1. Nuclear Magnetic Resonance Spectroscopy

2. 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

3. 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.

4. 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

5. 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

6. 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

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 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)

9. 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.

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 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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.

17. 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

18. 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

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

20. 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.

21. 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

22. 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

23. 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.

24. 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.

25. 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

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 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

28. Chemical Shifts

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

30. 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

31. 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

32. 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.

33. 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.

34. Chemical Shifts

35. 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

36. 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 = Gives us the ratio of protons.

37. 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.

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

39. 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

40. 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

41. Multiplicity
Consider an example where there are two protons on the adjacent carbon
There 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

44. 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

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

46. 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

47. 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

48. 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

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

50. 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

51. 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

52. 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

53. 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

54. 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

55. 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

56. 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

57. 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

58. 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

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 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

63. 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

64. 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

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 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

66. 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

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 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

70. 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

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

72. 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?

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