5.301 | January IAP 2012 | Undergraduate

Chemistry Laboratory Techniques

Labs

WARNING NOTICE

The experiments described in these materials are potentially hazardous and require a high level of safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented.

Legal Notice

This section includes course modules for mastering a series of chemistry laboratory techniques. Information on the original research project assignment and a listing of techniques guides are also provided. All materials may be found in the complete laboratory manual. (PDF - 2.4MB) (*Please note: for users on Mac machines experiencing problems viewing the images, downloading and saving the document should resolve this issue.) 

Video instruction tutorials from the Digital Lab Techniques Manual (DLTM) are recommended for some of the modules and are noted below.

Introductory Reading Assignment
Technique Modules
Technique Guides
Instrument Operation Guides

Introductory Reading Assignment

Before we get started in the lab on our first class meeting, there are several chapters in the text that you must read first. Our time in the lab will be intense, but of limited duration, so it is important that you complete the readings on time. Not only is the reading essential to your success in 5.301, but it also will help in your development as an experimental chemist.

So, before you begin your experiments, take some time to read over the following chapters in Zubrick & Mohrig. These texts were selected because they’re easy to read and very practical. For more in-depth reading on these and related topics, we recommend the listed selections in the text by Leonard, Lygo, and Procter. It is often difficult to fully grasp a laboratory concept by simply reading about it, but using the strategy of introductory reading, practicing in the lab, and post-lab review reading you will retain most of what we will cover in 5.301.

At the beginning of each lab period, there will be a short overview of that day’s topic, where Dr. Dolhun and your TA will facilitate a discussion of the assigned reading and the actual lab experiment. Much of this time will be set aside to answer questions that you have from the readings.

The following list is the bulk of the reading for the course. There will also be additional reading during IAP, but this introductory reading is meant to familiarize you with the typical chemistry laboratory.

Zubrick, James. The Organic Chem Lab Survival Manual: A Student’s Guide to Techniques. 8th ed. Wiley, 2010. ISBN: 9780470494370.
(Chapters 1 - Safety, 2 - Notebooks, 4 - Jointware, 6 - Interesting Equipment, 9 - Clean and Dry, 10 - Drying Agents, 11 - On Products, 15 - Extraction and Washing, 17 - Heat, 18 - Clamps, 27 – TLC, 28 - Column Chromatography, 30 - Gas Chromatography, 32 - Infrared Spectroscopy, 33 – Nuclear Magnetic Resonance 34 - Distillation)

Leonard, J., B. Lygo, and G. Procter. Advanced Practical Organic Chemistry. 2nd ed. CRC Press, 1994. ISBN: 9780748740710.
(1 - Introduction, 2 - Safety, 3 - Keeping Records, 4 - Equipping the Lab, 8 - Vacuum Pumps)

Mohrig, J.R., C. Noring Hammond, and Paul F. Schatz. Techniques in Organic Chemistry: Miniscale, Standard Taper Microscale, and Williamson Microscale. 3rd ed. W.H. Freeman, 2010. ISBN: 9781429219563.

(Part 1: Basic Techniques: Chapters 1 thru 16 pages 1-197 Part 3: Spectroscopic Methods: Chapters 20-24 pages 275-438.)

Technique Modules

The technique modules make up the bulk of the class. The modules fall under the topics of “Transfer and Extraction,” “Purification by Crystallization,” “Purification by Distillation,” “Purification by Flash Column Chromatography,” and “Protein Assays and Error Analysis.” It is important to note that the manual does not contain all of the information that you will need to complete these experiments. Some important information will be found in your pre-lab reading, while the rest will be covered during the pre-lab discussion. This three-pronged approach (the texts, the manual, and the discussions) will prepare you to tackle the experiments outlined in the technique modules.

An important part of the modules is the techniques checklist. Each module begins with a list of techniques that you will encounter during the experiment. When you have completed a technique module, you should return to the techniques checklist and check off all of the techniques that you have mastered. If you are still uncomfortable with a specific skill then you should practice it until you feel confident that you could apply it in a different experiment. In addition to various purification and manipulation techniques, this section will also introduce you to spectroscopic techniques like nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy, gas chromatography (GC), and ultraviolet-visible (UV-Vis) spectroscopy.

Note:

“CC” refers to the “Competent Chemist” modules.
“EE” refers to the “Expert Experimentalist” modules.

Transfer and Manipulation (CC)

Acids, Bases & In Between (EE)

Recrystallization—Mothball (CC), and Single Crystal (EE)

Distillation (CC, EE)

Column Chromatography (CC, EE)

Biochemistry (CC, EE)

Original Research Project

Technique Guides

Found in section eight of the Lab Manual, these guides will provide you with step-by-step instructions for some of the more common techniques encountered in a chemistry laboratory.

TOPICS FILES
NMR Sample Preparation (PDF)
GC Sample Preparation (PDF)
Thin Layer Chromatography Guide (PDF)
Extraction and Washing Guide (PDF)
No Air Technique Guide (PDF)
Two-Solvent Recrystallization Guide (PDF)
Guide to Growing a Single Crystal (PDF)
Distillation Guide (PDF)
Flash Column Chromatography (PDF)

Instrument Operation Guides

These guides will provide you with step-by-step instructions for some of the instruments commonly encountered in a chemistry laboratory.

INSTRUMENT FILES
NMR (PDF - 1.3MB)
IR Guide (PDF)
Refractometer (PDF)
UV (PDF)
Mass Spectrometer (PDF)
GC (PDF)

Acknowledgements

The laboratory manual and materials for this course were prepared by Katherine J. Franz and Kevin M. Shea with the assistance of Professors Rick L. Danheiser and, and Timothy M. Swager. Materials have been revised by J. Haseltine, Kevin M.Shea, Dr. Sarah A. Tabacco, Dr. Kimberly L. Berkowski, Anne M. (Gorham) Rachupka, and Dr. John J. Dolhun.

Introduction

Your advisor has carefully monitored your progress in 5.301. You are ready to move from the technique modules to an actual project. This project will require you to use many of the skills that you have learned over the past three weeks to address a specific question. In addition, you and your lab mates will learn to work as a research group in order to reach a definitive goal in a short period of time.

Overview

Our research group has a longstanding interest in the synthesis of derivatives of penicillin, which is classified into the β-lactam class of antibiotics having a common structural backbone as illustrated in Figure 1.

By changing the R group, we will synthesize a new penicillin derivative library, (a number of different R groups will be available)

Penicillin is used to treat many types of bacterial infections mostly Gram (+) and sometimes Gram (-) organisms. Since the discovery of penicillin there has been an ever-increasing resistance by the bacteria to many of the penicillin molecules presently on the market. In this research project, you will be synthesizing a new group of penicillin derivatives in order to overcome the antibiotic resistance to various strains of bacteria. Our research group will begin a comprehensive study, which will afford each student an opportunity to synthesize a fresh penicillin derivative. Go to the library and look through the literature, talk to the department graduate students on this project about the reactions that you will be running, and be sure to organize your efforts with your fellow lab mates.

In this research project you will each synthesize a penicillin derivative through acylation of 6-aminopenicillanic acid. This process is illustrated in Scheme 1 (PDF).

A wide selection of acyl chlorides will be made available to choose from. Progress of the reaction will be followed using TLC plates with additional characterization by NMR and MASS SPECTROSCOPY. The penicillin derivatives once synthesized will be purified by column chromatography and tested in a bioassay to determine and quantify their ability to kill bacteria. A broth dilution method will be developed to determine the concentration of penicillin that inhibits the growth of 50% of bacteria in vitro. Each of the penicillins will be tested using broth diluted with E. coli, a Gram (-) bacteria. After incubation of the diluted samples, optical density (OD) measurements will be made measuring their absorption at 600 nm on a Perkin-Elmer UV-VIS spectrometer. The results of the entire class will be made available to all participants so that the students in their final reports can develop structural and functional relationships among the various penicillin derivatives and discuss trends used in ranking the various derivatives based on their effectiveness at inhibiting bacterial growth. We will be synthesizing penicillins that may not yet be commercially available. The goal is to create and identify new antibiotics to stave off biological resistance by the bacteria.

What is unique about our project is the type of bacteria that we will be using. We have worked out the method and adapted it to (BL1) E. coli Gram (-) bacteria (Strain ATCC 10798). Since penicillin antibiotics are most effective against Gram (+) bacteria, we wanted to look carefully at the potential of their action against a Gram (-) strain. Our thinking here was that this is a less virulent strain to work with in terms of student safety (BL1) and secondly, if the results are promising for the Gram (-) strain we potentially could apply our most effective synthetic derivatives against the more dangerous Gram (+) bacteria in a (BL2) rated lab. See the MIT Undergraduate Lab safety considerations (PDF) for more information on our BL1 rated lab. 1 Adapted from: Whitaker, D. R., M. L. Truhlar, et al. Journal of Chemical Education 87 (2010): 634–6.

6.1 - Competent Chemist Rating

“What’s in a Cow’s Heart Anyway?”

Techniques Checklist

  • Pipetting with pipetman
  • Calibrating pipetman
  • Preparation of a standard curve
  • Serial dilution
  • UV-Vis Spectroscopy

Pre-Lab

  • Discussion of Protein Assays

Equipment

  • Pipettmen: 100 P, 1000 P
  • Pipette tips—large and small, 8 Test tubes
  • Eppendorf tubes and holders
  • Disposable UV-Vis cuvettes—5 mL

Goal

  • You will be given a sample solution of bovine heart cytochrome c. You will use the Coomassie® Plus Protein Assay from Pierce to determine the protein concentration of the sample.

Note

You will receive a tray of Eppendorf tubes: one containing stock solution, three containing 50 µL each of bovine heart cytochrome c, and several empty tubes for mixing solutions. You will also be provided with a bottle of 25mM MOPS buffer, pH 7.

Experiment Outline

Pipetman Calibration

Prior to beginning any experiment with a pipetman, it is necessary to first calibrate it. This procedure will determine exactly how much liquid is delivered when a certain amount is “dialed-in” to the instrument. To calibrate your pipetman, simply draw up a certain amount of water and then empty it into a tared container. You can then obtain a weight and, knowing that water has a density of 1.00 g/mL, you can perform a calculation to tell you the accuracy of your pipetman. Most instruments will need no correction, and the ones that are incorrect will usually be off by no more than 1 µL.

The Coomassie®—Protein Reaction Scheme

This protein assay works by forming a complex between the protein and the Coomassie® dye. When bound to the protein, the absorbance of the dye shifts from 465 nm to 595 nm (A595). You will first generate a standard curve using the protein Bovine Serum Albumin (BSA) by measuring the absorbance at 595 nm of a series of standards of known concentration. Next, you will measure the A595 of your sample and determine its concentration by comparison to the standard curve.

Protein + Coomassie®G-250 in acidic medium—> Protein-Dye complex (blue; measured at 595 nm)

  1. Preparation of diluted BSA standards 

    • Prepare a fresh set of protein standards by diluting the 2.0 mg/mL BSA stock standard (Stock) as illustrated below. There will be sufficient volume for three replications of each diluted BSA standard, if necessary.

  2. Mixing of the Coomassie® Plus Protein Assay Reagent 

    • Allow the Coomassie® Plus reagent to come to room temperature. Mix the Coomassie® Plus reagent solution just prior to use by gently inverting the bottle several times. Do not shake.

  3. The Standard Protocol 

    • Pipette 0.05 mL of each standard or unknown sample into appropriately labeled Eppendorf tubes. Prepare 3 unknown samples.
    • Use 0.05 mL of the diluent (25 mM MOPS buffer, pH 7. Provided by TA) to prepare one blank tube.
    • Add 1.5 mL of the Coomassie® Plus reagent to each tube including the blank, mix well. Allow 10 minutes at RT for color to develop.
    • Transfer standards, unknowns and blank to 1.5 mL UV cuvettes. Measure the absorbance at 595 nm of each tube vs. blank.
    • The computer will subtract the average 595 nm reading for the blank from the 595 nm reading for each standard or unknown sample. Follow the guidelines for opening the program and operating the UV (see 9.4 UV Operation Guide).
    • Prepare a standard curve by plotting the average blank corrected 595 nm reading for each BSA standard versus its concentration in µg/mL. Using the standard curve, determine the protein concentration for each unknown sample.

Helpful Hints

  • Keep all of your solutions until after you have plotted and analyzed your data.
  • You may need to re-do some of your UV absorptions.

Results

  • To obtain your “CC Rating” in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.930 correlation coefficient (R value) or higher. Additionally, the results from your absorbance values of the unknown should have a standard deviation of less than 0.048. Finally, you must determine the concentration of your unknown protein.

6.2 - Expert Experimentalist Rating

“A Heart as Strong as Iron”

Techniques Checklist

  • Use of a centrifuge

Equipment

  • Disposable UV-Vis cuvettes (1-mL capacity)
  • Pipettmen: 20 P, 100 P, 1000 P
  • Pipette tips
  • Eppendorf tubes (safe-lock)
  • Centrifuge
  • Boiling plate or rack to hold Eppendorf tubes
  • Large crystallizing dish

Goals

  • From the CC-level experiment, you should know the concentration of protein in your sample. Now you will determine the concentration of iron in bovine heart cytochrome c.

Experiment Outline

The Ferrozine Assay

Ferrozine is an iron-chelating agent. When it forms a complex with ferrous iron (FeII), it shows a characteristic UV-Vis absorption at 562 nm. By comparing the A562 of your sample to a calibration curve of iron standards, you will determine the concentration of iron in your protein sample.

Solutions provided by your TA:

  • Fe AA standard (AA = atomic absorption)
  • Buffer—25 mM MOPS, pH 7)
  • Ultrapure HNO3 (5 M)
  • 75 mM Ascorbic acid
  • 10 mM Ferrozine solution
  • Saturated ammonium acetate solution

  1. Preparation of Standards 

    • Prepare a fresh set of iron standards in 2 mL Eppendorf tubes, as illustrated below. Carefully label each tube. Also fill 3 tubes with 300 µL of your unknown protein sample. 

       

µL OF Fe AA STANDARD (100 µg/mL) µL OF BUFFER TO ADD
0 300
3 297
6 294
12 288
18 282
24 276
30 270

  • Add 30 µL of ultrapure HNO3 (5 M) to each standard and sample.
  • Place the closed Eppendorf tubes in a rack, and boil them for 15 minutes in a hot water bath (a large Pyrex dish over a heating plate).
  • Centrifuge for 1–2 minutes, making sure the centrifuge is properly balanced.
  • Remove 300 µL of the supernatant liquid from each tube, and transfer to fresh tubes (labeled!).
  • Add 1020 µL of distilled water.
  • Add 60 µL of 75 mM ascorbic acid.
  • Add 60 µL of 10 mM ferrozine.
  • Add 60 µL of saturated ammonium acetate.
  • Shake each tube and wait 10–15 minutes, (the solutions should become purplish in color).
  • Transfer to a 1.5 mL cuvette, and determine the A562 for each standard and your three samples against a milli-q water blank.
  • Generate a calibration curve of A562 vs. [Fe] from your standards.
  • Determine the [Fe] in your unknown.

Results

  • To obtain your “EE Rating” in Protein Assays and Error Analysis, the line fit for your standard curve must have a 0.995 correlation coefficient or higher. Additionally, the absorbance values for your unknown samples must have a standard deviation of 0.035 or less. Finally, you must determine the number of molecules of iron per molecule of protein.

5.1 - Competent Chemist Rating

“Looks Are Sometimes Deceiving”

Techniques Checklist

  • Analyzing mixtures by TLC
  • Assembling a silica gel column
  • Applying crude mixtures to a silica gel column
  • Separating simple mixtures with a silica gel column

Pre-lab Discussion

  • Theory of column chromatography—Reading Zubrick chapter 28, Mohrig chapter 18
  • TLC—polarity/solvent sys—Reading Zubrick chapter 27, LLP chapter 9.3.1, Mohrig chapter 17
  • Setting up silica gel column Reading Zubrick chapter 31, LLP chapter 11.6, Mohrig chapter 18
  • Applying crude mixtures to the column
  • Running a flash column

Digital Lab Techniques Manual

Equipment

  • Flash Chromatography Column
  • Air Flow apparatus (Stopper, T-valve, Screw clamp, tubing)
  • Round-bottomed flasks—1x100-mL, 2x500-mL
  • Test tubes—18x150 mm
  • Test tube racks
  • TLC plates—cut silica/glass plates and UV lamp
  • Large plastic funnels

Goal

  • Purify a contaminated compound using silica gel flash column chromatography.

Experiment Outline

  • You will be given 2 mL of an ether/pentane solution containing 1.00 g of benzylacetone contaminated with a small amount of guaiazulene.
  • Analyze this mixture by TLC—see TLC Guide, using 10% ethyl acetate/hexanes as the solvent system.
  • Record the Rf values.
  • Prepare the column in the hood, using 10% ether/pentane and 50 g (about 5’’) of of silica gel—see Flash Column Chromatography Guide.
  • Elute the column with 10mL of pentane—Apply your sample to the column, being careful not to disturb the top layer of sand. Rinse the sample flask three times with 1 mL pentane each, and use the rinses to wash the sides of the column.
  • Run the column, monitoring the fractions by TLC—See Flash Chromatography Guide and TLC Guide.
  • Concentrate the set of fractions containing pure benzylacetone.
  • Weigh the purified compound and prepare a GC and GC-MS sample.
  • Check product with TLC and obtain a GC and GC-MS spectrum.

Results

  • To obtain your “CC Rating” in Purification by Flash Column Chromatography, you should collect at least 0.95 g of benzylacetone. This sample must be at least 95% pure as demonstrated by GC spectroscopy. Your sample must also be submitted to the TA for possible weight and purity verification.

5.2 - Expert Experimentalist

Techniques Checklist

  • Picking the correct eluent then adsorbtion of a crude mixture onto silica gel
  • Separating complex mixtures—using gradient elution

Pre-lab Discussion

  • Suggest limited list of eluent solvent systems
  • Discuss sample adsorption and gradient elution strategies

Equipment

  • Identical to CC Level

Goal

Separate mixture of three compounds using gradient elution flash column chromatography.

Chemical Data

  • Benzylacetone—FW 148.21, bp 235 °C, d 0.989
  • Benzylideneacetone—FW 146.19, mp 39–41 °C
  • 3-Methylanisole—FW 122.17, bp 175–176 °C, d 0.969

Experiment Outline

  • You will be given 1.00 g of a mixture containing 0.60 g of the major ketone, 0.20g of the minor ketone, and 0.20g of methylanisole in 20 ml of an ether/ hexane solution.
  • Analyze this mixture by TLC using various solvent systems—see TLC Guide for hints.
  • Pick an eluent.
  • Decide on the silica gel to compound ratio.
  • Prepare the column.
  • Deposit the mixture on silica gel, dry completely, then apply to the column.
  • Run the column.
  • Concentrate pure fractions.
  • Weigh the purified compounds.
  • Analyze the pure ketones by NMR and TLC.

Note

  • The ketones are somewhat volatile, and 3-methylanisole, with its low molecular weight, is much more so. Therefore, do not concentrate it (or a mixture containing it) using the vacuum pump.

Results

  • To obtain your “EE Rating” in Purification by Flash Column Chromatography, you should get at least 0.45 g of the major ketone and 0.13 g of the minor ketone, and 0.13 g of methylanisole. These samples must be at least 95% pure as demonstrated by NMR spectroscopy. Your samples must also be submitted to the TA for possible weight and purity verification.

4.1 - Competent Chemist Rating

“How Did the Peach Get Inside the Banana?”

Techniques Checklist

  • Setting up distillation glassware correctly
  • Performing atmospheric pressure distillations
  • Using Gas Chromatography and Mass Spectrometry GC-MS to analyze samples

Pre-lab Discussion

  • Theory of distillation—Reading: Zubrick chapter 34, LLP chapter 11.3, Mohrig chapter 13
  • Distillation glassware and how to set it up—Reading: Zubrick chapter 19
  • Use of the GC—Reading: Zubrick chapter 30, GC-MS- Mohrig chapter 19

Digital Lab Techniques Manual

Equipment

  • 25-mL & 50-mL round-bottomed flask with stir bar
  • 3 Scintillation Vials or flasks that fit short path
  • Distillation Kits (distillation head)
  • Ground glass Thermometer and adapter
  • Keck clips
  • Glass wool and aluminum foil (optional)
  • Heating mantle w/sand
  • Variac

Goal

  • To purify a mixture of two liquids using distillation.

Experiment Outline

  • You will receive a vial containing 11.20 g of a mixture of two compounds whose boiling points differ by about 40 °C (See possible compounds below).
  • Analyze the mixture using the GC—see GC Sample Preparation and GC Operation Guides.
  • Perform atmospheric pressure distillation—see Distillation Guide.
  • Prepare a GC sample of your purified, low-boiling fraction.
  • Obtain a mass spectrum and a gas chromatogram of your purified low-boiling compound.

Helpful Hints

  • Make sure all your joints are lightly greased and sealed well. Otherwise, you will lose your product into the atmosphere.
  • Do not heat your mixture too fast, or your entire sample may end up in your collection flask.
  • Insulate your distillation head with cotton and foil to increase the rate of distillation.
  • Be aware that the temperature reading on the thermometer may not correlate accurately with the boiling point of the distilling liquid.

Results

  • To obtain your “CC Rating” in Purification of Liquids by Distillation, you must obtain at least 7.00 g of the low-boiling material that is 92% pure or better, as determined using GC analysis. You must also correctly identify the two components of your mixture. Think boiling points and smell.

4.2 - Expert Experimentalist Rating

“What’s With Those High-Altitude Recipes Anyway?”

Techniques Checklist

  • Glassware setup for reduced pressure distillation
  • Running reduced pressure distillation

Pre-lab Discussion

  • Differences between atmospheric pressure and reduced pressure distillation

Digital Lab Techniques Manual

Equipment

  • 25-mL Round-bottomed flask
  • 3x10-mL Pear-shaped flasks
  • Vigreux column-Vacuum Distillation Kits
  • Short path distillation head
  • Ground glass Thermometer and cow adapter
  • Keck clips
  • Glass wool and aluminum foil
  • Heating Mantle (w/sand) and Variac

Goal

  • To purify a mixture of two liquids by reduced pressure distillation.

Experiment Outline

  • You will receive a vial containing 7.50g of a mixture of alpha-ionone and octadecane. Repeat procedure for CC level distillation but using a Vigreux column and the vacuum line—see Distillation Guide.

Results

  • To obtain your “EE Rating” in Purification of Liquids by Distillation you must predict the boiling points of the compounds in your mixture at 0.5 torr. You must also obtain at least 4.00 g of alpha-ionone that is 93% pure or better as determined using GC analysis.

The picture below is a nomograph. using it and a ruler, you can determine at what temperature a liquid will boil under vacuum.

3.1 - Competent Chemist Rating

“How Do You Recrystallize a Mothball?”

Techniques Checklist

  • Solubility tests
  • Choosing a good solvent system
  • Decolorization
  • Inducing crystallization
  • Filtration

Pre-Lab Discussion

  • Theory of recrystallization Reading: Zubrick chapter 13; LLP chapter 11.2, Mohrig chapter 15

Equipment

  • Test tubes—five 13x100 mm
  • 2x50-mL 1x 125-mL Erlenmeyer flasks
  • Small magnetic stir bars
  • Stemless funnel and fluted filter paper
  • Büchner funnel and filter paper
  • Magnetic stirring/hot plate
  • 250-mL Filter flask and aspirator stopper
  • Rubber filter adapters
  • Large vial with white cap
  • Solid 7 rubber stopper
  • Test tube rack
  • Large crystallizing dish / Desiccator

Goal

You will be given 2.00 g of impure naphthalene (mothballs!); your job is to purify the naphthalene by recrystallization without losing a significant amount of your sample!1

Experiment Outline

Part I: Solubility Tests

Determine an appropriate solvent system for the recrystallization of naphthalene. For your tests try: water, methanol, acetone, hexane, and toluene. To understand how to find the appropriate solvent or solvent mixture for recrystallization, see Zubrick or Mohrig.

Part II: Recrystallization of Naphthalene

  • Transfer the material to a 50-mL Erlenmeyer flask equipped with a stir bar. Add about 20 mL of the solvent (determined in Part I) and heat to boiling on a stir/hot plate.
  • Remove any insoluble impurities by filtration, and recrystallize your product—see Two-Solvent Recrystallization Guide.
  • Collect your crystals on a small Büchner funnel by vacuum filtration, and rinse with the cold solvent mixture.
  • Your crystals should be colorless. If some orange or yellow color persists, wash your material with cold hexane. (Be careful: What is the solubility of naphthalene in hexane?).
  • Dry your compound well—see Two-Solvent Recrystallization Guide for tips.
  • Determine the yield and obtain a melting point.

Results

To obtain your “CC Rating” in Purification of Solids by Crystallization, you must obtain colorless crystals (no traces of yellow) weighing at least 1.30 g (well dried!) and melting over no more than three degrees with the lower range beginning no lower than 77 °C and the upper range ending no higher than 83 °C. This material must also be submitted to the TA for possible weight and melting point verification.

3.2 - Expert Experimentalist Rating

“The Single-Crystal Shakedown”

Overview

X-Ray diffraction is an important and powerful tool for determining the solid state structure of compounds. Modern advances have made data collection and structure solution almost routine for many small molecules. To use this technique, however, good quality single crystals are still needed. In this exercise, you will experiment with the art of growing single crystals.

Techniques Checklist

  • Manipulation of milligram quantities of material
  • Syringe use
  • Crystallization techniques for growing good quality single crystals

Pre-Lab Discussion

  • Use of different recrystallization techniques: vapor diffusion, solvent layering, temperature variation

Digital Lab Techniques Manual

Equipment

  • Magnetic stirring hot plate
  • 50-mL Round-bottomed flask
  • Condenser
  • Stir bar
  • Vials (3 large, 4 small) + 2 Glass jars
  • Glass frit (D)
  • Side-arm 250 mL Erlenmeyer flask / rubber filter adaptor
  • 2-mL Glass syringe
  • Variac and heating mantle

Goal

Synthesize Cr(acac)3,2 then perform several slow recrystallizations to obtain a single crystal of satisfactory quality.

Experiment Outline

Before coming to the lab, perform the necessary calculations to fill in the following table.

REAGENT SOURCE FORM. WT. DENSITY MASS/VOL. MMOLES EQUIV
CrCl3·6H2O         1.00 mmol 1
Urea           17
2,4-pentanedione           8
Cr(acac)3 Product          

Experimental

  • Dissolve CrCl3·6H2O in 2 mL of distilled water in a 50-mL round-bottomed flask, equipped with a stir bar.
  • Add the urea in one portion to the flask, and stir until completely dissolved.
  • Add the 2,4-pentanedione dropwise via syringe.
  • Attach the condenser to the flask, and heat the mixture to vigorous reflux (this is important!), with stirring, for about 1 hour.
  • Cool the reaction flask to room temperature, and collect the product by vacuum filtration on a size D glass frit funnel, washing with cold water.
  • Dry the product in vacuo (using the high-vac. desiccator provided by the TAs), and obtain a yield or dry the product in your desiccator overnight, and obtain a yield and melting point of the product.
  • Set up multiple crystallizations to grow single crystals—see Growing a Single Crystal Guide.

Note

  • Urea slowly hydrolyzes in the acidic solution used for this reaction, liberating ammonia (NH3), which controls the pH of the reaction. As more NH3 is generated, the solution becomes more basic, making it easier to remove the proton from the acac (acetylacetonate, also known as 2,4-pentanedione); it is the acac anion which then coordinates to the metal to form the desired Cr(acac)3 complex. What is the limiting reagent? Calculate your percent yield.

Helpful Hints

When using a saturated solution to grow crystals, filter the solution through a plug of glass wool in a pipette before setting up the crystallization.

Results

  • To obtain your “EE Rating,” you must obtain ≥ 45% yield of Cr(acac)3 and you must produce at least one single crystal that is suitable for X-ray analysis.

1Adapted from Fieser, L. F., and K. L. Williamson. Organic Experiments. 7th ed. D. C. Heath and Company, 1992, p. 40.

2Adapted from Szafran, Z., R. M. Pike, and M. M. Sing. “Synthesis of Metal Acetylacetonates.” In Microscale Inorganic Chemistry: A Comprehensive Laboratory Experience. Wiley, 1991, pp. 224–9.

2.2 - Expert Experimentalist Rating

“Acid, Base, and in Between”

Techniques Checklist

  • Separation of multi-component mixture using pKa
  • Planning an extraction and washing sequence
  • Careful transfer of solutions without loss of material
  • Solvent drying and concentration
  • Melting point determination

Pre-Lab Discussion and Required Reading

  • Same as CC

Digital Lab Techniques Manual

Equipment

  • Graduated Cylinder (100-mL)
  • Separatory funnel (125-mL)
  • Erlenmeyer flasks (4x250-mL)
  • Beaker (150-mL)
  • Round-bottomed flask (100-mL)
  • pH paper
  • NMR tube
  • Funnel
  • Filter paper
  • Rotary evaporator

Goal

To separate a three-component mixture using differences in pKa, with minimal loss of material.

Experiment Outline

You will receive a vial containing 100 mg each of benzoic acid, 4-nitroaniline, and naphthalene. Using the pKa, values of these molecules, carefully devise an extraction and washing sequence that will selectively separate the three components.1

  • Before beginning your extraction sequence, check with your TA or professor to make sure that it will work. You are free to use any or all of the following solvents:
    • Diethyl Ether
    • Methanol
    • Water
    • Saturated Sodium Bicarbonate Solution (Aqueous)
    • 6 M HCl
    • 1 M NaOH
  • Carry out your extraction and washing sequence, isolating each of the three components.
  • For each compound, remove the solvent by rotary evaporation to a constant weight and obtain a mass.
  • Obtain a melting point for each compound.

Results

To obtain your “EE Rating” in Transfer and Extraction Techniques, you must isolate at least 90 mg of two of the three compounds. In addition, the isolated compounds should melt over no more than three degrees, with the range beginning no lower than two degrees below the melting point values given above. 1Adapted from Gilbert, J. C., and S. F. Martin. Experimental Organic Chemistry: A Miniscale & Macroscale Approach. 3rd ed. Pacific Grove, p. 141.

2.1 - Competent Chemist Rating

“Ethyl Ester’s Excellent Adventure”

Techniques Checklist

  • Extraction
  • Melting point determination
  • Solvent drying and concentration
  • Nuclear Magnetic Resonance (NMR) spectrometer operation
  • Infrared (IR) spectrometer operation
  • Careful transfer of solutions without loss of material

Pre-lab Discussion

  • Extraction—Reading: Zubrick chapter 15, LLP chapter 10, Mohrig chapter 11
  • Theory of extraction—Reading: Zubrick chapter 35
  • Melting point determination—Reading: Zubrick pages 88–103, Mohrig 176–180
  • NMR theory and operation—Reading: Zubrick chapter 33, LLP chapter 15.2, Mohrig chapter 21
  • IR theory and operation—Reading: Zubrick chapter 32, LLP chapter 15.3, Mohrig chapter 20

Digital Lab Techniques Manual

Equipment

  • Separatory funnel—250 or 500-mL
  • Graduated Cylinder—100-mL
  • Erlenmeyer flasks—2x250-mL, 1x500-mL
  • Beaker 150-mL
  • Round-bottomed flasksv50-mL, 100-mL
  • NMR tubes
  • IR cards
  • Funnels—large, with long neck
  • Filter Paper
  • Rotary evaporator

Goal

To manipulate and purify a known amount of a contaminated sample and to record its 1H NMR and IR spectra, all with minimal loss of material.

Experiment Outline

  • You will receive a vial containing 100 mg of ethyl 3-hydroxybenzoate (mp 72–74 °C) contaminated with triethylamine. You will also receive four different 1H NMR spectra of: the mixture in your vial, pure ethyl 3-hydroxybenzoate, pure triethylamine, and diethyl ether.

  • Dissolve the sample in 50–75 mL of ether in a separatory funnel.

  • Remove the amine by extraction with a 10% HCl solution.

  • Continue with the “standard aqueous work-up” (including an ether back extraction)—see Extraction and Washing Guide.

  • Remove the solvent by rotary evaporation and concentration under vacuum to a constant weight and obtain a mass.

  • Take an NMR spectrum of the compound and compare to your earlier spectra.

  • Recombine the NMR sample with the remainder of the purified sample.

  • Obtain an IR spectrum using your IR card—see IR Sample Prep. Guide.

  • Remove the solvent for the final time to a constant weight.

  • Obtain a mass and a melting point.

Notes

  • When removing solvent with the rotary evaporator, keep the receiving flask cold and the water bath warm. Otherwise, your product will never solidify.
  • If you have trouble getting your product to solidify, add a few milliliters of methylene chloride to your flask and return it to the rotary evaporator.

Results

  • To obtain your “CC Rating” in Transfer and Extraction Techniques, you must end with at least 90 mg of ethyl 3-hydroxybenzoate. Additionally, this material must be of adequate purity as determined by IR and 1H NMR analysis (spectra should show only negligible amounts of impurities in the judgment of the professor and TA) and by melting point measurement (should melt over no more than three degrees with the lower range beginning no lower than 69 °C and the upper range ending no higher than 73 °C). This material must also be submitted to the TA for possible weight and melting point confirmation measurements.

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