CHEMISTRY 462
Techniques of Synthetic Organic Chemistry
Supplemental Laboratory Handout
Dr. Tomikazu Sasaki
University of Washington
Department of Chemistry
Autumn 2011
TABLE OF CONTENTS
Part I:
General Information
Summary
1-3
Safety
4-9
Chemical Waste Disposal
Laboratory Notebooks
Laboratory Reports
10
11-19
20
Part II: Techniques
Distillation
21-22
Syringe/ Inert Atmosphere
23-24
Flash Chromatography
25-26
Part III: Experiments
Experiment 1: Aspartame Synthesis
27-28
Experiment 2: Diels-Alder, Photochemical Cycloaddition
29-30
Experiment 3: The Ethylene Ketal Protecting Group
31-33
Experiment 4: Sythesis of Chyrsanthemic Acid
34-36
Experiment 5: Directed Aldol Condensation
37-39
Experiment 6: DNA Oligomer Synthesis
40-51
Literature Prep experiment will be handed out during the quarter
Part IV: Appendix
Perkin Elmer FT-IR
52-54
DPX-200 NMR
55
MestReC NMR Analysis
56
Agilent 5973 GC-Mass Spectrometer
57-58
HP 8452 UV-Vis Spectrophotometer
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Chemistry 462 Supplemental Laboratory Handout
CHEMISTRY 462 - TECHNIQUES OF SYNTHETIC ORGANIC CHEMISTRY
Summary
This course includes 2 types of experiments - trivial and easy. Trivial experiments require the
students to choose which techniques to use to determine whether their synthesis was successful
(e.g. NMR, IR or MS). It will also be up to the student to monitor the reactions and decide when
they are complete. Easy reactions involve more sophisticated reactions and again, it will be up to
the student to determine when the reactions are complete and if the intermediates are pure
enough to proceed onto the following steps.
Techniques
TLC, chromatography, NMR, IR, GC/MS, inert atmosphere manipulations, reduced pressure
distillation, HPLC and photochemical reactor
Trivial Experiments
(3 credits choose Experiment 1 or 2, 2 credits neither required)
#1: Aspartame Synthesis (Nutrasweet) - Peptide coupling using DCC
Cbz-Asp-(OBzl) + Phe-OMe HCl
DCC
Cbz-Asp(OBzl)-Phe-OMe
Pd on carbon
Asp-Phe-OMe (Aspartame
#2 Tandem Diels-Alder Photochemical cycloadditions - immersion photochemical reactor
O
hv
+
O
O
O
Acetone
O
O
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Chemistry 462 Supplemental Laboratory Handout
Easy Experiments
(2 and 3 credits - Do experiments #6, #7 and one of the following pairs of experiments: 3 and 4, 4 and 5 or 3 and 5)
#3 The Ethylene Ketal Protecting Group - Chromatography and reduced pressure distillation
O
O
CH 3CCH 2COCH 2CH 3 + HO
OH
O
O
O
CH 3- C -CH 2COCH 2CH 3 +
O
O
O
CH 3- C -CH 2COCH 2CH 3
TSOH
MgBr
II
H 3O +
III
The student is to determine the structure of the products II and III by spectral methods.
#4 Synthesis of Chrysanthemic acid – Reduced pressure distillation, inert atmosphere. This is
a multistep convergent synthesis that produces the natural product.
CO2H
Chrysanthemic Acid
#5 Directed Aldol Condensation - inert atmosphere, generation of LDA. The structures of I
and II are deduced by spectral methods.
NH 2
+
CH 3
N
O
H
1) LDA
2) PhCOPh
3) H 3O+
I
(CO 2 H) 2
II
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Chemistry 462 Supplemental Laboratory Handout
#6 DNA Oligomer Synthesis - water sensitive reaction, phosphoramidite method of
oligonucleotide synthesis. HPLC analysis of product. This reaction will be done in pairs.
NH2
C-Cytosine
N
HO
O
N
O
O
CPG resin A
phosporamidite T
N
O
phoshoramidite C
O
P
O
O
N
H
O
T-Thymine
O-
NH2
N
N
O
O
P
O
O
O-
N
N
A-Adenine
O
PO3-
#7
Literature prep – Reproducing a synthesis from a published article; specifics will be
announced later in the quarter.
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Chemistry 462 Supplemental Laboratory Handout
LABORATORY SAFETY
Non-compliance with the rules/guidelines below may result in the removal from lab and/or will
receive a deduction of ‘lab safety/clean up’ points.
Safety in a chemical laboratory is mostly a matter of common sense coupled with knowledge of
the hazards associated with the materials used by you and your neighbors. A good perception of
your surroundings is also very important in a chemical laboratory. This state of mind requires
your full attention. If there is anything that is unfamiliar or doesn’t seem right, stop what you are
doing and ask your TA or the support staff for guidance. Don’t just plow ahead if anything looks
wrong. No one will be criticized for asking. It is, however, critical that you arrive prepared for
the laboratory, having worked out the procedures in your own mind and lab notebook so you
know what you’re going to do. Safety is an important aspect of this class and we want you to
think about safety as you read this lab manual and, especially, as you work in the lab.
Approach this course with a communal spirit. The success of a laboratory course of this size
depends on the cooperation of each individual. Take care of yourself and your neighbors.
Immediately warn your neighbor if you see him/her doing something dangerous. Accidents
happen, even if you are using common sense, someone else in the lab probably isn’t. An
example of good communal spirit would be if you see your neighbor looking at their reaction by
putting their head in the hood, remind them to take their head out of the hood and lower the sash
to watch their reaction through the glass. They would much rather hear this message from you
than teaching staff and lose safety points. Respect the fact that other students use the common
laboratory equipment, such as balances, melting point apparatuses, hoods, etc. Maintain your
work area in a reasonable state of neatness so other students will walk into a clean/organized
space just as you did. For example, the balances must be kept clean, hood bench tops wiped
down, and waste jugs emptied. Reagents must be capped and left in their proper place so that
fellow students do not waste time looking for them.
The most important safety rule is to THINK! Safety rules will be strictly enforced with the
possible consequences of removal from the lab and/or a deduction of safety points. What
follows is a detailed description of the safety rules for this class. For additional information on
safety, see your text, PLKE pages 542-558.
SAFETY GOGGLES ARE TO BE PUT ON BEFORE ENTERING THE LAB AND
MUST BE WORN UNTIL YOU ARE OUT THE DOOR. State health regulations require
the wearing of soft goggles that shield the eyes from above, below, and both sides in the
laboratory. Eyes are too valuable to risk. Students will not be allowed to work in the laboratory
without approved standard laboratory goggles. Failure to observe this state health regulation
may result in removal from the laboratory and will result in a deduction of safety points.
Standard laboratory goggles that meet all state regulations may be purchased from the University
Bookstore and undergraduate stockroom in BAG 271. Safety glasses, goggles that have the air
vents removed, sports goggles, etc. are not acceptable. If you already have goggles, stockroom
personnel must first approve them before you can begin working. Because of health regulations,
goggles cannot be borrowed from the stockroom.
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Chemistry 462 Supplemental Laboratory Handout
DRESS APPROPRIATELY FOR THE LABORATORY. A lab coat is required to be worn
over your street clothes before entering the lab and not removed until after leaving the lab.
Lab coats must be full length as they must extend to your mid-thigh. Short length lab jackets are
not acceptable. Lab coats may be purchased at the bookstore and the undergraduate stockroom
in BAG 271.
You will not be allowed into lab if you are not dressed appropriately. All students in the
laboratory are required to have clothing coverage from neck to toe; there can be no exposure of
skin anywhere. Long pants, socks and closed-toed shoes that cover the whole foot are
required. Long hair should be tied back whether you are a guy or girl when in the laboratory so
that it will not catch on fire or come into contact with chemicals.
The laboratory is not a good place to wear your favorite clothes. Do not wear clothing so loose
or bulky that it hampers your work and causes a safety hazard. Extra long jeans, while
fashionable, can not drag on the ground. If your fashion sense is to have holes in your jeans,
carry a roll of duct tape because you will be asked to cover them with it. Do not wear
hosiery/tight leggings as they will “melt” upon contact with acid and some chemicals.
Closed-toed shoes that cover the whole foot with socks are the appropriate type of
laboratory footwear. Sandals, ballet shoes, Mary Janes, open holes in the shoes, flip flops, etc.,
are not allowed in the lab. If you are wearing inappropriate shoes for lab, you will be asked to go
to the undergraduate stockroom to purchase yellow booties and receive a deduction of lab safety
points. If you commonly wear the shoes not allowed in lab, it’s advisable to have a pair of
sneakers in your locker to change into before the lab period begins.
Failure to remain safely dressed (e.g., not wearing goggles correctly) will result in a loss of
safety lab points, and you will be sent out of lab to acquire the correct clothing. If you do not
return in time to complete your work, the absence will be unexcused.
GLOVES ARE AVAILABLE TO BE WORN FOR ANY EXPERIMENT. Remember,
gloves are only a temporary barrier to chemical exposure, and should be replaced when they
become too contaminated. Experiments that use hazardous materials and require gloves are
usually noted in the manual. Gloves are not to be worn while using the computers in CHB 121.
This spreads hazardous chemicals into common areas and increases the risk of exposure.
IMPORTANT NOTE: Do not wear gloves outside of the lab, if you have to open a door to go
through it, your gloves must be off! 10% of your lab grade for that day will be deducted if you
wear gloves outside of the lab. This will be enforced by all TAs, instructors, lab techs,
stockroom personnel, and anyone else you encounter in the halls.
WASH HANDS OFTEN WHEN WORKING IN LAB AND THOROUGHLY BEFORE
LEAVING. Do not taste any chemicals. Do not put your hands, pens, or pencils in your mouth
while working in the lab. If you must leave the lab for any reason such as to use the restroom
during your scheduled time, please inform your TA, friend, or neighbor before leaving the lab.
DO NOT EAT, DRINK, CHEW GUM, OR SMOKE IN THE LABORATORY. Do not
even bring these materials into the laboratory. Also, no make-up or lip balm is to be applied in
the lab.
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Chemistry 462 Supplemental Laboratory Handout
KEEP COATS, BACKPACKS AND OTHER NON-ESSENTIAL MATERIALS AWAY
FROM AREAS WHERE PEOPLE ARE WORKING. There are designated areas for the
storage of these items within the lab. If personal belongings are not stowed away they become a
trip hazard to your friends and colleagues. Additionally with improper storage, hazardous
chemicals may come in contact with your belongings increasing the risk of exposure outside of
the lab. Lockers are the best place for personal belongings that are not essential for lab.
Hall lockers: You may bring a lock from home and claim an empty hall locker for use
during the quarter. These are ideal for storing coats, backpacks, and other bulky items.
Also, a locker is the best place to store your goggles, lab coats, and proper shoes when
not in lab. Lockers must be emptied by the end of the quarter - between quarters the
locks will be cut off and the locker contents thrown away. They are available in the
hallway located on the 2nd & 3rd floor of Bagley Hall.
CELL PHONES AND HEADPHONES MAY NOT BE USED IN THE LAB. If you take
your cell phone out during lab it will be confiscated for the lab period and there will also be a
deduction of lab safety points. Cell phone use for texting, internet surfing, timing reactions,
calculations, etc is not permitted. Also, protect your cell phone from the chemicals; leave it in
your backpack. Headphones are not allowed in the lab for any reason. If you don't take your
headphones out of your ears, you will be removed from the lab and receive a deduction in safety
points.
DRUGS, ALCOHOL, OR MEDICATION THAT COULD IMPAIR NORMAL MENTAL
OR PHYSICAL FUNCTIONING ARE FORBIDDEN PRIOR TO OR IN THE ORGANIC
LAB. If you are taking prescription drugs that might fall in this category, please notify your TA
or Dr. Tracy Harvey before attempting any experiments. Anyone who displays questionable
behavior, in this or any other regard, will be removed immediately from the lab and subject to
mandatory meeting with Dr. Tracy Harvey.
LEARN THE LOCATION AND OPERATION OF THE SAFETY SHOWERS,
EMERGENCY EYEWASHES AND FIRE EXTINGUISHERS IN THE LABORATORY.
In case of a spill onto a person or clothing, IMMEDIATELY rinse with lots of water. Do not
hesitate to yell for help. Use the safety shower and/or eyewash and don’t worry about the
resulting mess. However, don’t use the safety showers for non-emergencies since they are
designed to deliver ~50 gallons of water before shutting off. Report accidents to your TA and
an incident report will be submitted to the University by your TA with your assistance. All
instructors have been certified to administer first aid. If you are not familiar with operation of the
fire extinguishers, ask your instructor to show you. Only use fire extinguishers for real
emergencies, since the chemicals they contain can cause considerable damage. For any
emergency that requires the fire department, aid cars, or police, send someone to the
stockroom for assistance.
BECOME FAMILIAR WITH ALL OF THE EXITS FROM THE LABORATORY. A
repeating siren and flashing of the FIRE indicator is the building evacuation signal. If this alarm
goes off while you are in the lab, turn off any open flames, grab your valuables, and leave the
building as quickly as possible.
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Chemistry 462 Supplemental Laboratory Handout
SCHEDULED LAB TIME: You are not allowed to be in the lab before your lab section
begins. Even if the lab door is open and another TA is present you cannot enter unless your TA
has arrived. Students are allowed to work in the laboratory only during their scheduled sections
and under the supervision of their assigned teaching assistant. Removal from the lab and a
deduction of safety points will be a consequence to breaking this rule.
NEVER ATTEMPT ANY UNAUTHORIZED OR UNASSIGNED EXPERIMENTS.
Follow the experimental procedures explicitly, checking and double-checking the identity of all
reagents before you use them. There are potentially hazardous combinations of chemicals
present in the laboratory. If you have an idea for further investigation, discuss it with your
instructor.
LAB CLEAN-UP: At the end of lab you should be sure to clean up your work area and the
areas assigned to you by your TA. In the back of each hood, there is a list stating the proper
clean up and hood shut down procedure. All equipment checked out at the stockroom must be
properly returned by the end of the period. Point deductions may be made if the lab clean up is
not done or insufficient.
WORKING WITH EQUIPMENT AND GLASSWARE
Fume Hoods: Do all experiments and keep all chemicals in the hood. The ventilation system
draws the fumes generated by an experiment away from the person working in the hood. The
walls of the hood enclose the experiment on five sides. Therefore, if on explosion or spill
occurs, the experiment can be contained. The sash should always be kept between the
individual's eyes and lowered as much as possible but with the ability to conduct the
experiment. Set up equipment at least 6 inches from the front edge of the hood. Close the sash
when you are not working in the hood. Never put your head inside the fume hood.
Do not leave a Bunsen burner or other heated apparatus unattended. The person working
next to you may not know what is involved with your setup and may be working with a
flammable material. Turn off open flames if you must leave your area. Make sure the gas taps
are completely off whenever the Bunsen burner is not lit.
Hot Plates, Bunsen burners & Aluminum blocks are hot and pose a significant burn and/or
fire hazard! Do not use flammable liquids near open flames. Most organic liquids are
flammable. Diethyl ether is especially dangerous. Flammable vapors can ignite when exposed to
hot plates. Keep papers and all combustibles away from the hot plate/aluminum block/Bunsen
burner. Turn off hot plates when not in use. Hot plates and aluminum heating blocks will
remain hot for a long period of time after being turned off. Neither hot plates nor aluminum
heating blocks give any visual indication that they are hot, so check by holding your hand a
couple of inches away while “feeling” for heat. Only after checking this way should you attempt
to pick up the aluminum heating block or hot plate. If your hot plate or aluminum block is still
cooling down, put a hot sign on them to warn others. Hot signs are located under the prep hood
in the marked drawer.
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Chemistry 462 Supplemental Laboratory Handout
Do not pick up hot objects with your bare hands. Be sure all apparatus is cool before picking
it up with your fingers. A hot glove is located in the red box within the lab if you need it.
Do not use cracked or chipped glassware. Examine your glassware for “star” cracks. Broken
glassware should be replaced immediately with new glassware from the stockroom. We can firepolish chipped glassware so it is usable, but we can’t fix cut hands. Never heat cracked, chipped
or severely etched glassware.
Do not adjust glass tubing connected to rubber stoppers. Severe cuts or puncture wounds
may result.
Lubricate rubber tubing. When slipping rubber tubing over connectors, such as filter flasks or
aspirators, lubricate with a drop of glycerin (balance area) or liquid soap (by the sinks in the lab).
Do not use mouth suction when filling pipettes with chemicals. Use a rubber suction bulb.
Do not force pipet bulbs onto pipets. Apply just enough pressure to maintain a seal between
the pipet and the pipet bulb. Forcing the bulbs may cause the pipet to slip and break, leading to
severe cuts or puncture wounds.
Broken glassware, used pipets, melting point capillaries, and TLC capillaries are to be
disposed of in laboratory glass boxes only. In each lab there are two. One is located in front
of the pillar by the balances and for the pipets that have been used with “smelly” chemicals,
dispose of these in the laboratory glass box in the prep hood. Instrument rooms have melting
point capillary tube waste bins on the island with the Mel-Temps as well as a laboratory glass
box in the room. No glass goes into the regular trash. Custodial personnel can be injured by
sharps and will stop collecting trash if they find them in the trash cans.
WORKING WITH CHEMICALS
General Chemical Safety: Horseplay and carelessness are not permitted. Add concentrated
acid to water. Waft fumes gently toward your face. Never point a heated test tube toward you or
your neighbor; the contents may erupt and cause serious burns. A separatory funnel must be
used in a hood, vented often, and pointed away from you and your neighbor. Don’t walk around
shaking separatory funnels, test tubes, or centrifuge tubes. Leave chemicals in your hood; if you
need advice from your TA, raise your hand or go to them, but leave the chemicals in the hood.
Proper chemical storage: The policy is that all chemicals need to be stored in the upright
position, clearly labeled, and capped, covered, or parafilmed. Beakers are a good tool to use to
keep vials in the upright position. Solids that are being dried until the next period need to be in a
labeled beaker loosely covered with a watch glass or parafilmed. Random drawer checks may be
done and safety point deductions will be made for improperly store chemicals.
Reagents: Read the label (contents and hazards) before using reagents. Take only as much
reagent as you need - they are expensive and time consuming to prepare. When taking reagents,
transfer the amount you need to a clean beaker or other suitable container for taking the material
back to your desk. Replace the cap. Let your TA know if a reagent stock bottle is empty.
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Chemistry 462 Supplemental Laboratory Handout
Never return unused reagents to their storage containers. If by accident you take an excess
amount of a reagent, share it with a fellow student or dispose of the excess properly.
Clean up spills immediately. The next person to come along has no way of knowing if the
clear liquid or white powder on the lab bench is innocuous or hazardous. Neutralize acid spills
with sodium bicarbonate before cleaning them up.
Keep the dispensing areas clean and pick up any spills immediately. Return all chemical
bottles to the proper location when finished with them. Hand brooms and dustpans are on top of
the flammable cabinets in the lab. Brushes are supplied at each balance. Clean off chemical
spills and keep the common areas clean.
Suggested Procedures for Cleaning Up Chemical Spills
Solid Reagents: Wipe up small spills with a damp paper towel; rinse the reagent out of the
towel with water, then dispose of the towel in the trash cans. Clean up large spills using the
broom and dustpan (located on top of the flame cabinet) and dispose of the reagent in an
appropriate waste container. If glass is present in the spill, separate the glass from the reagent
before disposal. DO NOT place solid chemicals in either the trash cans or the glass box. Spills
on the balances should be immediately brushed out using the camel’s hair brush provided; the
reagent may then be disposed as above.
Liquid Reagents (Non-organics of near-neutral pH): Wipe up the spill using a damp paper
towel or sponge; rinse the reagent out of the towel with water, then dispose of the towel in the
trash cans.
Acids: Neutralize the acid by sprinkling solid sodium bicarbonate over the area of the spill.
Clean up the bicarbonate residue with either a damp towel or the broom and dustpan, depending
upon the amount used to neutralize the acid. Dispose of the bicarbonate in the solid waste.
Organic liquids: Wipe up the liquid with paper towels. Do not rinse the paper towels or place
them in the trash. Instead, place them in a hood. Allow the liquid to evaporate and then dispose
of the paper towels in the trash cans.
Mercury: Inform your TA of the spill and they will assist you with the clean up procedure.
Obtain a “mercury sponge” from the instrument room. Moisten the sponge with water and then
rub it over the area of the spill (metal side down). The mercury should quickly become
amalgamated with the metal. When finished, place the sponge back into the plastic bag and
return it to the designated white bucket in the waste hood within the instrument room. During a
mercury spill, small droplets may spatter a surprising distance from the area of the spill,
especially if the mercury falls from the bench to the floor. Be sure to check a wide area around
the spill to be sure that all the mercury has been located and notify others in the lab to avoid the
spill area. If you have a large spill, a special mercury vacuum may be necessary; ask for
assistance.
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Chemistry 462 Supplemental Laboratory Handout
WASTE DISPOSAL
Dispose of chemical reagents and other materials properly. The proper disposal of chemical
wastes is essential for the protection of our environment. Improper disposal of chemical waste
puts at risk the health and safety of the University and the surrounding community. Chemical
wastes must be managed and discarded in the most responsible and environmentally sound
methods available. UW and the Seattle Metro expect your cooperation in reducing any
environmental impact. Your laboratory manual will specify how to dispose of chemicals used
during the laboratory period, make note of these instructions in your lab notebook. Do not put
chemicals into glass boxes or wastebaskets. Waste containers for other materials will be
provided. If you are unsure of how to dispose of a particular material, ask your instructor.
Waste disposal in CHM 220/241/242/346/347/462: In general, nothing can go down the drain
or into the trash! Use specific waste bottles (acetone, organic solvents, or aqueous acid/base)
located in your hood for collecting waste during your lab period. Empty and rinse waste bottles
into the corresponding waste jugs located in the “Instrument Room Waste Hood” On occasion,
there will be waste jugs designated for specific waste or experiments to use. When in doubt
where your waste should go, ask your TA or support staff.
Solid chemical waste has its own waste jug. This specific collection is for solid organic waste,
Drierite, sodium/magnesium sulfates. DO NOT put tlc plates, paper towels, filter paper, etc., in
this jug.
All non-chemical solid wastes used in this class go into the trash cans unless otherwise noted.
Paper towels, matches, pH paper, etc. should NOT be placed in the sinks.
Dispose of broken glassware and other sharp objects in the cardboard glass disposal boxes
as mentioned above. Cleaning up broken glass is greatly facilitated by using the broom and
dustpan (located on top of the flammable cabinet). Custodial personnel will stop collecting trash
after they find broken glass in the trashcans!
Hazard Identification: As part of the UW Laboratory Safety Manual, each laboratory has a
Chemical Hygiene Plan (CHP). This is available to all students in the lab at all times. As part of
the CHP, Material Safety Data Sheets (MSDS)** must be readily accessible to all students.
MSDS and chemical information are available at:
http://hazard.com/msds/index.php
www.fishersci.com – type compound name in "product search", then click on "MSDS" link
www.vwrsp.com/search – go to MSDS tab
http://www.sigmaaldrich.com/safety-center.html
The Merck Index - this reference book is located in the instrument room
**Material Safety Data Sheets: Material Safety Data Sheets (MSDS) are provided by the
manufacturer or vendor of a chemical. They contain information about physical properties of the
chemical and identify any hazards associated with the chemical.
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Chemistry 462 Supplemental Laboratory Handout
THE LABORATORY NOTEBOOK
A complete, accurate record is an essential part of laboratory work. Failure to keep such a record
means laboratory labor lost. An adequate record includes the procedure (what was done),
observations (what happened), and conclusions (what the results mean).
The laboratory notebook must be bound and have serially numbered pages. Record all
information in black ball-point pen. Allow space at the front for a table of contents, number the
pages throughout, and date each page as you use it. Never record anything on scraps of paper to
be recorded later in the notebook. Do not erase, remove, or obliterate notes; simply draw a
single line through incorrect entries.
The notebook should contain a statement or title for each experiment, followed by balanced
equations for all principle and side reactions, and, where relevant, mechanisms of the reactions.
Give a reference to the procedure used; do not copy verbatim the procedure in the laboratory
manual.
Before coming to the lab, prepare a table (in your notebook) of reagents to be used and the
products expected with their physical properties (see example on page 13). From your table, use
the molar ratios of reactants and determine the limiting reagent and calculate the theoretical yield
(in grams) of the desired product. Enter all data in your notebook.
When working in the laboratory, record everything that you do and observe as it happens. The
recorded observations constitute the most important part of the laboratory record as they form the
basis for the conclusions you will draw at the end of the experiment. Record the physical
properties of the product, the yield in grams, and the percentage yield. If the reaction was
unsuccessful, comments concerning the possible cause for failure and potential modifications
should be included.
When your record of an experiment is complete, another chemist should be able to read the
account with complete understanding, determine what you did, how you did it, and what
conclusions you reached. In other words, from the information in your notebook, a chemist
should be able to repeat your work.
Æ
Samples and spectra should be numbered as follows:
Your initials, notebook page number, and sample letter. For example, the fourth
sample or fraction obtained on page 53 by John Q. Chemist would be JQC 53D.
You should also include an outline of the method of purification of the product by means of a
flow sheet, which lists all possible products, by-products, unused reagents, solvents, etc., that
appear in the crude reaction mixture. On the flow sheet diagram, indicate how each of these is
removed, e.g., by extraction, distillation, crystallization, etc. With this information entered in the
notebook before coming to the laboratory, you will be ready to carry out the experiments with
the utmost efficiency.
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Chemistry 462 Supplemental Laboratory Handout
A common objection raised by students is “Why must I write down in a notebook all of the steps
in a procedure which I am following in a lab manual?” The reason for doing this is to provide an
orderly account into which your own data and observations can be incorporated. Moreover, it is
a habit that must be acquired. In later experiments, you will adapt a general procedure to your
own situation; your own specific case is unique, and the record of what you do is vital.
The laboratory notebook should not be:
• A verbatim transcription of a procedure from the laboratory manual or any other
source which you are following.
•
An ex post facto scrapbook of recollections and miscellaneous jottings.
It is impossible to reconstruct a record from isolated numbers and memory; on the other hand,
your experimental data cannot be recorded before it exists. It is necessary, therefore, to record
the salient operations, measurements, and observations insofar as possible as they are done or
made. This procedure usually will not result in a flawless copybook record, but it should be
coherent and legible.
Preparations: In general, for all reactions, the following preparations should be recorded in the
laboratory notebook and which will be spot checked by the teaching assistant at any time during
class.
1. Balanced reaction equation with data (formula, MW, mass and moles used, density
and physical properties) listed under each reactant and product
2. A detailed mechanism of the reaction, see page 14 for examples.
3. Method of starting material assay and/or necessary pre-treatment.
4. Sketch of any special apparatus to be assembled.
5. List of hazards and safety precautions.
6. Brief outline of procedure.
7. Method of assaying the reaction progress, if any.
8. Table of product mixture constituents with flow sheet for product isolation and
purification.
Starting Materials: The quality of starting materials, solvents, etc., should be considered. For
solids, a simple mp check may suffice. GC or appropriate spectral analysis may be necessary in
other cases. Attention should be given to likely contaminants that would interfere with the
desired reaction. Some procedures give specific reagent purification procedures that should be
followed (often the period BEFORE the actual experiment).
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Chemistry 462 Supplemental Laboratory Handout
Spectra: Students run their own spectra. Consult the list at the end of each procedure to
determine the spectra required for each prep. See the teaching assistant if you are unfamiliar
with any spectrophotometric equipment or techniques. Arrangements for spectra may be made
with the teaching assistant in special cases.
Products: In all synthetic experiments, it is expected that you will critically examine the crude
product at as early a stage as possible rather than proceed (optimistically) through a recipe for
purification. In most cases, it will be up to you to select appropriate methods (NMR, IR, GC,
TLC, etc.) for this examination of 1) desired product; 2) remaining starting material; 3) expected
side reaction products; and 4) other contaminants. Beyond this, an effort should be made
wherever possible to utilize an assay method in synthetic experiments to monitor the progress of
each reaction as it proceeds, again as opposed to blindly following a recipe.
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Chemistry 462 Supplemental Laboratory Handout
LABORATORY NOTEBOOK ADDENDUM
The following is material relevant to keeping your lab notebook that is not mentioned on the
previous pages. In addition, some material below may cover areas that are mentioned on the
previous pages, and in these cases, the material in this addendum replaces the material given
previously.
Labeling Samples: Properly labeling samples is one of the most important skills for you to
master this quarter. Samples must be labeled in a way that makes it possible for you to identify
the compound by having your notebook and the labeled compound. This should be true even if
you suffer total memory loss. Thus, each container of compound must bear a label and the label
name must appear in your notebook where the compound is described. Each unique substance
must have a unique label!!!!!!!!!!!!!!!!!!!!!! Here, uniqueness is based on chemical composition.
Here are some examples.
1. You take a substance and submit it to column chromatography. You collect 10
fractions and store them in 10 vials. Each vial is to be given a unique label.
2. At the end of the lab period, you remove solvent from your sample in a round bottom
flask; you cap the flask and store it until your next lab period. This flask should bear
a label.
3. You accidentally spill a portion of your sample on the floor. You decide to collect it
from the floor and keep it. This should be placed in a separate container from the one
that you spilled and both should bear a separate label (because you cannot guarantee
that both have the same composition since the spilled substance may contain
impurities from the floor).
4. You have too much substance to fit into a single vial, so you decide to put it into two
vials. These two vials should be given separate labels since you cannot guarantee that
the impurities, if any, present in the supposedly clean vials are the same.
You must label samples in the following way: MG-9-16-96-3. Here MG is your initials (Mike
Gelb), 9-16-96 is the date, and 3 is a number to distinguish different substances labeled on the
same day. This label must appear in your notebook next to the text that describes the compound.
You must also indicate in your notebook how the compound is being stored (i.e. in a capped vial,
under argon, at 4°C, in the dark). You should also give the location of the compound.
Why all this fuss? Suppose you want to locate a compound that you made 12 years ago. You
will have some notion of when it was made and thus you can go to your 1984 notebook and find
the description of the compound and its label and its location. You can then locate the vial and
verify that it is the desired compound. The only catch is that you may have used up the
compound for another purpose. What you should do (which is not required for this course) is
keep a list of all the compounds you have made. Each time you use up some compound, you
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Chemistry 462 Supplemental Laboratory Handout
should indicate this on your list. This way you can always go to your inventory list and figure
out what you have and where it is. Your notebook will tell you what it is.
Source of materials: It is not sufficient to say in your notebook what you used. You must also
say where you got it, the catalog, and lot numbers where appropriate. What does it mean to say
ethyl acetate in your reagents/solvents table? How wet is the ethyl acetate? To what extent do
impurities in the solvent affect the outcome of the reaction? Where can I try to find this reagent?
etc ..... For the purpose of this class, since it will be tedious for you to track down this info, just
indicate in your notebook that the item was obtained from the Chem 462 TA, stockroom, or lab.
But in a real research lab, you should indicate all the above info about each reagent.
What's wrong with catalog numbers? Why lot numbers? When a chemical company makes a
substance, they may run out and have to make more. There is no guarantee that the newly made
substance is compositionally identical to the first batch even though it was made the "same" way.
Thus, the company gives each synthesis product a unique batch name. What if your reaction
worked the first time with ethyl acetate from Aldrich but not two years later using ethy1 acetate
from Aldrich? Well, if you don't have the lot numbers in your notebook, there is not much you
can do when you call Aldrich complaining that your reaction didn't work this time.
Describing how you executed experiments: Here I am not looking for grammatically correct
text, just text that can be clearly interpreted by another human. Thus, it must make sense and it
must be legible. Now what do you write down and what don't you write down? The rule of
thumb is that someone with your abilities (i.e another chem 462 student) must be able to read
your protocol and understand what you did well enough so that this person could repeat the
experiment successfully having not done this experiment before. Therefore, virtually all details
must be indicated in your notebook, even if you think they are not required for a successful
experiment. You can be reasonable. You don't have to say what color pen you used to label
your vials, etc ...
Here is an example of an acceptable description of a lab protocol (the following assumes that all
reagents have been properly described, see above). Comments from Gelb are in italics:
50 mL of dry ethyl acetate (freshly distilled in glass from ketyl under argon) was placed in an
oven dried (overnight at 140°C) 250 mL, round bottom, flask fitted with an oven dried magnetic
stir bar. The ethyl acetate was added via an oven-dried syringe and the flask was maintained
under an atmosphere of argon (see drawing below):
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Chemistry 462 Supplemental Laboratory Handout
15 mL of n-butyl lithium (1.43 M in hexane) was added dropwise by
syringe to the ethyl acetate with constant stirring, no temperature bath
was used. The reaction flask was placed in a -20°C (CCl4, dry ice,
temperature monitored with an alcohol thermometer inserted into the bath). After stirring for 15
min under argon, 1.2 g of cyclohexanone in 5.8 mL of dry ether was added dropwise by syringe
with stirring over 30 minutes while the bath was kept at -20°C by periodically adding dry ice.
During this step, mild bubbling occurred and the solution turned faint yellow. The
cyclohexanone solution was made by quickly weighing the cyclohexanone, which was dried by
stirring for 4-5 hrs over anhydrous type 4A molecular sieves (prepared as described on page 45
of "Methods in Organic Synthesis" 3rd Ed. Gelb. M. H. Ed.) into an oven dried vial, quickly
capping the vial with a septum, and adding 5.8 mL of dry ether via syringe under argon.
After adding the cyclohexanone, the reaction flask was removed from the cold bath and stirring
under argon was continued as the flask warmed to room temperature (no bath). The septum was
removed and 10 mL of ice-cold 1M aq. NaCl was added with stirring over about 5 minutes. The
mix was poured into a sep. funnel, and extracted twice with 50 mL portions of aq. NaCl.
Emulsions developed during this process, but it clarified after leaving the sep. funnel for 1 hr
without stirring. After removal of the aq. wash, the organic layer was dried in a foil-capped
Erlenmeyer flask over about 2 g of anhydrous MgSO4 overnight at room temperature. The entire
flask was wrapped light-tight with foil to prevent photodecomposition of the product. This
material was labeled as MG-9-17-96-1.
The next day (top of this page should have the date), all of MG-9-17-96-1 was filtered by gravity
through Whatman type 5 filter paper to remove the dessicant into a tared (5.6789 g) round
bottom flask. Solvent was removed in a rotary evaporator (water aspirator, 40°C temp. bath).
The residue was further dried by placing the open flask in a dessicator under vacuum (oil pump,
about 1-3 mm Hg) for two hours. No protection from light was used except that the dessicator
was placed in a hood with the light inside the hood left off. This material was labeled MG-9-1896-2. The flask was weighed. 1.34 g of material was there. A small amount of this material,
about 5-10 mg was dissolved in about 0.5 mL of CDCl3 (with 1% TMS) and the sample was
submitted to NMR analysis. (Note the label name of this compound, MG-9-18-96-2 should be
written directly on the NMR; the NMR tube should have some mark on its cap so that you don't
get the sample mixed up with another sample.)
NMR indicated that the compound is the desired one (cyclohexanone) and I estimate its purity
based on NMR to be > 80%. The sample was also submitted to TLC analysis on a silica-G plate
(Merck cat. number) using ethyl acetate:CHCl3 (3:2, by volume) as solvent, compound was
detected by placing the plate in a tank of I2.
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Chemistry 462 Supplemental Laboratory Handout
Product appears pure by TLC. All of the MG-9-18-96-2 was transferred to a glass vial fitted
with teflon septum-lined screw cap. The vial was purged with argon (by inserting a needle of
flowing argon through the septum and letting the argon vent around the threading, cap left loose,
for 2-3 min, cap was tightened, and needle was removed immediately. The vial was wrapped
light-tight with foil and stored in a jar with dessicant (CaSO4) in the -20°C freezer, room 436
CHB.
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Chemistry 462 Supplemental Laboratory Handout
Sample Lab Notebook with Table of Reagents
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Chemistry 462 Supplemental Laboratory Handout
DRAWING DETAILED ELECTRON PUSHING MECHANISMS:
DOING THINGS THE PROPER WAY
In the field of organic chemistry there are a great many conventions which are accepted
throughout the entire world. These were adopted so that chemists could communicate easily
with one another. Here are some rules for drawing mechanisms which must be adhered to with
military rigor and precision. Severe penalties will be dealt to those who violate these rules.
We urge you not to test us on this point!
Rules for Mechanisms:
1.
Use electron pushing arrows to indicate electron flow in mechanisms.
2.
Draw arrows from electron rich centers to electron poor centers.
3.
Place formal charges closest to the charge-bearing atoms.
4.
No protons should be drawn under basic conditions and no hydroxide ions under acidic
conditions.
5.
Use double barbed arrows to indicate two electron processes and single barbed arrows to
indicate one electron processes.
Your strict adherence to these rules will help us make you the best synthetic chemists you can be.
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Chemistry 462 Supplemental Laboratory Handout
Chemistry 462
Advanced Organic Synthesis
LABORATORY REPORTS
To be turned in for intermediates as well as final products
Name of compound prepared
Inclusive dates of lab work
Literature reference
Student Name ___________________
Date ______________
Notebook pages __________
Balanced Equation
A + B → C + D
Mechanism (see page 14 for example)
Data Table--see page 13 for example
Properties:
mp or bp (include literature values)
tlc data (sketch tlc plate and note solvent and visualization used)
STC-I-3A
cospot
SM
5% MeOH/Acetone - Silica - UV
Spectra: Attach spectra to report
NMR
Fully analyze the spectrum — for all peaks list chemical shift, splitting pattern
and integration. Assign all peaks to the corresponding protons in the product.
Identify impurities.
IR
Fully analyze the spectrum — assign all major IR bands.
MS
Fully analyze the spectrum--assign all major m/z fragments
Comments and Conclusions:
Be brief, indicating how you knew that you made the desired compound. Mention purity and
any anomalies (e.g., low yield, unusual observations, impurities, etc.).
Questions: From the lab manual.
(Note: See syllabus for percentage of points allotted for each section.)
Notes:
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Chemistry 462 Supplemental Laboratory Handout
DISTILLATION TECHNIQUES
Setups for simple and fractional distillation at atmospheric pressure are shown (Figure 1). A 30
cm Vigreux column (Figure 1b) is convenient if the components boil at least 50° apart at
atmospheric pressure. For better separation, a column packed with glass helices is suitable. All
columns employed in fractional distillation should be wrapped or jacketed to minimize heat loss.
Figure 1. Setups for atmospheric pressure distillation
(a) for simple distillation, (b) Vigreux column for fractional distillation.
Heat sources for distillation must be closely controlled to prevent overheating or too rapid
distillation. The best heat sources are electrically heated liquid baths. Mineral oil or wax is a
satisfactory medium may be contained in a stainless steel beaker or sponge dish and heated by an
electric hot plate or immersion coil. The bath temperature (20-80°C above the boiling point) is
easily monitored by an immersed thermometer.
Distillation at reduced pressure is advisable with the majority of organic compounds boiling
above 150°C at 1 atmosphere. Aspirator pressure (20-30 mm depending on water temperature
and system leaks) is sufficient for many reduced pressure distillations. A liquid boiling at
200°C/1 atm., for example, will have a boiling point of approximately 100°C at 30 mm.
(Estimates of observed boiling points at reduced pressure can be made by use of the pressuretemperature alignment charts shown in Figure 2 or using the interactive pressure-temperature
nomograph at www.sigmaaldrich.com/chemistry/solvents/learning-center/nomograph.html). The
aspirator pump is simple and is not affected by organic or acid vapors. The pressure in such a
system is best monitored by a manometer.
A vacuum system employing an oil pump is shown schematically in Figure 3. Protection of the
pump requires that the system be well trapped between the pump and the distillation setup. The
pressure can be regulated by introducing an air leak through a needle valve (a bunsen burner
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Chemistry 462 Supplemental Laboratory Handout
needle valve is satisfactory). The pressure is monitored by use of a tipping McLeod gauge which
gives intermittent (as opposed to continuous) reading of pressure down to about 0.05 mm, of
sufficient precision for the purpose.
Figure 2. Nomograph: Pressure-temperature alignment chart
Figure 3. Schematic diagram of a vacuum system for distillation.
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Chemistry 462 Supplemental Laboratory Handout
SYRINGE TECHNIQUES
Small quantities (up to 50 mL) of air-sensitive reagents and dry solvents may be transferred with
a syringe equipped with a needle (length 1-2 ft). The long needles are used to avoid having to tip
reagent bottles and storage flasks. Tipping often causes the liquid to come in contact with the
septum. Contact of rubber septa with many organic liquids causes swelling and deterioration of
the septa, and should therefore be avoided.
A rubber septum in contact with organic vapors provides a positive seal for only a limited
number of punctures, depending upon the needle size. The lifetime of the septum may be
extended by always reinserting the needle through the existing hole. Ideally, the syringe and
needle should be dried in an oven prior to use. Naturally, the syringe body and plunger should
not be assembled before being placed in the oven. The syringe should be flushed with nitrogen
during the cooling. A syringe may also be flushed 10 or more times with dry nitrogen as
illustrated in Figure 4 to remove the air and most of the water adsorbed on the glass. A dry
syringe may be closed to the atmosphere by inserting the tip of the needle into a rubber stopper.
Figure 4. Flushing a syringe with nitrogen.
The syringe-needle assembly should be tested for leaks prior to use. The syringe is half-filled
with nitrogen and the needle tip is inserted in a rubber stopper. It should be possible to compress
the gas to half its original volume without any evidence of a leak. A small amount of stopcock
grease or a drop of silicone oil placed on the Luer lock tip will help ensure tightness.
Reagent Transfer with Syringe
The syringe transfer of liquid reagents is readily accomplished by first pressurizing the Sure/Seal
reagent bottle with dry, high-purity nitrogen followed by filling the syringe as illustrated in
Figure 5. The nitrogen pressure is used to slowly fill the syringe with the desired volume plus a
slight excess (to compensate for gas bubbles) of the reagent. Note that the nitrogen pressure
pushes the plunger back as the reagent enters the syringe. The plunger should not be pulled back
since this tends to cause leaks and creates gas bubbles. The excess reagent along with any gas
bubbles is forced back into the reagent bottle as illustrated in Figure 6. Once the correct volume
of reagent is obtained, let the syringe fill with a small volume of nitrogen to keep over the
headspace (keep the syringe tipped back as in Figure 6). The accurately measured volume of
reagent in the syringe is quickly transferred to the reaction apparatus by puncturing a rubber
septum on the reaction flask or addition funnel. Syringes with capacities up to 100 mL are
available. However, the large syringes become awkward to handle when completely full.
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Chemistry 462 Supplemental Laboratory Handout
Figure 5. Filling syringe using nitrogen pressure.
Figure 6. Removing gas bubbles and returning
excess reagent to the Sure/Seal bottle.
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Chemistry 462 Supplemental Laboratory Handout
FLASH CHROMATOGRAPHY
Find a solvent system to use in the flash column. To do this, test various solvents in TLC as
shown below. Ideally, one would like to have the compounds that need to be separated as far
apart as possible, that is, a large difference in Rf. It may be necessary to separate compounds
that don’t have a large difference in Rf, such as A and B below. In this case, the best solvent
system will be one that has the Rf of A and B between .1 and .4, and separated as much as
possible (see the lower part of 4).
50%
Hex/EtOAc
Hexane
A Rx
B
1
A Rx
2
B
75 Hexane
25 EtOAc
A Rx
3
B
90 Hexane
10 EtOAc
A Rx
B
4
solvent system #1: No good!
solvent system #2: No good, separation on TLC, but the Rf’s are to high to get
separation on a column.
solvent system #3: In this system, the high Rf spot could be separated from the
lower Rf spots but the lower spots could not be effectively
isolated.
solvent system #4: All components can be separated. A and B are as close
together in this system as they are in #3, but they are at lower
Rf, consequently they will be held in the column longer and
they should separate out.
When the solvent system has been determined, pack a column as described below and in the
diagram (Note: Load and empty columns in the Hood!). First, a cotton plug and about a ¼ cm
of sand are put in to support the silica. What would be about 6-7 in of silica in the column (you
may want to measure out about 6-7 in. of silica in the column before starting) is added to enough
solvent (in a flask) to make a slurry. Before adding the slurry to the column, add enough solvent
to fill the column ⅓ of the way full (See the figure on the next page, column #1) (add gently as
not to disturb the sand bottom). The slurry is then poured into the column and allow the silica to
settle and gently push the solvent line down to about an inch above the silica (2).
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Chemistry 462 Supplemental Laboratory Handout
solvent
solvent
sand
sand
settled silica
solvent line
cotton plug
1
2
3
4
Next, gently add about 0.5 cm of sand on top of the silica (3). This will prevent disturbing the
silica surface during solvent additions. Fill the column with solvent and push it through. The
silica should appear uniform. Air will cause white spots to appear in the packing. If it looks like
there is air in the silica, add solvent and repeat.
When the column is free of air, drain the solvent down to just below the sand level (4). Be
careful not to allow the solvent to fail below the silica level or the column will have to be flushed
with solvent to remove air again.
The sample to be chromatographed is added to, or dissolved in 0.5-2 mL of solvent (depending
on sample size) and dripped via pipet onto the sand in the column. Drain to just above the silica
level, and with a pipet, wash the inside of the column with 2 ml of solvent. Let this drain to just
above the silica level, and repeat. Fill the column with solvent, and start pushing the solvent
through at about 2 inches per min. Collect fractions and spot them on TLC.
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Chemistry 462 Supplemental Laboratory Handout
Experiment #1
ASPARTAME SYNTHESIS
CH 2 CO 2 CH 2 Ph
O
PhCH 2 O
N
H
Ph
+
CO 2 H
CO 2 CH 3
H 3N
+
Cl-
Cbz-Asp-(OBzl)
Phe-OMe HCl
DCC
Methylene Chloride, 0
C
CH 2 CO 2 CH 2 Ph
O
PhCH 2 O
o
Ph
CONH
N
H
CO 2 CH 3
Cbz-Asp(OBzl)-Phe-OMe
Pd on Carbon
Cyclohexadiene
Refluxing Methanol
CH 2 CO 2 H
Ph
H 2N
CONH
CO 2 CH 3
Aspartame--(Asp-Phe-OMe)
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Chemistry 462 Supplemental Laboratory Handout
DCC coupling of the Amino acids
Cbz-Asp(OBzl) + PheOMe
.
HCl
DCC
o
CH 2 Cl 2 , 0 C
Cbz-Asp(OBzl) PheOMe
A 5 mL conical vial equipped with spin vane, air condenser and drying tube (packed with
Drierite) is flame dried. Once the apparatus has cooled, 3 mL of dry methylene chloride is added
followed by 160 mg of Phe-OMe HCl, 260 mg Cbz-Asp(OBzl) and 0.1 mL of triethyl amine.
The solution is cooled to 0°C in an ice bath (most of the precipitate has dissolved). Prepare 1mL
of a 0.74 M solution of 1,3 dicyclohexyl carbodiimide (DCC) in methylene chloride and add this
to your reaction mixture. (Warning: DCC is a strong sensitizer, use gloves and caution). Let
the solution stir at 0°C. Monitor reaction by TLC with neat Ethyl Acetate.
Once the coupling is complete by TLC, add the reaction mixture to a filter pipet and filter off the
dicyclohexyl urea (DCU) by-product. (To prepare a filter pipet see figure 8.6, pg 623 of PLKE
4th edition). Collect the filtrate and add enough methylene chloride to make the volume
approximately 4 mL. Using separatory funnel, extract the methylene chloride solution with
saturated sodium bicarbonate followed by an extraction with 1M HCl. If an emulsion forms, add
a small amount of ethanol. The organic layer is then dried over sodium sulfate for 10 minutes.
Decant the organic layer from the sodium sulfate into a tared round bottom flask and evaporate
the solvent using rotary evaporation. The precipitate can be recrystallized with methanol/water.
mp 116-117°C. Collect all the analytical data (1H NMR, GC-MS, IR, mp).
Deprotection of the Dipeptide
Cbz-Asp(OBzl) PheOMe
Pd/C,
Refluxing Methanol
Asp-PheOMe
(Aspartame)
To a 5 mL conical vial equipped with a spin vane and air condenser, add 3 mL of methanol, 150
mg of the dipeptide, 3 drops of 1,4-cyclohexadiene and a spatula tip full (approx 3 mg) of 5% Pd
on carbon. Stir and heat this solution at reflux and monitor the reaction using TLC using a
developing solvent of 9/1/1 butanol, water, acetic acid. Add more drops of the cyclohexadiene
after 15 and 30 minutes of reflux (it is volatile and can easily evaporate from the solution).
Once deprotection is complete, remove the apparatus from the heat source and let the carbon
particles settle. Filter the methanolic solution through a pad of celite in a filter pipet (wash the
celite with 2 mL of methanol beforehand). Use of celite while filtering insures the removal of
carbon particles. Evaporate the methanol using rotary evaporation. Take a melting point of the
crude aspartame. Collect all the analytical data on crude (1H NMR, GC-MS, IR, TLC) then try to
recrystallize a small portion of it from water, but this step is very tricky. The melting point of
aspartame is 258-260°C. Collect all the analytical data on final product (1H NMR, GC-MS, IR,
mp).
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Chemistry 462 Supplemental Laboratory Handout
Experiment #2
TANDEM DIELS-ALDER, PHOTOCHEMICAL CYCLOADDITION
This sequence of reactions shows a Diels-Alder 4+2 cycloaddition followed by a photochemical
2+2 intramolecular cycloaddition to make a caged structure that will provide an interesting NMR
analysis.
O
hv
+
O
O
Acetone
O
O
O
Generation of Cyclopentadiene
Since two molecules of cyclopentadiene undergo a Diels-Alder reaction to form
dicyclopentadiene it is not possible to store cyclopentadiene. In this experiment you must first
"crack" dicyclopentadiene with heat and then to collect the cyclopentadiene by fractional
distillation.
Add about 15 mL of dicyclopentadiene to a Vigreux or Claissen fractional distillation apparatus
and heat to distill the solution. Make sure the distillate is kept cool during the entire process.
Cyclopentadiene should distill off between 40-50°C.
Fresh cyclopentadiene can be stored in the freezer for up to 5 days but is highly recommended
that you use cyclopentandiene immediately after it has been cracked. Note: Cyclopentadiene
has a strong odor--dispense the solution in the hood only. Also, thoroughly clean your
distillation apparatus before returning it to your cabinet.
The Diels-Alder Reaction
To a 100 mL round bottom flask with stirbar, add 2.0 g of purified benzoquinone* and 20 mL of
ethanol. Cool the slurry to 0°C. To this mixture, add dropwise and with stirring, 1.6 mL of
cyclopentadiene over a period of 5 minutes (The solution will become clear). Let the reaction
warm to room temperature and stir for an additional 20 minutes. Evaporate the solvent by rotary
evaporation. The Diels-Alder adduct can be recrystallized using hexane. The mp of pure product
is 71-73°C (green/yellow crystals). Collect all the analytical data on product (1H NMR, GC-MS,
IR, mp).
*Benzoquinone will need to be purified by recrystallization from hexanes. Dissolve in hot hexanes, filter off
insoluble impurity (green), and remove hexanes.
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Chemistry 462 Supplemental Laboratory Handout
Photochemical 2+2 Cycloaddition
Before proceeding, have the TA show you how to set up and use the photochemical apparatus.
Procedure: Dissolve 2 g of the Diels-Alder adduct in 500 mL of acetone. Add this solution to
the photochemical chamber and then set up for the photochemical reaction as instructed by your
TA. Turn on the UV lamp and let the reaction proceed for 30 minutes. At the end of this time
pour your reaction mixture into a pear shaped or round bottom flask and evaporate the solvent
using a rotary evaporator.
The white solid can be recrystallized using 85% hexane/ethyl acetate. mp 247-248°C. Take a
proton NMR and COSY of your sample and fully interpret and analyze your NMR spectra.
Collect all the analytical data on final product (1H NMR, COSY GC-MS, IR, mp).
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Chemistry 462 Supplemental Laboratory Handout
Experiment #3
THE ETHYLENE KETAL PROTECTING GROUP
One of the most useful procedures in organic chemistry is that of employing protecting groups in
organic synthesis. This procedure is encountered when one wishes to “protect” a functional
group from an undesirable reaction at some point in the synthetic scheme. The protecting group
may then be easily removed in a later step. Many organic laboratory texts illustrate this method
by depicting an acylation of an amine in a synthetic sequence.
Another common example of a protecting group is the use of an ethylene ketal to mask a
carbonyl group and to protect it from reaction in basic media. This lab demonstrates the use of
the ethylene ketal protecting group in a 3-step synthesis.
Also, in this lab the identity of the second intermediate and final product are withheld. It is left
to the student to analyze the experimental procedures and from them predict what products to
expect. Since there will be some element of uncertainty as to the outcome of the synthesis the
student will have to thoroughly analyze the intermediates and final product.
The reaction sequence starts with the preparation of the ethylene ketal of ethyl acetoacetate using
p-toluene-sulfonic acid as the acid catalyst.
O
O
CH 3CCH 2COCH 2CH 3 + HO
OH
TSOH
O
O
O
CH 3- C -CH 2COCH 2CH 3
Once the reactive ketone moiety has been masked, the less reactive ester group can be modified.
The ester is reacted with phenyl magnesium bromide to form an unknown structure, II. II is then
treated with aqueous acid (HCl) to give III which is ultimately purified by column
chromatography.
O
O
O
CH 3- C -CH 2COCH 2CH 3 +
MgBr
II
H 3O +
III
The student is to elucidate the structures of II and III by using techniques used in previous
experiments and their knowledge of organic chemical reactions.
Preparation of ethyl acetoacetate ethylene ketal (I)
A 250 mL round bottom flask is equipped with a reflux condenser and a Dean-Stark trap. To
this, add 30 g (0.231 mole) of ethyl acetoacetate, 15 g (0.240 mole) of ethylene glycol, 0.13 g of
p-toluenesulfonic acid monohydrate and 100 mL of heptane. Progress of the reaction can be
monitored by comparing the volumetric measurement of the aqueous phase in the Dean-Stark
trap to the theoretical yield of H2O. The reaction mixture is cooled to room temperature and
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Chemistry 462 Supplemental Laboratory Handout
washed with 35 mL of 5% sodium hydroxide, two 50 mL portions of water and dried over
anhydrous potassium carbonate. After filtration, the heptane is removed by rotary evaporation
and the residue distilled under reduced vacuum pressure (be sure to fit the claisen adapter into
the distillation apparatus or a short path) to give the ketal. If two layers form, heat and the crude
product can be used for the next step. (bp 135-138°C at 50 mm). Be sure to check the product by
1
H-NMR before moving on to the next step. Collect all the analytical data on product (1H NMR,
GC-MS, IR).
Reaction of the ethylene ketal with phenylmagnesium bromide (II)
A 500 mL round bottom flask is equipped with a reflux condenser, addition funnel and magnetic
stir bar. To this add 5.35 g of magnesium turnings. The flask is then flame dried and a Drierite
drying tube placed on top of the condenser (make sure the Drierite is actually dry - if any has
turned purple, you will need fresh Drierite). After the flask has cooled, 30 mL of anhydrous
ether is added to the flask. A solution of 31.4 g of bromobenzene in 50 mL of anhydrous ether is
added to the addition funnel. Begin by adding about 5 mL of this solution from the addition
funnel to the 3-neck flask by using the stopcock. Make sure the reaction mixture is stirring
vigorously.
When the reaction begins, it should become cloudy, turn slightly brown in color, and may give
off some heat. If the reaction fails to initiate, you can add a small crystal of iodine (about 10
mg). If this does not work, consult your TA, and they may apply some heat. Once the reaction
has begun, slowly add the solution of bromobenzene dropwise. This will require constant
attention; if the reaction goes too fast, it will give off too much heat and the ether may begin to
boil off violently. Have an ice bath ready in case cooling is needed.
After addition is complete, the mixture is heated under reflux for 25 minutes then cooled in an
ice bath. At this point, 17.4 g of the ketal in 50 mL of anhydrous ether is added to the addition
funnel, and then this is added dropwise to the 3-neck flask with stirring. Be aware that a solid
will form on the side of the flask. After the addition is complete, stir the reaction mixture at
room temperature for 30 minutes.
Before the end of the lab period, you must slowly add a mixture of 50 mL of water and 50 g of
ice to the reaction flask (this is done to quench any excess Grignard reagent leftover - because
Grignards will react violently with water, you must add water slowly). If there is no more time
left in the period, you may leave this mixture to stir until the next period. If there is still time,
then wait until the ice has melted and add 50 mL of ether. Stir the mixture until the gummy
residue dissolves (more water may be added if the residue fails to dissolve). The layers are
separated and the ether layer is washed with 50 mL of water and dried over anhydrous
magnesium sulfate. The ether is removed by rotary evaporation to give an orange liquid which
crystallizes upon cooling to give the crude product. If the separation produces an emulsion,
allow it to sit until the next lab period and it will solidify. Direct filtration and then washing the
emulsion on filter paper with cold ether will reduce the yield but will result in crystals. The
crude product is recrystallized from n-hexane (not hexanes) overnight to give pure (II), mp 9091°C. Collect all the analytical data on crude product (1H NMR, GC-MS, IR, mp)
32
Chemistry 462 Supplemental Laboratory Handout
Preparation of III
To a 250 mL round bottom flask, add 2.5 mL of concentrated HCl, 100 mL of acetone and
4.5 mL of water. 10 g of II is added. The mixture is refluxed one hour, diluted with 100 mL of
water and extracted with two 50 mL portions of ether. The combined ether extracts are washed
with 50 mL of saturated sodium bicarbonate and 50 mL of water then dried over anhydrous
magnesium sulfate. After filtration, the solvent is removed by rotary evaporation to give crude
III.
A sample of the crude product can be purified by flash column chromatography over silica gel.
Analysis
Collect all the analytical data on product (1H NMR, GC-MS, IR). Report all data that you
gathered in order to identify your compounds.
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Chemistry 462 Supplemental Laboratory Handout
Experiment #4
SYNTHESIS OF CHRYSANTHEMIC ACID
In this multi-step synthesis, you will synthesize the natural product chrysanthemic acid.
Chrysanthemic acid is derived from pyrethrin flowers and is an ecologically safe insecticide.
The reaction scheme is shown below. In this convergent synthetic plan, you will combine the
products of 2 synthetic routes in order to product your final product.
Path 2
Path 1
HO
CO2H
H2 SO4
CH3 OH
HBr ( 48%)
Br
CO2CH3
CH3
+
SO2- Na
O
S
CH3
O
NaOCH3
DMF
KOH
CO2CH3
Methyl Chrysanthemate
Ethanol
CO2H
Chrysanthemic Acid
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Chemistry 462 Supplemental Laboratory Handout
EXPERIMENTAL
Path 1: Synthesis of methyl 3-methyl-2-butenoate
Into a 100 mL round bottom flask equipped with a reflux condenser and magnetic stirrer, place
8 g of 3,3-dimethyl acrylic acid, 40 mL of absolute methanol, and 2 mL of concentrated sulfuric
acid. The mixture is refluxed for 1.5 hours. The cooled mixture is poured into 40 mL of ice
water and the resulting solution is extracted three times with 25 mL portions of ether. The
combined ether extracts are washed with 40 mL of saturated sodium bicarbonate and dried over
anhydrous magnesium sulfate. The ether extract is concentrated to approx. 20 mL on a rotary
evaporator. Do not heat the rotary evaporator as the product is volatile; watch your solvent
removal carefully. Additionally, use high vacuum on the class manifold to ensure complete
solvent removal. The residue, methyl 3-methyl-2-butenoate, is transferred to a smaller flask and
distilled under vacuum. Collect all the analytical data on product (1H NMR, GC-MS, IR).
Path 2: Synthesis of 3-methyl-2-butenyl p-tolyl sulfone
Into a 500 mL 3 necked round bottom flask equipped with a reflux condenser, addition funnel,
glass stopper and magnetic stir bar are placed 14 g of sodium p-toluenesulfinate dihydrate and
50 mL of dimethyl formamide. The slurry is stirred at room temperature while 1-bromo-3methyl-2-butene is prepared.
Into a separatory funnel is placed 25 mL of 48% hydrobromic acid and 4.1 g of 2-methyl-3buten-2-ol. The mixture in the separatory funnel is shaken vigorously for 15 minutes. After
shaking, the hydrobromic acid layer is separated, and the upper organic layer is washed with
25 mL of saturated sodium bicarbonate solution. The organic layer, now the bottom layer, is
separated, dried over anhydrous calcium chloride, and placed in an addition funnel.
The crude 1-bromo-3-methyl-2-butene is added over a 10 minute period to the stirred suspension
of sodium sulfinate via the addition funnel. The suspension is stirred for 1.5 hours while being
heated at 80-90°C. The mixture is poured slowly into 250 mL of water and stirred for 1 hour.
Leave the mixture until the next lab period.
The white crystals of 3-methyl-2-butenyl p-tolyl sulfone are separated by filtering on a Buchner
funnel. The sulfone may be recrystallized from isopropyl alcohol to afford colorless needles, mp
82-84°C. Collect all the analytical data on product (1H NMR, GC-MS, IR, mp).
Methyl Chrysanthemate: Convergence of path 1 and 2
Note: The procedure must be started on a Tuesday and not Thursday
Into a dry 100 mL round bottom flask equipped with a nitrogen inlet and magnetic stirrer, place
4.5 g of methyl 3-methyl-2-butenoate, 7.5 g of 3-methyl-2-butenyl p-tolylsulfone, and 40 mL of
dry dimethyl formamide. To the mixture is added 5 g of sodium methoxide and the mixture is
stirred at room temperature in a nitrogen atmosphere for 24-48 hours. If the reaction was
successful, the mixture will turn red. The mixture is poured into a beaker containing 12 mL of
concentrated hydrochloric acid, 25 mL of water, and 25 g of ice. The mixture is extracted five
times with 25 mL portions of hexane. If an oily layer forms in between the organic and aqueous
layers, remove it with the aqueous phase. The combined hexane extracts are washed
successively with 50 mL of saturated sodium bicarbonate, 50 mL of saturated sodium chloride
35
Chemistry 462 Supplemental Laboratory Handout
solution and dried over anhydrous magnesium sulfate. The hexane is removed on a rotary
evaporator, then the flask containing the crude products should be placed under high vacuum.
The major byproduct of this reaction will be chrysanthemic acid, which can form if moisture was
present during the reaction. If this is the only impurity present based on 1H NMR and GC-MS,
then no further purification will be required. If further purification is required, then the residue
can be fractionally distilled in vacuo to afford methyl chrysanthemate (bp 56-59°C at 1.2 mm
and chrysanthemic acid, bp 92-99°C at 1.2 mm). Collect all the analytical data on product (1H
NMR, GC-MS, IR).
Chrysanthemic acid
Into a 100 mL round bottom flask, place 2.5 g of methyl chrysanthemate, 2.5 g of potassium
hydroxide, and 40 mL of 95% ethanol. The mixture is refluxed for 2 hours. The ethanol is
removed on a rotary evaporator. 50 mL of water is added to the solid and the solution is
extracted with 25 mL of ether. The ether extract is discarded and the aqueous phase is acidified
to pH 1-2 with concentrated hydrochloric acid. The aqueous phase is extracted three times with
20 mL portions of ether. The combined ether layers are dried over anhydrous magnesium
sulfate. The solvent is removed by rotary evaporation, and the residue is distilled in vacuo to
yield an oil, bp 85°C at 0.5 mm. The oil may solidify upon standing. If not, store the oil in the
refrigerator until the next class period. The melting point of pure trans-chrysanthemic acid is 5052°C. Collect all the analytical data on product (1H NMR, GC-MS, IR, mp).
36
Chemistry 462 Supplemental Laboratory Handout
Experiment #5
DIRECTED ALDOL CONDENSATION
Purpose: This experiment is intended to illustrate 1) the use of an inert atmosphere for a
reaction which is sensitive to water and oxygen; 2) the preparation and use of lithium
diisopropylamide, a strong non-nucleophilic base; 3) the specific reaction of two different
carbonyl compounds with one another via the directed aldol condensation; 4) the formation and
hydrolysis of imines; and 5) the use of steam distillation as a method for separation and
purification.
Discussion: Until recently1 it has not been possible to control the aldol condensation so that the
enolate anion derived from an aldehyde can be condensed with a carbonyl group of a ketone
because of the rapidity of the self-condensation of the aldehyde. This problem can be
circumvented if the aldehyde is first converted to the corresponding imine. The anion derived
from this “protected” aldehyde can be added to another carbonyl group to give an easily
crystallized β-hydroxy imine adduct. Subsequent dehydration and concurrent removal of the
imino protecting group yields an α,β-hydroxy imine adduct. Subsequent dehydration and
concurrent removal of the imino protecting group yields an α,β-unsaturated aldehyde. This
overall procedure constitutes a new procedure for effecting an aldol condensation. This
procedure can be applied to a variety of aldehydes or ketones as carbonyl components.
In this experiment the student will have to elucidate the structures of I and II by spectral means.
NH 2
+
CH 3
N
O
H
1) LDA
2) PhCOPh
3) H 3O+
I
(CO 2 H) 2
II
1. See Newer Methods of Preparative Organic Chemistry, vol. 4 pp 48-66; G. Wittig and H. Reiff, Angew, Chem.
Internat. Ed. Engl. 7, 7 (1968).
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Chemistry 462 Supplemental Laboratory Handout
Ethylidenecyclohexylimine
The cyclohexylamine (bp 133-134°C) is purified by short path distillation from calcium hydride
and must be used fresh and if storage is needed, it must be air-free. A dried 25 mL round bottom
flask containing a magnetic stir bar is flushed with nitrogen and then stoppered with a rubber
septum. Cyclohexylamine (4.96 g, 50.0 mmol, 5.72 mL) is added to the flask and the flask is
cooled to approximately -10°C in an ice-salt bath.
Acetaldehyde boils at 21°C. The syringe should be precooled in the refrigerator. Avoid
breathing the acetaldehyde fumes. This reaction must be run in the hood. Acetaldehyde (2.21 g,
50.0 mmol, 2.80 mL) is added dropwise with stirring over 5 minutes via syringe , and the
reaction mixture stirred at -10°C to 0°C for 30 minutes. Anhydrous MgSO4 (2 g) and ether (5
mL) is added, and the reaction is allowed to warm to room temperature. The MgSO4 is removed
by filtration, washed with ether (5 mL), and the ether from the filtrate is evaporated by rotary
evaporation.
It is important that the imine is pure for the next step. The imine can be distilled at reduced
pressure using a short path distillation apparatus and aspirator in the hood or distillation cart. (bp
47-48°C at 12 mm).
Identify your product. If the product is not to be used immediately in the next step store it in the
refrigerator (5-10°C) under N2. Collect all the analytical data on product (1H NMR, GC-MS,
IR).
Preparation of (I)
See the T.A. before performing this reaction. A TA will show the proper technique for
dispensing the solvent and must be present while dispensing n-butyllithium in hexanes. The
preparation of (I) must be done air free.
A dry 50 mL round bottom flask containing a magnetic stir bar is flushed with nitrogen,
stoppered with a rubber septum, and equipped with a nitrogen filled balloon. Diisopropylamine
(bp 83-84°C) is purified by distillation from calcium hydride under nitrogen. Freshly distilled
diisopropylamine (1.32 g, 13.0 mmol, 1.84 mL) and dry ether (10 mL) (Obtain dry diethyl ether
from the prep hood with TA assistance or after proper training has occurred) is added to the flask
via syringe. The reaction mixture is cooled to 0°C, and a solution of n-butyllithium in hexane
(11.0 mmols, 5.0 mL of 2.2 M solution) is added dropwise via syringe with stirring. A TA must
be present while dispensing n-butyllithium, and a large beaker of dry ice must be present in
your hood in case of emergency.
The excess n-butyllithium that will remain in the syringe/needle must be quenched before any
other steps are taken. Fill a 50 mL Erlenmeyer flask with about 10-15 mL of hexanes. Place the
needle below the liquid level and gently pull hexanes into and out of the syringe several times.
Then slowly add isopropanol to the hexane solution. Note: this procedure should only be used to
quench very small volumes of n-butyllithium. For volumes exceeding 0.5 mL, the
38
Chemistry 462 Supplemental Laboratory Handout
n-butyllithium solution should be poured over dry ice (solid CO2). Never use or dispose of any
n-butyllithium without direct TA supervision.
The resulting solution of lithium diisopropylamide is stirred at 0°C for 5 minutes, and a solution
of ethylidenecyclohexylimine (1.25 g, 10.0 mmol) in dry ether (8 mL) is added via syringe
dropwise with stirring at 0°C. The mixture is stirred for 10 minutes, cooled to -78°C in a dry ice
acetone bath, and a solution of benzophenone (1.82 g, 10.0 mmol) in dry ether (10 mL) is added
via syringe over 5 minutes. The resulting solution is allowed to warm to room temperature and
stand for 24 hours during which time a white solid separates. The reaction mixture is poured into
ice water (20 mL) and extracted with methylene chloride (3 times with 15 mL). The combined
organic layers are washed with brine, dried (MgSO4), filtered to remove the drying agent, and the
solvents are removed on the rotary evaporator. The residue is crystallized from hexane to give
approximately 2.5 g of (I) as white needles (mp 127-128°C). Identify (I) and assess its purity.
Collect all the analytical data on product (1H NMR, GC-MS, IR, mp).
Preparation of (II)
A mixture of 1.54 g of (I) and 10 mg (0.11 mmole) of oxalic acid is subjected to steam
distillation. The steam distillation is most easily carried out using a 100 mL 3-neck flask with a
distillation head and an addition funnel filled with distilled water. The sample and about 150 mL
of water is added to the flask, and distilled. Water is added from the addition funnel to maintain
the solvent volume.
The steam distillation is continued until a clear distillate is obtained. The distillate is extracted
with ether (3 times with 20 mL) and the combined ether extracts are dried (MgSO4), filtered, and
the ether is removed on the rotary evaporator. The residual crude product (approximately 1.0 g,
m.p. 42-44°C) is recrystallized from pentane to separate 0.80-0.88 g (78-85%) of (II) as pale
yellow needles. Identify your product and assess its purity. Collect all the analytical data on
product (1H NMR, GC-MS, IR, mp).
39
Chemistry 462 Supplemental Laboratory Handout
Experiment #6
DNA OLIGOMER SYNTHESIS
In this experiment you will synthesize an oligonucleotide using phophoramidite chemistry. This
advanced procedure is run on micromole scale and is extremely water sensitive. You will be
working in pairs for this experiment.
In DNA synthesis, a reactive 3′ phosphorous group of one nucleoside is coupled to the 5′
hydroxyl of another nucleoside. The former is a monomer, delivered in solution, while the latter
is immobilized on a solid support. An internucleotide linkage is thus formed. After the
coupling, three other chemical reactions are necessary to prepare the growing chain of DNA for
the next coupling. In this way, a synthesis cycle is conducted, adding one nucleoside monomer
at a time. When a chain of desired length is complete, the crude DNA (oligonucleotide) must be
cleaved from the support and deprotected. This introduction will help you understand the
synthesis chemistry of the phosphoramidite method of DNA synthesis and in the lab you will
prepare a DNA piece consisting of 3-7 nucleosides (your choice on length). Once your DNA has
been made you will analyze the product by LC-MS to see if your couplings were successful.
The phosphoramidite method of oligonucleotide synthesis is the chemistry of choice for most
laboratories because of efficient and rapid coupling and the stability of the starting materials.
The synthesis is performed with the growing DNA chain attached to a solid support so that
excess reagents which are in the liquid phase can be removed by filtration. Therefore, no
purification steps are required between cycles. This support material is a form of silica,
controlled-pore-glass (CPG) beads. The particle size and the pore size have been optimized for
liquid transfer and mechanical strength. The synthesis cycle is depicted in Figure 7-1. The
starting material is the solid support derivatized with the nucleoside which will become the 3′hydroxyl end of the oligonucleotide. As shown in Figure 7-2, the starting nucleoside is bound to
the solid support (CPG) through a linker attached at the 3′-hydroxyl. The 5′-hydroxyl is blocked
with a dimethoxytrityl (DMT) group.
The first step of the synthesis cycle is treatment of the derivatized solid support with acid to
remove the DMT group (Figure 7-3). This frees the 5′-hydroxyl for the coupling reaction. An
activated intermediate is created by simultaneously adding the phosphoramidite nucleoside
monomer and tetrazole, a weak acid, to the reaction vessel. The tetrazole protonates the nitrogen
of the phosphoramidite, making it susceptible to nucleophilic attack (Figure 7-4). This
intermediate is so reactive that addition is complete within 30 seconds. Note that the
phosphoramidite is blocked at the 5′-OH with the dimethoxytrityl group.
The next step, capping, terminates any chains which did not undergo addition. Since the
unreacted chains have a free 5′-OH, they can be terminated or capped by acetylation. These
unreacted chains are also called “failure products.” Capping is done with acetic anhydride and
1-methylimidazole. Since the chains which reacted with the phosphoramidite in the previous
step are still blocked with the dimethoxytrityl group, they are not affected by this step. Although
40
Chemistry 462 Supplemental Laboratory Handout
capping is not required for DNA synthesis, it is highly recommended because it minimizes the
length of the impurities and thus facilitates product identification and purification.
The internucleotide linkage is then converted from the phosphite to the more stable
phosphotriester. Iodine is used as the oxidizing agent and water as the oxygen donor. This
reaction is complete in less than 30 seconds (Figure 7-5).
After oxidation, the dimethoxytrityl group is removed with a protic acid, either trichloroacetic or
dichloroacetic acid. The cycle is repeated until chain elongation is complete. At this point, the
oligonucleotide is still bound to the support with protecting groups on the phosphates and the
exocyclic amines of the bases A, G, and C. The oligonucleotide is cleaved from the support by a
one-hour treatment with concentrated ammoniuim hydroxide. Ammonia treatment also removes
the cyanoethyl phosphate protecting groups. The crude DNA solution in ammonium hydroxide
is then treated at 55°C for 8 to 15 hours to remove the protecting groups on the exocyclic amines
of the bases (Figure 7-6).
Summary of the DNA synthesis cycle
Each cycle of base addition consists of four steps:
1. Detritylation
2. Coupling
3. Capping
4. Oxidation
These reaction steps are repeated in the above order until all bases are added. Following
synthesis, the DNA chain must be cleaved and deprotected from the solid support. Each step
will be discussed in detail.
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Chemistry 462 Supplemental Laboratory Handout
Figure 7-1
Phosphoramidile method of oligonucleotide synthesis
Figure 7-2
Starting nucleoside bound to CPG at 3′
42
Chemistry 462 Supplemental Laboratory Handout
Figure 7-3
Removal of DMT protecting group
Figure 7-4
Activation and coupling of the next DNA base
43
Chemistry 462 Supplemental Laboratory Handout
Figure 7-5
Oxidation Step
Figure 7-6
Removal of protecting groups
44
Chemistry 462 Supplemental Laboratory Handout
Detritylation
The first step in oligonucleotide synthesis is to remove the acid-labile, dimethoxytrityl (DMT)
protecting group on the 5′-hydroxyl of the support-bound nucleoside. As shown in Figure 7-4,
treatment with the protic acids, trichloroacetic acid (TCA) or dichloroacetic acid (DCA), will
deprotect or detritylate the 5′ end. This will yield a reactive 5′ hydroxyl which can couple with a
phosphoramidite during the following addition step. The two acids can be used interchangeably
but in this lab you will use TCA.
Immediately before detritylation, the CPG support is washed with acetonitrile to eliminate traces
of the preceding reagent. Next, TCA is delivered to the reaction vessel to cleave the trityl group.
Detritylation under anhydrous conditions is a reversible reaction. The DMT cation is highly
reactive and can re-tritylate any reactive nucleophile. Detritylation is driven to completion by
the removal of the DMT cation from the reaction vessel. TCA is added two times and removed
in order to insure the completion of the detritylation step. Any residual TCA must be removed by
an acetonitrile wash to prevent detritylation of the incoming phosphoramidite. If the
phosphoramidite monomer becomes detritylated, an unwanted dimer can form in solution and
then couple to the support-bound nucleotide chain. Continued chain propagation would result in
some sequences being longer than the product, making purification difficult.
When the dimethoxytrityl, also referred to as DMT or trityl, protecting group is cleaved from the
nucleotide, it exists as a cation. When in acid solution, this cation is relatively stable and
produces a brilliant orange color. It has an absorbance maximum at about 498 nm and extinction
coefficient of about 70,000 in most solvents, such as acetonitrile or dichloromethane. It is easily
detected and quantitated spectrophotometrically.
To quantitate the trityl cation released at each detritylation step, the effluent from the TCA step
and the subsequent wash with acetonitrile is collected.
Next, the absorbance of the collected effluent is measured to quantify the trityl released in each
addition cycle. Since the trityl solution is very concentrated, it must be diluted before
quantitating or significant errors in the readings will occur. Typically, the trityl fraction is
brought up to a volume of 10 mL with 0.1 M p-toluenesulfonic acid in acetonitrile. The trityl
yield is then used to calculate the coupling efficiency of each addition step by devising the next
step absorbance value from the previous step value. From this data, an overall stepwise yield can
be determined and the expected product yield can be estimated. The molar amount of DMT
cation can be calculated using Beer’s Law:
A = εCl
where: A - absorbance
ε - extinction coefficient
C - concentration
l - path length
The overall yield is equal to the product of all the coupling yields.
45
Chemistry 462 Supplemental Laboratory Handout
Monitoring the trityl cation is very important, but the results must be interpreted with caution.
The trityl assay is only an indirect measure of synthesis efficiency. Certainly, high trityl cation
yields (>98%) must be present for a good synthesis. However, high trityl yields can be present
when a poor synthesis occurs. The reason for this discrepancy is that although this chemistry is
highly refined, it is not perfect. Unwanted side-reactions do occasionally occur. Some of these
side-reactions contribute to the trityl cation released each cycle. In particular, a low level of
extraneous chain growth other than the desired oligonucleotide occurs. Sites besides the 5′
hydroxyl group participate in coupling (e.g., imperfectly capped sites on the support or branched
sites on the nucleic bases).
Coupling
Phosphoramidites (shown in Figure 7-1) are chemically modified nucleosides which are used as
the building blocks for synthesizing DNA. They are added to the support-bound nucleotide
chain one at a time until all bases in the sequence are coupled. The cyanoethyl phosphoramidite
nucleosides have the following functional groups:
1. A diisopropylamino on a 3′ trivalent phosphorous moiety. The phosphoramidite is
very stable and is made highly reactive by the activator, tetrazole.
2. A β-cyanoethyl protecting group on the 3′-phosphorous moiety. This group prevents
side reactions and aids in solubility of phosphoramidites. It is removed upon
completion of the synthesis by using ammonia. In deprotection, ammonia acts as a
base to remove a proton on the methylene group bearing the nitrile group. This anion
is formed only in low concentration, but rapidly fragments by a β-elimination reaction
to form acrylonitrile and the deprotected internucleotide phosphodiester group.
Acrylonitrile then reacts irreversibly with ammonia to form 3-aminopropionitrile, an
inert compound.
3. A dimethoxytrityl (DMT) protecting group on the 5′ hydroxyl. The DMT is removed
during each detritylation step leaving a reaction 5′ hydroxyl available for coupling an
incoming phosphoramidite.
4. A benzoyl protecting group on the exocyclic amines of A and C (Abz, Cbz), and an
isobutyryl protecting group on the exocyclic amine of G (Gib). These amide groups
prevent side reactions and are removed upon completion of the synthesis with
ammonia. Since thymidine is unreactive and does not contain an exocyclic amine
moiety, it is not protected.
Before beginning the coupling step, the support is made anhydrous and free of nucleophiles (e.g.,
water) by an extensive wash with acetonitrile. Any extraneous nucleophiles will compete with
the support-bound 5′-hydroxyls for the activated phosphoramidite and will decrease coupling
efficiency. Tetrazole, the phosphor-amidite activator, is then delivered to the reaction vessel
followed by the Phosphoramidite.
When these reagents mix, the mild acid, tetrazole, (pKa = 4.8) transfers a proton to the nitrogen
of the diisopropyl group on the 3′ phosphorous (see Figure 7-4) This protonated amine makes a
46
Chemistry 462 Supplemental Laboratory Handout
very good leaving group upon nucleophilic attack by the tetrazole to form a tetrazolyl
phosphoramidite. This is the reactive intermediate which forms the internucleotide phosphite
with the support bound 5′ hydroxyl. A molar excess of tetrazole ensures that the
phosphoramidite will be activated. The excess phosphoramidite relative to free 5′ hydroxyl
ensures that the reaction is nearly quantitative (over 98% coupling).
Capping
Because coupling is not always quantitative, a small percentage (up to 2%) of support-bound
nucleotides can fail to undergo addition. These truncated, or failure sequences, will remain
attached to the support. If they remain in the hydroxyl form, they can propagate in subsequent
coupling steps. Failure sequences with one less base than the product would then be generated
making isolation of the product more difficult. Capping the remaining free hydroxyls by
acetylation eliminates this problem. The capped failure sequences are then prevented from
participating in the rest of the synthesis reactions.
Oxidation
The newly formed internucleotide linkage is a phosphite (trivalent phosphorous) triester. The
phosphite linkage is unstable and is susceptible to acid and base cleavage. Therefore,
immediately after capping, the trivalent phosphite triester is oxidized to a stable pentavalent
phosphate triester. This is shown in Figure 7-5.
Oxidation follows capping to eliminate the possibility of traces of water from the oxidizing
solution causing acetic anhydride to form acetic acid during capping. This would expose the
oligonucleotides to acid as well as make capping less effective.
Iodine is used as a mild oxidant in a basic tetrahydrofuran (THF) solution with water as the
oxygen donor. When the iodine-water-pyridine-THF mixture is delivered to the reaction vessel,
an iodine-pyridine complex forms an adduct with the trivalent phosphorous. This adduct is
decomposed by water with production of a pentavalent phosphotriester internucleotide group.
This is an extremely fast reaction, being quantitative in 30 seconds. The iodine solution is
removed and the support washed several times with anhydrous acetonitrile.
Other oxidizing agents such as sulfur can be used in place of oxygen to create nucleotide
phosphate analogs or to introduce radioactive atoms.
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Chemistry 462 Supplemental Laboratory Handout
Deprotection and Cleavage
Following the synthesis, the DNA remains covalently attached to the support. The diester
oligonucleotides are then cleaved from the support by a one-hour treatment with fresh,
concentrated ammonium hydroxide. As seen in Figure 7-6, the cleavage occurs at the base-labile
ester linkage between the linker of the support and the 3′ hydroxyl of the initial nucleoside. The
cleaved DNA has a free 3′ hydroxyl.
The DNA, now in solution, is collected in an Eppendorf tube. The tube contains the crude mix
(product and failure sequences) of base protected oligonucleotides in ammonium hydroxide.
Then, the protecting groups on the exocyclic amines of A, G and C must be removed.
Phosphate and Base Deprotection
The cyanoethyl protecting groups are removed by treatment with ammonium hydroxide. This
occurs at the same time as cleavage making phosphate deprotection very quick and simple. See
Figure 7-6.
The benzoyl and isobutyryl base protecting groups are removed by placing the vial of DNA at
55°C for 8 to 15 hours. This also cleaves the acetyl caps from the failure sequences. Base
deprotection is an ammoniolysis reaction, where ammonia is a nucleophile, attacking the
carbonyl of the amide protecting groups. For effective treatment, fresh, concentrated ammonium
hydroxide is used during cleavage.
Quantitation of the Oligonucleotide
Nucleic acids of any variety are most easily quantitated by UV spectroscopy, measuring at or
near their UV absorbance maxima, about 260 nm. A dilute aqueous solution of 1 mL or less,
depending on the cuvette size, is measured by either scanning a region of about 200-350 nm or a
single wavelength measurement. A scan of an oligonucleotide will show broad absorbance with
a maxima near 260 nm. Using Beer’s law, the concentration of the solution and absolute
quantity can be calculated. As a useful approximation, 1 optical density unit (odu) of singlestranded oligonucleotide consists of about 33 micrograms, by mass. An approximation to relate
absorbance to molar quantities is that a micromole of oligonucleotide has a number of odu equal
to 10 times the number of bases. For example, a micromole of a 20mer would be 200 odu.
Procedure for Phoshoramidite DNA Synthesis:
The following should be done the lab period before you begin the synthesis.
**Place the items below in the desiccator provided (use no drying agent) and then kept under
high vacuum overnight:
Æ
Special fritted funnel.
Æ
~10 disposable syringes.
Æ
Phosphoramidites: Add a 17 mg (1 µmole) portion of each phosphoramidite to a
glass vial (flame dried), seal with septum, and insert needle. You may prepare
anywhere from 3-7 phosphoramidite samples
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Chemistry 462 Supplemental Laboratory Handout
EXPERIMENTAL
1. Weigh out 1 µmole CPG resin (30-35 mg) and place resin in the fritted funnel. The
funnel should be capped with a septum and connected to a nitrogen line and bubbler. (see
Figure 8 on next page)
2. Wash resin with acetonitrile (0.5 mL)
3. Add 1 mL 3% trichloroacetic acid/methylene chloride solution. After 30 seconds drain
solution (using aspirator pressure while keeping the nitrogen pressure positive) into trityl
collection tube. Add another mL of 3% TCA/Methylene chloride for 15 seconds, and
then drain again into trityl collection tube. Combine trityl collections and save for UV
analysis. UV analysis must be done within 12 hours (see procedure at the end of this
experiment).
4. Wash residue 3 times with 1 mL portions of acetonitrile and discard.
5. Purge funnel with nitrogen for 3 minutes.
6. Add 0.5 mL of DRY acetonitrile to the vial containing the phosphoramidite.
7. Wash the CPG resin for 5 seconds with 0.5 mL DRY acetonitrile. Drain to waste.
Repeat.
8. To the CPG resin, add 100 µL 0.5 M tetrazole/MeCN followed by 0.5 mL of the
phosphoramidite solution prepared in step 6 above. Mix well for two minutes but don't
splash!
9. Filter off the solvent. Add 150 µL of 1/5 methyl imidizole/THF followed by 150 µL of
1/1/8 acetic anhydride/lutidine/THF. Mix for 10 seconds, drain to waste (this is the
capping step).
10. Add excess (1 mL) of iodine/water/pyridine/THF solution for 30 seconds and then drain
to waste.
11. Wash resin 8 times with MeCN (1 mL) and go back to step 3 to add another base. If you
have added your last base, go back and complete step 3 to remove the trityl group, and
then continue on with the removal of the DNA from the resin (see procedure on next
page). After adding the last base, the reaction is no longer water sensitive.
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Chemistry 462 Supplemental Laboratory Handout
Figure 8
Removal of DNA from resin: The lab technicians will do this procedure.
1. Put resin in 1 mL concentrated ammonia and let the solution stand 3 hour at room
temperature. At this point your TA or lab tech will perform the following two steps and
return your sample to you during the next lab meeting
2. Decant the solution (save) from the resin and wash resin with 2 mL of concentrated
ammonia. Combine ammonia portions.
3. Place the ammonia solution in a pre-weighed Eppendorf tube, heat in hot block at 55°C
for 12 hours. Evaporate to dryness using a lyophilizer or speed vac and weigh the
sample. Note: Your DNA sample can be stored in the ammonia solution at 0°C for up to
a week.
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Chemistry 462 Supplemental Laboratory Handout
UV analysis of the trityl collections
This is done the same day as the synthesis in BAG 83.
From the amount of the trityl groups isolated, you will calculate the coupling efficiency of each
phosphoramidite coupling step using Beer's law.
For each trityl fraction collected, dilute the sample to 4 mL using 0.1M p-toluenesulfonic acid in
acetonitrile (toluene sulfonic acid prevents decomposition of the trityl group). From this sample,
take 100 µL and dilute to a volume of 5 mL. Take the UV spectrum of the 5 mL samples at 498
cm-1. Using Beer's Law, calculate the coupling yield.
LC-MS analysis of the DNA sample.
You will sign up for an appointment with your TA for an LC-MS analysis of your product.
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Chemistry 462 Supplemental Laboratory Handout
PERKIN-ELMER FT-IR INSTRUCTIONS
WARNING: Do not use gloves on the controls of this machine.
Press enter if the screen is blank.
For the scanning process below you will sometimes use the softkeys which are the top row of
grey keys on the key board. On the lower portion of the screen you will see the softkey label and
below the label is the corresponding softkey.
Scanning your sample:
Place your salt plate into the 'v-groove' holder. Be sure to close the door of the sample
compartment.
On the key pad press the green 'scan' key followed by the softkey '4'.
Wait for the scanning to finish after about 20 seconds – the display will read “Ready”. You will
see your spectrum on the screen.
Manipulating the data:
Moving the spectrum: use arrow keys
Enlarging/reducing: use “< >” “> <” keys
SHIFT functions: use the shift and arrow keys to access the following functions:
Rerange
maximizes the horizontal range
Rescale
sets the %T (y-axis) range from 0 to 100
Autex
sets the %T so that your spectrum fills the screen
Peakcur
brings up a vertical line to mark peaks; move with arrow keys
Mark
peaks marked will show the wavenumbers when printed
If your spectrum does not look as you would expect, use the following sequence of functions to
reset the range and fill screen with the spectrum:
Shift -> Rerange -> Shift -> Autex
Plotting your spectrum:
Press the plot key (not the print key) located in the lower portion of the keyboard.
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Chemistry 462 Supplemental Laboratory Handout
INFRARED SAMPLE PREPARATION TECHNIQUES
Salt Plates, KBr pellet presses, and mortar & pestles are checked out from the stockroom.
Note: PLKE has excellent and more detailed instructions for the preparation of IR samples (see
Technique 25, Part A, pp.834-847).
Liquid Samples
Thin film
The fastest sample preparation technique is simply to place a drop of liquid sample between two
salt plates (KBr) and squeeze gently. If this is done properly, the film has enough surface tension
to hold the plates together. Caution should be used to prevent air from getting back into the
sample after it has been compressed. If the spectrum is too concentrated (many peaks bottoming
out at 0 %T) try adding a much smaller volume of sample to the salt plate.
Solid Samples
ATR (attenuated diffuse reflectance)
Please see your TA if you have not had instruction on this apparatus. Use the IR that is
designated "For Solid Samples Only”. Grind a small amount of your solid (~100 mg) in a mortar
& pestle. Carefully place this solid on top of the small zinc selenide crystal in the center of the
apparatus. Do not contact the crystal with metal (your spatula) or paper products (e.g. Kim
Wipes). Use a cotton Q-tip to maneuver your solid to fully cover the crystal. Once the solid is in
place gently lower the press onto your sample/crystal (rotate the two black circular knobs). Scan
the specta as you would for a liquid. For clean up, carefully sweep up your solid reside with the
brush and dispose of in the solid waste container. Finish cleaning the zinc selenide crystal using
a Q-Tip dipped in 2-propanol (not Kim Wipes)
Potassium bromide pellets (see PLKE, page 840)
In this method, a 1 mg solid sample is mixed with 80 mg of potassium bromide (located in the
oven) and pressed between two stainless steel bolts in a threaded barrel. The two materials are
ground to a find powder using a mortar and pestle. Screw one of the bolts into the barrel of the
KBr press leaving one to two turns left. Pour the mixture into the open end of the pellet press and
tap lightly on the benchtop to evenly distribute on the face of the bolt. The second bolt is then
carefully screwed in until it is finger tight. Place the head of the bolt into the hexagonal hole that
is attached to the benchtop. Using the torque wrench, making sure direction indicator on the head
of the wrench is pointed to the “R,” tighten the bolt system until the wrench makes a loud click
for the first time which is at 120 in/lb. Keep the bolts tight under pressure for approximately 60
seconds so that the crystals "settle". Be sure not to tighten the bolts too much–be firm, don't give
it too much muscle. If you heard the loud click, not the softer clicks of the ratchet mechanism,
you are done! Reverse the wrench by switching to “l” and rotating in the opposite direction.
Remove both bolts and place the cylindrical chamber containing your pellet into the sample
holder within the IR equipment. Some pellets will appear white. You may have used too much
sample. If the pellet is more than 1-2 mm thick, you probably should regrind and remake it with
less material. Your pellet could also have the freckled look. Tiny but distinct spots are apparent
throughout the pellet. This arises from insufficient grinding.
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Chemistry 462 Supplemental Laboratory Handout
Methylene chloride solution
Dissolve ~100mg of your solid in a small amount of methylene chloride (approximately 1 ml).
You may heat the solution to help it completely dissolve. Add 3-5 drops of this solution to a salt
plate and let the methylene chloride evaporate. Once evaporated, it should leave a thin film of
your solid on the plate that is ready to be analyzed.
Mineral oil (Nujol) mulls
A relatively simple sampling method for softer organic samples is a mull. The proper approach is
to use an agate mortar and pestle. Place a few milligrams of the sample into the mortar and grind
it until it looks like a thin film. At this point, add a drop of mineral oil and continue to grind. The
particle size must be reduced before the sample can be lubricated. Mineral oil has a considerable
spectrum of its own, being a hydrocarbon of high molecular weight. See the sample mineral oil
spectrum located by the IR.
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Chemistry 462 Supplemental Laboratory Handout
DPX-200 OPERATING INSTRUCTIONS
The most current DPX-200 operating instructions will be posted on the course website
separately.
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Chemistry 462 Supplemental Laboratory Handout
MESTREC NMR ANALYSIS INSTRUCTIONS
1. Locate the Raw Data File (FID file)
a. Right click on START and choose -> EXPLORE
b. Enter in the address bar at the top "ftp://phoenix" (without the quotation marks).
Please note….Outside of the OSC or CHB121, you may need to enter the full path
"ftp://phoenix.chem.washington.edu" for this to work. WAIT...and keep waiting until
the data directories are displayed (this can take a while).
c. Navigate through the directories DPX200. In the “DPX200” directory, you will see
the NMR user directories that were created for different courses in the NMR machine
located in CHB 118. You must identify the correct user name that was originally
used to collect the data, from this displayed list (unless otherwise instructed, this is
always in the form “chemXXX” with the XXX replaced by the course number, e.g.
“chem462”). Double-click it, find the data set that you created then drag the whole
directory to the desktop. Rename if desired.
2. Open the MestRe-C program by clicking on the icon on the right side of the screen
3. Click on
or File-Import Spectra
4. Open the file called ‘fid’ contained within the data folder that you copied.
5. Click on
or Process-Fourier Transform; press return to accept ‘along t1 axis’
6. Click on
or Process-Phase correction -Automatic Phase Correction
or going to Tools – Reference. Center the cursor
7. Set the reference peak by clicking on
on the rightmost peak (TMS) and select it. Set this peak to 0.
to Integrate or Tools – Integration – Integrate. Use the magnifying glass icon to
8. Click on
enlarge the spectrum as needed. Left click to mark the beginning and ending of the integral.
The integral values can be changed via right clicking on the integrated value and selecting
the Integration Manager.
Other helpful buttons:
The mouse wheel will enlarge or reduce the spectrum vertically
Peak picking (Tools – Peak Pick – Peak Pick) – Drag a box around the tops of the peaks that
you want to mark. The ppm will be marked at the top of the page. Coupling constants can be
determined by converting ppm differences into Hz.
Text – Insert a title or other notations into your spectrum
The salmon colored binders in CHB 121 and the organic study center contain a list of common
solvents and their chemical shifts to help identify peaks that do not belong to products or starting
materials Mestre-C is available on a trial basis from www.mestrec.com.
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Chemistry 462 Supplemental Laboratory Handout
AGILENT 5973 GC-MASS SPECTROMETER
Data Acquisition:
1. IF the software isn’t running, select the Mass Spectrometry icon from the desktop. Two
programs will start labeled ‘GC/MS MSTop/Enhanced’ and ‘GC – Mass Spec’. Note: These
programs should already be running.
IMPORTANT: If your sequence is already running, click on ‘Edit Sample Table’,
add your sample to the end of the list as detailed in step 2, and click ‘OK’. If a
sequence table from a different class is running, your data may be saved to a different
folder. Always put your samples on the end of the list.
2. If no sequence is running: From MS TOP, go to Sequence –> load and open sequence file
chem463.s (or chem346.s, etc). Return to sequence and select edit sample log table. Fill in
the appropriate data for each vial to be analyzed. Use “repeat” and “cut” to add or delete
entries.
Type – should always be ‘Sample’.
Vial number – corresponds to the numbered slots in the autosampler tray.
Method – There should be a method for your class: chem463, chem346 etc. Note –
no spaces or characters other than letters/numbers!
Data file – enter the designation for the sample. The data will be saved under this
name. If you use the same name your files will be overwritten. Any characters other
than letters and numbers will not save properly and possibly stop the sequence.
Press ‘OK’.
3. Make sure that the solvent vials A and B on the injector carousel are filled with solvent and
that the waste vials are emptied. The solvent vials usually contain methylene chloride (A)
and acetone (B).
4. Put your sample vials in the appropriate slots in the auto sampler tray. The samples should
be at least ½ full. Go to Sequence –> Run sequence. Check that the data is being sent to
D:\MSData\undergrad\<yourclass>. Click on ‘Run sequence’.
5. To restart a run from the middle of the sequence, choose Sequence –> Position and Run.
You will be prompted for the entry at which to start the run.
Data Analysis:
1. To analyze data from a run for which the acquisition is complete, select GC Data Analysis.
Click File –> Load and select the datafile that you want to look at. The data should be in
acropolis/MSDdata/Undergrad/ <your class>/<your datafile>. This drive should be available
in the drop down menu under either “m:” or “z:”.
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Chemistry 462 Supplemental Laboratory Handout
2. The total ion chromatogram (TIC) from the acquisition will be displayed when the data file
has loaded. To zoom in on a selected area of the TIC, drag a box over the plot with the left
mouse button. Double click with the left mouse button to display the previously selected
region.
3. To display a selected mass spectrum, double click with the right mouse on the TIC over the
desired scan. Drag the right mouse button over a retention time range on the TIC to display
the average of several scans.
4. To subtract the background from the spectrum, drag the right mouse button over the TIC
baseline adjacent to the peak displayed, and then select Subtract from the Spectrum menu.
5. To print a copy of the displayed spectrum select Print from the File menu, choose Selected
Window, then enter 1 as the window to print, or select TIC/Spectrum.
6. To generate a tabulated mass list of the displayed spectrum, select Tabulate from the
Spectrum menu. Select Print in the Tabulate window for a printed copy of the mass list,
and Done to clear the window.
7. Double right click in the lower pane (showing the mass spec) to do a library search of your
data. A new window will appear showing the spectrum that you took and the best match
found by the computer.
8. Click on File –> Print Autoreport to get a printout of your data and the best match.
9. Quit the Data Analysis window when you are done by selecting Exit from the File menu.
Library Search:
1. Return to the TIC window. The left mouse button can be used to draw a box around any
peaks of interest. This will enlarge this area. Using the right mouse button, draw a box
around a peak. Below will now be the mass spectrum (MS) of this peak. Go to ‘file’ and print
out the TIC and spectrum.
2. To identify this peak, double click anywhere on the MS (lower window) with the right mouse
button. This will initiate a library search that will match your MS to mass spectra contained
in a large electronic library. The results will be presented as several choices with the top one
being the most likely candidate. Look through the possibilities and select the most likely
possibility. On the right hand side there will be a structure of the compound you select. You
have the option of printing this out or recording the identity in your notebook. Click on
‘Done’ when finished with the search.
3. Go back to the TIC and make a box with the right mouse button around any smaller peaks to
generate its MS and subsequent library search.
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Chemistry 462 Supplemental Laboratory Handout
HEWLETT-PACKARD 8452 UV-VIS SPECTROPHOTOMETER
There is one HP 8452 spectrophotometer in the spectroscopy lab. The HP 8452 instrument is
calibrated for UV to visible spectroscopy and scans the spectral range from 190 to 820 nm.
Power Up - Set Up:
1. You should find the spectrophotometer, computer, printer, and monitor powered up and
ready to go when you sit down. Click on the software icon “UV Vis Instrument 1 online.”
If not seen immediately on the desktop, go into the all programs portion of the computer to
select the correct software.
2. A dialog box will pop up for an operator name which is always “default” and no password is
needed.
3. The mode used for the experiments in Chem 462 is standard.
4. Next select the Task that you would like to perform.
Spectrum/Peaks allows you to specify how many of the tallest peaks and deepest valleys
the software will show data for.
Set Up: Indicate the number of peaks and/or valleys that you are interested in. Most
likely, for this course, you only want the peaks to be displayed.
Data Type: Absorbance
Display Spectrum: Insert the range of the spectrum in nm that you would like the
software to display.
Fixed Wavelength will show information on the wavelengths that you indicate.
Set Up: Indicate the specific wavelengths that you are interested in.
Data Type: Absorbance
Display Spectrum: Insert the range of the spectrum in nm that you would like the
software to display.
5. Sampling is always set to manual.
A note can be made that the equipment will collect data from 190-820nm whether or not it is
selected so another scan is not needed to get information from other parts of the spectrum than
originally selected.
Data Acquisition:
1. Turn on the lamp by clicking the lamp icon in the software so it is illuminated (lamp on) and
not if not illuminated (lamp off).
2. Run a blank with solvent only by clicking blank.
3. To collect data for the sample, click sample. A table will list the absorbances for the tallest
peaks and deepest valleys. You may want to deselect the valley annotation.
4. Print your spec file by clicking print.
59