Nuclear Magnetic Resonance Spectroscopy

Intro to NMR Spectroscopy Nuclear Magnetic Resonance (NMR) spectroscopy provides structural information about organic compounds and biomolecules NMR involves an interaction between electromagnetic radiation and the nucleus of an atom We will focus on C and H nuclei. The structure (connectivity) of a molecule affects how the radiation interacts with each nucleus in the molecule

Intro to NMR Spectroscopy Protons and neutrons in a nucleus behave as if they are spinning If the total number of neutrons and protons is an ODD number, the atoms will have net nuclear spin Examples: The spinning charge in the nucleus creates a magnetic moment, perpendicular to spin axis.

Intro to NMR Spectroscopy If the normally disordered magnetic moments of atoms are exposed to an external magnetic field, their magnetic moments will align This interaction is quantized

Intro to NMR Spectroscopy The aligned magnetic moments can be either with or against the external magnetic field The 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

Intro to NMR Spectroscopy NMR requires a strong magnetic field and radio wave energy The 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

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.

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 field In most current NMR instruments, a brief pulse of RF energy (all relevant wavelengths) is used to excite the sample Each of the atoms is excited and then relaxes, emitting energy The emitted energy is recorded as a free induction decay (FID)

Acquiring a 1H NMR Spectrum The FID contains all of the information for each atom A mathematical treatment called a Fourier-transform separates the signals so an individual signal can be observed for each atom that was excited Such an instrument is called an FT-NMR Normally we collect many FIDs and average them. This reduces noise in the spectrum and enhances signals. Leads to cleaner spectra with sharper signals.

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 tube The sample is placed into the magnetic field and the tube is spun at a high rate to average magnetic field variations or tube imperfections

Characteristics of a 1H NMR Spectrum The NMR spectra provides information about the structure of the compound through: Number of signals Signal location – shift Signal area – integration Signal shape – splitting pattern

Number of Signals Protons with different electronic environments will give different signals, unless they are chemically equivalent Protons that are homotopic will have perfectly overlapping signals Protons 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

Number of Signals Another test for homotopic protons is to replace the protons one at a time with another atom If the resulting compounds are identical, then the protons that you replaced are homotopic

Number of Signals Protons that are enantiotopic will also have perfectly overlapping signals Protons are enantiotopic if the molecule has a plane of reflection that makes one proton the mirror image of the other

Number of Signals The replacement test is universal It will work to identify any equivalents protons whether they are homotopic or enantiotopic If the resulting compounds are enantiomers, then the protons that you replaced are enantiotopic

Number of Signals If the protons are neither homotopic nor enantiotopic, then the are NOT chemically equivalent Replacement of each of these 2 H with D would produce diastereomers.

Number of Signals There are some shortcuts you can take to identify how many signals you should see in the 1H NMR The 2 protons on a CH2 group will be equivalent if there are NO chirality centers in the molecule The 2 protons on a CH2 group will NOT be equivalent if there is a chirality center in the molecule

Number of Signals There are some shortcuts you can take to identify how many signals you should see in the 1H NMR The 3 protons on any methyl group will always be equivalent to each other Multiple protons are equivalent if they can be interchanged through either a rotation or mirror plane

Number of Signals Identify all the groups of equivalent protons in the molecules below and describe their relationships

Number of Signals Recall that cyclohexane chairs have 6 equitorial and 6 axial protons Axial 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.

Chemical Shifts Tetramethylsilane (TMS) is used as the standard for NMR chemical shift In many NMR solvents, 1% TMS is added as an internal standard The shift for a proton signal is calculated as a comparison to TMS For benzene on a 300 MHz instrument

Chemical Shifts The shift for a proton signal is calculated as a comparison to TMS For benzene on a 60 MHz instrument The Hz of the signal is different in different instruments, but the shift relative to TMS (δ) is constant

Chemical Shifts The shift for a proton signal is calculated as a comparison to TMS The shift relative to TMS (δ) is a dimensionless number, because the Hz units cancel out Units 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 spectrometer Most 1H signals appear between 0-10 ppm Same scale applies regardless of the strength of the instrument.

Chemical Shifts Early NMRs analyzed samples at a constant energy over a range of magnetic field strengths from low field strength = downfield to high field strength = upfield Shielded protons required a stronger external magnetic field to be excited at the same energy as deshielded protons.

Chemical Shifts Current NMRs analyze samples at a constant field strength over a range of energies Shielded protons have a smaller magnetic force acting on them, so they have smaller energy gaps and absorb lower energy radio waves Higher Energy Lower Energy

Chemical Shifts Alkane protons generally give signals around 1-2 ppm Protons can be shifted downfield when nearby electronegative atoms cause deshielding.

Chemical Shifts To 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

Chemical Shifts

Chemical Shifts Handbooks can be used for functional groups beyond table 16.1

Klein, Organic Chemistry 2e Chemical Shifts Predict chemical shifts for all of the protons in the molecule below Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Klein, Organic Chemistry 2e

Chemical Shifts When the electrons in a pi system are subjected to an external magnetic field, they circulate a great deal causing diamagnetic anisotropy Diamagnetic anisotropy means that different regions in space will have different magnetic strengths

Chemical Shifts The 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.

Chemical Shifts The result of the diamagnetic anisotropy effect is similar to shielding for protons that extend into the pi system External protons in [14] Annulene appear at 8 ppm, while internal protons are shielded, and appear at 1 ppm.

Chemical Shifts

Integration The integration or area under the peak quantifies the relative number of protons giving rise to a signal A computer will calculate the area of each peak representing that area with a step-curve The curve height represents the integration

Integration The computer operator sets one of the peaks to a whole number to let it represent a number of protons The computer uses the integration ratios to set the values for the other peaks 1.48 1.56 1.00 1.05 Numbers are calculated by dividing each integrated area, by the smallest area (ex. 40.2/27.0 = 1.48. Gives us the ratio of protons.

Integration Integrations represent numbers of protons, so you must adjust the values to whole numbers If the integration of the first peak is doubled, the computer will adjust the others according to the ratio 2.96 3.12 2.00 2.10 The actual number is usually given, or it can be calculated from the molecular formula.

Multiplicity When a signal is observed in the 1H NMR, often it is split into multiple peaks Multiplicity or a splitting patterns results

Multiplicity Multiplicity results from magnetic affects that protons have on each other Consider protons Ha and Hb We already saw that protons align with or against the external magnetic field Hb will be aligned with the magnetic field in some molecules. Other molecules in the sample will have Hb aligned against the magnetic field Some Hb atoms have a slight shielding affect on Ha and others have a slight deshielding affect

Multiplicity The resulting multiplicity or splitting pattern for Ha is a doublet A doublet generally results when a proton is split by only one other proton on an adjacent carbon

Multiplicity Consider an example where there are two protons on the adjacent carbon There are three possible affects the Hb protons have on Ha

Multiplicity Half of the Ha atoms will not experience a signal shift. ¼ of the Ha atoms will be shielded and ¼ deshielded

Multiplicity Ha appears as a triplet The three peaks in the triplet have an integration ratio of 1:2:1

Klein, Organic Chemistry 2e Multiplicity Consider a scenario where Ha has three equivalent Hb atoms splitting it Explain how the magnetic fields cause shielding or deshielding Copyright © 2015 John Wiley & Sons, Inc. All rights reserved. Klein, Organic Chemistry 2e

Multiplicity Ha appears as a quartet Relative peak intensities are 1:3:3:1.

Multiplicity Signal 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

Multiplicity Remember three key rules Equivalent protons can not split one another No splitting patterns observed for 1,2-dichloroethane, since all protons are equivalent To split each other, protons must be within a 2 or 3 bond distance

Multiplicity Remember three key rules The n+1 rule only applies to protons that are all equivalent The splitting pattern observed for the proton shown below will be more complex than a simple triplet Complex splitting will be discussed later in this section

Multiplicity Predict splitting patterns for all of the protons in the molecule below

Multiplicity The degree to which a neighboring proton will shield or deshield its neighbor is called a coupling constant The coupling constant or J value is the distance between peaks of a splitting pattern measured in units of Hz When protons split each other, their coupling constants will be equal Jab = Jba

Multiplicity The coupling constant will be constant even if an NMR instrument with a stronger or weaker magnetic field is used Higher field strength instruments will give better resolution between peaks, because the coupling constant is a smaller percentage of the overall Hz available

Multiplicity Sometimes recognizable splitting patterns will stand out in a spectrum An isolated ethyl group gives a triplet and a quartet Note the integrations The triplet and quartet must have the same coupling constant if they are splitting each other

Multiplicity A peak with an integration equal to 9 suggests the presence of a tert-butyl group An isolated isopropyl group gives a doublet and a septet Note the integrations

Multiplicity Complex splitting results when a proton is split by NONequivalent neighboring protons If Jab is much greater than Jbc, the signal will appear as a quartet of triplets In the molecule shown, Hb is split into a quartet by Ha and into a triplet by Hc

Multiplicity Complex splitting results when a proton is split by NONequivalent neighboring protons If Jbc is much greater than Jab, the signal will appear as a triplet of quartets If Jbc is similar to Jab, the signal will appear as a multiplet

Multiplicity Splitting is not observed for some protons. Consider ethanol The protons bonded to carbon split each other, but the hydroxyl proton is not split

Multiplicity The hydroxyl proton and other labile or exchangeable protons undergo rapid exchange with trace amounts of acid. Show a reasonable mechanism Such exchange blurs the shielding/deshielding affect of the neighboring protons giving a singlet that is often broadened If ethanol is rigorously purified to remove traces of acid, then hydroxyl proton splitting is generally observed Aldehyde protons also often appear as singlet because their coupling constants are sometimes too small to cause observable splitting

Multiplicity Signals 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

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.

Analyzing a 1H NMR Spectrum With a given formula and 1H NMR spectrum, you can determine a molecule’s structure by a 4-step process Calculate the degree or unsaturation or hydrogen deficiency index (HDI). Consider the number of NMR signals and integration to look for symmetry in the molecule Analyze each signal, and draw molecular fragments that match the shift, integration, and multiplicity Assemble the fragments into a complete structure like puzzle pieces

Acquiring a 13C NMR Spectrum Because 1H is by far the most abundant isotope of hydrogen, 1H NMR signals are generally strong 13C only accounts for about 1% of carbon atoms in nature, so a sensitive receiver coil and/or concentrated NMR sample is needed In 1H NMR, shift, splitting, and integration are important In 13C NMR, only the number of signals and the shift will be considered

Acquiring a 13C NMR Spectrum In 13C NMR, the 1H-13C splitting is often so complex that the spectrum is unreadable To 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

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 ppm Each signal on the 13C spectra represents a carbon with a unique electronic environment Planes and axes of symmetry can cause carbon signals to overlap if their electronic surroundings are equivalent

Chemical Shifts in 13C NMR Spectra Note how symmetry affect the number of signals for the molecules above How many 13C signals should be observed for the molecule below

Chemical Shifts in 13C NMR Spectra Like 1H signals, chemical shifts for 13C signals are affected by shielding or deshielding

Chemical Shifts in 13C NMR Spectra Predict the number of signals and chemical shifts in the 13C NMR spectrum for the molecule below

DEPT 13C NMR Spectra 13C spectra generally give singlets that do not provide information about the number of hydrogen atoms attached to each carbon Distortionless Enhancement by Polarization Transfer (DEPT) 13C NMR provides information the number of hydrogen atoms attached to each carbon Full decoupled 13C spectrum: shows all carbon peaks DEPT-90: Only CH signals appear DEPT-135: CH3 and CH give (+) signals, and CH2 give (-) signals

DEPT 13C NMR Spectra Full decoupled 13C spectrum: shows all carbon peaks DEPT-90: Only CH signals appear DEPT-135: CH3 and CH = (+) signals, CH2 = (-) signals

DEPT 13C NMR Spectra Explain how DEPT 13C spectra could be used to distinguish between the two molecules below

Medically Speaking MRI (magnetic resonance imaging) instruments are essentially 1H NMR spectrometers The body is analyzed rather than a sample in an NMR tube Different 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?

Additional Practice Problems Predict the chemical shift, integration, and splitting patterns for all of the protons in the following molecule

Additional Practice Problems Predict the number of signals and chemical shifts in the 13C NMR spectrum and DEPT spectrum for the molecule below