Nuclear Magnetic Resonance Spectroscopy

Introduction NMR is the most powerful tool available for organic structure determination. It is used to study a wide variety of nuclei: 1H 13C 15N 19F 31P

Introduction: NMR is the most powerful tool available for organic structure determination. Studying a molecules by NMR we obtained following information. 1. The numbers of signal: How many different kinds of protons in molecules 2. Position of signal: Electronic environment of each kind of protons 3. Intensity of signal: Resolution of signal 4. Splitting patterns: Numbers of peaks neighboring protons & its environments 5. Coupling constant: molecular structure and its stereo chemical studies E.M Purcel and F. Bloch the two physicest shared the noble prize 1952. It is used to study a wide variety of nuclei: 1H 13C 15N 19F 31P => Chapter 13

NUCLEAR SPIN The nuclei of some atoms have a property called “SPIN”. ….. we don’t know if they actually do spin! Each spin-active nucleus has a number of spins defined by its spin quantum number, I. NMR Discovered by F. Bloch and E. Purcell

THE ENERGY SEPARATION DEPENDS ON Bo - 1/2 = kBo = hn DE degenerate at Bo = 0 + 1/2 Bo increasing magnetic field strength

g-rays IR X-rays UV Microwave Radio Visible The entire electromagnetic spectrum is used by chemists: Frequency, n in Hz ~1019 ~1017 ~1015 ~1013 ~1010 ~105 Wavelength, l ~.0001 nm ~0.01 nm 10 nm 1000 nm 0.01 cm 100 m Energy (kcal/mol) > 300 300-30 300-30 ~10-4 ~10-6 g-rays X-rays UV IR Microwave Radio nuclear excitation (PET) core electron excitation (X-ray cryst.) electronic excitation (p to p*) molecular vibration molecular rotation Nuclear Magnetic Resonance NMR (MRI) Visible

What is NMR? NMR – Nuclear Magnetic Resonance is a branch of spectroscopy that deals with the phenomenon found in assemblies of large number of nuclei of atoms that possess both “magnetic moments” and “angular momentum” is subjected to external magnetic field. Resonance – Implies that we are in tune with a natural frequency of the nuclear magnetic system in the magnetic field.

External Magnetic Field When placed in an external field, spinning protons act like bar magnets. => Chapter 13

PRINCIPLE: When energy in the form of Radiofrequency is applied the nuclei are said to be in resonance, and the energy they emit when flipping from the high to the low energy state can be measured and the obtained absorption of energy occurs and a NMR signal is recorded . Applied frequency = Precessional frequency frequency of the incoming radiation that will cause a transition √ ὰ Bo strength of the magnetic field The Larmor Equation!!!

Reference Std. Tetramethylsilane(TMS) Chemically inert Magnetically Isotopic Sharp single only one peak on NMR spectrum Molecule must be soluble in most of the organic solvent Must be relatively volatile High electronic density of H in TMS. (Almost all the H peaks of organic compounds appear on the left of the TMS peak.) Since silicon is less electronegative than carbon, TMS protons are highly shielded. Signal defined as zero.

√sample– √ref X 106  ppm √o CHEMICAL SHIFT: () The separation between the frequencies for the same nucleus in different chemical environments is known as chemical shift. OR The difference in absorption of a particular proton (nuclei) from the absorption position of reference protons called chemical shift. √r= Frequency of the reference √s= Frequency of the sample √o= The frequency at which the Spectrometer operates √sample– √ref X 106  ppm √o

NMR Spectrum A Spectrum of Absorption of Radiation Vs. Applied Magnetic Strength is called as NMR Spectrum. Number of Signal- shows how many different kinds of protons are present. Intensity of the Signal- shows the number of protons of each kinds. Location of the Signals- shows how shielded or deshielded the proton is. Signal splitting- shows the number of protons on adjacent atoms.

Diamagnetic shielding In a magnetic field, the six  electrons in benzene circulate around the ring creating a ring current. The magnetic field induced by these moving electrons reinforces the applied magnetic field in the vicinity of the protons. The protons thus feel a stronger magnetic field and a higher frequency is needed for resonance. Thus they are deshielded and absorb downfield. Aromatic Protons, 7-8

Protons in a Molecule Depending on their chemical environment, protons in a molecule are shielded by different amounts. Chapter 13

FACTORS INFLUENCING CHEMICAL SHIFT Both 1H and 13C Chemical shifts are related to the following major factors: Depends on Hydrogen bonding Depends on adjacent group Depends on carbon group attached Depends on hybridization Depends on anisotropy

Delta Scale

Shielded Protons Magnetic field strength must be increased for a shielded proton to flip at the same frequency. => Chapter 13

Location of Signals More electronegative atoms deshield more and give larger shift values. Effect decreases with distance. Additional electronegative atoms cause increase in chemical shift. => Chapter 13

Typical Values

Diamagnetic shielding: In a magnetic field, the six  electrons in benzene circulate around the ring creating a ring current. The magnetic field induced by these moving electrons reinforces the applied magnetic field in the vicinity of the protons. The protons thus feel a stronger magnetic field and a higher frequency is needed for resonance. Thus they are deshielded and absorb downfield Aromatic Protons, 7-8

Vinyl Protons, 5-6 1H NMR—Chemical Shift Values 4.5-6 ppm In a magnetic field, the loosely held  electrons of the double bond create a magnetic field that reinforces the applied field in the vicinity of the protons. The protons now feel a stronger magnetic field, and require a higher frequency for resonance. Thus the protons are deshielded and the absorption is downfield.

Acetylenic Protons, 2.5 In a magnetic field, the  electrons of a carbon-carbon triple bond are induced to circulate, but in this case the induced magnetic field opposes the applied magnetic field (B0). Thus, the proton feels a weaker magnetic field, so a lower frequency is needed for resonance. The nucleus is shielded and the absorption is upfield.

POPULATION AND SIGNAL STRENGTH The strength of the NMR signal depends on the Population Difference of the two spin states Radiation induces both upward and downward transitions. induced emission resonance For a net positive signal there must be an excess of spins in the lower state. excess population Saturation = equal populations = no signal

A Simplified 60 MHz NMR Spectrometer hn RF (60 MHz) Oscillator RF Detector absorption signal Recorder Transmitter Receiver MAGNET MAGNET ~ 1.41 Tesla (+/-) a few ppm N S Probe

Instrumentation: Main features of a basic NMR include: A radio transmitter coil that produces a short powerful pulse of radio waves A powerful magnet that produces strong magnetic fields The sample is placed in a glass tube that spins so the test material is subject to uniform magnetic field. A radio receiver coil that detects radio frequencies emitted as nuclei relax to a lower energy level A computer that analyses and record the data

The NMR Spectrometer:

O-H and N-H Signals Chemical shift depends on concentration. Hydrogen bonding in concentrated solutions deshield the protons, so signal is around 3.5 for N-H and  4.5 for O-H. Proton exchanges between the molecules broaden the peak. => Chapter 13

Carboxylic Acid Proton, 10+

Number of Signals Equivalent hydrogens have the same chemical shift.

Intensity of Signals The area under each peak is proportional to the number of protons. Shown by integral trace. =>

How Many Hydrogens? When the molecular formula is known, each integral rise can be assigned to a particular number of hydrogens. =>

Spin-Spin Splitting It is unlikely that a 13C would be adjacent to another 13C, so splitting by carbon is negligible. 13C will magnetically couple with attached protons and adjacent protons. These complex splitting patterns are difficult to interpret. => Chapter 13

1,1,2-Tribromoethane Nonequivalent protons on adjacent carbons. => Chapter 13

Doublet: 1 Adjacent Proton

Triplet: 2 Adjacent Protons

The N + 1 Rule If a signal is split by N equivalent protons, it is split into N + 1 peaks.

Range of Magnetic Coupling Equivalent protons do not split each other. Protons bonded to the same carbon will split each other only if they are not equivalent. Protons on adjacent carbons normally will couple. Protons separated by four or more bonds will not couple. => Chapter 13

Splitting for Ethyl Groups

Splitting for Isopropyl Groups

Coupling Constants Distance between the peaks of multiplet Measured in Hz Not dependent on strength of the external field Multiplets with the same coupling constants may come from adjacent groups of protons that split each other. =>

Values for Coupling Constants

Complex Splitting Signals may be split by adjacent protons, different from each other, with different coupling constants. Example: Ha of styrene which is split by an adjacent H trans to it (J = 17 Hz) and an adjacent H cis to it (J = 11 Hz). =>

Splitting Tree =>

Spectrum for Styrene

Stereochemical Nonequivalence Usually, two protons on the same C are equivalent and do not split each other. If the replacement of each of the protons of a -CH2 group with an imaginary “Z” gives stereoisomers, then the protons are non-equivalent and will split each other. =>

Some Nonequivalent Protons =>

Resonance Frequencies of Selected Nuclei Nuclie Percentage Abundance Applied field in Tesla Precessional frequency in MHz 1H 99.98 1.0 42.6 2H 0.0156 6.5 13C 1.108 10.7 19F 100 40.0

Time Dependence Molecules are tumbling relative to the magnetic field, so NMR is an averaged spectrum of all the orientations. Axial and equatorial protons on cyclohexane interconvert so rapidly that they give a single signal. Proton transfers for OH and NH may occur so quickly that the proton is not split by adjacent protons in the molecule. =>

Hydroxyl Proton Ultrapure samples of ethanol show splitting. Ethanol with a small amount of acidic or basic impurities will not show splitting. =>

N-H Proton Moderate rate of exchange. Peak may be broad. =>

Identifying the O-H or N-H Peak Chemical shift will depend on concentration and solvent. To verify that a particular peak is due to O-H or N-H, shake the sample with D2O Deuterium will exchange with the O-H or N-H protons. On a second NMR spectrum the peak will be absent, or much less intense. =>

MODERN INSTRUMENTATION PULSED FOURIER TRANSFORM TECHNOLOGY FT-NMR requires a computer

FOURIER TRANSFORM n1 + n2 + n3 + ...... A mathematical technique that resolves a complex FID signal into the individual frequencies that add together to make it. ( Details not given here. ) converted to DOMAINS ARE MATHEMATICAL TERMS TIME DOMAIN FREQUENCY DOMAIN FID NMR SPECTRUM FT-NMR computer COMPLEX SIGNAL n1 + n2 + n3 + ...... Fourier Transform individual frequencies a mixture of frequencies decaying (with time) converted to a spectrum

Fortunately, different types of protons precess at different rates in the same magnetic field. N Bo = 1.41 Tesla EXAMPLE: 59.999995 MHz 59.999700 MHz hn To cause absorption of the incoming 60 MHz the magnetic field strength, Bo , must be increased to a different value for each type of proton. 59.999820 MHz 60 MHz S Differences are very small, in the parts per million range.

PULSED EXCITATION (n1 ..... nn) n2 n1 n3 N S BROADBAND RF PULSE contains a range of frequencies n3 (n1 ..... nn) S All types of hydrogen are excited simultaneously with the single RF pulse.

Spin Quantum Numbers of Some Common Nuclei Element 1H 2H 12C 13C 14N 16O 17O 19F Nuclear Spin Quantum No 1/2 1 0 1/2 1 0 5/2 1/2 ( I ) No. of Spin 2 3 0 2 3 0 6 2 States Elements with either odd mass or odd atomic number have the property of nuclear “spin”.

n1 n2 n3 FREE INDUCTION DECAY ( relaxation ) n1, n2, n3 have different half lifes

COMPOSITE FID “time domain“ spectrum n1 + n2 + n3 + ...... time

The Composite FID is Transformed into a classical NMR Spectrum : “frequency domain” spectrum

IN THE CLASSICAL NMR EXPERIMENT THE INSTRUMENT SCANS FROM “LOW FIELD” TO “HIGH FIELD” LOW FIELD HIGH FIELD NMR CHART increasing Bo DOWNFIELD UPFIELD scan

NMR Spectrum of Phenylacetone NOTICE THAT EACH DIFFERENT TYPE OF PROTON COMES AT A DIFFERENT PLACE - YOU CAN TELL HOW MANY DIFFERENT TYPES OF HYDROGEN THERE ARE

COMPARISON OF CW AND FT TECHNIQUES

CONTINUOUS WAVE (CW) METHOD THE OLDER, CLASSICAL METHOD The magnetic field is “scanned” from a low field strength to a higher field strength while a constant beam of radiofrequency (continuous wave) is supplied at a fixed frequency (say 100 MHz). Using this method, it requires several minutes to plot an NMR spectrum. SLOW, HIGH NOISE LEVEL

PULSED FOURIER TRANSFORM (FT) METHOD FAST LOW NOISE THE NEWER COMPUTER-BASED METHOD Most protons relax (decay) from their excited states very quickly (within a second). The excitation pulse, the data collection (FID), and the computer-driven Fourier Transform (FT) take only a few seconds. The pulse and data collection cycles may be repeated every few seconds. Many repetitions can be performed in a very short time, leading to improved signal …..

IMPROVED SIGNAL-TO-NOISE RATIO By adding the signals from many pulses together, the signal strength may be increased above the noise level. signal enhanced signal noise 1st pulse 2nd pulse add many pulses noise is random and cancels out nth pulse etc.

Carbon-13 12C has no magnetic spin. 13C has a magnetic spin, but is only 1% of the carbon in a sample. The gyromagnetic ratio of 13C is one-fourth of that of 1H. Signals are weak, getting lost in noise. Hundreds of spectra are taken, averaged. =>

Fourier Transform NMR Nuclei in a magnetic field are given a radio-frequency pulse close to their resonance frequency. The nuclei absorb energy and precess (spin) like little tops. A complex signal is produced, then decays as the nuclei lose energy. Free induction decay is converted to spectrum. =>

Hydrogen and Carbon Chemical Shifts =>

Combined 13C and 1H Spectra

Differences in 13C Technique Resonance frequency is ~ one-fourth, 15.1 MHz instead of 60 MHz. Peak areas are not proportional to number of carbons. Carbon atoms with more hydrogens absorb more strongly. =>

Spin-Spin Splitting It is unlikely that a 13C would be adjacent to another 13C, so splitting by carbon is negligible. 13C will magnetically couple with attached protons and adjacent protons. These complex splitting patterns are difficult to interpret. =>

Proton Spin Decoupling To simplify the spectrum, protons are continuously irradiated with “noise,” so they are rapidly flipping. The carbon nuclei see an average of all the possible proton spin states. Thus, each different kind of carbon gives a single, unsplit peak. =>

Off-Resonance Decoupling 13C nuclei are split only by the protons attached directly to them. The N + 1 rule applies: a carbon with N number of protons gives a signal with N + 1 peaks. =>

Off-Resonance Decoupling 13C nuclei are split only by the protons attached directly to them. The N + 1 rule applies: a carbon with N number of protons gives a signal with N + 1 peaks. =>

Interpreting 13C NMR The number of different signals indicates the number of different kinds of carbon. The location (chemical shift) indicates the type of functional group. The peak area indicates the numbers of carbons (if integrated). The splitting pattern of off-resonance decoupled spectrum indicates the number of protons attached to the carbon. =>

Two 13C NMR Spectra

MRI Magnetic resonance imaging, noninvasive “Nuclear” is omitted because of public’s fear that it would be radioactive. Only protons in one plane can be in resonance at one time. Computer puts together “slices” to get 3D. Tumors readily detected. =>