lab 3: it’s all fun and games until someone loses an eyesites.fas.harvard.edu/~lsci1a/lab3.pdf ·...

Life Sciences 1a Laboratory – Fall 2006

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Lab 3: It’s all fun and games until someone loses an eye (AKA Chemistry lab safety) Goals of the lab:

1. To introduce you to safe laboratory practices 2. To orient you to your new lab surroundings 3. To prepare you for techniques you will use in lab 4 4. To 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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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 latex-free 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 on-site 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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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, 365-days 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.

drawlines spot 1. develop

ab

c

Rf bottom spot = a/c

Rf top spot = b/c

solvent front

2. visualize

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 non-polar 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 TF: Anne Radcliffe Partner: Frank Westheimer July 11, 1953 Wednesday 6-11PM Prelab Purpose: To synthesize phenacetin from p-ethoxyaniline Overall Reaction:

NH2

OEt

HCl

NH3

OEt

NaOAc

Ac2O

H2O

HN

OEt

CH3

O

Reagent Table1

Reagent

MW Mp (°C) g used mL used mols equiv.

phenetidine 137.18 2.4 3.4 3.2 0.025 1 sodium acetate 82.03 2.5 0.030 1.2 acetic anhydride 102.04 3.1 2.9 0.030 1.2

conc. HCl (11.6M) 2.1 0.025 1 phenacetin

179.22 137.8

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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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 137-138°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

lab 3: it’s all fun and games until someone loses an eyesites.fas.harvard.edu/~lsci1a/lab3.pdf ·...

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