The reaction continues when Br
reacts with another hydrocarbon molecule, and so on. This cycle is repeated hundreds or
thousands of times until termination occurs. Termination occurs when any two radicals collide with
each other forming a bond, breaking the chain.

Br Br

Init iation:

P ropagat ion:

Net react ion:

heat

R H

Br

Br Br

R H + BrBr

2 Br
+

Br

Br +

Br

Br

RBr

The first propagation step is the rate-determining (slower) step. This step is usually endothermic,
and the rate of this step is highly dependent on the strength of the C-H bond being broken. As the
strength of the C-H bond decreases, the energy of activation also decreases, causing the rate of
this step (and of the overall reaction) to increase.
Bromine radicals are highly selective, which means that they have a high preference for
abstracting the hydrogen with the weakest C-H bond, no matter how many other C-H bonds are
in the molecule. This is reasonable because breaking the weakest C-H bond has the lowest
energy of activation; breaking this bond will be fastest. If one knows all of the C-H bond
strengths in a hydrocarbon, one knows which particular hydrogen will react fastest with bromine.
If one compares the various C-H bond strengths from one molecule to another, whichever
compound has the weakest C-H bond will react faster. Applying this same reasoning to a series
of hydrocarbons, the order of reactivity from fastest to slowest can be predicted. The reactivity
of the hydrocarbons is not based on the total strength of C-H bonds, but is based on the
strength of the bond that must be broken for the reaction to occur. The reaction requires
breaking a C-H bond, so therefore, the compound with the weakest C-H bond will react fastest,
followed by the compound with the next-weakest C-H bond, and so forth. Put another way, the
weakest link in each compound determines its relative order of reactivity.
Carbon-hydrogen bonds are classified by the nature of the carbon atom to which they are attached.
Alkyl hydrogens (those hydrogens bound to sp 3 hybridized carbons) may be classified as being
primary (1), secondary (2), or tertiary (3) depending upon the number of other carbon atoms that
are attached to the reference carbon atom.
C-H Bond Type

Typical B.D.E.,

C-H Bond Type

Typical B.D.E.,

69

kcal/mol
1 alkyl
2 alkyl
3 alkyl
Vinyl
(attached to double
bond)
Aryl
(attached to aromatic
ring)

98
95
92
108

110

kcal/mol
1 benzylic or allylic
2benzylic or allylic
3benzylic or allylic
Acetylenic

85
82
79
125

Allylic = next to double bond

Benzylic = next to aromatic ring

In this experiment, you will predict the order of reactivity of six hydrocarbons toward
bromination, then test your predictions by experiment. At the instructors discretion, you may
also be directed to test one or more additional hydrocarbons.
In this reaction, Br2 is the only colored species. If the reaction is carried out with bromine as the
limiting reagent, one can follow the progress of the reaction. When the red-brown color of
bromine fades, the reaction is essentially complete. If equal amounts of two hydrocarbons are
allowed to react with equal portions of bromine, the mixture that fades faster is the more
reactive. By implication, this hydrocarbon should have the weaker C-H bond.
Pre-lab Work (Write this in your notebook, as well as on a separate sheet of loose-leaf paper.
You may be asked to turn the separate sheet at the start of lab.)
For each of the hydrocarbons shown below, identify all of the types of C-H bonds and list
their bond strengths. For example, toluene has aromatic hydrogens (110 kcal/mol) and
primary benzylic hydrogens (85 kcal/mol. Use the table on the previous page to help you.
Use this information to predict which of your compounds would react fastest, then
second fastest, and so forth.
CH 3

CH2CH3

CH(CH3 )2

C(CH3)3
CH 2
CH 2

T oluene

CH 2
CH 2

CH2 H C
2
CH2 H C
2

CH 3
CH
CH 2

CH2
CH2

Cyclohexane
Isopropylbenzene
(Cumene)
Methylcyclohexane
Ethylbenzene
tert-Butylbenzene

Safety Alert!
1. Bromine is corrosive and a severe respiratory irritant. Do not breathe its vapors or
allow it to contact your skin. Perform all transfers of bromine in a hood, and wear
rubber gloves. If you get bromine on your skin, wash the area immediately with soap
and warm water and notify your instructor.
2. Dispense the bromine solution in the hood.
3. Bromine reacts with acetone to produce the powerful lachrymator (tear producer),
bromoacetone, CH3COCH2-Br. Do not rinse glassware containing residual bromine
with acetone. Add a drop or two of cyclohexene to consume the bromine, after which
you can rinse with acetone.

70

4. Avoid excessive inhalation of the vapors of any of the materials being used in this
experiment.
Procedure
IMPORTANT! Record your predictions in your notebook before you carry out the experiment!
Obtain 12 clean, dry test tubes and label six of them, one for each hydrocarbon being tested.
Add 10 drops of each hydrocarbon to its labeled tube. Add 10 drops of a 1.0 M solution of Br2 in
CH2Cl2 to the remaining six tubes. Try to add the same amount of bromine to each tube
because variations in the amounts of bromine can lead to misleading results. With the aid of a
partner, quickly pour the bromine portions into the hydrocarbon tubes and agitate the tubes to
mix the contents. Record the start time. When the characteristic bromine color fades completely
(or nearly so), record the time elapsed. Some of the reactions are quite fast and take less than
30 seconds, so you may have to repeat the analysis of some tubes to accurately measure the
time.
Monitor the test tubes at room temperature for 30-40 minutes. Those tubes that have not
reacted after this time should be heated in a beaker of water warmed to about 50 C. Do not
heat the water above 50 C because bromine is volatile and the fumes are irritating and toxic.
The experiment can be stopped when only one colored solution remains, or when the last two
tubes have a significant difference in color, because it is now obvious which hydrocarbon reacts
the slowest.
Cleanup: Decolorize any solutions in which the color of bromine is visible by the drop-wise
addition of cyclohexene; then discard the resulting solutions together with all other solutions in
the designated container for halogenated organic liquids.
Analysis of your results
List the compounds in order of reactivity from fastest to slowest. For each compound, list the
weakest C-H bond present. What correlation, if any, is there between the order of reactivity and
the strength of the weakest C-H bond in each compound?
Things to consider in your Discussion
How closely do your results correspond to your predictions? How many results are in the relative
same order as predicted? Double-check the bond strengths for each C-H bond in each compound
to verify that your predictions were based on accurate information. If your predictions and results do
not match, attempt to identify likely cause(s). Sources of error include misidentification of C-H bond
types, (leading to inaccurate predictions), poor timekeeping or inconsistency in measuring out
reagents. If you did not identify the bond types correctly, determine what the correct order of
reactivity should have been. Do your results fit the revised order of reactivity more closely? Were
your predictions correct and the results flawed, or vice versa?
If you also carried out the reaction of one or more unknown hydrocarbons, answer these questions:
1. The reactivity of your unknown most closely resembles which hydrocarbon(s)?
2. What can you deduce about the structure of your unknown?

Experiment 8B: Relative Rates of Nucleophilic Substitution

Reactions of Alkyl Halides
Background

71

One of the most major factors affecting the rates of S N1 and SN2 reactions is the structure of the
alkyl halide. You will use two qualitative tests to determine the relative rates of reactions of
several alkyl bromides under S N1 and SN2 conditions. Use your knowledge of the effect of
structure on SN2 and SN1 reactions to make your predictions before you carry out the
experiment. After the experiment is completed, compare your experimental findings to your
predictions.
Important! Answer these questions before coming to lab. Write the answers in your
notebook and on a sheet of paper to turn in at the start of lab.
Predictions prior to the experiment:
1. Predict the reactivity of the alkyl halides in the Table with NaI/acetone from most reactive to
least reactive. Briefly explain your reasoning.
2. Predict the reactivity of the alkyl halides in the Table with AgNO 3/ethanol from most reactive
to least reactive. Briefly explain your reasoning..
3. Which compounds, if any, are there that you would expect to be unreactive under either set
of conditions? Briefly explain your reasoning.
SN2 test: (Sodium iodide in acetone, also known as the Finkelstein reaction)
When an alkyl halide reacts with NaI/acetone, the following reaction occurs (by an S N2
mechanism):
I + RX

X + RI

(X = Cl, Br)

Sodium iodide will dissolve in acetone, but sodium bromide is much less soluble in acetone, so
NaBr forms as a white precipitate. If a precipitate is observed, this indicates that the alkyl halide
can react in an SN2 fashion. Historical note: This reaction is also known as the Finkelstein test. It
can be used in synthesis to prepare alkyl iodides from alkyl chlorides or alkyl bromides.
SN1 test: (Silver nitrate (AgNO3) in ethanol)
When an alkyl halide reacts with AgNO 3 in ethanol, the following reactions occur:
First step:

RX

R+ + X

(X = Cl, Br)

Second step: Ag+ + X AgX (solid)

The first step is aided by the polar solvent, ethanol. The leaving group, X , then reacts with Ag+
to form AgX, which appears as a white precipitate. The first step is also the rate-determining
step in an SN1 reaction. A positive test (formation of a white precipitate) indicates that the alkyl
halide can readily react in an S N1 fashion.
Procedure
Note: Some compounds will react very quickly while others will take minutes or even a few
hours to react. Some will not give a positive test at all. Note that contamination by water or
acetone can lead to false positive results. Acetone and ethanol are both quite volatile, so if the
test solutions are heated too strongly or for too long, some of the solvent will evaporate.
Evaporation of the solvent will cause the AgNO3 and NaI to deposit on the sides of the tubes,

72

which is often mistaken for a positive result. Note the level of liquid in the tubes when you begin
heating, and top off the tubes with solvent if evaporation is a problem.
SN2 Reaction Conditions: Label your test tubes before obtaining any reagents. Place 3 drops,
measured of each halide in its own dry test tube. Add 1.0 mL of NaI/acetone solution to each
test tube and shake the tube to ensure complete mixing. Monitor the tubes and record the time
required for any precipitates to form. Tubes showing no precipitate after 5 min. at room
temperature should be heated in a warm-water (50 C) bath for about 15 min. A negative result
after 15 minutes means that the compound reacts very slowly or not at all.
SN1 Reaction Conditions: Repeat the above procedure, using 1.0 mL portions of AgNO 3/ethanol
solution as the test reagent.
Cleanup: Pour the contents of all test tubes in the container for halogenated organic waste.
Pour any remaining silver nitrate solution in the container for inorganic waste.
Record your observations, reaction times and how much, if any, heating is required for each of
the compounds you test. Summarize your findings in your notebook in a table similar to the one
shown. Your instructor may include additional compounds for you to test.
Sample layout for notebook data tables
Compound
Class of substrate
(1, 2, 3, aryl)
1-Bromobutane
2-Bromobutane
2-Bromo-2-methylpropane
Bromobenzene
Additional compounds
provided by instructor:

Time and temp. for positive result:

NaI/Acetone
AgNO3/Ethanol

List the alkyl halides from the most reactive to the least reactive with NaI/acetone. List the alkyl
halides from most reactive to least reactive with AgNO 3/ethanol. Which compounds, if any, fail to
react under either set of conditions?
Topics for Discussion
Do your results match your predictions? Account for any discrepancies between what you
predicted for each set of reactions and what you observed. Check your predictions against
information in Chapter 6 of your lecture textbook. Which of your predictions for no reaction were
valid? Were there any unexpected instances where no reaction occurred? Explain these
discrepancies. Do your predictions fit what the textbook says? What are some sources of error
that could have led to the discrepancies between the predicted reactivities and the observed
reactivities? How would these errors affect your results? Suggest specific ways to eliminate
these errors (not just being more careful.)
Follow-up Questions
1. The NaI/acetone test gives a precipitate with 1-chlorobutane and 1-bromobutane, but not 1iodobutane. Why?
2. Why is it important to use a polar solvent such as ethanol in the silver nitrate test?

73

3. Suppose that
organobromides:

organochlorides

(R-I)

were

used

in

this

experiment

instead

of

a. In the NaI test, would organochlorides react faster, slower or at the same rate as the
corresponding organobromides? Explain. Would the order of reactivity change? Explain.
b. In the AgNO3 test, would the organochlorides react faster, slower or at the same rate as
the corresponding organobromides? Explain. Would the order of reactivity change?
Explain.

74

EXPERIMENT 9: Medicinal Chemistry

Conversion of Acetaminophen into Phenacetin
Reference: Palleros, D.R. Experimental Organic Chemistry, 2001, J. Wiley and Sons, New
York, pgs 327-333.

O
OH

CH3
COOH

salicylic acid

CH3

HN

HN

OH

OCH2 CH3

acetaminophen

O
O

phenacetin

CH3
COOH

O
HO

acetylsalicyclic acid

ibuprofen

Introduction: In this experiment, you will prepare by the Williamson ether synthesis, phenacetin
from acetaminophen contained in Tylenol tablets. Actaminophen and phenacetin are chemically
related. Both have an acetamido group, but there is a free OH group in acetaminophen,
phenacetin has an ethoxy group in its place. Phenacetin is a pain reliever and a less potent
antipyretic than acetaminophen. It has little value as an anti-inflammatory agent. Phenacetin
was found to cause liver and kidney damage when administered in large doses. Experiments
on laboratory animals showed that phenacetin can induce cancer when taken orally. Its use in
medicinal preparations was banned in the US in 1981.
PROCEDURE:
WEEK I
Weigh an Extra-Strength Tylenol tablet. Pulverize the tablet with mortar and pestle.
Weigh out 0.22 g and place it in a dry 25-ml round-bottom flask along with 0.28 g of finely
pulverized K2CO3 (mortar and pestle) and 3.0 ml of butanone.
Carefully add 0.28 ml of ethyl iodide with a syringe.
Add a stir bar, attach a water-cooled condenser to the flask and heat the mixture until reflux
directly on a hot plate at medium setting for one hour.
Obtain the IR of acetaminophen using a nujol mull or by diluting and using NaCl plates.
Turn off the heat. Allow the mixture to cool down.

75

Add 4 mL of water to the flask, and transfer its contents to a 10 mL concentrator tube with
cork. Rinse the round-bottom flask 4 times with 1 mL of tert-butyl methyl ether (t-BME) and
add the rinsings to the tube.
Cork the tube and shake the layers. Vent to release pressure by unscrewing the cap
momentarily.
With a Pasteur pipette, remove the lower (aqueous) layer. Transfer the aqueous layer to a
12 x 100 mm corked test tube. Keep the organic layer in the concentrator tube.
Extract the aqueous layer with 2.5 mL of t-BME. Remove the aqueous layer with a Pasteur
pipette. Transfer the organic layer to the concentrator tube with the original organic layer.
Add 2.5 ml of 5% aqueous NaOH to the combined organic layers and shake well. Let the
layers settle and remove the lower aqueous layer with a Pasteur pipette.
Repeat the extraction with another 2.5-mL portion of 5% NaOH. Save the aqueous layers
until the end of the experiment.
Extract the organic layers with 2.5 mL of saturated sodium chloride solution.
WEEK II
Dry the organic layer by adding magnesium sulfate little by little with a microspatula until the
solid runs freely in the liquid. Let the system stand for 5 minutes with occasional swirling.
Filter the mixture using a Pasteur pipette and a cotton plug and receive the filtrate in a dry,
pre-tared, 25-mL round-bottomed flask. Evaporate off the solvent using a rota-vap.
Weigh the flask with the product. Calculate the crude % yield. Determine its melting point.
Obtain the IR spectrum of phenacetin using a nujol mull or dilute with CH 2Cl2 for NaCl
plates. Compare the IRs of phenacetin and acetaminophen.
Ferric Chloride Test:
This is a test for phenols. Acetaminophen is a phenol while phenacetin is not, it is an ether.
Place 1 ml of 1% aqueous solution of ferric chloride in each of four small, labeled test tubes. To
each one, add a microspatulaful of one of the three compounds: acetaminophen, phenacetin
and salicylic acid. Add a drop of water to the fourth tube, which is used as the blank.
Observe the change in color. Colors ranging from green to red are considered a positive
test; yellow is negative.
Analysis of Analgesics by TLC
Pulverize a tablet of each analgesic (Bayer aspirin, Tylenol and Anacin) using a clean
mortar and pestle (use a little acetone and tissue paper to clean mortar and pestle).
Transfer about one-eighth of each solid analgesic to a labeled test tube. Add 2 ml of
acetone to each tube and stir with a clean spatula. The insoluble solid is the binder of the
tablet.
Dissolve about 1-2 mg of phenacetin (your compound) in 0.5 ml of acetone.
Spot your sample on silica gel plates with fluorescent indicator. Do not overload the spots.
One or two applications will be enough.
For a precise analysis, the unknown should be run side by side on the same plate with the
other analgesics.
Use ethyl acetate as the developing solvent. Use the UV lamp to visualize spots. Make sure
you record a sketch of the plate as well as colors under the lamp.
Calculate the Rfs in your notebook and identify each component.
Clean Up

76

Dispose of aqueous waste and ferric chloride solutions in the analgesics-aqueous waste
bottle.
Used Pasteur pipettes go in the broken glass container.
MgSO4 goes in the solid waste jar labeled solid organic waste.
Place phenacetin, acetaminophen and aspirin into the analgesics solid waste jar.

In your lab report, include the following:

1. Calculate how many mmol of reactant were used. Determine which one is the limiting
reagent. Take into account the weight of the tablet and the amount of acetaminophen
per tablet.
2. Calculate theoretical yield of phenacetin in mg.
3. Report the M.P.of phenacetin. Compare with the literature value.
4. Report and discuss the results of the ferric chloride tests.
5. Assign as many bands as possible to the IR of phenacetin and starting material.

77

Experiment 10: Preparation of Nylon [6,10] and the Rope

Trick
References
Step-Growth Polymerization Basics and Development of New Materials He, Z.; Whale, E. A.;
Davis, F. J. In Polymer Chemistry A Practical Approach, Davis, F., Ed., Oxford, 2004, 127
For Nylon basics: http://en.wikipedia.org/wiki/Nylon
For understanding polymer basics: http://www.pslc.ws/macrog/maindir.htm
Introduction
A polymer (from Greek meaning made up of many parts) is a large molecule (macromolecule)
composed of repeating structural units typically connected by covalent chemical bonds. While
polymer in popular usage suggests plastic, the term actually refers to a large class of natural
and synthetic materials with a variety of properties.
Nylon is a thermoplastic silky material, first used commercially in a nylon-bristled toothbrush
(1938), followed more famously by womens stockings ("nylons"; 1940). It is made of repeating
units linked by peptide bonds (another name for amide bonds) and is frequently referred to as
polyamide (PA). Nylon was the first commercially successful synthetic polymer. There are two
common methods of making nylon for fiber applications. In one approach, molecules with an
acid (COOH) group on each end are reacted with molecules containing amine (NH 2) groups on
each end. The resulting nylon is named on the basis of the number of carbon atoms separating
the two acid groups and the two amines. These are formed into monomers of intermediate
molecular weight, which are then reacted to form long polymer chains.

H
H
N R N

O
R'

Repeat structure of a polyamide

Nylon was intended to be a synthetic replacement for silk and substituted for it in many different
products after silk became scarce during World War II. It replaced silk in military applications
such as parachutes and flak vests, and was used in many types of vehicle tires. Nylon fibers
are used in many applications, including fabrics, bridal veils, carpets, musical strings, and rope.
Aramids are another type of polyamide with quite different chain structures which include
aromatic groups in the main chain. Such polymers make excellent ballistic fibres.
In this lab, two molecules called monomers, one with an acid chloride (COCl) group on each
end is reacted with another monomer with two amine (NH 2) groups on each end. This process
is called a step-growth condensation polymerization and generates nylon [6,10], whereby the
diamine has six carbon atoms and diacid chloride has ten carbon atoms. As a consequence to
this polymerization, a condensate is produced, in this case, hydrogen chloride.

78

O
H2 N

NH2

Cl

Cl
O

N
H

H
N

+
O

HCl

Nylon [6,10]

Procedure
Dissolve about 1 g of hexamethylene diamine in 25 ml of water in a 100 ml beaker.
Make solution of about 1 g (or 1 mL of solution) of sebacoyl chloride in 25 ml hexane.
Gently pour the sebacoyl chlorie/hexane solution on top of the hexamethylene
diamine/water solution in the beaker, using a glass rod to pour down. A film will form at
the interface. At the interface of the two phases, the diacid chloride and diamine meet
each other and will polymerize there.
Nylon rope trick: Draw a thread out of this interface using a glass rod, and draw the
thread out of the beaker. Using a second 100 ml beaker as a spool, slowly wind up the
thread as you draw it out.
After allthe polymer has been collected, wash it thoroughly with water, dry it superficially
with a towel, then let it air dry.
Unwind the dry thread and let the students examine its physical properties. Rarely will
this material display any significant strength.
Clean-up
The washed and dried solid polymer is disposed in the regular garbage.
The remaining hexamethylene diamine and sebacoyl chloride are disposed in the
labeled organic waste container.

Appendix A: Gas Chromatography Background

79

Introduction
Gas chromatography - specifically gas-liquid chromatography - involves a sample being
vaporized and injected onto the head of the chromatographic column. The sample is transported
through the column by the flow of inert, gaseous mobile phase. The column itself contains a
liquid stationary phase which is adsorbed onto the surface of an inert solid.
Have a look at this schematic diagram of a gas chromatograph:

Instrumental components
Carrier gas
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium,
argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of
detector that is used. The carrier gas system also contains a molecular sieve to remove water
and other impurities.
Sample injection port
For optimum column efficiency, the sample should not be too large, and should be introduced
onto the column as a "plug" of vapor - slow injection of large samples causes band broadening
and loss of resolution. The most common injection method is where a microsyringe is used to
inject sample through a rubber septum into a flash vaporizer port at the head of the column. The
temperature of the sample port is usually about 50C higher than the boiling point of the least
volatile component of the sample. For packed columns, sample size ranges from tenths of a
microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample,
typically around 10-3 L. For capillary GC, split/splitless injection is used. Have a look at this
diagram of a split/splitless injector;

80

The injector can be used in one of two modes; split or splitless. The injector contains a heated
chamber containing a glass liner into which the sample is injected through the septum. The
carrier gas enters the chamber and can leave by three routes (when the injector is in split
mode). The sample vaporizes to form a mixture of carrier gas, vaporized solvent and vaporized
solutes. A proportion of this mixture passes onto the column, but most exits through the split
outlet. The septum purge outlet prevents septum bleed components from entering the column.
Columns
There are two general types of column, packed and capillary (also known as open tubular).
Packed columns contain a finely divided, inert, solid support material (commonly based on
diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in
length and have an internal diameter of 2 - 4mm.
Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of
two types; wall-coated open tubular (WCOT) or support-coated open tubular (SCOT). Wallcoated columns consist of a capillary tube whose walls are coated with liquid stationary phase.
In support-coated columns, the inner wall of the capillary is lined with a thin layer of support
material such as diatomaceous earth, onto which the stationary phase has been adsorbed.
SCOT columns are generally less efficient than WCOT columns. Both types of capillary column
are more efficient than packed columns.
In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT)
column;

81

These have much thinner walls than the glass capillary columns, and are given strength by the
polyimide coating. These columns are flexible and can be wound into coils. They have the
advantages of physical strength, flexibility and low reactivity.
Column temperature
For precise work, column temperature must be controlled to within tenths of a degree. The
optimum column temperature is dependant upon the boiling point of the sample. As a rule of
thumb, a temperature slightly above the average boiling point of the sample results in an elution
time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If
a sample has a wide boiling range, then temperature programming can be useful. The column
temperature is increased (either continuously or in steps) as separation proceeds.
Detectors
There are many detectors that can be used in gas chromatography. Different detectors will give
different types of selectivity. A non-selective detector responds to all compounds except the
carrier gas, a selective detector responds to a range of compounds with a common physical or
chemical property and a specific detector responds to a single chemical compound. Detectors
can also be grouped into concentration dependant detectors and mass flow dependant
detectors. The signal from a concentration dependant detector is related to the concentration of
solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will
lower the detectors response. Mass flow dependant detectors usually destroy the sample, and
the signal is related to the rate at which solute molecules enter the detector. The response of a
mass flow dependant detector is unaffected by make-up gas. Have a look at this tabular
summary of common GC detectors:

Detector

Type

Support
gases

Selectivity

Detectability

Dynamic
range

82

Flame
ionization
(FID)

Mass flow

Thermal
conductivity
(TCD)

Hydrogen
and air

Most organic cpds.

100 pg

107

Concentration Reference

Universal

1 ng

107

Electron
capture
(ECD)

Concentration Make-up

Halides, nitrates, nitriles,

peroxides, anhydrides,
organometallics

50 fg

105

Nitrogenphosphorus

Mass flow

Hydrogen
and air

Nitrogen, phosphorus

10 pg

106

Mass flow

Hydrogen
and air
possibly
oxygen

Sulphur, phosphorus, tin,

boron, arsenic,
germanium, selenium,
chromium

100 pg

103

Aliphatics, aromatics,
ketones, esters,
aldehydes, amines,
heterocyclics,
organosulphurs, some
organometallics

2 pg

107

Flame
photometric
(FPD)

Photoionization
(PID)

Concentration Make-up

Hall
electrolytic
conductivity

Mass flow

Hydrogen,
oxygen

Halide, nitrogen,
nitrosamine, sulphur

The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds
burning in the flame produce ions and electrons that can conduct electricity through the flame. A

83

large electrical potential is applied at the burner tip, and a collector electrode is located above
the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs
are mass sensitive rather than concentration sensitive; this gives the advantage that changes in
mobile phase flow rate do not affect the detector's response. The FID is a useful general
detector for the analysis of organic compounds; it has high sensitivity, a large linear response
range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.

84

Appendix B: Infrared Spectroscopy Background

Introduction and a Little Bit of Theory
Infrared (IR) spectroscopy is a convenient and effective method for determining the presence or
absence of a range of functional groups in molecules. Textbooks usually treat molecules as if
they consist of rigid bonds (a ball-and-stick approach), but, in truth, the atoms in a molecule
are in constant motion, and the bonds within molecules can be stretched and bent as if the
atoms were connected by stiff springs. In the same way that an objects motion can be broken
down into x, y and z components, the motion of atoms in a molecule can be broken down into
six vibrational modes involving bond stretching and bending. The six possible vibrational modes
are depicted in Figure 1.
Figure 1. The vibrational modes that can be observed with IR spectroscopy
H

H
Symmetric
Stretch

H
Scissoring Bend
(in plane of paper)

H
Asymmet ric
Stretch
C

H
Rocking Bend
(in plane of paper)

(int o
paper)

H
C

T wist Bend

(out of
paper)

C
H
H
Wagging Bend
(parallel t o paper)

The frequencies of these vibrations are roughly 10 12 Hz, the same frequency range as infrared
radiation. This means that when molecules are irradiated with infrared light, the radiation with
frequencies matching the vibrational frequencies of bonds can be absorbed by those bonds. IR
radiation with frequencies not corresponding to bond vibrations is not absorbed. As a result IR
spectrum can be considered to be a shadow cast by the bonds within a molecule. In other
words, the portions of the IR spectrum that are absorbed reveal the presence of certain
functional groups. Conversely, if absorption is not observed in certain parts of the spectrum,
groups that absorb in those regions of the spectrum are not in the sample being analyzed.
Only about two dozen functional groups are important for most organic chemistry analysis.
Fortunately, the absorption frequencies of these functional groups are usually only mildly
affected by nearby bonds or groups. For example, the C=O bond found in aldehydes, carboxylic
acids, esters, amides, etc. absorbs at a well-defined range. What is more, the absorption
frequency for each of the types of carbonyl compounds has characteristic absorptions that help
identify the particular group. The frequencies of these vibrations do not vary significantly from
one molecule to another, and we use the known absorption ranges of groups to determine the
presence or absence of these groups.
As a result, we can obtain the IR spectrum of a sample and compare observed frequencies of
the absorptions to those that particular functional groups are known to have. This allows one to
determine which functional groups are present or absent. We can describe the absorption
bands in terms of the wavelength ( ) of the absorbed IR radiation, but absorption frequencies
are usually described in terms of wavenumbers. The wavenumber () is simply the reciprocal of

85

the wavelength of the IR radiation absorbed expressed in cm, and the units are in reciprocal
centimeters (cm-1), as shown in Equation 1. The range from 4000 cm-1 to 600 cm-1 is generally
the range of interest to organic chemists
(Equation 1)
Two principal factors determine the frequencies of vibrations (and thus the locations of the
absorptions in the IR spectrum): the masses of the atoms in the bond and the stiffness of the
bond. Bonds containing light atoms (CH, NH, OH) vibrate faster than bonds containing
heavier atoms (C-C, C-O, C-N, C-Cl, etc.). The vibrational frequencies of bonds are also
proportional to bond strength. Stronger bonds vibrate faster because the bonds are more rigid,
and stiffer bonds absorb at higher frequencies. For example, the C=O bond absorbs at a
frequency of 1650-1800 cm-1, and the C-O bond vibrates at a range of 1050-1200 cm-1. Similarly, the C-H bond of a sp3-hybridized carbon is longer and weaker than the C-H bond of an
alkyne. C-H stretches in alkanes absorb at about 2850-3000 cm-1 but alkyne C-H bonds absorb
at about 3300 cm-1.
Not all bond vibrations are useful for identifying compounds. Some absorptions fall outside the
typical range used for IR spectra, and some bonds absorb relatively little energy, which
produces weak absorptions that can be obscured by absorptions from other groups. Some
bonds actually cannot be observed by IR spectroscopy. A vibration that causes no change in
dipole momentsuch as the CC triple bond stretch in 2-butyne, CH3-CC-CH3, (note the
symmetry around the triple bond) cannot be observed by IR spectroscopy. Only vibrations that
cause a change in the dipole moment of a bond can be observed. The greater the polarity of a
bond, the more intense the absorption. For example, the absorption caused by the stretching of
the highly polar C-O bond is quite intense, whereas the absorption caused by the less polar C-N
bond is usually weaker. There are many specialized textbooks that you may consult for more
detailed information concerning IR spectroscopy. Your lecture textbook has useful information,
too.
Stretching vibrations often produce intense absorptions (sometimes called peaks or bands)
because the changes in dipole moment tend to be larger for stretching vibrations. (This is
especially true for N-H, O-H, and C-O bonds.) As was mentioned above, some vibrations give
rise to absorptions that are too weak to observe. In addition, absorptions from various bonds
may overlap, especially in the fingerprint region below 1500 cm -1.
Some bonds give rise to overtone absorptions arising from particularly intense absorptions.
The two most common examples of overtone bands are those caused by the carbonyl (C=O)
stretch and those caused by the out-of-plane (o.o.p) C-H bending vibrations in aromatic C-H
bonds. Overtone bands for carbonyl groups show up as a fairly weak absorption near 3400 cm1
. This absorption is often misinterpreted as traces of water or alcohols because the OH group
absorbs at a similar frequency. However, OH absorptions tend to be broader and stronger. The
overtone bands from the o.o.p bends of aromatic C-H bonds appear between near 1800-2000
cm-1.

IMPORTANT NOTES:
When analyzing a known compound, you should list the peaks that you would expect to see in a
spectrum of pure material. (You may wish to obtain a copy of an IR spectrum of an authentic
sample for comparison purposes. There are several databases containing IR spectra on the

86

Internet. Consult your instructor for more information.) Knowing what your spectrum should
show will help you to pinpoint extra absorptions that are signs of impurities. The Table of
Infrared Absorption Frequencies (next page) lists the most common absorptions that are
encountered in this course.
When interpreting an IR spectrum, you should start with the most intense and easily identified
bands, then move to the weaker ones that are often used for confirmation. Bear in mind that the
values given in the table are not absolute, and frequencies can vary by 10-30 cm-1. If your
interpretation requires that several absorptions lie outside expected ranges, you have probably
misinterpreted the spectrum.
Your interpretations must be reasonable and logical. Reject any interpretations suggesting the
presence of groups could not be present. For example, absorptions near 3050 cm -1 cannot be
from the C-H stretch of an aromatic ring if it is known that the sample contains no aromatic ring!
(The absorption is the alkene C-H stretch instead.) Do not force an assignment on peaks. If a
sample is known to be a hydrocarbon, but the interpretation identifies some absorptions as
arising from C-O bonds or NO2 groups, then something is wrong.
When interpreting a spectrum, you should consider which functional groups are likely to be
present, then check the spectrum to verify the presence or absence of bands consistent with
these groups. Remember: you do not have to identify every absorption in the spectrum.
However, you should identify all of the easily recognized functional groups and bonds. You
should be able to identify all of the following basic absorptions: C-H (alkyl, aryl and alkene), CO, C=O, C=C, N-H, O-H. You should also be able to recognize the presence of nitriles, alkynes,
aldehydes, amides, and esters. Do not worry if about identifying absorptions from C-C bonds,
because these can vary considerably in frequency and intensity. Remember to consult your
instructor if you have any questions.
Table 1. Important Infrared Absorption Frequencies.

Functional Group, vibration

Alkanes and alkyl groups
alkyl CH, stretch
alkyl CH, bend
CH3, bend

Alkenes
=CH, stretch
C=C, stretch

Frequency, cm-1

Intensity

2960-2850
near 1450
near 1450 and 1380

medium-strong
medium-strong
medium-strong

3095-3010
1680-1620

medium
variable

See Table 2 for information on o.o.p. bending and its use to determine substitution patterns of
alkenes
Alkynes
CH, stretch
CC, stretch

near 3300
2260-2100

sharp, strong
sharp (weak for

87

Aromatic rings
CH, stretch
C=C, stretch
Overtones of o.o.p. C-H bending

near 3030
1600 and 1500
1800-2000

medium
medium to weak
(several weak bands)

See Table 2 for information on o.o.p. bending and its use to determine substitution patterns of
aromatics
Alcohols, phenols, carboxylic acids (OH stretching)
alcohols or phenols (dilute solutions)
3650-3590
alcohols or phenols (H-bonded OH)
3550-3200
carboxylic acids (will be H-bonded)
3300-2500

variable, sharp
strong, broad
strong, very broad

Carbonyl groups (C=O stretching)

1780-1630
strong
Aldehydes
1740-1690
strong
(Look for C-H stretches at about 2815 and 2715 to confirm aldehyde)
Ketones
Esters
Carboxylic acids
Amides

1750-1680
1750-1735
1780-1750
1660-1640

strong
strong
strong
strong

Sometimes, the C=O group produces a weak overtone band near 3400 cm-1. Do not confuse
this for an OH or NH band.) Also, the C=O stretch in amides is very strong, so it is not easily
confused with C=C stretching.
Amines, Amides
NH stretch (amines)
NH stretch (amides)
NH bend

3500-3300
3500-3300
1600

weak to medium, broad

medium to strong, can be sharp
weak-med., broader than C=C

Primary amines (R-NH2) show two bands. Secondary amines (RRNH give a single band. The
N-H stretch in amides is much more intense than the N-H stretch in amines. Be careful not to
mistake the N-H bend for the aromatic C=C stretch.
Other Important Bonds
CO stretch in alcohols and ethers 1050 (1), 1100 (2)
1200 (3, aryl carbons)
CCl
NO of nitro group (NO2)
S=O of sulfonyl group (R-SO2-R)
CN (nitrile)

800-750
1570-1500 and 1380-1300
1320-1420 and 1130-1200
2250-2100

strong
strong
strong
strong
strong
medium, sharp

88

Table 2. C H Out-of-Plane Bending Absorptions of Benzene Rings and Alkenes

Band 1 (cm-1)

Band 2 (cm-1)

671

Monosubstituted benzenes

770-730

710-690

1,2-Disubstituted

770-735

1,3-Disubstituted

810-750

710-690

1,4-Disubstituted
1,2,3-Trisubstituted
1,2,4-Trisubstituted

835-810
780-760
825-805

745-705
885-870

1,3,5-Trisubstituted
1,2,3,4-Tetrasubstituted

865-810
810-800

730-675

1,2,3,5-Tetrasubstituted
1,2,4,5-Tetrasubstituted
Pentasubstituted

850-840
870-855
870

Band 1 (cm-1)

Band 2 (cm-1)

alkene:

1000-985

920-905

geminal
disubstituted:
R2C=CH2
transdisubstituted:
RCH=CHR
cis- disubstituted: RCH=CHR

900-880

975-960

730-675

Substituted Benzene
Benzene

Substituted Alkene
monosubstituted
RCH=CH2

Systematic Method for Assignment of Infrared Absorptions

This flow-chart starts with the carbonyl (C=O) group, because it often the most intense peak in
the IR spectrum, and it is found in a wide range of functional groups. This scheme is not
foolproof, but should allow identification of the most common functional groups you are likely to
encounter. Where possible, the conclusions reached by this scheme should be confirmed by
other means such as NMR spectroscopy or chemical tests.
Step 1 If there is an intense peak (typically one of the most intense in the spectrum) in the
1820-1650 cm-1 range, there is a carbonyl group present. Proceed to Step 2 to identify the
specific functional group. If not, proceed to Step 5.

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Step 2 Is there a very broad absorption ranging from 3300-2500 cm-1? If so, a carboxylic acid
group is present (OH stretch). Proceed to Step 6 to get more information about the compound.
If a broad peak is not observed within the above range, move to Step 3.
Step 3 Are there bands near 2720 (and possibly 2820 cm -1? These absorptions reveal an
aldehyde group (O=CH stretch). Go to Step 6. If these absorptions are absent, then the
compound is either an ester or a ketone. Proceed to Step 4.
Step 4 Generally speaking, esters absorb at higher frequencies than do ketones. Saturated
esters absorb from 1750-1735 cm-1. Conjugated esters absorb from 1730-1710 cm-1. Esters can
be confirmed by looking for a CO peak around 1200-1100 cm-1. Saturated ketones absorb from
1725-1705 cm-1. Conjugated ketones absorb from 1685-1665 cm-1. Move to Step 6 for more
information.
Step 5 Is there a broad peak in the 3650-3200 cm-1 range? If so, an OH bond or an NH bond
is indicated. Alcohols and phenols generally give stronger and broader peaks than NH peaks.
In addition, the presence of an alcohol or phenol can be confirmed by looking for a CO peak
around 1200-1050 cm-1. Alcohols and phenols can be distinguished by Step 10. Primary amines
(R-NH2) give a pair of peaks in the NH range. Go to Step 6 for more information about the
compound.
Step 6 Look for sharp peaks in the area around 2250 cm-1. Peaks here indicate CN or -CC-.
The nitrile stretch is of medium intensity. The -CC- is weak, except for terminal alkynes.
Terminal alkynes will also give a sharp peak near 3300 cm-1 (CH stretch) Go to Step 7.
Step 7 Alkenes will exhibit absorptions in the 1680-1620 cm-1 (C=C stretch) as well as =CH
stretches between 3095-3010 cm-1. In addition, peaks from out-of-plane =CH bending (See
Table 1) will also be visible if any alkenyl =CH bonds are present. Go to Step 8.
Step 8 Aromatic compounds will exhibit peaks at near 1600 and 1500 cm-1 (C=C stretch) as
well as =CH stretches near 3095-3010 cm-1. Also, peaks from out-of-plane =CH bending
(See Table 1) will also be visible if any aromatic =CH bonds are present. For mono- or disubstituted aromatic compounds, the out-of-plane vibrations can also be used to determine the
type of substitution present. Go to Step 9.
Step 9 The CO stretch is found in alcohols, phenols and ethers. This absorption is useful
because it be used to determine whether the carbon is primary, secondary, tertiary, or aryl
(phenols or aryl ethers). Primary carbons usually absorb at 1050 cm -1. Secondary carbons
usually absorb near 1100 cm-1. Tertiary and aryl carbons usually absorb near 1200 cm -1.
(Obviously, if a compound is a phenol or aryl ether, it must also show an aromatic ring is
present). Go to Step 10.
Step 10 The CH stretches of saturated carbons (such as those found in alkanes and
cycloalkanes) are found between 2960 and 2850 cm -1. The peaks are usually strong. The CH
bending vibrations are visible at near 1450 cm -1. Methyl groups show peaks at both 1450 and
1380 cm-1. The peaks from bending vibrations are usually of medium intensity. Go to Step 11.
Step 11 Assemble the inferences gained from the previous ten steps to deduce which
functional groups are present and which are absent. For example, a compound which gives an
OH peak for an alcohol (but not for an acid) and which also has an C=O group cannot be a
carboxylic acid, so any possible structures containing the carboxylic acid functional group can

90

be rejected. Similarly, a compound that has a sharp peak near 2250 cm -1 contains either a nitrile
or alkyne functional group, so examination of the molecular formula should be able to eliminate
one or the other. If you are given the molecular formula, be sure to calculate the Index of
Hydrogen Deficiency, because this provides valuable information about how many rings and/or
multiple bonds may be present. At this point, you should be able to write out some possible
structures for the compound. Go to Step 12.
Step 12 For each possible structure you come up with, you should be able to examine it and
predict where IR absorptions would occur. If these predictions do not fit the actual spectrum you
have, then the structure is incorrect. For example, if you predict you have an aromatic ring with
para-substituted groups, the spectrum should show the expected absorptions for
para-substitution. If not, then it cannot be that structure. In addition, if there are large peaks that
are unexplained by your structure, it is probably incorrect, too. Structure determination is an
exercise in deductive reasoning; structures are proposed and are eliminated if their predicted
spectra do not correspond with the actual spectra. It takes practice, but it does become easier
with a bit of experience.

References
1. R.J. Fessenden, J.S. Fessenden, Organic Laboratory Techniques, Brooks/Cole, Pacific
Grove, CA, 1884, Chapter 16.
2. R.J. Angelici, Synthesis and Technique in Inorganic Chemistry, 2nd ed., Saunders,
Philadelphia, 1977, pp. 19-23.
3. W.L. Jolly, The Synthesis and Characterization of Inorganic Compounds, Prentice Hall,
Englewood Cliffs, N.J., 1970, Chapt. 21.
4. K. Nakamato, Infrared and Raman Spectra of Inorganic and Coordination Compounds,
4th ed., Wiley, New York, 1986.
5. F. A. Miller, C. H. Wilkins, Anal. Chem., 1952, 24, 1253.
6. R.S. Drago, Physical Methods in Chemistry, Saunders, Philadelphia, 1977, Chap. 6.
7. R.W. Hannah, J.S. Swinehart, Experiments in Techniques of Infrared Spectroscopy,
Perkin-Elmer, 1974

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Appendix C: Nuclear Magnetic Resonance Spectroscopy

Introduction
One of the important spectroscopic aids that organic chemists use to determine the structure of
molecules is nuclear magnetic resonance (NMR) spectroscopy. The foundation of NMR
spectroscopy lies in the magnetic properties of atomic nuclei such as hydrogen, carbon,
nitrogen, phosphorus, and many others of chemical interest.
With the purpose of studying some characteristics of a nuclear resonance spectrum, we shall
utilize the proton NMR spectrum of ethyl formate shown below in Figure 1. The following
observations should be noted upon examination of this spectrum.

O
H

O-CH2 -CH3

Figure 1. NMR spectrum of ethyl formate.

1. Several signals are found for the various protons in the molecule. Each absorption signal is
measured at a particular frequency of chemical shift which is relative to a standard
reference frequency.
2. The area under each signal is proportional to the number of equivalent protons (hydrogen
atoms) that give rise to the signal. The area under each signal may be measured by
integration.
3. Not all of the signals shown in Figure 1 are simple (single peaks which are called singlets).
Here, one signal consists of a triplet (three peaks) and another signal consists of a
quartet (four peaks). In other NMR spectra, doublets (signals consisting of two peaks), or
multiplets (signals that contain overlapping or complex patterns of peaks) are sometimes
observed.
The absence of an absolute energy scale makes the comparison of NMR spectra difficult if
there is not a universal point of reference. For this reason, an energy scale has been
established using tetramethylsilane (TMS, Si(CH 3)4) as a standard. The twelve protons in TMS
give rise to a single peak in the NMR spectrum which has been assigned a value of = 0.00.
The chemical shifts of hydrogen atoms usually produce signals between 0 and 10 . There are
a few exceptions, such as the hydrogen atoms in carboxylic acid or enol functional groups.
Almost invariably, most NMR spectra will display an absorption at 0.0 due to the presence of

92

the standard, TMS. (Often, TMS is contained in the solvents used in obtaining an NMR
spectrum, in order to make things more convenient.)
Chemical Shifts and Chemical Equivalence
Chemically equivalent hydrogen atoms in a molecule have the same chemical shift. For
example, all six protons in benzene are indistinguishable, and the NMR spectrum of benzene
shows a single signal at about 7.15 ppm as shown in Figure 2.

10

Figure 2. NMR spectrum of benzene.

Determining if two hydrogens are chemically equivalent is quite simple. Draw out the
molecule twice. In one structure, replace one hydrogen atom with something elsesay as a
bromine atom. Then do the same thing to a different hydrogen atom in the other structure. Then
compare the compounds. If the compounds are the same, then the two hydrogen atoms are
equivalent. If the compounds are different, the hydrogens are different.
On the other hand, hydrogens that are chemically distinct from one another (not chemically
equivalent) give rise to different absorption signals. This is the case for the NMR spectra of
toluene (Figure 3) and t-butyl acetate (Figure 4, next page).
Notice that the ortho, meta, and para hydrogens in toluene are chemically different, but their
chemical shifts are so similar that they overlap. (This is often the case regarding hydrogen
atoms on benzene rings bearing alkyl substituents.) Overlapping peaks can complicate the
interpretation of some NMR spectra, it is not a major issue with modern NMR instruments,
because they can resolve these peaks into distinct and separate signals. See Chapter 16 of
Klein for more information.

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CH3
H

10

Figure 3. NMR spectrum of toluene.

CH3
O C CH3

H3C

CH3

10

Figure 4. Integrated NMR spectrum of t-butyl acetate.

Integrals of Peaks
An NMR spectrum shows how many types of protons are in the molecule, and it also shows
how many hydrogens cause each peak. This information is reflected by comparing the areas of
the signals causing the peaks. The common term for the area of an NMR peak is its integral.
The more hydrogens that cause a given signal, the larger the signal appears. The area of a
signal caused by a CH3 is three times larger than that caused by a C-H or O-H group. The
ratios of all of the signals in the spectrum match the ratios of the corresponding hydrogens in
the compound. For example, the ratio of the integrals of the signals at 2.0 ppm and 0.96 ppm is
1:3. A 1:3 ratio is the same as a 3:9 ratio, which corresponds to a methyl group causing the
signal at 2.0 ppm and three chemically-equivalent methyl groups causing the signal at 0.96
pp.m. Modern NMR instruments can determine the integrals of peaks rapidly and reliably. The
steps involved in obtaining and comparing the integrals of NMR peaks are sometimes referred
to as obtaining the integration of a spectrum. Integrals are often shown by thin S-shaped curves
that straddle the peaks. The vertical length of the curves usually correspond to the integrals of
the signals.
Characteristic Chemical Shifts

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Different types of protons have different chemical shifts. The value of each chemical shift is
characteristic of the type of proton(s) within a molecule. For instance, note the three different
types of protons present in ethyl formate (Figure 1) give rise to peaks with three different
chemical shifts. Students should become familiar with the ranges of chemical shifts for the most
common types of protons. It is recommended that the beginner start by learning the general
chemical shift ranges illustrated in Figure 5 shown below.

O-H in alcohols and phenols,

N-H in amines (T hese vary widely due to solvent
effects and hydrogen bonding. )

12

O
C

O
C

O-H

11

10

CH2 -C=O
CH2 -aryl
CH2 -C=C

CH2 -OCH2 -Halogen

"T ypical"
Alkyl
Hydrogens

Figure 5. NMR chemical shifts associated with the various types of protons.
The N+1 Rule
A more detailed look at of the NMR spectrum of ethyl formate reveals that there is not only a
difference in the position of the signals and the intensity of the signals, but there is also a
difference in the multiplicity of the signals. Visible at the far right is a triplet (set of three peaks)
and in the middle of the spectrum is a quartet (set of four peaks). These two sets of peaks are
related because the hydrogens in these peaks are adjacent to each other. Hydrogens that are
adjacent to each other (and very occasionally hydrogens that are farther apart) give rise to
spin-spin splitting.
O
H

O-CH2 -CH3

Figure 6. Integrated NMR spectrum of ethyl formate, showing the splitting of the ethyl
hydrogens.
Empirically, spin-spin splittings may be predicted by the N+1 Rule. This rule states that each
type of proton "senses" the number of protons (N) that are adjacent to it. The signal due to the

95

former proton is split into the NMR spectra of many compounds, the reader should be cautioned
that it may not always apply. Apply the N+1 Rule to the NMR spectrum of ethyl formate shown
in Figure 6.
In Figure 6, the methyl hydrogens (a) are adjacent to two (N = 2) methylene hydrogens (b).
Hence the signal due to the methyl hydrogens is split into (2+1) peaks. The triplet signal at 1.3
is the result. The methylene protons (b) are adjacent to three (N = 3) methyl hydrogens (a).
Hence the signal for the methylene protons consists of (3+1) peaks. The quartet signal at 4.2 is
the result. The hydrogen atom designated (c) gives rise to a singlet at 7.9. This is because there
are no adjacent hydrogen atoms in the molecule (N = O). The result is a single peak.

96