2 Intro to NMR Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy provides structural information about organic compounds and biomoleculesNMR involves an interaction between electromagnetic radiation and the nucleus of an atomWe will focus on C and H nuclei.The structure (connectivity) of a molecule affects how the radiation interacts with each nucleus in the molecule
3 Intro to NMR Spectroscopy Protons and neutrons in a nucleus behave as if they are spinningIf the total number of neutrons and protons is an ODD number, the atoms will have net nuclear spinExamples:The spinning charge in the nucleus creates a magnetic moment, perpendicular to spin axis.
4 Intro to NMR Spectroscopy If the normally disordered magnetic moments of atoms are exposed to an external magnetic field, their magnetic moments will alignThis interaction is quantized
5 Intro to NMR Spectroscopy The aligned magnetic moments can be either with or against the external magnetic fieldThe β spin state is higher in E than the α state (ΔE is the energy gap between the 2 states).When an atom with an α spin state is exposed to radio waves of just the right quantized energy, it can be promoted to the higher energy β spin state – atoms are in a state of resonance
6 Intro to NMR Spectroscopy NMR requires a strong magnetic field and radio wave energyThe stronger the magnetic field, the greater the energy gap (ΔE)The amount of radio wave energy necessary for the α β energy transition depends on the electronic environment for the atom
7 Intro to NMR Spectroscopy Magnetic moment of electrons generally reduces effect of the external field (i.e., shielding) on nuclei.The more shielded a nucleus is with electron density, the smaller the α β energy gap because proton is influenced by external magnetic field and effects of electron fields.
8 Acquiring a 1H NMR Spectrum The strong magnetic field is created when a high current is passed through a superconducting material at extremely low temperature (≈4 Kelvin)The greater the current, the greater the magnetic fieldIn most current NMR instruments, a brief pulse of RF energy (all relevant wavelengths) is used to excite the sampleEach of the atoms is excited and then relaxes, emitting energyThe emitted energy is recorded as a free induction decay (FID)
9 Acquiring a 1H NMR Spectrum The FID contains all of the information for each atomA mathematical treatment called a Fourier-transform separates the signals so an individual signal can be observed for each atom that was excitedSuch an instrument is called an FT-NMRNormally we collect many FIDs and average them. This reduces noise in the spectrum and enhances signals. Leads to cleaner spectra with sharper signals.
10 Acquiring a 1H NMR Spectrum NMR samples prepared neat or in a liquid solution (usually deuterated, like chloroform-d) and placed in a small NMR tubeThe sample is placed into the magnetic field and the tube is spun at a high rate to average magnetic field variations or tube imperfections
11 Characteristics of a 1H NMR Spectrum The NMR spectra provides information about the structure of the compound through:Number of signalsSignal location – shiftSignal area – integrationSignal shape – splitting pattern
12 Number of SignalsProtons with different electronic environments will give different signals, unless they are chemically equivalentProtons that are homotopic will have perfectly overlapping signalsProtons are homotopic if the molecule has an axis of rotational symmetry that allows one proton to be rotated onto the other without changing the molecule
13 Number of SignalsAnother test for homotopic protons is to replace the protons one at a time with another atomIf the resulting compounds are identical, then the protons that you replaced are homotopic
14 Number of SignalsProtons that are enantiotopic will also have perfectly overlapping signalsProtons are enantiotopic if the molecule has a plane of reflection that makes one proton the mirror image of the other
15 Number of Signals The replacement test is universal It will work to identify any equivalents protons whether they are homotopic or enantiotopicIf the resulting compounds are enantiomers, then the protons that you replaced are enantiotopic
16 Number of SignalsIf the protons are neither homotopic nor enantiotopic, then the are NOT chemically equivalentReplacement of each of these 2 H with D would produce diastereomers.
17 Number of SignalsThere are some shortcuts you can take to identify how many signals you should see in the 1H NMRThe 2 protons on a CH2 group will be equivalent if there are NO chirality centers in the moleculeThe 2 protons on a CH2 group will NOT be equivalent if there is a chirality center in the molecule
18 Number of SignalsThere are some shortcuts you can take to identify how many signals you should see in the 1H NMRThe 3 protons on any methyl group will always be equivalent to each otherMultiple protons are equivalent if they can be interchanged through either a rotation or mirror plane
19 Number of SignalsIdentify all the groups of equivalent protons in the molecules below and describe their relationships
20 Number of SignalsRecall that cyclohexane chairs have 6 equitorial and 6 axial protonsAxial and equitorial protons have different electronic environments; should produce 2 different signals in the 1H NMR.Because chair interconverts rapidly at room temp., only 1 signal observed.Separate signals could be observed by cooling sample to -100 °C.
21 Chemical ShiftsTetramethylsilane (TMS) is used as the standard for NMR chemical shiftIn many NMR solvents, 1% TMS is added as an internal standardThe shift for a proton signal is calculated as a comparison to TMSFor benzene on a300 MHz instrument
22 Chemical ShiftsThe shift for a proton signal is calculated as a comparison to TMSFor benzene on a 60 MHz instrumentThe Hz of the signal is different in different instruments, but the shift relative to TMS (δ) is constant
23 Chemical ShiftsThe shift for a proton signal is calculated as a comparison to TMSThe shift relative to TMS (δ) is a dimensionless number, because the Hz units cancel outUnits for δ are often given as ppm (parts per million), which simply indicates that signals are reported as a fraction of the operating frequency of the spectrometerMost 1H signals appear between 0-10 ppmSame scale applies regardless of the strength of the instrument.
24 Chemical ShiftsEarly NMRs analyzed samples at a constant energy over a range of magnetic field strengths from low field strength = downfield to high field strength = upfieldShielded protons required a stronger external magnetic field to be excited at the same energy as deshielded protons.
25 Chemical ShiftsCurrent NMRs analyze samples at a constant field strength over a range of energiesShielded protons have a smaller magnetic force acting on them, so they have smaller energy gaps and absorb lower energy radio wavesHigher EnergyLower Energy
26 Chemical Shifts Alkane protons generally give signals around 1-2 ppm Protons can be shifted downfield when nearby electronegative atoms cause deshielding.
27 Chemical ShiftsTo predict chemical shifts, start with the standard ppm for the type of proton (methyl, methylene, or methine)Use table 16.1 to adjust the ppm depending on proximity to certain function groups
31 Chemical ShiftsWhen the electrons in a pi system are subjected to an external magnetic field, they circulate a great deal causing diamagnetic anisotropyDiamagnetic anisotropy means that different regions in space will have different magnetic strengths
32 Chemical ShiftsThe result of the diamagnetic anisotropy effect is similar to deshielding for aromatic protons.Ethylbenzene has 3 different types of aromatic protons, but the peaks overlap.
33 Chemical ShiftsThe result of the diamagnetic anisotropy effect is similar to shielding for protons that extend into the pi systemExternal protons in  Annulene appear at 8 ppm, while internal protons are shielded, and appear at 1 ppm.
35 IntegrationThe integration or area under the peak quantifies the relative number of protons giving rise to a signalA computer will calculate the area of each peak representing that area with a step-curveThe curve height represents the integration
36 IntegrationThe computer operator sets one of the peaks to a whole number to let it represent a number of protonsThe computer uses the integration ratios to set the values for the other peaks1.481.561.001.05Numbers are calculated by dividing each integrated area, by the smallest area (ex. 40.2/27.0 = Gives us the ratio of protons.
37 IntegrationIntegrations represent numbers of protons, so you must adjust the values to whole numbersIf the integration of the first peak is doubled, the computer will adjust the others according to the ratio2.963.122.002.10The actual number is usually given, or it can be calculated from the molecular formula.
38 MultiplicityWhen a signal is observed in the 1H NMR, often it is split into multiple peaksMultiplicity or a splitting patterns results
39 MultiplicityMultiplicity results from magnetic affects that protons have on each otherConsider protons Ha and HbWe already saw that protons align with or against the external magnetic fieldHb will be aligned with the magnetic field in some molecules. Other molecules in the sample will have Hb aligned against the magnetic fieldSome Hb atoms have a slight shielding affect on Ha and others have a slight deshielding affect
40 MultiplicityThe resulting multiplicity or splitting pattern for Ha is a doubletA doublet generally results when a proton is split by only one other proton on an adjacent carbon
41 MultiplicityConsider an example where there are two protons on the adjacent carbonThere are three possible affects the Hb protons have on Ha
42 Multiplicity Half of the Ha atoms will not experience a signal shift. ¼ of the Ha atoms will be shielded and ¼ deshielded
43 Multiplicity Ha appears as a triplet The three peaks in the triplet have an integration ratio of 1:2:1
45 Multiplicity Ha appears as a quartet Relative peak intensities are 1:3:3:1.
46 MultiplicitySignal will split into n+1 peaks, where n = neighboring protons.By analyzing the splitting pattern of a signal in the 1H NMR, you can determine the number of equivalent protons on adjacent carbons
47 Multiplicity Remember three key rules Equivalent protons can not split one anotherNo splitting patterns observed for1,2-dichloroethane, since all protons are equivalentTo split each other, protons must be within a 2 or 3 bond distance
48 Multiplicity Remember three key rules The n+1 rule only applies to protons that are all equivalentThe splitting pattern observed for the proton shown below will be more complex than a simple tripletComplex splitting will be discussed later in this section
49 MultiplicityPredict splitting patterns for all of the protons in the molecule below
50 MultiplicityThe degree to which a neighboring proton will shield or deshield its neighbor is called a coupling constantThe coupling constant or J value is the distance between peaks of a splitting pattern measured in units of HzWhen protons split each other, their coupling constants will be equalJab = Jba
51 MultiplicityThe coupling constant will be constant even if an NMR instrument with a stronger or weaker magnetic field is usedHigher field strength instruments will give better resolution between peaks,because the coupling constant is a smaller percentage of the overall Hz available
52 MultiplicitySometimes recognizable splitting patterns will stand out in a spectrumAn isolated ethyl group gives a triplet and a quartetNote the integrationsThe triplet and quartet must have the same coupling constant if they are splitting each other
53 MultiplicityA peak with an integration equal to 9 suggests the presence of a tert-butyl groupAn isolated isopropyl group gives a doublet and a septetNote the integrations
54 MultiplicityComplex splitting results when a proton is split by NONequivalent neighboring protonsIf Jab is much greater than Jbc, the signal will appear as a quartet of tripletsIn the molecule shown, Hb is split into a quartet by Ha and into a triplet by Hc
55 MultiplicityComplex splitting results when a proton is split by NONequivalent neighboring protonsIf Jbc is much greater than Jab, the signal will appear as a triplet of quartetsIf Jbc is similar to Jab, the signal will appear as a multiplet
56 MultiplicitySplitting is not observed for some protons. Consider ethanolThe protons bonded to carbon split each other, but the hydroxyl proton is not split
57 MultiplicityThe hydroxyl proton and other labile or exchangeable protons undergo rapid exchange with trace amounts of acid. Show a reasonable mechanismSuch exchange blurs the shielding/deshielding affect of the neighboring protons giving a singlet that is often broadenedIf ethanol is rigorously purified to remove traces of acid, then hydroxyl proton splitting is generally observedAldehyde protons also often appear as singlet because their coupling constants are sometimes too small to cause observable splitting
58 MultiplicitySignals for exchangeable protons such as those shown below disappear completely when the 1H NMR sample is prepared for analysis in a deuterated solvent such as chloroform-d. WHY?Protic compounds have exchangeable protons
59 Using 1H Spectra to Distinguish Between Compounds The three molecules below might be difficult to distinguish by IR or MS, but can be differentiated by NMR.
60 Analyzing a 1H NMR Spectrum With a given formula and 1H NMR spectrum, you can determine a molecule’s structure by a 4-step processCalculate the degree or unsaturation or hydrogen deficiency index (HDI).Consider the number of NMR signals and integration to look for symmetry in the moleculeAnalyze each signal, and draw molecular fragments that match the shift, integration, and multiplicityAssemble the fragments into a complete structure like puzzle pieces
63 Acquiring a 13C NMR Spectrum Because 1H is by far the most abundant isotope of hydrogen, 1H NMR signals are generally strong13C only accounts for about 1% of carbon atoms in nature, so a sensitive receiver coil and/or concentrated NMR sample is neededIn 1H NMR, shift, splitting, and integration are importantIn 13C NMR, only the number of signals and the shift will be considered
64 Acquiring a 13C NMR Spectrum In 13C NMR, the 1H-13C splitting is often so complex that the spectrum is unreadableTo elucidate the 13C spectrum and make it easier to determine the total number of 13C signals, 13C NMR signals are generally decoupled from proton splitting.In the vast majority of 13C spectra, all of the signals are singlets
65 Chemical Shifts in 13C NMR Spectra Compared to 1H, 13C atoms require a different frequency of energy to excite (resonate)Compared to the standard TMS, 13C NMR signals generally appear between 220 and 0 ppmEach signal on the 13C spectra represents a carbon with a unique electronic environmentPlanes and axes of symmetry can cause carbon signals to overlap if their electronic surroundings are equivalent
66 Chemical Shifts in 13C NMR Spectra Note how symmetry affect the number of signals for the molecules aboveHow many 13C signals should be observed for the molecule below
67 Chemical Shifts in 13C NMR Spectra Like 1H signals, chemical shifts for 13C signals are affected by shielding or deshielding
68 Chemical Shifts in 13C NMR Spectra Predict the number of signals and chemical shifts in the 13C NMR spectrum for the molecule below
69 DEPT 13C NMR Spectra13C spectra generally give singlets that do not provide information about the number of hydrogen atoms attached to each carbonDistortionless Enhancement by Polarization Transfer (DEPT) 13C NMR provides information the number of hydrogen atoms attached to each carbonFull decoupled 13C spectrum: shows all carbon peaksDEPT-90: Only CH signals appearDEPT-135: CH3 and CH give (+) signals, and CH2 give (-) signals
70 DEPT 13C NMR SpectraFull decoupled 13C spectrum: shows all carbon peaksDEPT-90: Only CH signals appearDEPT-135: CH3 and CH = (+) signals, CH2 = (-) signals
71 DEPT 13C NMR SpectraExplain how DEPT 13C spectra could be used to distinguish between the two molecules below
72 Medically SpeakingMRI (magnetic resonance imaging) instruments are essentially 1H NMR spectrometersThe body is analyzed rather than a sample in an NMR tubeDifferent tissues have different concentrations of protons, based on density of water in tissues.The MRI gives a 3D image of different tissues.Would you expect there to be side-effects from exposure to either radio waves or a magnetic field?
73 Additional Practice Problems Predict the chemical shift, integration, and splitting patterns for all of the protons in the following molecule
74 Additional Practice Problems Predict the number of signals and chemical shifts in the 13C NMR spectrum and DEPT spectrum for the molecule below