EXPERIMENT #4 Fischer Esterification

EXPERIMENT #4 Fischer Esterification MASSACHUSETTS INSTITUTE OF TECHNOLOGY Department of Chemistry 5.310 Laboratory Chemistry EXPERIMENT #4 Fischer Esterification: The Organic Synthesis, Isolation, Purification, and Characterization of a Natural Flavoring Agent The Use of Boiling Point, Density, Refractive Index, NMR and Mass Spectrometry as Laboratory Tools for Analyzing the Product obtained Using Wet Chemistry Techniques I. PURPOSE OF THE EXPERIMENT This is an integrated experiment, which combines techniques from Organic, Thermodynamics, Physical, and Analytical chemistry. It has been designed to introduce the student to: • Organic Synthesis: Synthesizing an ester from an acid catalyzed reaction of a carboxylic acid and alcohol • Fundamental Chemical Engineering Separation Process principles on refluxing • Two phase solvent extraction using a Separatory Funnel • Distillation at atmospheric pressure • Precise handling and measurement techniques for volatile organic liquids • Correct handling and operation of a Mass Spectrometer & NMR Spectrometer • Thermodynamics, solution chemistry, intermolecular bonding, and organic nomenclature II. Safety of Chemicals1 You will be handling a number of chemicals during this experiment, all of which must be treated with care in order to avoid harm to yourself or to the surroundings. None of these chemicals should be ingested or allowed to come in contact with your skin and eyes. You must take extra precautions to avoid breathing the vapors from these chemicals. Assume that all of these volatile organic chemicals are flammable and each could be irritable to the eyes, skin and respiratory system. Descriptions of each of the chemicals follow with a brief summary of their properties. 1 Various Material Safety Data Sheets: Mallinckrodt Chemicals, J. T. Baker, Phillipsburg, NJ; Fisher Scientific, MSDS, Pittsburgh, PA Fischer Esterification-1 EXPERIMENT #5 Fischer Esterification Methanol Flammable liquid and vapor. Harmful if inhaled. May be fatal or cause blindness if swallowed. May cause central nervous system depression. May cause digestive tract irritation with nausea, vomiting, and diarrhea. Causes respiratory tract irritation. May cause liver, kidney and heart damage. Target Organs: Kidneys, heart, central nervous system, liver, eyes. Ethanol Flammable liquid and vapor. May cause central nervous system depression. Causes severe eye irritation. Causes respiratory tract irritation. Causes moderate skin irritation. This substance has caused adverse reproductive and fetal effects in humans. Warning! May cause liver, kidney and heart damage. Target Organs: Kidneys, heart, central nervous system, liver. 1-Propanol Flammable volatile organic liquid. Harmful if swallowed, inhaled or absorbed through the skin, may affect central nervous system. Causes irritation to skin, eyes and respiratory tract. Aspiration may cause lung damage. 2-Propanol May cause central nervous system depression. May form explosive peroxides. Flammable liquid and vapor. Hygroscopic. Causes respiratory tract irritation. Aspiration hazard if swallowed. This material has been reported to be susceptible to autoxidation and therefore should be classified as peroxidizable. Causes eye irritation. Breathing vapors may cause drowsiness and dizziness. Prolonged or repeated contact causes defatting of the skin with irritation, dryness, and cracking. Target Organs: Central nervous system, respiratory system, eyes, skin. 1-Butanol An eye and skin irritant. A sensitizer, which is hazardous in case of contact with skin. Contact may be characterized by itching, redness and sometimes blister formation. 2-Butanol Avoid direct heat, open flame. Avoid contact with strong oxidizing agents and strong acids and bases. Exposure can cause irritation to the respiratory tract, dizziness, headache, nausea, and narcosis. Liquid and high vapor concentrations can be irritating, and can cause conjunctivitis and corneal burns. 2-Methyl-1-Propanol Flammable. Irritating to respiratory system and skin. Vapor may cause drowsiness and dizziness. 1-Pentanol Hazardous irritant in case of skin (permeator) or eye contact. Hazardous in case of ingestion or inhalation. Fischer Esterification-2 EXPERIMENT #5 Fischer Esterification 1-Hexanol An irritant if exposed to skin, eyes and respiratory tract. May cause inflammation in the eyes with characteristic redness, watering and itching. Repeated exposure can result in target organ damage. 1-Octanol Skin, eye and respiratory tract irritant. Prolonged repeated skin contact may cause dermatitis. 3-Methyl-1-butanol Highly flammable. High vapor concentrations are irritating to eyes and upper respiratory tract. Contact with skin may cause irritation. Benzyl alcohol Sensitizer slightly hazardous in case of skin contact. Repeated prolonged exposure can produce target organ damage. May be toxic to liver and central nervous system (CNS). Benzoic Acid Eye, skin and respiratory irritant. Dust and liquid may cause mild irritation and redness. Ingestion of large amounts can cause coughing, nausea and vomiting. Formic Acid Contact with the eyes causes severe burns. Could result in scarring and ulceration. Causes severe burns on skin contact. Can sensitize the skin causing an allergic reaction. Could result in blistering of the skin on contact. Ingestion or breathing in the vapor could cause sever burning in the digestive tract resulting in abdominal pain, vomiting and possible death. Vapors can cause dizziness, nausea, itching, burning, and swelling of the eyes. Butyric Acid Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Propanoic Acid Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Eye inflammation will show up as redness, itching and watering. Skin inflammation is generally characterized by redness, itching and blistering. Acetic Acid Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Eye inflammation will show up as redness, itching and watering. Skin Fischer Esterification-3 EXPERIMENT #5 Fischer Esterification inflammation is generally characterized by redness, itching and blistering. Repeated or prolonged breathing of the mist or vapor can result in respiratory tract irritation eventually leading to frequent attacks of bronchial infection. Isobutryric Acid Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Eye inflammation will show up as redness, itching and watering. Skin inflammation is generally characterized by redness, itching and blistering. Repeated or prolonged breathing of the mist or vapor can result in respiratory tract irritation eventually leading to frequent attacks of bronchial infection. Pentanoic Acid Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Eye inflammation will show up as redness, itching and watering. Skin inflammation is generally characterized by redness, itching and blistering. Repeated or prolonged breathing of the mist or vapor can result in respiratory tract irritation eventually leading to frequent attacks of bronchial infection. Iso-Pentanoic Acid (3-Methyl-butanoic) (Isovaleric Acid) Hazardous in terms of contact with skin, eyes or respiratory system. Contact with skin could cause burns. The vapor or mist can produce tissue damage targets include the mucous membranes of the eyes, mouth, and respiratory tract. Inhalation could cause coughing, choking and shortness of breath. Eye inflammation will show up as redness, itching and watering. Skin inflammation is generally characterized by redness, itching and blistering. Repeated or prolonged breathing of the mist or vapor can result in respiratory tract irritation eventually leading to frequent attacks of bronchial infection. Severe over exposure can result in death. Sulfuric Acid Concentrated sulfuric acid is used in this lab and causes severe burns on skin and clothing. Do not inhale the vapor severe lung damage can result. An extremely corrosive acid handle carefully. Causes sever eye burns which could result in irreversible eye injury. A corrosive oxidizer may be fatal if inhaled, ingested or absorbed through the skin. If inhaled may cause severe damage to respiratory tract resulting in coughing, shortness of breath and delayed lung edema. Destructive to tissue in mucous membranes. Can cause severe burns to the respiratory tract. Prolonged breathing of the vapors may cause nosebleeds, chest pain, and bronchitis. Contact with many organic chemicals can cause fire and explosion. Fischer Esterification-4 EXPERIMENT #5 Fischer Esterification Table 1. Physical Properties of Some Organic Esters 20 Density Aroma nD 3 g/cm Amyl acetate 149 0.876 1.4023 Banana Benzyl acetate 212 1.054 1.5232 Peach Butyl acetate 126.1 0.883 1.3941 Banana/Apple Ethyl acetate 77.1 0.902 1.3724 Pear Hexyl acetate 171.5 0.867 1.4092 Plum Isoamyl acetate 142 0.876 1.4000 Banana Isobutyl acetate 118 0.875 1.3902 Fruity/floral Octyl acetate 203-211 0.868 ±1.4205 Orange Prenyl acetate 149.7 0.917 ±1.4290 Juicy Fruit Propyl acetate 102 0.888 1.3844 Pear Ethyl benzoate 211-213 1.045 1.5040 Fruity Amyl butyrate 186 0.883 1.4100 Apricot Ethyl isobutyrate 112-113 0.865 1.387 Fruity Butyl butyrate 165 0.869 ±1.4060 Pineapple Ethyl butyrate 120-121 0.879 1.392 Pineapple Isopentyl butyrate 184-185 0.862 1.411 Fruity Methyl butyrate 102 0.898 1.385 Apple Ethyl formate 54 0.921 1.3597 Rum Isobutyl formate 98.4 0.885 1.385 Raspberry Methyl Isovalerate 116-117 0.881 ±1.3950 Perfumes Methyl pentanoate 126 0.890 1.397 Fruity Isoamyl valerate 204 0.872 ±1.4140 Apples Ethyl propanoate 98.9 0.888 1.3844 Pineapple Isobutyl propanoate 136.8 0.869 1.397 Rum Methyl propanoate 80 0.915 1.3775 Rum like Propyl propanoate 122-124 0.881 1.393 Pineapple th Sigma Aldrich & Handbook of Chemistry and Physics, 84 Edition, CRC Press Ester BP 0 C Fischer Esterification-5 EXPERIMENT #5 Fischer Esterification III. INTRODUCTION General References These provide background information on the techniques and theory and are provided to improve your understanding and provide practical hints that may help you avoid mistakes that may prove costly in terms of laboratory time. • • • • • • • • • • • • • • Measuring Mass & Volume Using Pasteur Pipettes to Transfer Thermometer Calibration Refluxing a Reaction Mixture Extraction: Separatory Funnel Boiling Point & Distillation Refractive Index Nuclear Magnetic Resonance Mass Spectrometry Infrared Spectroscopy Distillation Boiling Esters Clausius-Clapeyron Equation MHS, Chapter 5 pp. 52-61 MHS, Chapter 5 pp. 55-62 MHS, Chapter 5.4 pp. 63 MHS, Chapter 7 pp. 59-60 MHS, Chapter 11 pp. 113-124 MHS, Chapter 12 pp. 174-176 MHS, Chapter 13 pp. 206-211 MHS, Chapter 22 pp. 350-405 MHS, Chapter 24 pp. 441-462 MHS, Chapter 21 pp. 311-344 ATKINS, Chapter 10, pp. 404-406 ATKINS, Chapter 10.4 pp. 373-374 ATKINS, Chapter 20 pp. 828-829 ATKINS, Chapter 10.3 pp. 371-373 Videos: Digital Technique Manual • • • • • • #1 Volumetric techniques #5 Reaction Work-Up I #7 Filtration #11 Using a Balance #15 Distillation I: Simple & Fractional Distillations #17 Refluxing a Reaction Fischer Esterification-6 EXPERIMENT #5 Fischer Esterification A. Esterification—Background Esters are found naturally in a variety of fruits, vegetables, herbs and spices. Odors can be complex and generally pleasantly smelling usually the odor of the ester stands out. Esters are made, by condensing an alcohol with a carboxylic acid. The reaction is generally catalyzed with concentrated sulfuric acid (H2SO4). Esters have a general formula as illustrated in (5.1). O C R (5.1) OR' There are many possible combinations possible between an alcohol and various acids each combination producing different esters with characteristic identifiable odors. See Table 1 on page 5 of this manual. The forward / reverse reaction is rapid and is illustrated in (5.2). O C R + HO OH O H+ R' C R Fischer Esterification-7 R' O + H2O (5.2) EXPERIMENT #5 Fischer Esterification Generally, equilibrium exists between reactants on one side and products on the other. This equilibrium predominantly favors the reactants. Under acidic conditions using concentrated sulfuric acid as a catalyst, the water on the product side is removed causing the equilibrium to shift in favor of the products. The mechanism for the fisher acid catalyzed esterification reaction is shown in (5.3). (5.3) Reaction mechanism © V8rik at English Wikipedia. Wikimedia Commons, License CC BY-SA. All rights reserved. This content is excluded from our Creative Commons license. For more information, see https:// ocw.mit.edu/help/faq-fair-use. In this classic reaction scheme, the H+ acts as a Lewis acid activating the carbonyl group, which becomes a good electrophile. At the same time, the -OH is converted into a good leaving group as water, which breaks off. The pathway presented lowers the energy barrier speeding up the reaction to products. Fischer Esterification-8 EXPERIMENT #5 Fischer Esterification B. Esterification Lab—Overview Day 1— “Introduction to Refluxing, Extraction and Drying the Product” Assembling a ground-glass reflux apparatus in the hood for Fisher synthesis of an ester from an unknown alcohol and unknown carboxylic acid. After refluxing remove any acid impurities from the ester by extraction with aqueous base separating out the ester from the reaction mixture. Dry the ester with an anhydrous chemical drying agent and store in a clean scintillation vial for Day 2. Day 2— “Introduction to Atmospheric Distillation” Assemble a distillation apparatus. Product Characterization using Infrared Spectroscopy (IR) Day 3— “Product Characterization using Boiling Point (BP), Density, Refractive Index (20nD) and NMR Spectroscopy.” Day 4— “Further Product Characterization using Mass Spectrometry (MS). Final Product Identification.” Fischer Esterification-9 EXPERIMENT #5 Fischer Esterification C. Hewlett Packard GC/Mass Spectrometer The GC-Mass Spectrometer has become one of the most sensitive and powerful instruments for the identification of organic compounds. Analysis of trace quantities of organic compounds has advanced to a stage greater than 1 part in 1015. Many impurities and contaminants found in food and water can now be identified unambiguously in trace amounts. To make the process easier many thousands of spectra including insecticides and industrial pollutants have been compiled into databases and are now readily available as packages that can be purchased and downloaded to computers hooked directly to GC/MS systems. The Organic volatile unknown will be injected neat into the heated injector port of the GC/MS using a special custom-designed 0.01-microliter syringe. The volatilized compound is carried in the helium flow to a splitter with a split-out ration set at 1:300 whereby only 0.33% of the sample is diverted to the GC column (the rest is vaporized and exits the system). The GC column used in this instrument is a capillary column, which is 10-30 meters long and only 0.4 mm in diameter. The conditions above insure that no more than 10-9 gram of the sample is applied to this column. Schematic Diagram of HP GC/MS Quadrupole Mass Spectrometer In the Hewlett Packard GC/MS as the organic compounds elute from the GC, they are introduced immediately into a Quadrupole Mass Spectrometer and a series of mass spectra are repeatedly recorded for each two-second interval. In the ionization chamber, electrons traveling across a 70-eV potential bombard the molecules. When a molecule is struck by one of these high-energy electrons, it loses one of its own electrons and becomes a radical cation M. +. Many of the molecular ions will be formed with enough excess energy to undergo subsequent reactions in the ionization chamber. From the ionization chamber, the positively charged ions are attracted through the magnetic field of a quadrupole ion chamber maintained under high vacuum. For a given strength of the magnetic field, only those ions having the correct charge/mass ratio (m/e) will be deflected through the magnetic field and reach the detector. The instrument varies the magnetic field continuously to produce a spectrum of m/e peaks. Fischer Esterification-10 EXPERIMENT #5 Fischer Esterification In the next section are detailed mechanisms for common fragmentation and ionization reactions. It is much more difficult to solve the structure of an organic compound from the mass spectrum alone than it is to match the spectrum with that of the known compound. With the help of NMR and IR spectroscopy, mass spectrometry can play an important role in structure determination. Fischer Esterification-11 EXPERIMENT #5 Fischer Esterification D. Reactions Observed in Mass Spectroscopy A Fragmentation Reaction involves the cleavage of one bond of the radical cation M.+ to form a cation A+ and a radical B.. The reaction is especially facilitated by substituents, which stabilize the cation A+. a-Cleavage Reaction. The a-cleavage reaction is driven by the formation of a stable carbocation as shown in the examples listed below. In the first case, ethers readily undergo the a-cleavage reaction forming a stable oxonium ion (also common for similar N, S compounds). In the second example, alkenes undergo a-cleavage reaction to form a stable allylic cation (also common for aryl compounds and alkynes). In the third example, ketones readily undergo acleavage to produce stable acylium ions. Inductive Cleavage Reaction. Alkyl halides often undergo the inductive cleavage reaction in which the halogen atom (e.g. Cl.) simply breaks off the initial radical cation. This is most prevalent for bromides and iodides and for compounds, which can produce a stable carbocation. Fischer Esterification-12 EXPERIMENT #5 Fischer Esterification In each case, fragmentation reactions above produce both a radical and a cation from the initial radical cation M.+. Also, as a consequence of the nitrogen rule: For molecules that do not contain nitrogen, fragmentation reactions of the even-massed radical cations will produce oddmassed A+ ions. This can be a useful method for finding which ions are produced by one of the above reactions. A Rearrangement Reaction involves the cleavage of more than one bond of the radical cation M.+ to ultimately form a new radical cation A.+ and a neutral compound B. Often, rearrangement reactions may proceed by complementary pathways so that the mass numbers for both parts of the molecule can be found in the mass spectrum. Rearrangement of Cyclic Structures. When a cyclohexene derivative is ionized in the mass spectrum, a particularly facile cleavage of the ring is reminiscent of a retro-Diels-Alder reaction. Fischer Esterification-13 EXPERIMENT #5 Fischer Esterification Radical Site Rearrangements (McLafferty Rearrangement). Carbonyl derivatives readily undergo a rearrangement reaction in which the M+. undergoes intramolecular H atom transfer to the carbonyl oxygen atom. The best geometry involves a six-membered transition state and subsequent a-cleavage or inductive cleavage leads to the fragment ions of each piece. Fischer Esterification-14 EXPERIMENT #5 Fischer Esterification For each example of rearrangement reactions, the observed ions are radical cations. For a molecule which does not contain nitrogen, the McLafferty rearrangement of the even-massed M+. ion produces an even mass A+. ion and so are easily distinguished from the odd-massed ions from a simple fragmentation reaction. Also, many molecules display a peak in the mass spectrum arising from a McLafferty +1 rearrangement. For the ester above, the mechanism involves a second H atom transfer. Fischer Esterification-15 EXPERIMENT #5 Fischer Esterification D. NUCLEAR MAGNETIC RESONANCE I. Purpose The Nuclear Magnetic Resonance portion of the course is designed to introduce 5.310 students to the basic concepts of nuclear magnetic resonance (NMR) spectroscopy including spin, energy levels, absorption of radiation, and a number of other NMR spectral parameters. Most importantly to provide experience in the identification of unknowns from 1H (proton) NMR spectra. All students will be given a tour of the Varian 300 MHz operation room in the Undergraduate lab, which will include observing the running of a sample (subject to the availability of the instruments). You will then receive a recorded spectrum of your Unknown volatile organic sample and will use this spectrum along with your mass spectrum and wet chemistry results to determine the structure and identity of your unknown sample. II. Safety One of the main safety considerations that you should be aware of when walking into the instrument room is the field generated by the 300 MHz NMR magnet. The field is strong enough to wipe out data on magnetic credit cards (Master charge, VISA, American Express and others). Thus, when working in the vicinity of an NMR spectrometer, it is a good practice to leave these items in some alternate location. In fact, bringing any type of metal objects near a strong magnet is not a good idea because they could be sucked away ending up under the NMR. III. Theory: Energy Levels of Nuclei in a Magnetic Field Many isotopes of elements in the periodic table, but not all, possess spin angular momenta, which results from coupling the spin and orbital angular momenta of the protons and neutrons in the nucleus. In Principles of Chemical Science, 5.111, you learned that the orbital angular momentum, !, of an electron can be oriented in 2! + 1 discrete states (e.g. a p electron (!=1) can have m! = -1, 0, +1 orientation states). With NMR, we will be conducting experiments on the nuclei of atoms, not the electrons. Nuclear spin angular momenta behave the same way. Nuclei with spin I can have 2I + 1 values of the spin orientation, characterized by quantum numbers, mI, which are [-I, -(I-1)……(I-1), I]. For I = ½ the allowed mI are +1/2 and -1/2 and for I = 1 the allowed mI are -1, 0, +1. If I is non-zero, the nucleus has a magnetic moment, and when placed in a magnetic field, the component of the moment along the field direction (conventionally taken as the z direction) is µz = g"mI, where g, which is a constant specific for each isotope, termed the gyro magnetic ratio; " is the Planck’s constant divided by 2p (" = 1.055 x 10-34 J s), and mI is the quantum number. Some g values for chemically important nuclei are given in Table I. Note that nuclei such as 12C and 16O are not included in the table, since they have I=0. Fischer Esterification-16 EXPERIMENT #5 Fischer Esterification The rules for determining whether a nucleus has a net spin (I) are fairly simple: 1. If a nucleus has an odd number of protons and an odd number of neutrons then the effective spin of the nucleus will be an integer number such as 1, 2, and 3. 2. If in a nucleus, the sum of the protons and neutrons is odd, then the effective spin of the nucleus will be on the order of a half-integer such as ½, 3/2, 5/2. 3. If the sum of the protons and neutrons is even then the effective spin of the nucleus will be zero. Table 1. Spin, gyro magnetic ratios, NMR frequencies (in a 1 T (10 kG) field) and the natural abundance of selected nuclides)* * T stands for Tesla (1 Tesla = 104 Gauss. The earth’s magnetic field is around 0.5 Gauss) ** What is the physical meaning of a negative gyro magnetic constant? In a laboratory field B0, the nuclei can assume 2I + 1 orientations corresponding to the values of mI, each value of mI corresponds to an energy given by (Figure 1): EmI = - µ B0 = - µz B0 = - " g B0 mI Fischer Esterification-17 (1) EXPERIMENT #5 Fischer Esterification Which can be rewritten as EmI = - mI " w0 (2) Where the Larmor frequency is w0 = g B0 z B0 µ µz Figure 1. The relationship between the laboratory magnetic field B0 and, the magnetic moment of the nucleus, µ, and its µz component (µ component along the z axis) Fischer Esterification-18 EXPERIMENT #5 Fischer Esterification As shown in Figure 2, the energy separation between the two levels a and b of an I = ½ system with the magnetic field turned on is shown below. Note in the absence of the magnetic field the two energy levels are degenerate resulting in the same energy, with the field applied the two levels move apart: DE = E(b) - E(a) = 1/2" g B0 - (-1/2) " g B0 = " g B0 (3) m I = - 1/2 β (h/2π) γB0 mI = + 1/2 α Zero field Magnetic field on Figure 2. The nuclear spin energy levels of a spin-1/2 nucleus (e.g. 1H or 13C) in a magnetic field. Resonance occurs when the energy separation of the levels, as determined by the magnetic field strength, matches the energy of the photons in the electromagnetic field. Thus, when the sample resides in a magnetic field and is bathed with radiation of frequency w0/2p = no (energy = " w0) matching the difference between the levels resonance occurs—i.e. energy is absorbed causing the excited nucleus to jump for the a to the b, energy level, as illustrated schematically in Figure 2. When the nucleus in the higher b energy level drops back down to the lower a energy state, a quantum of energy is released in the radio frequency range and it’s this energy that produces the NMR signal. Equation (3) is the resonance condition. Using the values for g in Table 1, we can calculate that for 1H in a 4.7 T field, the resonance frequency is 200 MHz, and for an 11.7 T field no = 500 MHz. The method of obtaining the NMR spectrum that we use includes FT (Fourier Transform NMR). A short pulse of RF energy is applied to the sample tilting the net magnetization vector off its equilibrium. The decaying oscillations of the magnetization vector are recorded in real time with a magnetic coil as it decays to equilibrium (FID). A computer program uses Fourier Fischer Esterification-19 EXPERIMENT #5 Fischer Esterification transform mathematics to convert the FID, which is a function of time, to an NMR spectrum as a function of frequency. The amount of energy absorbed (and therefore the NMR signal strength) is proportional to the population difference between the a, and b energy levels. The NMR signal that we are plotting is basically the energy difference between the energy absorbed by the spins that causes them to move from the lower parallel a energy level to the upper antiparallel b energy level and the energy reemitted by the spinning nucleons as they make the transition back down from the higher b energy level to the lower a energy level. In the Charles River Experiment, we used the Beer-Lambert law to calculate the amount of light transmitted by an absorbing sample: % T = I/Io = e-A = e-ecl The absorbance A=ecl can be written as A = kabs Nabs l where kabs is an absorption coefficient and Nabs represents the number of absorbing molecules in the beam path. Because excited molecules can re-radiate light, the effective Nabs is actually N (lower a level) – N (upper b level). The ratio of the population of nuclear spins in the upper b state, N(b), to the population of nuclear spins in the lower a state, N(a), can be calculated using the Boltzmann equation: P= ΔE − Nβ = e kT Nα (4) where, DE equals the energy difference between spin states, k is the Boltzmann constant (1.3805 x 10-23 J/K-mole), and T is the absolute temperature in K. At room temperature, the number of spins in the a lower energy state, N(a), just slightly outnumbers the number of spins in the b higher energy state N(b). For a proton, in an applied field with a strength of 7.0 T (e.g. the magnetic field of the Varian 300 MHz), using DE = hn, the difference " g B0 between two spin states of the proton is 1.99x10-25J per molecule. The kT at room temperature (T = 298 K) is 4.11 x 10-21 J, therefore DE/kT = 4.84 x 10-5 substituting, ΔE − Nβ = e kT Nα Nβ = e −4.84 xE −5 Nα Nβ = 0.999952 N β = 0.999952 Nα Nα With such small population differences, highly sensitive detection techniques are required. The NMR signal depends on the N(b) / N(a)a population ratio. As the temperature decreases the ratio decreases, and as the temperature increases the ratio approaches 1. Fischer Esterification-20 EXPERIMENT #5 Fischer Esterification IV. Chemical Shifts Every nucleus in a molecule is surrounded by an electron cloud, which possesses an electronic angular momentum induced by the laboratory field, B0. This angular momentum results in a local magnetic field, Bloc , at the nucleus, which is opposed to the applied field because it arises from negatively charged electrons. Putting a molecule into a magnetic field causes the electrons to circulate inducing a current that has a magnetic field associated with it. Thus, the induced magnetic field from the micro current of circulating electrons shields the nucleus from the applied field according to the following formula where s is the shielding constant. Bloc = (1 - s) B0 (5) So, the net field felt at each nucleus (Bloc) is less than the applied field. The feature that makes NMR so interesting and extremely important to chemists is that s varies markedly from chemical group to chemical group. Thus, s for a -C1H3 is different from s for a -C1H2-, and that in turn is different from s for an -O1H. These features of NMR spectra, illustrated for the 1H's in ethanol in Figure 3, are referred to as chemical shifts. They are important since they facilitate the identification of each type of 1H (or 13C) present in a molecule. All of the shifts in Figure 3 are referenced to the shift of the standard tetramethylsilane (TMS) and are calculated from the formula: δ= ν 0 − ν ref x10 6 ν ref (6) where, n0 and nref is the frequency of the unknown and reference (TMS), respectively. As mentioned above, the amount of shielding, and therefore the frequency separation between two lines, will increase with increasing field. However, the chemical shifts calculated from Equation (6) are field independent. Thus, shifts measured at 100 MHz can be compared directly with those obtained at 500 MHz. Note that positive d values correspond to higher frequencies or lower magnetic fields. Fischer Esterification-21 EXPERIMENT #5 Fischer Esterification Figure 3 – The proton NMR spectrum of ethanol. TMS (the rightmost signal) stands for tetramethylsilane (Si(CH3)4). TMS is the widely-used reference signal. The step-like curve is the integrated signal, proportional to the number of spins in each signal. J.T. Arnold and co-workers first recorded this spectrum in 1951 [Journal of Chemical Physics 19, p. 507]. Figure 4 illustrates the dependence of proton chemical shifts on the various functional groups in more detail. Note that Figure 4b also shows chemical shifts for 13C, another important NMR nucleus. (a) Fischer Esterification-22 EXPERIMENT #5 Fischer Esterification (b) Figure 4: Bar graph illustrating the range of chemical shifts expected for various types of (a) 1H resonances and (b) 13 C resonances, referenced to TMS. V. J-Couplings and Spectral Intensities The spectral lines of ethanol in Figure 3 exhibit fine structure -- i.e. the -CH3 line consists of three lines and the -CH2- has four components2. This coupling occurs because each magnetic nucleus also contributes to the local field of adjacent nuclei -- an effect referred to as spin-spin coupling. The strength of this coupling is described by a field independent coupling constant, J, measured in Hertz (Hz). 2 J.T. Arnold, Phys. Rev. 102, 136 (1955). Fischer Esterification-23 EXPERIMENT #5 Fischer Esterification X2 Intensity X3 Intensity 1 1 2 3 1 3 1 Figure 5: (a) Orientations of the spins in the X2 group (-CH2-) of an AX2 spin system which give rise to a 1:2:1 triplet in the A resonance. The two nuclei may have the 22 = 4 spin arrangements shown. (b) The 23 = 8 arrangements of the three spins in an AX3 group, which lead to the 1:3:3:1 quartet in the A resonance. The spectral pattern in Figure 3 can be understood by examining the simple diagrams shown in Figure 5. If two spins that correspond to the two 1H's on a -CH2- are present, they can be aligned in four different ways. Each spin is either parallel or opposed to the field. Therefore, two spins can be aligned both up, both down, and one up and one down (in two different ways: spin one up/spin two down, and spin one down/spin two up). Thus, the local field at the -CH3 due to the adjacent -CH2- should have three components with an intensity ratio of 1:2:1, corresponding to both up, one up and one down (2 ways), and both down. Correspondingly, there are eight ways to align the three spins on the methyl group, as illustrated in the figure, resulting in a quartet for the -CH2- with intensities of 1:3:3:1. By extending these arguments to larger numbers of spins, it is possible to show that N equivalent spins split the resonance of a coupled group into N+1 lines, with intensities corresponding to the coefficients of a binomial expansion or Pascal's Triangle. A Pascal's triangle is shown on the following page, where N is the total number of equivalent spins. The size of J-couplings depends on the structure and the nuclei involved. 1H-1H couplings are ~10 Hz, while 1H-13C and 31P-1H couplings can be ~100 Hz. and ~600 Hz, respectively. Another noteworthy feature of Figure 3 is the integrated spectral intensity of each set of lines. The integrals, which are performed electronically in an experiment, are shown as the steplike curves in the figure and are in the ratio 1:2:3, corresponding to the number of each type of 1H present. Thus, intensities in 1H spectra can be employed to determine the relative numbers of each type of 1H present in a molecule. In summary, chemical shifts and spectral intensities provide information on the types and number of nuclei, respectively, associated with each functional group present in a molecule. Further J-couplings permit a determination of which groups are chemically bonded. Together these parameters permit the structures of most organic molecules to be determined by simply recording and interpreting their NMR spectra. Fischer Esterification-24 EXPERIMENT #5 Fischer Esterification PASCAL’S TRIANGLE N INTENSITY DISTRIBUTION 0 1 2 3 4 · · N 1 1 1 1 2 1 1 3 3 1 1 4 6 4 1 Expand (1+x)N and select the coefficients VI. Experimental Procedure: Sample Preparation General The NMR sample tubes should be clean and free of dust and particulate matter. Solid samples should be dissolved in a deuterated solvent and liquid samples can be run neat or diluted in a deuterated solvent as described further below. A good 1H NMR sample contains about 10 mg of compound. The solution should contain no solids or paramagnetic impurities. Your deuterated NMR solvent should be free of water, and your NMR spectrum should contain no solvent peaks. An NMR sample tube is typically 175 mm in length with a 5 mm O.D. Minimum filling level is a distance of approximately 2 cm up from the bottom of the tube, which is equal to a volume of about 0.3 mL. However, the optimum filling level is a distance of approximately 4-6 cm up from the bottom of the tube (0.6-0.9 mL). You must determine the minimum height of a sample by checking the depth gauge in the NMR room. Over filling can distort the homogeneity and lead to lower resolution. When the tube is inserted into the probe, the position of the transmitter coil is about 1 cm from the bottom of the tube. As mentioned above, the solvent must dissolve the sample material. It is generally desirable to use as concentrated a solution as possible -- i.e. about 10%. In difficult cases where the sample is not very soluble, it may be necessary to find an alternative solvent. Hydrogencontaining solvents should be avoided whenever possible. The selected solvent should not produce strong signals of its own in the spectral region of interest. Some solvents commonly employed for proton NMR spectroscopy include: deuterochloroform -- CDCl3 deuterated water—D20 hexadeuteroacetone (acetone-d6) -- (CD3)2C=O hexadeuterodimethylsulfoxide (DMSO-d6) -- (CD3)2S=O deuterodacetonitrile – CD3CN deuterotetrahydrofuran – C4D8O Fischer Esterification-25 EXPERIMENT #5 Fischer Esterification Chloroform is by far the most popular and will be used almost exclusively in 5.310. The TA will prepare the bottle of CDCl3 that you will use for your samples. The CDCl3 will be spectral grade already treated with TMS. Just keep in mind that you don’t want water getting into your deuterochloroform, so keep the bottle open to the atmosphere as little as possible. As long as the bottle remains open, water from the air will dissolve in your NMR solvent. For preparing your NMR tube with an unknown liquid it is recommended that you take 50 uL of sample and dilute with 900 uL of deuterated chloroform in a small scintillation vial. Mix well and using a 9-inch glass pastuer pipette place the pipette into the NMR tube as far in as it will go then gently release the sample up to the tip of the pipette. The NMR tube should now be prepared perfectly with the correct volume of sample. The TA’s will demonstrate the technique for you during the pre-lab lecture. When your sample has finished running the NMR tube should be opened up and turned over into a glass beaker located under the hood, provided and labeled by the TA’s. It is most important that the sample tube to be absolutely clean within and without; its surfaces must be completely dust-free. Particles just visible with the unaided eye may degrade the resolution if they are within the irradiation region. Reference Material It is usual to provide a reference line in each spectrum from which the chemical shifts of other signals can be measured. The normal method is to add a reference material (known as an internal standard) directly to the sample. The reference material should not interact with the test sample. If a symmetrical and non-polar type of molecule is used as the reference material, these effects will be very small. For this purpose, tetramethylsilane (TMS) is most suitable. An additional advantage of using TMS is that the spectral line it produces is at a higher field position than almost all other signals, so that the chemical shifts of nearly all lines measured with respect to TMS are of the same low field direction. As an alternative method, the reference material may be contained in a sealed capillary tube, which is inserted into the sample tube. There can be no intermolecular effects by this method. TMS will already have been added to the deuterochloroform, which we will be using. Fischer Esterification-26 EXPERIMENT #5 Fischer Esterification Day 1 Assembling a Ground-Glass Reflux Apparatus for Synthesis of an Ester, Extraction of Acidic Impurity from Product, Drying the Product. The apparatus for synthesis of the unknown ester requires assembling a ground-glass reflux apparatus similar to that shown in Figure 5-1. Obtain from the stockroom a kit containing a 250mL ground-glass round bottom flask, 14/20 condenser, and an electric heating mantle which fits the 250mL round bottom flask. Secure an unknown organic alcohol and acid from your instructor and record the unknown number into your laboratory notebook. Please remove the cotton ball from the drying tube and refill with drying agent (CaCl2), and a new cotton ball before assembling. Figure 5-1 Reflux Apparatus Fischer Ester Synthesis ©�� AUS-e-TUTE. All�� rights�� reserved.�� This�� content�� is�� excluded from�� our Creative�� Commons�� license. For more�� information, see�� https://ocw.mit.edu/help/faq-fair-use/.� Fischer Esterification-27 EXPERIMENT #5 Fischer Esterification When pouring the reactants into the 250mL round bottom flask do not allow the reactants to make any contact with the ground glass opening of the flask (See Figure 5-2). Rather, use a funnel this will keep the reactants from coming into contact with the ground glass opening of the flask. Both the sulfuric acid and the carboxylic acid are corrosive and should be handled carefully. Please Do Not Open the unknown tubes outside of the hood because the smells will fill the lab. The TA will come around and collect the empty unknown tubes please keep them capped and under the hood. Figure 5-2 Use a Funnel for Pouring Reactants into the Reflux Flask To a 250mL round bottom flask set up in a laboratory hood, add 40 mL of the unknown carboxylic acid followed by 30mL of the unknown alcohol. Gently, swirl the flask to mix the alcohol and carboxylic acid then, carefully add 3 mL of concentrated sulfuric acid H2SO4. (There are approximately 20 drops in 1 mL) Obtain the sulfuric acid from the small dropper bottles provided. Swirl the flask during the addition of the sulfuric acid the reaction is exothermic and heat will be Fischer Esterification-28 EXPERIMENT #5 Fischer Esterification released. Use caution in handling the acid it is highly corrosive and the liquid and vapors are harmful. Add a small magnetic stir bar to the flask, then, attach the reflux condenser with lightly greased joints as shown in Figure 5-3. Attach a drying tube to the top of the condenser. Make sure you replace the old drying agent with new fresh CaCl2. Figure 5-3 Reflux Apparatus & Heating Mantle Fischer Esterification Fischer Esterification-29 EXPERIMENT #5 Fischer Esterification Using a heating mantel connected to a rheostat apply heat (approximately 75 electrical units) bringing the solution to a boil, and reflux the solution for 1 hour. Objective is to achieve a slow rate of reflux with liquid boiling and condensed vapor dripping back into the flask. While the reaction is refluxing set up a 250 mL separatory funnel with a ring clamp support as shown in Figure 5-4. Figure 5-4 Separatory Funnel with Ring Stand Support Once the reflux has completed, cool reaction mixture to room temperature. Transfer the reaction solution to a 250 mL separatory funnel and add 60 mL of cold distilled water. Use an additional 20 mL of water to rinse out the reaction flask and add that to the separatory funnel as well. Swirl the solution, allowing the layers to separate and remove the aqueous layer. Pour the Organic layer into a 400mL clean dry beaker. Extract the Organic layer with two 40 mL aliquots of 5% sodium bicarbonate solution to remove any traces of the carboxylic acid. CO2 gas is released during the extraction and this can cause a high pressure inside the separatory funnel. Because of this the first addition of 40 mL 5% sodium bicarbonate should be carried out in the 500-mL beaker slowly adding the sodium bicarbonate solution while stirring with a glass stirring rod. Once the fizzing subsides transfer the contents of the beaker back to the separatory funnel and continue with the remaining extractions. DO NOT INSERT THE GLASS Fischer Esterification-30 EXPERIMENT #5 Fischer Esterification STOPPER into the separatory funnel until the bubbling subsides. Test the second extracted aqueous layer with litmus paper and make sure that it turns litmus paper blue and is basic. If not, repeat the extraction with two more 40 mL portions of 5% sodium bicarbonate solution until the aqueous extract tests basic. Once the aqueous extraction layers have been removed extract the organic layer with two 10mL portions of saturated aqueous sodium chloride solution. The sodium chloride will help to pull any trace amounts of water out of the organic layer. Pour off the top organic layer into a 125 mL Erlenmeyer flask and dry with an anhydrous chemical drying agent such as MgSO4 or Na2SO4. Add approximately 4 grams of the drying agent swirling the flask gently, allowing the drying agent to clump up indicating that it is pulling water out of the sample. Add an additional 1-2 grams of the drying agent if needed until it stops clumping (caking) up in the flask. Typical set up is shown in Figure 5-5. After drying, the liquid should be very clear. Decant the organic layer into a clean scintillation vial and store it in your desk drawer until the next lab. Take great care to make sure that the drying agent stays in the original Erlenmeyer flask. Figure 5-5 Drying Sample with an Anhydrous Drying Agent Fischer Esterification-31 EXPERIMENT #5 Fischer Esterification Day 2 Atmospheric Distillation & Check Product Fractions with IR Spectroscopy Set up a simple distilling apparatus as shown in Figures 5-6. Figure 5-6 Atmospheric Distillation Apparatus with Vigruex Column Fischer Esterification-32 EXPERIMENT #5 Fischer Esterification Now carefully, loosen the condenser clamp and disconnect the condenser and the distilling head from the 100mL distilling flask. Insert a funnel containing a small loose cotton plug into the 100mL distilling flask. Using the funnel, carefully pour the saved ester product into the clean dry 100 mL distilling flask. Add a magnetic stir bar to the distilling flask and re-attach to the distilling apparatus. Be sure that the adapter is open to the atmosphere. Have the system checked by your instructor prior to starting the distillation. Collect the distillate in large labeled test tubes that are cooled in ice (See Figure 5-7). Fill a beaker with ice chips and use it to hold the test tubes during collection of the distillate. Make sure the test tube is positioned to capture any drops, which drip from the condenser. Figure 5-7 Test Tube Receiver Fischer Esterification-33 EXPERIMENT #5 Fischer Esterification Re-check that the cooling water has been turned on. When all has been checked, then carefully begin heating the distilling flask. For heating your apparatus start off at 40 electrical units on the rheostat control and go up 5 units every 5 minutes until you start collecting product. Try to aim for a distillation rate of approximately 1 drop of distillate every 2-3 seconds. Initially it may appear that nothing is happening, check to make sure the heating element is working and getting warm. Gradually as the heat is increased, condensation should begin to appear on the interior of the vigreux column and distilling head. Keep track of the thermometer readings as the vapor begins to condense off the tip of the thermometer bulb. At that point, the liquid should begin to collect inside the condenser and the first drop of distillate should reach your receiver test tube. At this point, carefully adjust the rate of heating on the rheostat such that you are collecting about 1 drop of distillate every 2-3 seconds. Continue at this heating rate and regularly record the temperature on the thermometer in the laboratory notebook. After collecting about 10-15 drops of distillate into the first test tube, change the collection flask by inserting and collecting into a second test tube. Collect the distillate in this second test tube as long as the temperature remains constant within a few degrees. If the temperature changes or if the test tube is more than half filled with product, change to another third clean test tube. Keep records of the temperature range that each fraction was collected at in the laboratory notebook. Make sure each of the tubes is clearly labeled in the order that product was collected. Continue the distillation until about 2-3 mL remains in the distillation flask. The clear colorless product should have an intense fruity odor. Under no circumstances should you distill your product to dryness. Once finished, shut off and unplug the rheostat from the wall, turn off the condenser water and allow the entire apparatus to cool to room temperature. Using one of the flat pan balances in the lab tear 150 mL beaker on the balance then place your first test tube receiver into the beaker recording the mass of the test tube and contents. Then, it might be good to also make a note on the odor of your product. Repeat this procedure for each receiver test tube that was used in collecting your product, recording the mass and odor of each fraction. Once the distillation apparatus is cool, disassemble the entire apparatus and wash and clean all of the parts with soap, hot water, and acetone air drying all components. Similarly, clean the separatory funnel and the reflux apparatus. Make sure all the glassware is absolutely clean and dry with no trace of any ester smell in the kit. Take the kit to the TA and have the TA initial the kit is being returned clean. Once you obtain the signature of your TA return the kit to the stockroom. Do NOT store the equipment in your desk drawers. IR Spectroscopy Run fractions on the Bruker Alpha II IR Spectrometer (see Appendix I) Fischer Esterification-34 EXPERIMENT #5 Fischer Esterification Day 3 Characterize the Product by Boiling Point (BP), Density, and Refractive Index (20nD) and NMR. Day 3 will begin with the TA demonstrating the sample preparation and operation of the refractometer, and NMR spectrometer. Students will then prepare an NMR sample tube and run their spectrum on the 60MHz NMR spectrometer. Following this student’s will proceed to determine the BP, Density, Refractive Index, and run an IR spectrum on the Unknown Ester. The thermometer calibration part of the experiment is performed to check the accuracy of the digital thermometer or regular thermometer that will be used in this experiment. The boiling point and freezing point of water can be used to calibrate the thermometer for this experiment. Take two readings on the thermometer one for the freezing point of water and one for the boiling point and compare those readings with the true values. For the freezing point fill a 150 mL beaker with ice and add distilled water. Let it sit for 10-15 minutes until it’s cold then take the temperature of the ice water mixture. Record the measured value and note the theoretical value, which should be 0oC. While waiting for the water to get cold fill a 150 mL beaker about ¾ full with water and heat it up on a hot plate to boiling (cover the beaker with aluminum foil to speed up the process). This may take 20 minutes. While the water is heating, take a reading for the barometric pressure in the lab from the barometer in inches of mercury. Do a quick calculation to find the correct theoretical boiling point of water at that pressure. Temperature corrections to the boiling point of water can be calculated using the following formula: Theoretical B.P. correction = (atmospheric pressure – 760 mm Hg) x (0.037oC / mm) Theoretical B.P. = 100 0C ± correction above Check the temperature of the boiling water as it is heating up. Be sure not to touch your thermometer to the sides or bottom of the beaker. Record the experimental boiling point of the water. Using excel, construct a thermometer-calibration curve by plotting observed temperature values versus true theoretical values. Boiling point can be measured very accurately using an electronic digital thermometer and the apparatus pictured in Figure 5-8a,b. This device can be used Fischer Esterification-35 EXPERIMENT #5 Fischer Esterification to measure the boiling point of the unknown organic liquid. Please set up the reaction apparatus under the hoods. Pour about 0.3 mL of liquid into a 12 x 75 mm test tube. Add a boiling chip to the test tube. Note in particular the position of the thermometer probe, which should be placed about 5 mm above the liquid and is positioned so that it does not touch the test tube at any point. The test tube sits in an 80 x 40 pyrex heating dish filled with sand, which is heated by an electric hotplate. Heat the sand until the liquid boils gently in the test tube and the hot vapor begins to condense and drip off the tip of the thermometer probe. Keep heating as the droplets drip from the tip of the thermometer probe and watch the temperature carefully as it continues to rise. After a few more minutes the temperature should stop rising and stabilize while the drops continue to drip from the probe.. When the temperature stops rising, this is the boiling point of your volatile unknown organic liquid record it into your laboratory notebook. Avoid unnecessarily breathing and inhaling the vapors from your system the unknown liquids should be assumed to be flammable and toxic. FIGURE 5-8a Photo of Boiling Point Apparatus Fischer Esterification-36 EXPERIMENT #5 Fischer Esterification Figure 5-8b Schematic Drawing of Boiling Point Apparatus 1. 13x100mm test tube 4. white sand 2. 80x40 pyrex heating dish 3. digital thermometer 5. electric hot plate Fischer Esterification-37 EXPERIMENT #5 Fischer Esterification To determine the density of the unknown students should use the new Rudolph 2910 automated density meter set up in the lab. (Please see instructions in Appendix V) For the density do a detailed error analysis with 95% confidence limits. Measure the refractive index of the sample on the new Rudoph J357 refractometer, follow the procedure demonstrated by your TA and outlined in Appendix IV. Remember, refractive index varies inversely with temperature (T). Make sure to record the temperature from the refractometer. The Rudolph J357 refractometer should automatically correct the refractive index to the temperature you have designated. The machine makes the temperature adjustments to the refractive index. If you need to compare your refractive index with a literature value that was obtained at a different temperature, you can use the following formula for calculating the correction: Dn = 4.5E-4 (T1-T2) where T1= temperature of the refractometer and T2= temperature you are comparing to. Then, add the correction to the measured refractive index obtained from the instrument. Instructions for the Rudolph Refractometer can be found in Appendix IV. NMR Sample Tube Preparation: Obtain a clean dry 5mm NMR tube from the stockroom. Check the tube carefully for any cracks and/or chips. Obtain a bottle of deuterated chloroform (CDCl3) from the stock room. Dissolve 50 µL of the ester in 900 µL of CDCl3. If the solution is clear add the solution to the NMR tube using a 9” glass Pasteur pipette. Insert the pipette filled with your sample into the NMR tube as far as it will go, then release the sample until it reaches the opening (tip) of the pipette. At that mark withdraw the glass pipette and the NMR sample tube now contains the correct amount of liquid. If the solution is cloudy filter using a small piece of kim wipe inserted into a glass Pasteur pipette. Cap the NMR tube and label with your name. See Appendix III for the operation of the NMR 60. Fischer Esterification-38 EXPERIMENT #5 Fischer Esterification Day 4 Identification of the Unknown Ester followed by running a Mass Spectrum From the observed boiling point, density, refractive index and NMR spectrum of the unknown ester, students must fill out the Preliminary Identification of Unknown Ester form and turn it in to your instructor at the start of the lab on Day 4 (Form is on Stellar under General). The first identification is worth 5 points. Once the identification form has been turned in to your instructor, run a mass spectrum on the previously identified ester. Then, fill out the final Identification of Unknown Ester form and turn it in to your instructor prior to the end of lab on Day 4. The second identification is worth an additional 5 points. If it is correct you are ready to begin writing your report. If it is not correct you may try to determine the source of your error and verify it with your TA. All of the laboratory work must be completed in the allotted four days for this experiment and incorrect attempts at identification in no way affects the written report due date. If you missed the identification you will be given the name of the correct ester for work up and analysis in your report. Mass Spec Sample Preparation: Obtain a small scintillation vial from the stockroom. Dissolve (150µL) of the ester in 1 mL of Pentane. Proceed to the Mass Spectrometer and inject 0.01 µL of your sample into the GC-MS. Each run takes 10 minutes + 3 minutes for data analysis. Your TA will have a sign-up sheet for the GC-MS. Please follow the direction of the TA. Operating procedure for the GCMS can be found in Appendix II. Fischer Esterification-39 EXPERIMENT #5 Fischer Esterification Analysis and Discussion 1. Indicate the structural formula of the ester and how it was obtained from the proton NMR. Describe how the structure is consistent in terms of the proton NMR spectrum. Compare the calculated chemical shifts with the actual values obtained from the NMR. If you missed the identification of the unknown ester, present reasoning and indicate what it should have been in terms of being consistent with the data collected. 2. Summarize the data in table form that indicates the identity of the ester. Talk about the IR spectrum and what peaks were present/absent that indicated an ester. Which features of the IR spectrum can be assigned to the ester? Be sure to identify and label as many functional groups as you can. 3. Compare the boiling point, density, refractive index and composition of the ester identified from your data with literature values. Explain any discrepancies. 4. Comment on the quantities of alcohol/acid used in the experiment as well as the sulfuric acid in terms of Kinetics and/or Thermodynamic reasoning. Discuss the yield of the product in terms of both of the above. How would each of the following shift the equilibrium in the esterification reaction (a) removal of H2O as its formed (b) adding more H2SO4 catalyst. 5. Write out the synthesis steps for the ester that you identified. Include all of the names, structures, catalysts, products and reaction conditions that were used in the synthesis. Identify the limiting reagent and show calculations including a table or results. Calculate the theoretical yield and percent yield of product formed. 6. Comment on the odor of the ester (this should be carefully obtained and you should not inhale or breathe the ester directly but waft the air to obtain the scent. Research the ester and determine if the smell observed was accurate in addition describe clearly where this ester is found and how it is used. What are the association of acid/alcohol and the odor of your ester? Fischer Esterification-40 EXPERIMENT #5 Fischer Esterification 7. Talk about sources of error in this experiment and how the experiment could be improved. 8. Aside from any improvements in experimental techniques used in this experiment describe how the % yield of the product might be increased. 9. Write out the full mechanism (with arrows) for the formation of the ester that was synthesized. 10.Analyze the IR spectrum by reporting the observed bands in a table and assigning (labeling) those bands on the spectrum. Identify and label as many functional groups as you can. 11.Analyze the NMR spectra by reporting tabulated results and assigning (labeling) those peaks to H atoms in the identified molecules. Identify, label and explain all of the peaks. 12.Analyze the MS spectrum reporting on the major peaks and assigning (labeling) those peaks. 13.Your well written discussion should address the following (within the text, NOT as a numerical series of answers to questions): • Comparison of your distillation fractions (temperature ranges) collected. • Explanation of how the separation of components could be improved. • Comparison of your results with literature values (boiling points/compositions/yields) • Discussion what information gas chromatography provides. • Discussion what information infrared spectroscopy provides. • Discussion what information mass spectroscopy provides. • Discussion what information nuclear magnetic resonance spectroscopy provides. • Describe the odor of your carboxylic acid and alcohol prior to the reaction Fischer Esterification-41 EXPERIMENT #5 Fischer Esterification APPENDIX I BRUKER ALPHA II IR OPERATING INSTRUCTIONS 1. IR Stays on all the time do not turn off 2. Turn on Computer 3. Double click on OPUS 8.1 software 4. USER: Student PASSWORD: student 5. Click OK on message that comes up version of software 6. Look at computer screen make sure green light at lower right-hand corner is on indicates computer and IR are connected 7. To begin and take a measurement click on the green test tube 8. Routine Measurement dialogue box comes up—Fill in sample description 9. Click background single channel 10.. If Solid, place solid on the diamond ATR—then rotate arm over sample—then use pressure clamp lever all the way down onto the solid—click on sample single channel Axis will appear as Absorbance v. wave number with peaks pointed up (this is what you want for a solid sample)—to change to transmittance v. wave number (liquid samples) click the button AB to TR on top of screen, you can toggle back and forth. There are two ways to label the spectrum, single peak pick which labels individual peaks and the standard peak pick which opens a dialogue box which can label all of them or some of them. Undo changes to file will undo what you just did. If you want to enlarge part of the spectrum use the Zoom button creating a box place on the area and click. You can always scale the Y axis to adjust and always click the undo button to get back to the original. Click the print icon to print the spectrum. Be sure to darken the color (black) is best for printing. 11. To run a new spectrum click on the test tube icon—run a background—then follow instructions above. The previous spectrum on the screen will automatically disappear. 12. All the data from the spectrum is located on C drive in the folder alpha FTIR data—you can open a file and insert a USB drive to copy the data. 13. If running a Liquid sample, move arm counterclockwise out of the way (no pressure necessary from the arm on the liquid). Place one drop of liquid on the diamond ATR and follow the steps in 10 above. 14. To clean diamond ATR use brush to collect solid sample into a weigh boat then for solids and liquids spray a little isopropanol alcohol on a kim wipe and clean the diamond ATR after you have ran the sample. Fischer Esterification-42 EXPERIMENT #5 Fischer Esterification APPENDIX II3 Operating Instructions for HP 5890 GC & 5971 MS (Have students prepare samples 200 microliters sample to 1 mL Pentane) 1. 2. 3. 4. 5. 6. 7. 8. 9. Plug in Needle Block Heater on Counter bake out both needles before class On GC click Detector B and touch ON wait until temperature reaches 290°C At same time Click Oven Temperature, type in 150°C Press Enter bakes out column Once Detector A has reached 290°C, click column type in 40°C press Enter Turn on Computer, Monitor, and Ion Gauge, Printer is always on. Once computer loads it stops blinking waiting for a command: Type, WIN Double Click HP GCMS this opens the Top Program Opens-Default On computer click TUNE MS then click STANDARD AUTOTUNE Message sometimes appears Tune Stopped, MS Fault, click OK, then retry Step 8, the tune process take about 5 minutes when done a printout automatically prints, click OK 10. GO to METHODS on computer click Load and Run Method double click disc5301.m press OK 11. Data File name appears DO NOT CHANGE we will overwrite the file EVALDEMO.D, click Run Method 12. Parameters change in GC to run method when all lights on GC go out ready for injection 13. Inject 0.01 microliters of sample septum is very hot keep fingers away from it, insert needle until metal touches metal, then quick injection, quickly pull out and press START on GC 14. Message appears on computer screen Override Solvent Delay? Answer NO 15. After two minutes peak appear on the screen you can click + by abundance to resolve peaks 16. After 10 minute run screen disappears some data print out which is not needed. Click Done and then click OK on the Printing Problem Error 208 that appears. 17. Click Data Analysis, Main Panel, File, Load Data File, EVALDEMO.D appears click OK, GC trace appears. 18. Move mouse so the cursor is under the ester peak and RIGHT DOUBLE CLICK, Mass Spectrum opens 19. Click FILE click Print Trace and Spectrum, spectrum prints 20. Now Click Spectra, Tabulate Spectrum, then click Done 21. Go up to Data Analysis -----bar in left grey box, left click on bar, Close then YES to exit 22. Go back to 10. Above and set up the machine for the next sample 23. To Shut Down, click on Detector B on GC and Press OFF temperature starts to decrease 24. Close Programs in Computer go to highest gray bar----left click Close then EXIT YES 25. Keep closing until you get message Window Sessions End, shut off computer and monitor 26. Pull plug from wall on the heating block 3 Written by John J. Dolhun Fischer Esterification-43 EXPERIMENT #5 Fischer Esterification APPENDIX III Department of Chemistry Undergraduate Lab 60 MHz NMR Operation Guidelines John Dolhun adopted from NMReady User’s Manual & DCIF NMR Guide Objective: Obtain well-shimmed NMR spectra to determine: chemical shifts, integration (1H only), splitting patterns, coupling constants. This document is intended as an introductory guide to the NMReady 60MHz NMR acquisition and processing software catering to the needs of the MIT Undergraduate Lab. Magnet Safety: • • • • CAUTION: NMR Magnets are ALWAYS ON! People with pacemakers, cochlear and other medical implants should not enter the NMR room without a doctor’s permission. Keep all metal objects, electronic and magnetic devices, at least 5 feet from the magnet. This includes cell phones, iPods, jump drives, chairs, wallets, keys, hair clips and pins. If a tube breaks inside the magnet, contact the Undergraduate Lab Staff immediately. Fischer Esterification-44 EXPERIMENT #5 Fischer Esterification Fig. 1 NMR Equipment: (A) NMR Console with Magnet, (B) Sample Warmer Fischer Esterification-45 EXPERIMENT #5 Fischer Esterification Sample Preparation: • Use a clean and dry 5 mm NMR tube that is free of cracks and chips. • Choose an NMR solvent by testing the solubility of the compound in the non-deuterated solvent. Unless otherwise noted use deuterated chloroform. • Dissolve 50 uL of the compound in 900 uL of the chosen deuterated4 solvent. • If the solution is clear, add the solution to the NMR tube using a 9” glass Pasteur pipette. Insert the pipette filled with your sample into the NMR tube as far as it will go, then release sample until it reaches the opening (tip) of the pipette. At that mark withdraw the glass pipette and you have the correct amount of sample in the NMR tube. • If the solution is cloudy, filter using a small piece if a Kim Wipe inserted into a glass Pasteur pipette. • Cap and label the NMR tube. Sample Warmer: The sample warmer is used to keep the NMR samples and solvent reference tubes at the same temperature as the instrument’s magnet. While the instrument is warming up, turn on the sample warmer (switch on the back) and place the solvent reference tubes in it. Any student samples that will be tested should also be placed in the warmer for at least 5 minutes before running in the machine. Performance can be degraded if un-heated samples and/or references are used. 4 Deuterated solvent is used for two purposes: 1) Serve as internal reference (lock) for the spectrometer to correct for magnetic field drift. 2) Prevent large solvent 1H signals. Fischer Esterification-46 EXPERIMENT #5 Fischer Esterification Placing a sample in the NMReady The NMReady uses standard 5mm NMR tubes. There is no air, no sample spinners, and no additional sample requirements so the tube glides directly into the magnet bore until it is located in the coil and then the operator will fee resistance. When you guide the sample in hold the tube straight up and insert it gently. When pulling it out pull it out carefully straight up until it is completely out of the magnet bore. It is strongly recommended that you warm your sample in the warmer. NdFeB magnets can be affected by temperature, so to prevent line broadening due to temperature gradients; it is recommended that the sample equilibrate to the same temperature as the magnet for several minutes prior to introduction into the magnet coil before acquiring NMR data on the sample. To begin an experiment, simply take out the reference sample, insert your sample, and press ‘GO’ button and the NMR will obtain your spectrum. If needed you can change some of the acquisition parameters easily using the scan calculator if the defaults are not suitable for your needs. Solvent Selector The first person using the NMR ready will power up the system, taking it off standby. Then, the solvent being used must be selected. The instrument will not be correctly calibrated until a solvent is chosen. After solvent selection, the system will prompt for a color-coded (blue, yellow or red) shim standard to be inserted into the magnet. Once the reference is inserted into the spectrometer, the appropriate configuration files are automatically loaded and the NMReady performs internal system tests (e.g. lock, receiver gain, auto-shim). A notification message will confirm that the solvent change was successful. If running a series of samples in the same solvent, the solvent does not need to be re-selected between each sample. To acquire a spectrum simply insert the sample into the magnet bore and press ‘GO’. This is One-touch-NMR. Fischer Esterification-47 EXPERIMENT #5 Fischer Esterification Once the ‘GO’ button has been activated, the spectrometer will begin to acquire the data. This includes a quick lock and automatic receiver gain and before the NMR pulse protocol begins. The first scan will be presented (post Fourier transform) on the screen and the spectrum will be updated with S/N improvements every additional four scans. Once the spectrum has appeared you are ready for analysis, saving or exporting your spectrum. Visual Appearance Full Screen The two-headed arrow in the lower right-hand corner of the data display area can be used to toggle to full screen mode if it is necessary to increase the viewing area for data processing. Ppm / Hz Toggle A spectrum can be viewed with the chemical shift displayed in ppm (parts per million) or in Hz. Grid Toggle A grid can be turned on/off by pushing the Grid button in the upper right hand corner of the screen. Adjust The ‘Adjust’ menu enables 1) baseline adjustment 2) spectrum phasing and 3) spectrum referencing. These adjustments are beneficial in improving the accuracy of the integrations. View Menu Zoom Width To zoom into a specific chemical shift range along the x-axis of a spectrum, push ‘Zoom Width’ then touch the screen on either side of the area you want to zoom to. Zoom Height To adjust the height of the displayed peak(s), first press the ‘Zoom Height’ button (vertical zoom) and then touch the screen above the peak that you want to extend. How far above the peak that you touch will determine the extent of the vertical zoom. Fischer Esterification-48 EXPERIMENT #5 Fischer Esterification Show All The show all button restores the original size of the spectrum. Cursor Touching the ‘Cursor’ toggle button places a cursor on the screen, adjustable by touch. The cursor can be used to quickly ascertain the position of a resonance or, by switching the Hz mode, used to quickly determine coupling constants. Integrate Touching the integrate button shows the automatically generated integrals of the peaks in the current spectrum. The integrals give ‘area under the curve’ information and can be used to determine relative concentrations of protons in the sample at analysis. Peaks Touching the peaks button places a list of all detected peaks on the screen. The peak list can be used for comparative purposes or to measure coupling constants. Process The process functions enable dynamic changes to be applied to the spectrum during processing. Depending on the complexity of the spectrum and the quality of the data, the spectrum can be smoothed (apodization) or enhanced (zero-filling). Apodize L/B Line broadening increases the smoothness of the lines, so for a well resolved spectrum, increasing this number can improve the look of the spectrum. However, this smoothness also results in line broadening and can therefore reduce the resolution. Zero Fill Increasing the zero filling enhances the digital resolution of the spectrum. Enhanced digital resolution will make feature-to-feature measurements more accurate. Increased zero filling is recommended for accurate coupling constant determination. Fischer Esterification-49 EXPERIMENT #5 Fischer Esterification Saving and Exporting Data After data processing is complete, the data can be saved (as either a. jdx or a .png) directly to the NMReady, exported to a network folder or printed to a network laser printer. Print Push the print button to send the spectral image (including integrations, peaks and coupling constants) to the laser printer in the room. The image will print in a frame and will be accompanied by experimental parameters. JDX Export Push the JDX to export the current spectrum directly to a USB drive that you insert into the NMReady. DO NOT PRESS SAVE and DO NOT PRESS Screenshot as these will save your spectrum on the spectrometer which we don’t want. You can however export your file to our network folder, which is hooked up on the computer next to the NMR. The Dell computer is equipped with the latest Mestrelab software to analyze the spectrum further if you so desire. It is however, not necessary to export your spectrum here once you get your printout and are satisfied. Fischer Esterification-50 EXPERIMENT #5 Fischer Esterification APPENDIX IV RUDOLPH J357 REFRACTOMETER OPERATING INSTRUCTIONS Always use soft disposable flexible pipettes to apply sample to prism. Never use glass pipettes which might scratch the prism. Turn on switch located on left front of instrument. Screen may appear dark for 15 seconds. 2. Wait 30 to 45 seconds for temperature to stabilize 3. Open the presser place deionized distilled water on the black prism until covered then close the presser. Make sure sample just covers black prism and stays within white circle. 4. Check sample quality blue bar should be at least 80% towards good if not add more sample. 5. Make sure the temperature of the refractometer is reading 20°C 6. Look at the live reading of water on the screen it should be 1.33294 to 1.33304. If its within that range prism is clean and you can continue with your sample. If not rinse prism again with water and dab off with tissue wipes. 7. Open the presser place sample on the black prism until covered then close the presser. Make sure sample just covers black prism and stays within white circle. 8. Press START—machine is set to take five readings each takes about 5s. When Start screen appears again measurement is complete. 9. Print results print key located bottom right—In the event printer does not print no worries data is stored and can be retrieved by date/time— from DATA and printed from there. 10. Clean off sample first using tissue dab off then put on few drops of methanol and dab off. 1. Fischer Esterification-51 EXPERIMENT #5 Fischer Esterification APPENDIX V RUDOLPH DENSITY METER Model 2910 OPERATING INSTRUCTIONS 1. Turn on density meter switch in top right rear—instrument goes dark for 15s then allow 45-60 seconds for machine to equilibrate. 2. Load Density Method. 3. To verify system is clean press START to take an air blank—this takes around 20 seconds if machine is clean you should get a reading of 0.0011 to 0.0012. If reading for air is higher you must clean and dry the machine U tube before loading sample. 4. To clean take acetone bottle with special connecting nipple and insert and squeeze delivering acetone through the tubing, then, attach air-line tube, press AIR PUMP and it dries the tubing for 100 seconds. 5. Disconnect tube to air pump and press START again to verify it is clean and dry running on Air. If result is 0.0011 to 0.0012 you may proceed with your sample. 6. Fill a 3mL syringe with sample make sure there is no bubble, hold syringe vertically with tip up and remove bubble with kim-wipe while pushing plunger gently. 7. Now insert syringe into U tube and inject sample slowly to avoid introducing bubbles. Inject slowly while watching live U tube picture on screen, once filled continue introducing sample until you see sample begin to appear in the waste line connected to the small waste bucket. Click on the U tube to check for bubbles (visible on the screen), click on U tube again to scan for bubbles. You will use about 1.5 mL from the 3.0 mL syringe leave syringe in the introduction port during the run. 8. Press START in around 90s machine will give you density and specific gravity. 9. Go to RESULTS , pick most recent your sample, it turns yellow then print. 10. Remove the syringe then take the acetone bottle with special connecting nipple and insert and squeeze delivering acetone through the tubing, then, attach the airline tube, press AIR PUMP and it dries the tubing for 100 seconds. 11. Disconnect air-line press start and verify air reading to make sure U tube is clean. Each student is responsible for cleaning the machine and verifying the system is clean by taking an air blank (step 3). Fischer Esterification-52