Lab Manual CHML 2210 Fall 14

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Sample Organic Laboratory Report 20
Characterization and Purification 23
EXPERIMENT 4: Extraction 48
EXPERIMENT 5: Distillation 53
EXPERIMENT 7: Identification of Organic Compounds by Qualitative Analysis 62
EXPERIMENT 8A: Effect of Structure on the Free-radical Bromination of Hydrocarbons 69
EXPERIMENT 8B: Relative Rates of Nucleophilic Substitution of Reactions of Halides 72
EXPERIMENT 9: Medicinal Chemistry 75
EXPERIMENT 10: Nylon Experiment 78
Appendix A: Gas Chromatography 80
Appendix B: Infrared Spectroscopy 85
Appendix C: Nuclear Magnetic Resonance Spectroscopy 92
8/18/2019 Lab Manual CHML 2210 Fall 14
LABORATORY SAFETY RULES
Chemistry laboratories are places with unusual hazards, which for the most part, are associated with the chemicals in them. Chemicals are unforgiving: if you mishandle them, you can be seriously hurt. TREAT THE LABORATORY AND EVERYTHING IN IT WITH RESPECT. The following rules will minimize risks in the lab:
1. Safety goggles MUST be worn. When anyone is working, EVERYONE must wear goggles. There are NO exceptions. Eyes are priceless and irreplaceable. When goggles fog, step outside to mop them. Goggles on top of the head do not protect the eyes.
2. An apron or lab coat MUST be worn when working. This adds an extra layer of protection between chemicals and your body as well as protects your clothes from discoloration or mutilation.
3. Shoes MUST cover the entire foot. NO exposed toes. NO flip flops, sandals or extreme ballet flats that expose the entire top of the foot. You may carry your shoes in a pack to lab.
4. Contact lenses tend to complicate eye injuries. The use of contact lenses during lab periods is strongly discouraged.
5. Wearing long pants to lab is required. Tie back long hair to keep it out of reactions. 6. Absolutely no smoking, eating or drinking in the lab . 7. Locate the fire extinguishers, eye washes and safety showers BEFORE you begin any
experimental work. 8. Do not run. Do not push. Do not work and socialize. IF you’re sure yo ur sample can be
left safely you can talk. If you are not sure about the safety of your sample or reaction — give it your attention and save the conversation for later.
9. Do not alter the procedure for the experiment you are doing. Follow all directions carefully.
10. Unauthorized experiments will guarantee an “F” for CHML 1045 for any student involved. (Note on academic honesty: failure to perform analyses that you report, will result in a zero for the experiment.)
11. Never taste or smell any chemical (to detect an odor, use your hand to waft the chemical towards your nose). Do not touch chemicals with your hands. At the least, you will get a skin rash. Many chemicals are absorbed through the skin and some can cause serious burns.
12. WARNING: All ethanol in this laboratory is denatured. Denatured means that it is toxic as it contains poisons, which cannot be removed by distillation. If you take any ethanol from the laboratory for drinking purposes, you will kill or seriously injure yourself or whomever you give it to.
13. Never heat a sealed apparatus —it WILL explode. The flying glass will cause serious injuries.
14. Never heat a sample or reaction rapidly, always gradually. NEVER leave a reaction unattended.
15. Your instructor will clean up broken glass (after you inform the instructor); don’t pick it up yourself. Please treat glassware with care.
LABORATORY SAFETY PROCEDURES
Safety is one of the major concerns in any lab. Safety for humans is always a primary concern. The location of safety equipment should be common knowledge to anyone working in the lab. In order to become familiar with the safety equipment in this lab, make a map on the reverse side of this sheet showing the location of the following safety items. Indicate the fire escape with an arrow.
1. Emergency shower and eye wash station
2. Fire extinguisher
3. Fire blanket
8. Electrical power cut off switch
9. First aid box
I have read and agree to follow ALL of the listed safety procedures and precautions.
_________________________________________ ________________ Signature Date
_________________________________________ ________________ Instructor Section
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Note: The next few pages are a general description of how to prepare for lab, write up your experiments, write lab reports, etc . Each lab instructor has personal preferences concerning these topics. These preferences may differ somewhat from what is presented here, but your instructor will tell you what their preferences are. You should use the following information as a guideline, but follow any specific directions your instructor gives you.
THE LABORATORY NOTEBOOK Keeping organized, accurate records of procedures, observations and data in a lab notebook is the cornerstone of all kinds of scientific work. This includes organic chemistry lab. A lab notebook is your record of procedures, observations, amounts of chemicals and other information as you complete experiments. Your lab notebook should give a step-by-step record of what you do and observe and measure. Writing your lab reports is easier for you to do with a well-organized lab notebook. It also makes studying for the final exam easier.
Why bother with a lab notebook if I'm not majoring in chemistry? In most scientific careers, graduate work, clinical studies, or forensic analysis, etc., it is vital to properly record procedures, data and results in a laboratory notebook. Improperly documented procedures and data have resulted in patents being denied, research findings being rejected or forensic tests being thrown out of court. Maintaining a lab notebook takes some work, but many instructors in upper-division science courses (and most supervisors in the “real” world) will assume that you can properly use and maintain a laboratory notebook.
What should be recorded in the lab notebook? Everything relating to carrying out the experiment (title, objective(s), reaction, reagent table, procedures, observations, raw data, etc.) should be written directly into the notebook. At a minimum , all steps that must be done in the lab (procedure, measuring mass, obtaining spectra, etc.) must be recorded directly into the notebook.
Notebook guidelines Write all observations and data in ink directly into your notebook as you carry out the experiment . (Yes, you have to take your notebook to the balance or laboratory instrument to record masses and/or measurements!) If you make an error in your notebook, cross it out with a single line and re-enter your data nearby. Do not obliterate your error with a large “blob” because if the first entry was right, it is now unreadable. Do not write the correction directly on top of the error; some notebooks use carbon paper, and if the original copy is hard to read — then carbon copy is usually impossible to read!) Write data only in your notebook. Never write data on scratch paper or in the margins of the lab manual! This very poor lab practice can (and does) lead to lost or garbled data. The instructor reserves the right to confiscate materials used to improperly record data and/or deduct points from an offender. Please find below a summary of some guidelines for keeping a laboratory notebook for organic chemistry lab courses. These are taken from the UCL Journal of Technology (Jan 2000).
1. DO use bound books . Permanently bound books should be used. They should be consecutively numbered and each page should be dated, signed and witnessed.
2. Do use ink . Notebook entries should be made in ink and in chronological order. Entries should not be erased or ‘whited out’. If an entry contains an error, a line should be drawn through the error and new text should continue in the next available space.
3. Don’t leave blank spaces . Blank gaps between entries should be avoided. If a blank space is left on a page, a line or cross should be drawn through the blank space and the pages dated to prevent subsequent entries.
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4. Don’t modify . Prior entries should not be modified at a later date. If data were omitted, the new data can be entered under a new date and cross-referenced to the previous entry. Record experiments when they are performed.
5. Do use past tense . Use the past tense (e.g., ‘was heated’) to describe the experiments that were actually performed.
6. Do explain abbreviations and special terms . Explain all abbreviations and terms that are nonstandard. Explain in context, in table of abbreviations or in glossary.
7. Do staple attachments . Attachments such as graphs or computer printouts should be permanently affixed in the notebook (by stapling) and both the attachment and the notebook page signed and dated.
8. Don’t remove originals . No original pages should be removed from the notebook. 9. Do outline new experiments . When a new project or experiment is started, the
objective and rationale should be briefly outlined e.g., in a short paragraph or by providing a flowchart.
10. Do provide detail . Record experimental descriptions, including operating conditions, test results, and all explanation of the results, and thin layer chromatography sketches of the results. Any conclusions should be short and supported by factual data. DO NOT COPY THE DIRECTIONS IN THIS MANUAL. DO NOT wait until the end of the experiment to write down observations – write them down as you make them. Also, write directly into your notebook. NEVER take notes on a paper towel or other loose sheet of paper.
If carbon copies are used, remember that your instructor will grade the carbon copies so make sure they are readable! Whatever you write in your notebook must give legible carbon copies. Make it easy for the instructor to read what you write. Print in your notebook; cursive writing often gives carbon copies that are very difficult to read. Make sure that you press hard enough to produce clear copies, and make sure the plastic shield is in place (for a carbon copy notebook). Remember that tapping your pen, writing on another paper resting on your notebook, or forgetting to put the shield behind your carbon page will cause marks on the carbons that will obscure what you
write. Periodically check your carbons to confirm that your writing is clear and legible. Use several paragraphs instead of large blocks of text because small paragraphs are easier to read . Keep solvents and chemicals away from your notebook because they may dissolve the ink or destroy the pages.
Your notebook is not expected to be flawless, but it should be easy to follow. Cross-outs and corrections are expected. Do not skip pages, and do not tear original pages out of your notebook, even if they are spoiled. If you run out of room on a page, do not try to cram your writing into the available space, because this typically leads to unreadable carbon copies. Instead, use “Continued on page...” and “Continued from page…” to help the instructor follow your report. Useful tip: Leave a few blank lines between steps of your procedure, so you can insert any changes or forgotten steps. Also, leave space between sections of your report in case
you need to add something you forgot to include or if you need to correct or better explain something you previously mentioned.
GOAL: You are keeping a good laboratory notebook if a student who has taken a similar organic lab course at another university could carry out the experiment based on your notes. You should work toward this goal.
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These sections should be prepared and in your notebook before you come to lab:
Title, lab meeting time, date of experiment: This is self-explanatory.
Objective: Write one to three sentences about the goal(s) of the experiment. This could be the identification of an unknown compound, the preparation of a compound, isolating a pure compound from an impure sample, etc.
Main reaction (if applicable): All preparative experiments should include the balanced reaction. Include reaction conditions, catalysts, etc.
Table of reagents, solvents and products: Prepare a table listing the chemicals used in the experiment. List headings such as compound names, molecular weights, densities, melting and boiling points, refractive index, etc. Include the relevant properties in the table. Irrelevant information such as the boiling point of a solid or the density of a gas can be excluded. You can find information in your textbook, online (chemfinder.com is a good place to start), in chemical catalogs (Aldrich, ACROS, Lancaster, etc.) or the CRC Handbook.
Procedure: Do not just copy out of the manual. Summarize the steps you will use. Example: “A miniscale fractional distillation apparatus was set up as in Figure X in the manual page X. Glass beads were used as packing, as in Figure Y in the manual .” You do not have to describe how you clamped each piece of glassware. (Recipes in a cookbook do not explain how to turn on an oven or boil water.) Similarly, you do not have to explain each step of obtaining a refractive index or setting up a filtration because this information is freely available in your manual. Leave one or two blank lines between steps to give you room to insert any steps you left out or to include any changes announced by the instructor. Avoid using first person (I/me/my) in your report. Record the exact amounts of chemicals that you use in the Observations section.
A very convenient and effective way to organize your Procedure and Observation sections is the “two column” method shown in the Sample Laboratory Notebook on pages 14-20. You may
also want to create a flow chart if the procedure is complicated or has multiple steps with several fractions.
These two sections must be also entered in your notebook as you carry out the experiment:
Changes to the procedure: If you deviate from the written procedure (even if the instructor announces the changes), you need to write the changes in your notebook. Any changes such as using different reagents, different amounts, using an additional portion of solvent to wash a solution, heating for a different time, etc. must be recorded in your notes. Even if you have to redo a step because of a mistake, you still have to write the changes down. (This is why leaving a few blank lines between steps is a good idea!)
Observations and Data: Record the exact amounts of chemicals actually used in the experiment. (The procedure may say to use 0.0100 moles of cyclohexanol. This amount is 10.0 g of cyclohexanol, but you need to record the amount you used (10.03 g, 9.86 g or whatever). Remember to base your yield calculations on the amounts of reagents you actually used , not on the amounts called for in the procedure. You should describe the colors and appearances of the reagents (before and after mixing!), color changes of solutions, temperature changes as a chemical is added to a solution and so forth. Make as many observations as you can. A surprisingly large number of students forget to record the appearance of their products.
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Sometimes, the cause of an unsuccessful experiment can be pinpointed using observations. (True story: When half of the students failed to get a reaction to occur, checking the notebooks showed they used one bottle (with grey powder) and the others used another bottle containing white powder. It became clear that the first bottle had been mislabeled, so the students unknowingly used the wrong material.
REMEMBER: Unwritten observations and data never happened!
Data Table: This is for “raw” numbers such as masses or volumes of reagents, uncorrected melting points and uncorrected refractive index values or masses of products. Important! All of this information should have been previously recorded in the Observations section. Remember to use significant figures appropriately.
The following sections can be entered in your notebook outside of lab time:
Interpretation of Spectra: Report the information (peak values, intensity, interpretation, etc .) in tables in your notebook. See the Sample report for examples. Do not write on the spectrum itself except for your name and a title (e.g. “John Doe, IR Spectrum of product of Exp. 13 , thin
film”). Be sure to note the presence (or absence) of significant peaks. For example, if you are preparing an alcohol from a ketone, you should look for peaks in your spectrum that indicate that could show the presence of the alcohol product as well as any unreacted ketone.
Calculations: This is where numerical data is processed. Thermometer readings are corrected, measurements on a TLC plate are converted into R f values, masses of reactants and products are converted to moles and so forth. Use significant figures (sig figs.) properly in your calculations! NOTE: In any preparative lab, you need to determine the limiting reagent and calculate the theoretical and actual yield —even if you obtain no product at all! If you do not know or do not remember how to calculate yields, consult your instructor.
Do not “create” or “discard” significant figures. If you measured to only hundredths of grams,
your calculations must reflect this. Thus, if your product weighs 3.13 g, do not add a trailing zero (3.130 g). On the other hand, if a beaker weighed 45.217 g full of reagent and 41.137 g after adding the reagent to the flask, you added 4.080 g to the flask, not 4.08 grams. The zero in the thousandths place is a significant figure here.
Discussion of Results: This is the most important section of the laboratory report. Here is where you explain and interpret the results of your experiment. The length and specific content will depend on the experiment, but the Discussion section is usually at least 10-15 sentences in length. The section will be somewhat longer in preparative experiments. You should, of course, always use correct grammar, spelling and terminology in the lab report, but doing so is especially important in the Discussion section. Why? Because the quality of your discussion indicates how clearly you understand the objectives of the experiment and how you interpreted
your results. If you make inaccurate or poorly supported claims, use illogical reasoning, omit relevant information, or use the wrong terminology, it gives the impression —whether true or not —that you do not understand the experiment. This impression will impact your score for that report. Likewise, unclear wording, misspelled words and poor grammar give the impression of sloppiness or that you did not proofread your work. Even if an experiment goes poorly, a well- written, well-reasoned Discussion will often earn more points than a badly written Discussion describing a successful experiment. More information concerning what should be included in the Discussion section is given on the next page. In contrast to the Procedure, using first-person
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pronouns (I/me/my) is more acceptable in the Discussion section. See the following page for more information.
Summary of Experiment: The key word is “summary”. This section should be brief (typically 3 - 5 sentences). The summary should state the outcome of the experiment and a couple of important details. You are expected to include specific information such as vial numbers, measured properties, yields, etc. Do not introduce any new information in this section. This section should only include information that is mentioned elsewhere in the report. The key topics from the Discussion section will probably be part of your Summary, but remember that the Discussion and Summary are separate sections of the report.
Example of a poorly-written Summary (The reader has no idea what the result was or what the data means.):
I successfully accomplished the objective of this experiment. My data was very close to the expected values, and there were no significant problems in carrying out the experiment.
Example of a much better summary (A reader can clearly see what was done and what the results meant.):
My unknown liquid (code B-23) was identified as tetralin. The measured boiling point was about 5 C lower than the listed value for tetralin, but the measured refractive index was very close to the value in the manual. Tetralin was the only compound with a boiling point and refractive index that were both fairly close to the measured values, so I am confident that my identification is correct.
Example of a summary for an experiment involving the preparation of a compound : Benzhydrol was prepared by the reduction of benzophenone by NaBH 4. The yield of purified product was 65.8%. The IR spectrum closely matches that of a pure sample and the melting point is only 1- 2 C below the reported value, indicating that the product is proba bly quite pure. The loss of mass during recrystallization shows that this is the step where the major loss of product occurred.
REMEMBER! Focus on the highlights. Think: “What in terms of “What I did in lab in 50 words or less.”
Answers to follow-up questions: Your instructor may assign some or all of these questions.
Additional Guidelines for Writing Your Discussion Section The most important rule is: do not make claims in unless they are backed up with specific information! For example, if you state that the sample you obtained was pure, you must support this by giving specific evidence that you have recorded such as a narrow melting range, a melting range or appearance that that matches an authentic sample, etc. Use specific data, IR absorptions, etc . to support your claims. See these three examples.
Good: “My sample melted at 113 -114 C, which is close to the m.p of pure acetanilide (114 - 115 C).”
So- so: “My sample melted at 113 -114 C, which is close to the m.p of acetanilide.”
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Poor: “My sample had the same melting point as acetanilide.” (The reader must then look up the values.)
Consider all relevant information, even some seems contradictory. For example, if a product looks identical to a known pure sample, but the melting range data suggests the sample is impure, the Discussion must address both pieces of evidence. Remember that contradictory data may be due to misreading an instrument, incorrect calculations, misinterpreting the instructions or many other sources. If you find contradictions, double-check all of your readings, calculations, etc. (Having a friend look at your data can be helpful here.) If you cannot find a problem, but you still have contradictions, then you must judge the importance or reliability of each piece of data and the “weight” to give it as you draw conclusions about the experiment. When you have contradictory data, it is a good idea to consult your instructor before you begin to write your Discussion.
Examples of appropriate topics for Discussion: Example #1. In Experiment Y, you will purify a solid by recrystallization, and identify it by melting point determination. The Discussion section should address issues such as:
1) What is the identity of your solid, and how do you know that the identification is correct? 2) Is the recrystallized material pure? What evidence supports your claim that the product is
pure or not? 3) At which steps was product lost? If possible, point out where major loss of product
occurred.
Example #2. In Experiment Z, you will prepare and purify an alkyl halide and analyze it using refractometry and infrared (IR) spectroscopy. Good topics for your Discussion include the following:
1) Did you successfully prepare and isolate the desired product? Was the product pure? 2) What does the IR spectrum indicate about the identity of the product and/or the presence
of impurities? 3) What is the percent yield of isolated product? At which steps were significant amounts of
product lost?
Three things you should n o t do in your Discussion 1. Do not introduce new data, or observations. Never refer to anything concerning the experiment that is not recorded in your notebook. For example, your Discussion should not refer to the color of your product unless it is recorded in your observations. Likewise, if you spill a third of your product at the end of the experiment, but you fail to document it in your observations, it is not appropriate to cite spillage as a source of product loss in your Discussion. Note: it is appropriate to refer to sources such as the Merck Index or the CRC handbook for information about your product, but you must properly cite the references you use.
Remember: Any unwritten observations or data never really happened! Therefore, you cannot discuss something that never happened!
2. Do not re-state the procedure as part of the Discussion. Use the Discussion section to explain and interpret your data and results.
NOTE: It is okay to mention steps that affect the yield or purity of your product. For example: “I started the lab late, so I was only able to heat the mixture for 40 minutes, instead of 90. The
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reaction probably did not go to completion.” In short, discussing how the procedure affects the results is fine; just restating the procedure is not.
3. Do not combine your Discussion and Summary sections. The Discussion section is where you evaluate and explain your results. The Summary section is where you wrap up the experiment.
Keep this in mind when you are sick of preparing your notebook and writing lab reports: Clear communication of information through writing is the backbone of science. Strive to make your writing as clear, complete, concise and correct as possible. Employers out in the “Real World” place a high value on sound writing skills in their employees . Also, many employers will expect new employees to have experience in properly maintaining a laboratory notebook and writing clear, concise lab reports.
Twelve Key Points to Remember as You Prepare for Lab and Write Your Notebook 1. Preparing for lab is absolutely essential . Know what you are supposed to do, what
chemicals you will use, what techniques you will employ and so forth. Being prepared when you begin the experiment makes it much easier to keep your observations, data, etc. organized. This, in turn, makes it easier to find the information you need when you write your Discussion section.
2. Summarize procedural steps when you write them. This saves time for you and the reader as well. You may assume that the reader is familiar with basic lab techniques, so you do not have to fully explain them. Always include any additions or changes to the procedure — including things like re-doing steps, performing additional extraction, re-distilling your product or even restarting the whole experiment.
3. The amounts of chemicals you list in the Procedure are approximately what you will need. Therefore you will need to record the exact masses and volumes in your Observations.
Always record the appearances of your solvents, reagents, the colors of solutions, changes in appearance or texture during heating or cooling, colors of the layers when performing extractions, colors and appearance of products, etc.
4. When performing calculations, use significant figures correctly.
5. Present data from IR and NMR spectra in tables. Try to interpret all relevant signals. “Relevant” is loosely defined as a signal or peak that is probably from either the product, starting material or a solvent. You must develop your skills in interpreting spectra so you can determine whether a given peak or signal is from the product, starting material, contaminants, etc . Feel free to consult your instructor if you need some help.
6. The Discussion section should focus on the results. (What did you make/isolate? What is the purity? What is the % yield/recovery? Where did losses occur? What problems did you have?) Support all your claims.
7. Some material usually remains behind when pouring or transferring chemicals. For example, scraping solid out of flasks or off of a piece of filter paper usually fails to remove all of it. Unavoidable losses caused by transferring materials from one container to another are often
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referred to as “mechanical losses.” Spills and similar mishaps are not mechanical losses, and they should be addressed in the Discussion section.
8. Use correct scientific terms. For example, The plural of “spectrum” is spectra , not spectrums . Use “percent yield” and “percent recovery” appropriately.
9. Carefully check your spelling and grammar to make sure what you say makes sense. You may want to have a friend or classmate read your report to help locate mistakes and parts that should be revised. Terms that are in Mohrig or the manual should always be correctly spelled.
10. Keep your Summary section brief, but remember to include specifics such as % yield, code numbers of unknowns, etc.
11. Use common sense in your Discussion. For example, if your yield of crude product was 52.4% and after recrystallization your purified yield was 39.3%, you should not claim that the major source of product loss was recrystallization. It is true that you lost a quarter of the crude product’s ma ss during recrystallization, but more remember that nearly half of the theoretical yield was lost even before you isolated the crude product! Other sources of loss are important and should be considered. Similarly, if some product is spilled or left as residue on glassware, and if the amount lost is small compared to the amount you actually isolated, then the transfer losses are not a major source of loss of product. Critically analyze your Discussion and your reasoning to avoid making contradictory or nonsensical claims. Reading it aloud —or even better —having someone else read it aloud can help spot problems such as flawed reasoning, contradictory sentences, poor grammar and so forth. Hint: A clearly written report with sound reasoning and analysis shows that the writer really understands the experiment. Similarly, a report with contradictory reasoning, unreasonable (or unsupported) claims or that ignores important data or results gives the impression that the writer did not really understand what they did.
12. Remember to make sure that your carbon copies are legible!
A Sample Preparative Laboratory Notebook The following pages show an example of how a preparative experiment should be written up in a notebook. Remember that if your notebook is organized, your carbon copies can serve as the majority of your report.
Ben Zeen Sunday, Feb. 30
Experiment 6: Preparation of Ethyl 4-Aminobenzoate (Benzocaine)
Purpose: The goal of this experiment is to prepare benzocaine from p-aminobenzoic acid. Theproduct will be purified by recrystallization and the purity checked by m.p., IR, and NMR.
Reaction:
Compound MW m.p., C
comments
p- Aminobenzoic acid (PABA) 137.136 187-9 white crystals* Ethanol (EtOH) 46.068 78 0.789 flammable
Sulfuric acid, conc. (18 M) corrosive Ethyl p -aminobenzoate 165.189 92 Off-white crystals* Ether 35 0.71 very flammable
Other materials used: *11 th Ed. Merck Index
water 10% Na2CO3 solution saturated NaCl solution anhydrous MgSO 4
Procedure Observations 1. Add 0.020 mol of p -aminobenzoic acid to
a 100-mL round-bottom flask Add 40 mL of 95% EtOH and swirl to mix. Not all
solid will dissolve.
PABA is white fine crystals. 0.020 mol ~2.7 g. 2.67 g was actually used. EtOH is clear liquid. Used 41 mL Swirling
dissolves most solid to give pale yellow sol ’n.
2. Cool flask in ice-water bath, then slowly add 2.5 mL of concentrated sulfuric acid. Lots of precipitate will form, but it will dissolve during reflux.
Acid was thick clear liquid. Adding acid caused some spattering. Lots of white-yellow precipitate formed and sank to the bottom of the flask. Solution turned pale yellow
3. Add a small magnetic stir bar as boiling stone. Assemble simple reflux set-up with flask and reflux for 75-90 minutes. Stop the reflux every 15 minutes to vigorously
swirl the flask to help solid to dissolve.
As solution boiled, it turned yellowish and solid began to dissolve. Some solid was crust
just above liquid. Crust dissolved when flask was swirled after 15 minutes of reflux. All
solid dissolved after about 25 min. of reflux.Sol’n stayed yellow. Reflux stopped at 75 min. 4. After reflux period, remove heating
mantle and let mixture cool for 10 minutes, then cool with cold-water bath.
No changes visible after initial cooling. No changes after cooling in ice bath.
5. Pour contents of flask into 250-mL beaker. Rinse flask with 2-3 mL of ethanol
Had to use about 5 mL of ethanol to wash out the flask. Solution in beaker is yellow.
6. Slowly add 10% Na2CO3 solution (with lots of stirring) until gas evolution ceases and the
solution is strongly alkaline (pH of 9-10 asshown by test strips) About 35 mL of base should be needed. Ppt. should form.
Na2CO3 solution is water-like. Lots of foaming as added to the beaker. Some solid particles
deposited on sides of beaker. Fizzing stoppedafter about 30 mL of solution added. Lots of ppt formed then.
7. Pour mixture into separatory funnel. Add 40 mL of ether to dissolve as much of the solid benzocaine as possible. Stopper and shake funnel. VENT! Separate layers.
Ether is clear liquid. Ether solution after shaking is slightly yellow. Aqueous layer (bottom) is sort of yellow, but cloudy, too.
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8. Pour aqueous layer back into sep. funnel and extract with 20 mL of ether in same way as previous step. Separate layers and add ether layer to earlier ether solution.
Ether solution after second extraction is nearly colorless. Combined ether solution is pale yellow.
9. Wash ether layer with 10 mL of sat’dNaCl solution, then dry using MgSO 4 in Erlenmeyer.
No change in appearance of organic solution.MgSO4 made the “snow globe” when swirled.
10. Gravity filter solution into 24/40 joint round-bottom flask. Rinse flask and filter paper with a little ether.
Used 5 mL of ether to rinse flask and filter paper.
11. Add a glass bead as boiling stone and remove ether and ethanol using rotary evaporator. Scrape solid from flask. Weigh and obtain crude melting point.
Removal of solvent gave pale yellow residue stuck to sides of flask. Easy to scrape from sides but some left. Yellowish powder. 2.55 g 2.05 g crude product (damp). m.p = 86- 90 C.
12. Purify solid by mixed-solvent recrystallization using just enough boiling- hot ethanol to dissolve product. (10-13 mL)
Used 11 mL of hot ethanol. Gave yellowish solution.
13. After solid dissolves, add warm water drop-wise with stirring until the cloudiness does not disappear. Cool on wood block 10 min. Then chill in ice bath for 15 min.
Cloudiness formed after adding about 1 mL of water. More solid formed as solution cooled. Solid was whiter in color than the crude solid.
14. Vacuum filter, rinsing w/ ethanol. Obtain
final mass and m.p. Obtain IR, NMR, and submit sample in labeled vial.
Expect yield of purified product of 40-60%
Solid was creamy white. Filtrate was
yellowish. 1.39 g of dry pdt. obtained. m.p. was 90- 91 C. IR, NMR spectra obtained using CDCl3 solvent and attached to report.
The IR was obtained by allowing a CH 2Cl2 solution to evaporate on a salt plate.
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Ben Zeen IR Spectrum of Benzocaine (CH 2Cl2 solution evaporated on a salt plate)
Analysis of the IR spectrum of the product gave the following information:
(The absorptions that were intense or which were easily identified are listed.)
Absorption, cm -1 intensity / shape Interpretation
3424 medium / fairly sharp Overtone of C=O 3346 + 3225 2 med-strong bands N —H stretches of NH 2
3047 sharp / weak aromatic C –H stretch 2986-2900 several weak bands C-H stretches in alkyl groups 1687 very strong / sharp C=O stretch of ester 1634 very strong N —H bending (probably) 1601 strong / sharp C=C stretch of aromatic ring
1518 strong / sharp C=C stretch of arom. ring (probably)1476 weak / sharp (unsure) 1450 weak / sharp C-H bend of alkyl groups 1367 weak / sharp C-H bend of CH 3 group 1312, 1174 very strong C —O of ester 849 medium / sharp o.o.p. C-H bends in 1,4-disub arom. ring
Summary of IR interpretation I expected benzocaine to show two bands from the NH 2 group at around 3300 cm -1, plus absorptions from the aromatic ring at near 3050, 1600, 1500 and around 835 cm -1. This is basically what was seen. The absorptions at 3000 and at 1450 and 1380 cm -1 were from the alkyl groups. The carbonyl group was not at 1735 cm -1, but instead it was at 1687 cm -1. Two strong absorptions at 1312 and 1174 cm-1 are probably the C —O stretches from the ester. There also is an unexpected, intense absorption at 3424 cm -1. I did not observe any strong, broad O —H near 3400 that would have been visible if water or residual alcohol had been present. The 3424 band is fairly sharp, so I believe it is from something else. It is almost exactly twice the frequency of the C=O band, so I think it is an overtone of the C=O stretch, but this is far more intense than any I have seen one before. Maybe the amine group affects the C=O group somehow.
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I believe that the C=O is the band at 1687 cm -1, but I do not know why it is below 1700. Wade, chapter 21.4 says the C=O for conjugated esters is around 1710-1725 cm -1, and the book shows C=O of methyl benzoate at 1723 cm -1. For now, I am assuming that the band at 1687 is the C=O, because there is nothing else that seems to fit. Maybe the amine group lowers the stretching below 1700. I was unable to find any information in Wade or Mohrig that explained the result.
NMR SPECTRUM Ben Zeen 1H NMR spectrum of my Benzocaine. Solvent is CDCl 3.
3H, triplet
Table of NMR Data Chemical Integration Splitting Assignment Shift, ppm
7.8 2H doublet A 6.6 2H doublet B 4.3 2H quartet C 4.1 2H br. singlet D 1.3 3H doublet E
Data TableMass of 4-aminobenzoic acid (PABA): 2.67 g Volume of ethanol: 41 mL Mass of crude product: 2.05 g Mass of recrystallized product: 1.39 g
Calculation of amounts of reagents used and determining the limiting reagent:
H 2 N O
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Moles of PABA: 2.67 g of PABA x 1 mole PABA = 0.0195 moles PABA 137.136 g PABA
Moles of EtOH
41 mL of EtOH x 0.95 x 0.789 g EtOH x 1 mole EtOH = 0.70 moles EtOH 1 mL of EtOH 46.068 g EtOH
The “0.95” corrects for the fact that the ethanol used contained 95% EtOH, 5% H 2O.
PABA is the limiting reagent.
Calculation of the theoretical yield of Benzocaine:
0.0195 moles PABA x 1 mol of Benzocaine = 0.0195 moles benzocaine
1 mol of PABA = 0.0195 moles benzocaine x 165.189 g benzocaine = 3.22 g benzocaine
1 mole benzocaine
Calculation of crude yield of benzocaine (assuming it is pure, which it isn’t):
2.05 g crude yield x 100% = 63.7 % crude yield 3.22 g theoretical yield
Calculation of the actual yield of purified Benzocaine:
1.39 g actual yield x 100% = 43.1 % yield of purified product 3.22 g theoretical yield
Discussion of results: I believe that I successfully prepared a sample of fairly pure benzocaine. The melting range of my product was 90- 1 C, which is narrow and close to the accepted value for pure benzocaine (92 C). This is one indication that my product is rather pure. The physical appearance of my product also fits the description in the Merck Index.
The IR spectrum fits what would be expected for benzocaine. The NH 2 group is visible as two absorptions at 3346 and 3225 cm -1. The ester group is shown by the strong C=O band at 1687 cm -1 and the C —O bands at 1312 and 1174 cm -1. The aromatic ring is shown by the C-H stretching band at 3047, the C=C stretches at 1601 and (probably) 1518 and the o.o.p C-H bend at 849 cm -1. The o.o.p bend also showed that the compound was 1,4- disubstituted, as would be expected. There were no broad O-H stretches near 3300 cm -1
that would be present if any unreacted PABA, residual ethanol or water were present, so I know that these are not in my product.
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I do not know why the C=O stretching frequency is below 1700 cm -1, but my spectrum does closely match the one the IR spectrum of benzocaine found at the Sigma-Aldrich website (www.sigmaaldrich.com/spectra/ftir/FTIR000308.PDF) . The online IR spectrum also has the band at 1690 cm -1, and nearly matches my IR spectrum. Therefore, I believe that the IR suggests that my product is quite pure.
The NMR spectrum is what would be expected for benzocaine. The ethyl group is shown by the 3H triplet and 2H quartet. The broad singlet at 4.1 ppm is the amine hydrogens. The aromatic H’s are the two 2H doublets at 6.6 and 7.8 ppm. The pattern indicates a 1,4 - disubstituted aromatic ring. The carbonyl group deshields the H’s ort ho to it, so they are the ones that are further downfield. There were no unexplained peaks present, so the NMR spectrum also indicates that my product is pure.
My 43.1 % yield of purified product is within the 40-60% range suggested by the lab manual, but this means that over half of the potential amount of product was lost. The
most obvious source of loss was the recrystallization step. Some benzocaine remained dissolved in the ethanol, so it was lost in this step. About 0.66 g of mass was lost, but the crude product was still damp (probably ether and/or ethanol), so the amount of solid lost here is less than 0.66 g, but it is not possible to know how much it was.
Also, the reaction was probably incomplete. I stopped the reflux at 75 minutes, instead of letting it go 90 minutes. The Introduction in the Lab Manual says the reaction was a Fischer esterification reaction, and these reactions are equilibria that do not go to completion. If I stopped too soon, then the reaction may not even have reached equilibrium. The crude yield was less than 64 %, so about a third of the potential yield of
product was lost even before isolating the crude product. However, there is no way tocalculate how much of the product never was formed by stopping the reflux so soon.
Some product was probably lost in the extractions, drying and filtering, but I took care to rinse my glassware to try to keep this to a minimum. Mechanical losses, such as not being able to scrape all of the product from the flask and traces left on the glassware and filter and filter paper, probably added up to a few percent of the loss. I saw no large deposits of residue on any glassware that would suggest greater losses.
Summary:
Benzocaine was successfully prepared in 43.1 % yield. The product appeared to be quitepure, as judged by the narrow melting point range, appearance, as well as the IR and NMR spectra. Recrystallization was a major source of product loss, but it was clear that the reaction did not proceed to completion.
[Note: Any additional spectra (such as those used for comparison or provided by the instructor), along with any assigned homework problems, are attached to the lab and turned in as well.]
WRITING LABORATORY REPORTS
The cornerstone of science is communicating experimental results. For each experiment, you will submit a written report that describes the reaction you carried out, the procedures you followed, the observations you made and the data you obtained and your interpretation of the results. Lab reports are made up of several sections as described below. Your instructor will provide you specific information about what is to be included for particular experiments. If you are unsure what to include something in your report, just ask your instructor.
The following is an example of an excellent organic lab report. Use it as a guideline for your own reports, but don’t plagiarize!
Abstract Observing the separation made by the spinach extract on the thin layer chromatography plates, a solution of 7:3 toluene:acetone was identified as the best eluent for calculating retardation factors. Compounds and Rf values were experimentally calculated as follows. The following compounds separated; B- carotenes, pheophytin a, pheophytin b, chlorophyll a, and chlorophyll b, along with their respective Rf values; .98, .90. .68, .61, and .53. Two fractions were collected from the column chromatography experiment, along with a sample of the original spinach extract. Fraction one resulted in a single compound with Rf value of .98. Fraction two resulted in two compounds with Rf values of .45 and .10. The original spinach extract resulted in four compounds with Rf values of .98, .63, .45 and .22. TLC was used for the identification of compounds in an unknown pain reliever based on Rf values for Tylenol and Bayer. The unknown resulted in three compounds with Rf values of .64, .46, and .24. Tylenol was experimentally found to have a Rf value of .45. Bayer was experimentally found to have an Rf of .5.
Introduction Thin-layer chromatography is a technique used to separate compounds with in mixtures. This is useful for evaluating the purity of compounds and monitoring the progress of a reaction. TLC consists of two
phases, a mobile phase and a stationary phase. TLC functions by the differences in polarity of the compounds with the solvent used, result in varying migration in well defined and separated spots.
Reagents and Equipment Reagents used in this experiment include methanol, hexanes, water, sodium sulfate, toluene, acetone, spinach, spinach extract from column chromatography experiment, and pain relievers. The equipment used includes a mortar and pestle, beakers with glass watches, glass funnel, cotton plug, 125 mL separatory funnel, Erlenmeyer flask, Pasteur pipets, and test tubes.
Procedure and Observations Ten grams of spinach was cut, dried and drained before weighing. The spinach was added to the mortar with twelve milliliters of methanol and crushed for three minutes resulting in a bright green mixture. The methanol was then discarded. Fifteen milliliters of hexanes and five milliliters of methanol was added to the mortar with the leaves and crushed for five minutes resulting in a dark green mixture. The liquid was
then transferred to a beaker and filtered with a cotton plug into the separatory funnel. Three layers formed. A dark green layer formed on top followed, by a lighter green layer followed, by an almost clear layer. Five milliliters of water was added to the separatory funnel. Funnel was inverted and periodicall y vented. The dark green organic layer stayed on top. The lower aqueous layer was extracted and discarded. The organic layer in the separatory funnel was then rinsed three more times, resulting in the aqueous layer becoming more clear with each rinse. The organic layer was then separated and transferred to an Erlenmeyer flask. Anhydrous sodium sulfate was added to absorb remaining water. Solution was then filtered with a cotton plug and funnel into a test tube. The test tube was sealed for one week. With the test tube concentrated down to one to three milliliters the experiment was continued one week later.
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Five development chambers were prepared in beakers. Solvents used included hexanes, toluene, 9:1 toulene-acetone, 7:3 toulene-acetone, and acetone. TLC plates were prepared for each development chamber. The 9:1 toulene-acetone was used to determine the correct size of compound to be used. The 7:3 toulene-acetone was found to have the most separation. The spinach extract from the column chromatography experiment was then dissolved in a small amount of the solvent used to collect the fraction. Each fraction and remaining extract was placed on a TLC plate in the 7:3 toulene-acetone developing chamber. Tylenol, Bayer, and an unknown pain reliever was respectively crushed to a fine
powder and placed in separate test tubes. Each compound was dissolved with a 1:1 mixture of methylene chloride and ethanol solution. Using .5% acetic acid in ethyl acetate as the solvent, TLC plates were labeled and ran. The unknown was paired with each comparative pain reliever on a separate TLC plate. The Unknown resulted in three separate compounds. The Tylenol and Bayer each resulted in one compound. Ultraviolet light was used to visually see the compounds and mark the TLC plate appropriately.
Data The following is a table of the spinach extract with 7:3 v/v toluene:acetone used as the eluent. TABLE 1 Compound Rf B-carotenes .98 Pheophytin a .90 Pheophytin b .68 Chlorophyll a .61 Chlorophyll b .53
The following is a table of the fractions and extract from column chromatography experiment with 7:3 v/v toluene:acetone used as the eluent.
TABLE 2 Compound Rf
.10
.63
.45
.22 The following is a table representing the unknown pain reliever, Tylenol, and Bayer with .5% acetic acid in ethyl acetate used as the eluent. Ultraviolet light was used to visually see the compounds. TABLE 3 Compound Rf Unknown .64
.46
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Results Based on the separation of the spinach extract using hexanes, toluene, 9:1 toluene-acetone, 7:3 toluene acetone and acetone as the eluents, 7:3 toluene acetone was found to have the best separation of compounds. Acetone was found to not be a good solvent to separate the pigments due low polarity. Taking the Rf values of each compound and comparing to the polarity of each pigment, the correlation is a direct relation. As the polarity of the compound decreases so does the Rf value of the compound. Using the spinach extract from part one as a basis of comparison for the spinach extract from the column chromatography experiment, fraction 1 was found to contain B-carotene. Based on the polarity of the carotenes and the pigment separations of the other eluents tested, a mixture of hexanes and toluene would
provide the best separation. Hexanes provided a minor movement of the carotenes with little movement of the rest of the pigments due to the differences in polarity. Toluene provided much more movement up the TLC plate of the carotenes and remaining pigments but not enough separation. To achieve an acceptable Rf value, close to but not greater than .75, a mixture of the two solvents is necessary. Fraction 2 was found to contain chlorophyll b. Chlorophyll a has a methyl group in the position chlorophyll b has an aldehyde group, making chlorophyll b more polar. Based on this assessment, the original appearance of the fraction and the proximity of Rf values, chlorophyll b was determined as the compound. The additional Rf compound found in fraction 2 is too far from the range of experimental Rf from any data collected in table 1 to be determined. The column chromatography spinach extract was found to contain B-carotenes, chlorophyll a chlorophyll b and an additional compound that was too far from the range of experimental Rf values in table 1 to be determined. The unknown pain reliever was found to have three separate compounds with similar components of both Tylenol and Bayer. The Rf values for Tylenol and Bayer were very close in proximity, along with a component in the unknown. This indicates that the unknown contains a component of both the Tylenol and the Bayer. The overall TLC plates of the pain relievers were not as clean in separation as the spinach plates. The compounds came out as large smears indicating overloading of the TLC plate. For future experiments, a technique of using less compound on the TLC plate will prove to provide better data.
Follow up questions (deleted so that you can come up with your own answers)
References Techniques for Characterizing and Purifying Organic Compounds. CHML 2210-70B Brevard Community, Palm Bay Campus. Fall 2013 Edition.
Characterization and Purification
There are four problems that confront organic chemists on a daily basis: 1. is the sample I have pure? 2. if it is not pure, what can I do to remove the contaminants? 3. if it is pure, what properties does the sample have which will enable me to recognize the
same compound if I obtain it again from a different reaction source? 4. what is this stuff anyway?
Virtually any method, which can be used to characterize a compound, will also serve as another check of the purity of the sample in question. No single means of characterization is sufficient to establish the identity of a previously known compound and no single means for indicating purity is sufficient to establish purity. Good experimental work in the characterization and purification area systematically includes several indicators of good purity and an array of sample properties to characterize the compound in question. Multiple redundancies in information make characterization possible.
In this course, the first half of organic chemistry, you will learn many basic techniques. All of these techniques have application beyond the chemistry laboratory (biology uses chromatography extensively).
Washing Dishes
In order to characterize and purify compounds successfully and to carry out actual reactions, one must start each experiment with clean glassware. The easiest way to obtain this is to do your dishes at the end of the previous experiment since many procedures need to be done under dry conditions. This isn’t trivial because many organic compounds are not very soluble in water. Suggested steps are as follows:
1. Remove grease (if any) from ground-glass joints. If stopcock grease has been used, it must be removed before washing is attempted, otherwise grease distributes itself over everything. To de-grease joints, moisten a paper towel with ether or dichloromethane and wipe the grease off the joint. Ether is marginally better but is flammable.
2. Scrub the glassware with soap and water. Use a brush. 3. Rinse the soap off with water. 4. Visually inspect the glassware to see if it’s clean. If so, you’re done. If not, a little more
work is needed. 5. If the glass isn’t clean, get it soapy again; skirt acetone onto the s tain and scrub again.
Use a brush. Wet, soapy acetone is a pretty good general solvent for slime. 6. Continue steps 3-5 until glass is clean.
C. Heath and Co. Lexington, MA.
COOH
napthalene cinnamic acid
Introduction: The melting point and boiling points of a pure solid organic compound are characteristic physical properties, along with molecular weight, refractive index and density. Both melting point and boiling point are affected by the forces that attract one molecule to another: ionic attraction, van der Waals forces, dipole-dipole interactions and hydrogen bonding.
A pure solid will melt reproductively over a narrow range of temperatures, less than 1 0C. Using less than 1 mg of material, a melting point can be determined. The melting point apparatus is very simple, consisting of a thermometer, a capillary tube to hold the sample and a heating bath. Melting points are determined for three reasons: 1) if the compound is known, the melting points will help characterize the sample in hand 2) if the compound is new then the recording of the melting point is done to allow for further characterization by others 3) The range of the melting point is indicative of the purity of the compound; an impure compound will melt over a wide range of temperatures. Boiling point is used to characterize a new organic liquid and knowledge of the boiling point helps to compare one organic liquid with another. Comparison of boiling points with melting points is instructive but process for determining boiling points is more complex. Boiling point determination requires more material and is affected less by impurities, it is not as good an indicator of purity as melting point.
Procedure: Part I: Calibration of the thermometer: Determination of the melting point of standard substances over the range of interest gives the difference between the values found and those expected. This difference constitutes the correction that must be applied to future temperature readings.
IMPORTANT! The liquid in the thermometers is very thin and can collect in the top part of the thermometer, resulting in inaccurate readings. Always store your thermometer with the bulb end lowermost. Also, check your thermometer before use to make sure that the thread of liquid is continuous and that the top is free of liquid.
Thermometer Calibration (Your instructor may tell you to skip this part; check your syllabus.) The thermometers used in this course are fairly accurate (±1 C) and usually do not nee d to be calibrated, so your instructor may have you skip this step. If you are instructed to calibrate your thermometer, follow the instructions given below.
Why carry out a thermometer calibration at all?
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When measuring temperatures, the upper part of the thermometer is cooler than the bulb, and the liquid in the upper part of the thermometer contracts slightly. The resulting reading can be 1-3 degrees below the temperature of the bulb. Many thermometers are accurate enough to need no correction, but a calibration curve corrects for less-accurate thermometers.
To prepare a calibration curve, record the identities and true melting ranges of three or four compounds provided by the instructor. Obtain reliable melting ranges for these solids and record them in your notebook. Determine the average values for both the observed and the true melting ranges for each compound. .
To prepare your calibration curve, label the X- axis as “Observed Temperature” and the Y -axis as the “Corrected” or “True” Temperature. Start numbering your graph at 30 or 40, using 1 per division on the
paper. Next, plot the melting points of your known compounds on the graph. Use small dots for the data points. Draw a straight line which best fits these points (use pencil first). Your line will probably not pass through the origin. Show your calibration curve to the instructor for checking. Save your approved calibration curve for use throughout the semester, and label your thermometer with your name and locker number. If you break or lose your thermometer, you will need to prepare a new calibration curve for your new thermometer.
Converting measured temperatures to corrected values is simple: The corrected temperature (Y-axis) is where the measured temperature (X-axis) intersects with the calibration line. You need to do this with
both the lower and upper values of a temperature range. Note: Do not use averages of temperatures in Part B or any future experiments; average temperatures are only used when constructing a calibration curve!
Take a 10 mg sample of trans -cinnamic acid and prepare a melting point tube by pushing a melting point capillary into the powder and force the powder down in the capillary by tapping the capillary or by dropping it through a long glass tube held vertically and resting on a hard surface. The column of solid should be no more than 2-3 mm in height and should be tightly packed. Take a melting point range of this compound using the Mel-Temp apparatus in the lab. The temperature can be raised rapidly until the temperature is about 20 0C below this point. Then slow down heating considerably so that rate of increase is no more than 1 0C per minute while the sample is melting. Compare the melting point range with the published melting point of t-cinnamic acid. Record the difference between published MP and the MP you obtained. Repeat the same procedure using urea. Record the differences. If the differences recorded for t-cinnamic acid and urea agree within 1 0C, use this correction for every melting point you perform in this laboratory, provided that the same thermometer is always used. Write this correction in your lab report.
Part II: Melting Points of urea and cinnamic acid mixtures. Make mixtures of urea and cinnamic acid in the approximate proportions 1:4, 1:1, 4:1 by putting side by side the correct number of equal-sized small piles of the two and then mixing them. Grind the mixtures thoroughly for at least a minute with a mortar and pestle. Not the ranges of the three mixtures and use the temperatures of complete liquefaction to construct a rough diagram of mp versus composition.
Record this information in your notebook:
Unknown Number: __________ Appearance of Unknown: ___________________ m.p. of Unknown (uncorrected):__________C (If applicable): Corrected m.p. value: __________C
Determine the melting point of the unknown given by your instructor and on the basis of that melting point, identify that substance. Run two capillaries of the unknown. Run a very fast determination on the first to obtain approximate melting point and then use the second to do a slow, careful determination. Record your results and identify the compound you tested in your lab report.
Table 1.1 Melting Points of Unknowns Compound Melting Point ( C)
Benzophenone 49-51 Naphthalene 80-82 Benzoic Acid 121.5-122 Urea 132.5-133 Salicylic Acid 158.5-159
Clean Up: Place all leftover solids in the solid waste bottles provided. Capillary tubes can be placed in the broken glass box in the laboratory.
Suggested topics for Discussion What is the identity of your unknown? What is your rationale for your identification and how confident are you in your identification of your unknown? What problems (if any) did you have during the experiment? Feel free to include additional topics as appropriate.
Follow-up Questions (It is usually helpful to consult your lecture text when answering these.) 1. A student prepared a mixture of two compounds. The mixture showed a narrow melting
range, but the value is considerably lower than the melting range of either component in the mixture. Explain.
2. A student attempts to obtain a melting range, but he filled the capillary with five times as much sample as necessary. How might this affect the observed melting point? Explain.
3. A student was trying to obtain the melting point of borneol. While heating the sample, he stepped away from the apparatus for a few minutes. He saw that the capillary tube was empty, even though the temperature was well below the listed boiling point of borneol. What probably happened? What should the student do to obtain the melting range of his solid?
Chlorophyll a Chorophyll b Blue-green, polar Green, polar C55H72MgN 4O5 C55H70MgN 4O6 M.W. 893.5026 M.W. 907.4862
-carotene Yellow, nonpolar C40H56 M.W. 536.8824
CH3 CH 3 CH 3 CH 3
CH 3 R
Objective(s): To separate the color pigments in spinach by column chromatography.
Technique(s) used: Isolation of a natural product; extraction; preparation and use of column chromatography.
Zubrick 7 th Ed. – Column Chromatography and TLC page 221 and page 239 Microscale column chromatography Zubrick 7 th page 243
CAUTIONS : Avoid inhaling the solvent fumes; keep the stoppers in the test tubes.
Procedure:
At the beginning of the lab set up a hot water bath on a hot plate and watch the water level so it doesn’t get too low in the beaker (hot water bath).
Extraction of the pigments:
Weigh out 5 - 6 grams of spinach. Remove as many of the stems as possible, then put the leaves in a mortar with a scoop of
sand and 5 mL of methanol. Grind the mixture with a pestle for 10 minutes. Pour the ground mixture into a large test
tube and add about 10 mL of hexane. With a spatula or stirring rod, mix the hexane with the ground spinach.
Decant the hexane into another test tube. Be sure to press the spinach to get out as much of the hexane as possible. To the hexane in the second tube, add about 10 mL of DI water, stopper the tube and shake, but not too hard. Remove the lower aqueous layer with a pipet and repeat the extraction. This will remove most of the methanol from the hexane.
Dry the hexane by doing the following: set up a vacuum filtration with a Buchner funnel and filter paper. Place a scoop of drying agent, the anhydrous sodium sulfate, on the filter paper. Turn on the water to the aspirator to do a vacuum filtration. Carefully pour the green hexane solution into the Buchner funnel over the drying agent and filter it. Alternate procedure: Put a scoop of anhydrous sodium sulfate drying agent in the flask. Swirl and let sit 5 minutes. If the drying agent is still clumping, add more until it moves like sand in bottle. Using a small funnel, put a small piece of a cotton ball loosely over the hole to the stem. Gravity filter the solution through the cotton ball to separate the extract from the drying agent. Save the green hexane spinach extract solution in a clean test tube.
Add a boiling chip to the test tube and evaporate the solution to about 0.5-mL in a hot water bath on a hot plate. This is the green hexane spinach extract that you will use with your column.
Figure 1. Pasteur pipet wet-column chromatography
Set up a microscale column as shown in Figure 1. Obtain a chromatography tube such as a Pasteur pipet. Shorten the tip of the Pasteur pipet; if it is not already shorten. Gently press a small glass wool plug into the tip of the tube - being careful not to pack it too
tight. Add about 50 mg of sand on top of the glass wool.
Weigh about 500 mg of alumina and break up any clumps with the stirring rod (if necessary,use a mortar and pestle to make a fine powder of alumina). Slowly add the alumina to the column. Every so often, thump the column with your finger to aid in settling the alumina.
After all the alumina has been added, place about 50 mg of sand on top of the alumina.
The chromatography separation: Clamp the column on a ring stand. In four large test tubes add the following elution solvents:
1 st test tube 10 mL of hexane 2 nd test tube 7 mL hexane + 3 mL toluene (7 / 3 hexane/toluene) 3 rd test tube 3 mL toluene + 7 mL ethyl acetate (3/7 toluene/ethyl acetate) 4 th test tube 10 mL of methanol.
Take two pipets; one for the green hexane spinach extract solution and one for solvents. Prepare to run the column. The microscale column (Pasteur pipets) doesn’t have a stopcock. Once it starts running, it goes until it runs out. You can’t let the column dry
out. Have all your elution solvents and the green hexane spinach extract solution ready and be prepared to run the column once you start it.
Now slowly add some hexane into the column and let it wet the entire column. From this point on, it is extremely important that the liquid level never be allowed
to get below the top of the sand.
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DO NOT EVER LET THE COLUMN RUN DRY! NEVER LET THE SOLVENT LEVEL FALL BELOW THE LEVEL OF THE ALUMINA!
Carefully pipet the green hexane spinach extract (from the extraction of the pigments section above) to the top of your column. Don’t fill the column with liquid; drip the solution onto the sand. Leave a small amount for the TLC experiment.
When the green hexane spinach extract is all on the column, just below the sand, add a small amount of hexane, the first elution solvent.
Watch your column! Do not ever let the column run dry. Collect the drops off the end of the column in a small beaker. As long as the first color band is seen moving down the column, continue with hexane. As the color band reaches the glass wool plug, place a small test tube in place of the
beaker to catch the colored solution in the smallest possible volume. Note in your lab book, the color, solvent, and approximate volume.
When no color bands are seen moving with your present elution solvent, change to the next more polar elution solvent.
Continue the process until all the solvents have been used. Collect each color band in small test tubes as a series of fractions and label all test
tubes. Any color left at the top of the column cannot be removed .
Store the reserved extract and fractions collected off the column in OPEN test tubes to be used next week in the TLC experiment .
Notebook Report: Keep a detailed record of all your observations during the extraction of the pigment and the chromatographic separation. In tabular format record the number of the fractions being collected, the time over which the fractions were collected, the concentration of the solvent (10 mL hexane; 7/3 hexane to toluene…..) on the column, the appearance of colors on the column, and the color of the solution in the fraction being collected. Based upon color and polarity, identify each fraction either as ß-carotene, chlorophyll a , chlorophyll b, or xanthophyll. Number
of Fraction
Color of Fractions in Test Tube
1. 2. 3. 4. 5. 6.
Under optimum separation, the fractions collected show the following pigments in spinach: ß-Carotenes (yellow-orange) nonpolar Pheophytin a (gray, may be nearly as intense as chlorophyll b) Pheophytin b ( gray, may not be visible) Chlorophyll a (blue-green, more intense than chlorophyll b) Chlorophyll b ( green) more polar than chlorophyll a Xanthophylls (yellow)
A characterization method and criterion for purity
Thin layer chromatography (TLC) uses glass, aluminum or plastic plates covered with a layer of
adsorbent. Common adsorbents are silica gel, alumina and cellulose. The sample is applied asa dilute solution to a position near the bottom of the plate. The plate is stood on end in a shallow pool of solvent (called the eluant), which will run up the plate.
As the eluant passes over the applied sample, the molecules, which are adsorbed on (stuck to) the adsorbent, will tend to dissolve in the eluant, move ahead in solution, and re-adsorb further up the plate. The sample will constantly re-dissolve and re-adsorb while the plate is running (being developed). The more strongly the sample adsorbs on the adsorbent, the less it will tend to dissolve, and the more slowly it will move up the plate. Different compounds will, in general, tend to dissolve, and the more slowly it will move up the plate. Different compounds will, in general, adsorb with different strengths, so that different compounds usually move different distances up a give plate.
Spotting and Developing TLC Plates: To apply a sample to a TLC plate, it is dissolved in a suitable solvent (try CH 2Cl2 first) at a concentration of 100 mg per mL of solvent. The solution is applied to the plate with a double- open ended capillary tube, ~3/8” from the bottom of the plate so that a spot of ~1/8” diameter is formed (Figure 1).
Figure 1. TLC plate with starting material product and a mixture of both spotted on the plate.
The appropriate solvent (eluant) for development is poured into a TLC chamber (either a special chamber or a beaker). The solvent should be 1/8” deep, so that the spot will wash the sample off the chamber (otherwise, solvent will wash the sample off the plate). The plates are then stood in the chamber (or beaker) and the chamber is covered (with a watch glass). Once the eluant reaches the top of the plate, the plate is removed from the chamber. Upon removal, mark, in pencil, where the solvent stopped on the plate. Lay the plate flat to dry.
Silica gel is very polar. Therefore, polar compounds tend to stay near the origin, while non-polar compounds tend to move much faster up the plate. A suitable eluant is chosen by trial and error, and often differs from the solvent used to spot the sample. If a compound runs too fast
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(too far up the plate) your next plate should be run with a less polar eluant. If the compound runs too slowly, your next plate should be run with a more polar eluant. See Table 1.
Table I. Comparative Polarities of Solvents LEAST POLAR
hexane * cyclcohexane
pyridine acetone
1-propanol ethanol
methanol* water
MOST POLAR
*indicates the “big four” solvents which can be used in mixtures to separate most organic compounds.
Visualizing Spots . A TLC plate, which has just been developed, looks (unless the sample is colored) just like an undeveloped plate: a plain white surface. To locate the spots of compounds, they must be visualized. Visualization can be done either destructively or non- destructively.
a) Non-destructive Visualization . The most common non-destructive technique requires that the TLC plate be prepared with an adsorbent, which contains a fluorescent indicator (a compound that fluoresces when you shine UV light on it. The TLC plates that we will be using have this indicator, which fluoresces green under 254-nm light. If such a plate is spotted and developed, UV light lets you see the spot(s). The beauty of this kind of visualization is that you can wash your sample back off the plate and recover it unchanged; for the closely related techniques thick-layer chromatography, this advantage is very important.
b) Destructive Visualization . The usual techniques for destructive visualization involve spraying the plate with a reagent which immediately reacts with adsorbed sample to give a product which either I) differs in color from the background color of the sprayed plates or II) has low volatility, so that vigorous heating will lead to charring of the sample, giving black spots on a white background. A wide variety of general-use and special-purpose sprays exist. Spray visualization is destructive because the sample is irreversibly converted into another compound. Every method is unable to detect some samples (for example, 254- nm UV visualization can’t detect a sample that doesn’t absorb 254 -nm
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still “clean”, you can have good evidence, but not proof, that the two compounds are the same (Figure 5). It the middle spot splits in two, you have proved that the compounds are different.
A Measure of Impurity . If your sample is a mixture, and if the components have different R f values under your TLC conditions, you will see several spots on the plate after visualization. TLC alone never tells you how many components a mixture has---it tells you the MINIMUM number of compounds, which must be present. It is a real possibility that your visualization method may not show all the spots present, or that one spot may represent a mixture of compounds, which have the same R f value (co-elution).
TLC Procedure (http://orgchem.colorado.edu/hndbksupport/TLC/TLC procedure.html)
1. Prepare the developing container.
The developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top:
In the teaching labs, we use a beaker with a watch glass on top.
Pour solvent into the beaker to a depth of ust less than 0.5 cm.
To aid in the saturation of the
TLC chamber with solvent vapors,line part of the inside of the beaker with filter paper.
Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you
repare your TLC plate.
2. Prepare the TLC plate.
TLC plates used in the organic chem teaching labs are purchased as 5 cm x 20 cm sheets. Each large sheet is cut horizontally into plates which are 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be.
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Shown in the photo to the left is a box of TLC plates, a large un-cut TLC sheet, and a small TLC plate which has been cut to a convenient size.
Plates will usually be cut and ready
for you when you come to lab.
Handle the plates carefully so that
you do not disturb the coating of
adsorbent or get them dirty.
easure 0.5 cm from the bottom of the late. Take care not to press so hard
with the pencil that you disturb the adsorbent.
Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin : the line on which you will "spot" the plate.
It's kind of hard to see the pencil line in the above photos, so here is a close-up of how the plate looks after the line has been drawn.
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Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised.
Use a pencil and do not press down so hard that you disturb the surface of the plate. A close-up of a
plate labeled "1 2 3" is shown to the right.
3. Spot the TLC plate
The sample to be analyzed is added to the plate in a process called "spotting". If the sample is
not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes,ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak ; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, sometimes only a process trial and error (as in, do it over ) will result in well-sized, easy to read spots.
add a few drops of solvent . . .
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The solution is applied to the TLC plate with a capillary tube or a microcap, as seen in the photos.
Take a tube and dip it into the solution of the sample to be spotted. Then, touch the end of the tube gently to the adsorbent on the origin in the place which you have marked for the sample. Let all of the contents of the tube run onto the plate. Be careful not to disturb the coating of adsorbent.
dip the microcap into solution - the arrow points to the microcap, it is tiny and hard to see
make sure it is filled - hold it up to the light if necessary
touch the filled microcap to TLC plate to spot it - make sure you watch to
see that all the liquid has drained from the microcap
do this rinse
process 3 times!
rinse the microcap with clean solvent by first illing it . . .
. . . and then draining it by touching it to a paper towel
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If the microcapillary tube breaks or clogs, you may obtain a new one.
here's the TLC plate, spotted and ready to be developed
4. Develop the plate.
Place the prepared TLC plate in the developing beaker, cover the beaker with the watch glass, and leave it undisturbed on your bench top. Run until the solvent is about half a centimeter below the top of the plate (see photos below).
place the TLC plate in the developing container - make sure the solvent is not too deep
The solvent will rise up the TLC plate by capillary action. In this
photo, it is not quite halfway up the plate.
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In this photo, it is about 3/4 of the way up the plate.
The solvent front is about half a cm below the top of the plate - it is now ready to be
removed.
Remove the plate from the beaker.
quickly mark a line across the plate at the solvent front
with a pencil
Allow the solvent to evaporate completely from the plate. If the spots are
colored, simply mark them with a pencil.
5. Visualize the spots
If your samples are colored, mark them before they fade by circling them lightly with a pencil (see photo above).
Most samples are not colored and need to be visualized with a UV lamp. Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil.
Beware! UV light is damaging both to your eyes and to your skin! Make
sure you are wearing your goggles and do not look directly into the lamp.Protect your skin by wearing gloves .
If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate.
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this is a UV lamp here are two proper sized spots, viewed under a UV lamp
(you would circle these while
viewing them) The plate to the left shows three compounds run at three different concentrations. The middle and right plate show reasonable
spots; the left plate is run too concentrated and the spots are running together, making it difficult to get a good and accurate R f reading.
Here's what overloaded plates look like compared to well-spotted plates. The plate on the left has a large yellow smear; this smear contains the same two compounds which are nicely resolved on the plate next to it. The plate to the far right is a UV visualization of the same overloaded plate.
NOTE: Your instructor will indicate which parts of the experiment are to be performed during the laboratory period.
PART ONE: Identification of Components in Spinach by Thin-layer Chromatography (Week 1- follow Instructor’s directions)
PROCEDURE:
Reference: Palleros, D. A. Experimental Organic Chemistry . 2001, J. Wiley and Sons, New York, pgs. 193-195.
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Vegetable Extract Weigh out 10 g of spinach leaves and chop them up using scissors. If they were previously frozen, drain as much water out of the leaves as possible before weighing.
Place the spinach in a large mortar, add 12 mL of methanol, and crush the leaves with a pestle for about 3 minutes. With the aid of the pestle or spatula, squeeze the spinach against the side wall of the mortar to remove as much methanol as possible. Transfer the liquid to a 250-mL Erlenmeyer flask, labeled “methanol -water” and set aside. The contents of the flask will eventually be discarded.
Extract the remains of the leaves with a mixture of 15 mL hexanes and 5 mL methanol, crushing the tissue with the pestle for about 5 minutes. The extract should be deep green. Leaving behind as much solid as possible, transfer the liquid (a mixture of hexanes and methanol) to a 100-mL beaker. Filter it using a glass funnel (4-6 cm diameter) with a cotton plug. Collect the filtrate directly into a 125-mL separatory funnel supported on a ring stand (make sure the stopcock is closed).
Add 5 mL of water to the separatory funnel, shake, vent and allow the layers to separate. Which layer is the aqueous layer? Collect the aqueous layer in the Erlenmeyer flask labeled “methanol -water.” Extract the remaining hexane layer with 5 mL of water in the separatory funnel. Collec t the aqueous layer along with any emulsion present in the “methanol -water” flask.
Transfer the organic layer to a clean and dry 50-mL Erlenmeyer flask; add a small spatulaful of granular sodium sulfate (Na 2SO 4) to dry the organic layer. Cap the flask with a stopper and swirl occasionally. After about 5 minutes, filter the suspension by using a clean and dry micro- funnel (about 2.5 cm in diameter) and a cotton plug. Collect the filtrate in a dry 50-mL round- bottom flask. Using the rota-vap, evaporate the solvent until the volume is approximately 2-3 mL. The color of the final extract should be deep green. With the aid of a Pasteur pipet, transfer the liquid to a labeled test tube.
TLC Analysis
Clean and thoroughly dry five 150-mL beakers and five watch glass covers. Label them with the names of the solvents to be used: 1) hexanes 2) toluene 3) toluene-acetone (9:1) 4) toluene- acetone (7:3) 5) acetone.
Determining the Optimum Number of Applications. Take the plate labeled “toluene - acetone” (9:1). This plate will be used to determine the optimum number of applications needed to visualize the separation of pigments. On this plate, the sample will be applied on three different spots, each with a different number of applications (1, 4 and 7). Immerse a double open-ended capillary tube in the extract; the liquid will rise through capillary action. Apply the
liquid to the plate by touching the plate with the tip of the capillary tube and applying slight pressure. If necessary, gently wiggle the capillary to make the liquid flow. The spot should be about 0.5 cm from the side edge of the plate. Raise the capillary tube to stop the flow of liquid when the diameter of the spot is about 3 mm. On the same plate, apply a second and a third spot at a distance of about 0.5 cm from each other. The number of applications should be 4 and for the third spot, 7. Allow the solvent to evaporate between applications. Failure

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Lab Manual CHML 2210 Fall 14

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