Life Sciences 1a Laboratory – Fall 2006
Lab 3: It’s all fun and games until someone loses an eye
(AKA Chemistry lab safety)
Goals of the lab:
1.
2.
3.
4.
To
To
To
To
introduce you to safe laboratory practices
orient you to your new lab surroundings
prepare you for techniques you will use in lab 4
be able to pass the lab safety quiz
Please make sure that you are intimately familiar with the content below before
arriving in the laboratory for the first lab.
When you do arrive for your first day of lab, please arrive with:
• this lab manual
• your lab notebook
• clothes that cover your legs
• shoes that cover your entire feet
• a calculator
Visit
www.courses.fas.harvard.edu:80/~orgolabs/PreLab_Videos/PreLab_Videos.tlkt
and view the Column Chromatography and TLC prelab video. If prompted,
name: orgolabs password: aldol. You will not be carrying out column
chromatography in this lab, however the general principles of how to carry out
thin layer chromatography (5 min 2 sec into the video) will be especially
relevant.
I. Safety
General Safety Considerations
A chemistry lab can be a dangerous place to work. There are flammable
solvents, toxic and corrosive chemicals, electrical equipment, delicate glassware,
and more. The potential for injury from these dangers can be minimized if you
are aware of and follow proper safety procedures. The most important
safety precaution you can take is to come to lab prepared and to think
about what you are doing. The most dangerous thing in a lab is an
unprepared student trying to carry out a procedure he or she does not
understand.
Eye protection
Safety goggles, located just inside the entrance to Science Center 117, must be
worn in the lab at all times. They must be worn from the second you walk in
until the second you leave, even if you are not doing chemistry. There are
absolutely no exceptions. The American Chemical Society has determined that
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Life Sciences 1a Laboratory – Fall 2006
only safety goggles, not safety glasses, provide adequate protection for students
in a chemistry laboratory.
The American Chemical Society has determined that contacts, when worn in
conjunction with safety goggles, are permissible in a chemistry laboratory. We
have adopted their recommendation as our policy. Should you get something in
your eyes while wearing contacts, make sure to tell your TF or medical personnel
that you are wearing contacts.
Clothing
Wear long pants, preferably something sturdy like jeans, and closed-toed shoes.
No shorts or open-toed shoes are permitted in the laboratory. You will be
immediately sent home if you enter the lab wearing shorts, sandals, flip-flops,
open-toed shoes, etc. There are lab coats available on the coat racks for those
who want to use them. The possibility of ruining your clothing exists so do not
wear clothes you cannot live without. Most damage to clothing shows up after
you’ve washed them (so don’t take it out on the machine). If you have long
hair, tie it back.
Gloves
You should wear gloves any time you’re working in the lab. You will find latexfree disposable purple nitrile “Microgrip” gloves (S, M, L, XL) in the lab. They are
only designed to protect you from splashes and other incidental contact with
chemicals. Acids, bases, and organic solvents will penetrate quickly through
these gloves, so you should not continue to wear a pair of gloves if you have
spilled anything on them. Even if you haven’t spilled anything on them, you
should change gloves periodically since they will tend to pick up traces of
chemicals when you handle reagent bottles, glassware, and equipment.
Changing gloves periodically will prevent you from spreading these chemicals
onto your lab book and everything else you touch. Never rub your eyes while
you’re wearing gloves. In rare instances, the purple nitrile gloves may irritate
your skin. Let your TF know if this should happen.
You should wash your hands several times while in lab, especially just before
leaving.
Food and Drink
It is important to eat before coming to lab. You’ll be on your feet for several
hours and without providing energy for your body, you may become tired and/or
light-headed. Food or drink in the laboratory is prohibited. Smoking is not
allowed in the Science Center, especially in the laboratory. If you start to feel ill
during lab and think some food or a drink may be the remedy, let your TF know
and you may go to the Science Center Food Court.
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Chemicals
If you spill anything on your skin, immediately wash the area with lots of water,
and notify your TF. If you spill a chemical all over your hood or the floor, tell
your TF, who will help you clean it up. If you spill a large amount of chemicals
or a particularly dangerous chemical on your body and/or your clothes, you may
need to use a safety shower. Get a TFs attention by yelling and proceed directly
to a safety shower and pull the chain. Remove the affected clothing (trust us,
modesty should be the last thing on your mind if you need the safety shower).
The TFs will direct other student’s attention away from you and provide a blanket
and a lab jacket for you when the rinsing is done.
Wash your hands thoroughly before you leave the lab. There is soap at each
sink.
Keep the lab clean. If everyone cleans up after themselves, the lab will be safer
and easier to work in.
Put waste in the appropriate waste containers.
Material Safety Data Sheets (MSDS’s) are available for every chemical that you
will be using in the lab. They can be accessed online via the safety link on the
Chemistry Department website (www.chem.harvard.edu). MSDS’s are intended
to give toxicity information, safe handling, and information on what to do in case
of exposure. In general, it is often difficult to ascertain how harmful chemicals
actually are. Err on the side of caution. Your limited exposure is well under the
suggested dosage limits if you use safety procedures.
Glassware
Be aware of the dangers of handling glassware. The most common laboratory
injury are cuts received from a piece of broken glass. Be especially careful when
connecting pieces of glassware together and taking them apart. Don’t force
anything. If you can’t get two pieces of glassware together or apart, ask your TF
for help.
Injuries
If you are injured, no matter how small the injury, tell your TF. In the case of
minor injuries (small cuts, minor burns, splinters, etc.) first-aid can be given onsite with laboratory first-aid kits. In the case of anything worse than a minor
injury, you will be escorted to UHS by a TF. Never leave for treatment without
notifying your TF. After treatment of any injury, you will be asked if you wish to
continue that day. You will not be penalized if you are unable to finish that day,
even if it’s because you simply feel uncomfortable returning to the bench.
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Fires and Serious Chemical Spills
The dangers from fire and spills can be minimized and effectively handled by
observing the following procedures:
Think! Common sense should prevent most fires and spills. Is it a good idea to
heat flammable solvents on a hot plate? If a fire does occur, immediately step
away from it and notify a TF. Most fires occur inside of a fume hood and will
burn themselves out on their own. The TFs are trained to use a fire extinguisher
if it is necessary. You are not responsible for putting out a fire.
There’s a fire alarm on the wall outside the main entrance if the fire gets out of
hand.
Notify your TF of all spills. Small spills are easily contained, and your TF can tell
you how to deal with them. If the spill looks large or is of something particularly
nasty, notify your TF and get out of the area.
Evacuation
If the fire alarm goes off, immediately walk out of the lab and exit the Science
Center. If a TF tells you to get out of the lab or a certain area, do it! In year’s
past we have had a number of students continue to work in areas where there
were fires and chemical spills. Dedication is a valuable asset to have in the
laboratory, but we would much prefer to have you safe than finished ten minutes
early.
II. Safety Equipment
Know where the nearest safety equipment is at all times; you never know when
you or your neighbor will need it. In case of any incident, notify a TF
immediately.
First Aid Kits
There are first aid kits in Sci Center 117. In these you’ll find band-aids, burn
ointments, Tylenol, ibuprofen, antacids, tweezers, eye drops, etc.
Eye Washes
There are emergency eye wash stations attached to some of the lab benches
and eyewash faucets along the back wall of the room. If you get a chemical in
your eyes, active an eye wash and hold your head such that your eyes are in the
streams of water and blink for at least 15 minutes. If you notice your neighbor
has gotten something in their eyes, calmly escort them to the eyewash and help
them rinse out their eyes. It is not the end of the world if you need to use the
eye wash station; if you feel a burning sensation in your eyes, you may want to
give them a rinse for a few minutes just to be safe.
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Life Sciences 1a Laboratory – Fall 2006
Safety Showers
Safety showers are located in three places along the back wall of the lab, directly
opposite three of the eyewashes. To operate, pull the handle down. The
shower puts out a massive deluge of really cold water, so be prepared.
Immediately strip off any contaminated clothing while under the shower. Don’t
be modest, be safe. The shower is not self-locking, so you’ll have to pull the
handle down every 20 seconds or so.
Fire Blankets
If someone’s clothing is on fire, either douse them with water under a shower or
wrap them in a fire blanket and roll them on the floor.
Electrical Disconnect
Do not touch a person in contact with a live electrical circuit. Disconnect the
power first by pressing one of the “Emergency Electrical Disconnect” red buttons
located along the front, back, and right-hand walls. These buttons cut off all
power to the lab.
Fire Extinguishers
Fire extinguishers are located along the front wall and are also attached to the
sides of the rows of benches that face the front/back wall. In case of fire, notify
a TF immediately. If the fire is small, put the fire out by aiming the extinguisher
at the base of the fire and by sweeping the spray back and forth until the fire is
extinguished. If you don’t think you can put out the fire, notify your TF and exit
the lab.
Spill Control Pillows
Any spills should be reported to your TF and the Head TF immediately. They will
supervise the clean-up or obtain professional assistance if deemed necessary.
Large spills of relatively non-volatile or non-toxic chemicals can be contained
using the absorbent pillows, which are located throughout the lab. Minor spills
of acid or base solutions can be cleaned up with the use of sodium bicarbonate
(for acids) or Hazorb (for acids or bases).
Fire Alarm
A fire alarm is located centrally in the room
Operations Center Telephone
A telephone that automatically connects to the Operations Center is located near
the main entrance to the lab. The Harvard University Operations Center is a
communications, dispatch and response center providing 24-hour per day, 365days per year Standard and Emergency Response Services to the Harvard
community. Regular phone calls cannot be made from this phone. The
Operations Center can be called to dispatch police, fire, ambulance, and facilities
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maintenance personnel. The TFs will be the ones who typically use this phone,
however you should be aware of its function.
III. Disposing of Chemical Waste
Massachusetts and Federal law require Harvard to adhere to strict regulations for
the identification, storage and disposal of hazardous wastes. Operations which
do not meet regulatory requirements can result in substantial penalties, including
fines of up to $27,500 per day, per violation. You as a student are the most
important element of our laboratory’s hazardous waste management system.
Proper handling of waste will keep you, your colleagues, and the environment
safe. Adhere to the following guidelines and ask a TF when you have a question
regarding the disposal of waste in the laboratory.
No organic chemicals, including acetone, ever get poured down the sink.
Occasionally, aqueous solutions of inorganic acids or bases (eg. HCl or NaOH)
can be disposed of down the sink if the pH is in the range of 5 - 9. Your TF will
provide explicit instructions when this is a possibility.
Regular Trash
Paper towels, paper, disposable gloves, tissues, etc. that are not contaminated
with organic chemicals can be disposed of in the regular trash barrels at the end
of each row of benches.
Glass Disposal Boxes
All glass, broken or unbroken, should be disposed of in the blue and white
cardboard Glass Disposal Boxes at the end of each row of benches. These boxes
help prevent the Science Center staff from getting cut when disposing broken
glass. Only glass that is clean and has been rinsed of all chemicals can be
disposed of in these boxes. Pipettes, vials, test tubes, bottles, and broken
glassware are the most common items disposed of this way.
Blue Buckets for Chemical Waste
There are several waste hoods throughout the lab that contain 5 gallon blue
buckets for chemical waste. The different types of chemical waste generated in
the laboratory are segregated to facilitate proper disposal, thus you should
dispose of any chemical waste you generate in the appropriately labeled blue
bucket. They are labeled as follows:
Aqueous Waste
Dispose of…
… aqueous solutions contaminated with organic material. This includes the
aqueous layers from your separatory funnel extractions.
… alcohol based solutions (eg. methanol and ethanol).
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… acetone rinse solutions. Water is very soluble in acetone, thus acetone should
go into the aqueous waste container.
… any mixture of solvents that include acetone or water.
Organic Waste
Dispose of…
… organic solvents such as ethyl acetate, hexane, and THF.
… solutions of organic compounds dissolved in organic solvents.
Solid Waste
Dispose of…
… paper towels, gloves, weigh boats, and filter paper contaminated with
chemicals.
… used drying reagents such as sodium sulfate.
… chemicals that are in a powder or crystalline physical state.
Now read the procedure for lab #4 and prepare a pre-lab in your lab
notebook as described in lab #3 appendix 2.
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IV. Amino Acid TLC
Thin Layer Chromatography (TLC)
Thin layer chromatography (TLC) is used to qualitatively identify compounds
and determine their purity. In this experiment you will use TLC as a tool to
follow the progress of your reaction and determine when all of your starting
material has been consumed. A TLC plate consists of an adsorbant, a porous
material that can bind to your compounds of interest, applied to a solid support.
We will be using silica gel (silicon dioxide) power applied in a thin layer onto
glass plates. Step-by-step instructions on how to perform a TLC analysis yourself
follows.
Preparing the Plate
Obtain a TLC plate and mark the level of your soon-to-be spot(s) (about a
centimeter from one end) using a pencil and a ruler. Do not use pen! Press
lightly with the pencil, making sure not to cut a channel in the silica. In our
example below only one spot (which will travel vertically upward) is being
illustrated, however you can fit several equally spaced spots on a plate.
Spotting the Plate
Using a glass capillary tube, spot a dilute solution of a compound or a mixture
of compounds onto the bottom of a TLC plate. Do not use the entire solution;
one small spot is sufficient. Spots no larger than 1-2 mm are optimal, especially
for distinguishing between two spots with similar polarities. Almost all students
new to lab tend to overspot, which actually makes a developed plate difficult to
read. Your objective is to make the smallest, most concentrated spot possible.
solvent front
draw
lines
spot
1. develop
2. visualize
c
b
a
Rf bottom spot = a/c
Rf top spot = b/c
Developing the Plate
Using tweezers, place the plate vertically in a TLC chamber (a beaker covered
with a watchglass is fine) with a pool of solvent at the bottom. The solvent level
must be below the height of the spots so that they are not dissolved in the
solvent pool.
The solvent will slowly rise by capillary action up the plate. As the solvent
front travels upward, it is carrying the spotted compounds through the silica gel
along with it to some extent. The ease with which a compound travels through
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the adsorbent depends on the polarity of the compound as well as the polarity of
the solvent. Silica gel is quite polar. More polar compounds interact with the
silica matrix more strongly than less polar ones. As a result, more polar
compounds move more slowly on a silica gel matrix than less polar compounds,
regardless of solvent. In our diagram above, we know that the bottom spot is
more polar than the top spot because it traveled more slowly through the polar
silica gel. By changing the polarity of the solvent that is used (e.g. varying
compositions of ethyl acetate and hexanes) you can control how far the
compounds you are working with will travel on the TLC plate. A very polar
solvent system will carry your compounds up near the solvent front while a nonpolar solvent system will leave your compounds down near the baseline of the
TLC plate. Usually solvents systems are selected so that the compounds of
interest move between one third and one half of the way up the plate.
Figure. Required components for TLC on the left, and an assembled
TLC apparatus on the right
When the solvent has traveled almost to the top, remove the plate and mark
the solvent front with a pencil line. Allow the solvent to evaporate from the
plate.
Visualization and Interpretation
You probably will have noticed by now that there is nothing to see on the
plate. Most organic compounds are not visible to the naked eye when spotted
on a TLC plate. It is for this reason that the silica gel on TLC plates is often
impregnated with a fluorescent dye. This dye will glow green under ultraviolet
light (254 nm). Many compounds (especially those with double bonds and
aromatic rings) absorb UV light, leaving dark spots on the plate.
Take your developed plate to a UV lamp box and visualize your spots. Use a
pencil to carefully circle any spots you see on the plate. UV light is harmful to
the eyes and skin. Always wear gloves and goggles, and do not look directly into
the UV lamp.
Measure the distance that the solvent has traveled (from where the spots
started to where the solvent ended). Measure the distance the individual spots
have traveled (from where each spot started to where it stopped). Dividing the
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distance the spot traveled by the distance the solvent front moved gives you a
number called the Retention Factor (Rf.) A compound always has the same Rf in
the same solvent (using the same adsorbent), independent of plate size.
Rf = (distance the spot moved) / (distance the solvent moved)
Draw your TLC plate to scale in your lab notebook and make note of the
solvent, visualization method, and the Rf of each spot.
In this brief lab module, you will practice TLC by separating amino acids in a
mixture. This will be key for next week’s experiment when you will be
performing TLC on your chemical reaction. Please record your procedure
and all of your notes and results in your lab notebook.
Procedure:
Please obtain the following items from your TF:
1 glass beakers
1 watch glasses
1 TLC plate
1 ruler
3 capillary tubes
1. In your beakers, add the TLC running solution
2. Cover both beakers with a watchglass to prevent evaporation of the
liquids
3. Take a TLC plate and draw a line in pencil 1 cm from the end
4. Using the capillary tube, place 3 separate spots from the samples your TF
will provide you along the line
5. Allow the spots to dry on the TLC plate (perhaps a minute)
6. Place the TLC plate in the beaker with the TLC running solution with the
spotted end in first.
a. Be careful not to submerge the spots into the liquid!
7. As the liquid travels along the paper, your sample will migrate up the
plate. Before the solvent front reaches the top of the plate, remove the
TLC plate and mark the solvent front in pencil.
8. Observe your plates in the UV light box.
9. Attempt to determine the identity of each spot on your TLC plate based
on your understanding of the differences in amino acid polarity.
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V. Extraction Practice
Obtain the following from your TF:
2 Scintillation vials and caps
Pasteur pipet
Pasteur pipet bulb
1. To your scintillation vial, add 2 ml of water and 2 ml of ethyl acetate
2. Cap your vial
3. Observe and record the appearance of the two liquids in the vial shake
the vial vigorously
4. Observe and record the appearance of the liquid in the vial
5. Place your vial on your benchtop and allow the liquids to separate.
6. Once the liquids have separated, using your Pasteur pipet and bulb,
extract the organic top layer without sucking up the aqueous bottom layer
of liquid.
7. Transfer the liquid from this layer into your second scintillation vial and
observe to see if any water was transferred.
VI. Laboratory Set-up
Please obtain the following items from your TF:
1 beaker
1 scintillation vial and cap
1 stir bar
1 beaker
1 clamp
1. Prepare an ice water bath as shown in your lab manual (lab #4) on a stir
plate
2. Add 2 ml of water to your scintillation vial
3. Place your stir bar into the scintillation vial and cap
4. Clamp your vial onto one of the posts on your bench and position it in
your water bath above the stir plate
5. Turn on the stir plate and adjust the speed to slowly spin the stir bar
6. When you have successfully assembled your apparatus, please show your
TF before cleaning up your set-up.
While cleaning-up, please do not throw away your stir bar. Transfer it
to the dirty stir bar container.
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Appendix 1: How to Clean Up Your Bench
Before leaving the lab
At the end of each lab, take a few minutes to make sure you are leaving the
lab the way you found it (or should have found it). Here’s a list of what you
should do:
Clean and Replace your glassware
Having clean, spotless glassware makes your laboratory experience all the
more pleasurable. You must thoroughly clean all of the glassware used and
return it where you got it from.
How to Wash Glassware
1. Empty the contents of the glassware into the appropriate waste.
2. Rinse the glassware with acetone to remove all organic chemicals. Collect
the rinse in an acetone rinse beaker.
3. Wash the glassware with hot, soapy water.
4. Rinse glassware at least 3x with hot water. This removes all soap residue.
Acetone causes soap to precipitate onto glass, causing those nasty white
spots to appear. This precipitate is very difficult to remove.
5. Rinse both sides (i.e. inside and outside) of glassware with acetone and
collect the rinse in your acetone rinse beaker. This removes water, which
can cause other spots.
6. Place glassware on drying rack for a few minutes to dry.
7. Pour your acetone rinse beaker into aqueous waste.
No acetone should be poured down the drain!
Clear off your lab bench
The lab bench (around the sink) should be left clean and empty.
Wipe out your bench area
You should leave your work space clean and safe for the next student. Use
acetone and paper towels to clean up any organics that may have spilled, then
wipe down the entire area with damp paper towels or a sponge.
Common laboratory equipment
Make sure all of the common laboratory equipment you have used is
returned.
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Appendix 2: Sample Synthetic Chemistry Lab Report
Lab X: The Synthesis of Phenacetin
R. B. Woodward
Radcliffe
Partner: Frank Westheimer
July 11, 1953
11PM
TF:
Anne
Wednesday 6-
Prelab
Purpose: To synthesize phenacetin from p-ethoxyaniline
Overall Reaction:
O
NH3
NH2
CH3
NaOAc
HCl
OEt
HN
Ac2O
H2O
OEt
OEt
Reagent Table1
Reagent
MW
Mp (°C)
g used
mL used
mols
equiv.
phenetidine
sodium acetate
137.18
82.03
2.4
3.4
2.5
3.2
0.025
0.030
1
1.2
acetic anhydride
conc. HCl
phenacetin
102.04
(11.6M)
179.22
3.1
2.9
2.1
0.030
0.025
1.2
1
137.8
Theoretical Yield2
0.025 mol p-phenetidine ==> 0.025 mol phenacetin
0.025 mol phenacetin x 179.22 g phenacetin / mol phenacetin = 4.48 g phenacetin
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Life Sciences 1a Laboratory – Fall 2006
Procedure2
• Add 75 mL water and p-phenetidine to 125 ml Erlenmeyer
• Add acid; swirl to dissolve.
• Decolorize if necessary.
• Make sodium acetate solution: 2.5 g sodium acetate and 15 mL water.
• Add anhydride to p-phenetidine•HCl solution from above, swirl a few seconds, add sodium
acetate
solution.
• Collect precipitate via filtration and recrystallize from ethanol.
• Check crystal appearance and take mp.
Observations3
• Put 3.4 g phenetidine and 75 ml water into 125 ml Erlenmeyer; stirred.
- white suspension resulted
• Added about 2.1 ml conc. HCl (may have added too much); white vapors filled the flask.
• Stirred for 5 minutes (until phenetidine disappeared).
• Solution looks purplish, so decolorized by boiling with activated charcoal until clear.
• Filtered; washed charcoal.
- Solution clear and colorless now
• Added 2.9 ml acetic anhydride to Erlenmeyer; reaction heats up.
• Made solution of 2.5 g sodium acetate in 15 ml water. Added to above flask. Oops - spilled
a bit.
• White crystals fell out of solution as soon as acetate added.
• Stirred until crystals stopped forming, about 5 minutes to be sure.
• Collected crystals, scraped off paper - lost some.
• Recrystallized from ethanol, filtered, let air-dry, and weighed
mass: 2.60
• Took mp.
range: 125-132°C
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Conclusions and Results4
Our reaction yielded 2.60 g (58%) of phenacetin, which melted from 125-132°C. The
crystals formed were long white spars, with no visible impurity. There had to have been
some impurity, however, for our product melted below the reported melting range of 137138°C; this also means that we got something less than 2.6 g of phenacetin, as that is the
weight of phenacetin + impurity. The impurity could have been ethanol, the recrystallization
solvent, which disrupts the crystalline lattice and lowers the melting point (melting point
depression).
Our yield of 58% could be due to a few things. First, the decolorization process is
known to decrease yields (called a “yield killer” by my TF), perhaps due to the number of
filtrations required. Second, adding excess acid and not enough sodium acetate (due to
spilling some of the solution) could have led to some of our phenacetin remaining in the
reaction solution as the protonated amide salt, which is water soluble. This amide salt would
not completely crash out of solution, and would not be trapped by the filter, thus leading to
a lower yield. Neutralization of the liquid remaining after filtration may have yielded more
phenacetin. Third, water hydrolyzes acetic anhydride, and if the sodium acetate wasn’t
added quickly enough, some of the anhydride could have been lost before the p-phenetidine
had a chance to react with it.
Note 1: It is fine to put only the relevant information. It will vary. Sometimes mp (for
products and unknowns), bp (for solvents to be refluxed and compounds to be distilled)
and density (for solvents used in extractions) will be very important.
Note 2: This should be as concise as possible. If you can do the lab without your
manual, you’ve written enough.
Note 3: Make note of important numbers, color changes, etc. as you go. Be clear and
concise. Briefly write what you did, and what you see, so that some other person—who
knew their way around the lab—could repeat what you did.
Note 4: Must contain yield, mp, product appearance, relevant discussion and
explanations.
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Appendix 3: Periodic Table of the Elements
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