Nmr 5k61

NUCLEAR MAGNETIC RESONANCE A branch of spectroscopy where the spin changes at the nuclear level when a radiofrequency energy is absorbed in the presence of magnetic field is studied.

It uses radio-frequency waves to induce transitions between nuclear energy states in a magnetic field.

Principle of NMR: 

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A nucleus with odd mass no. or an odd atomic no. has a nuclear spin which generates a magnetic field along the axis of spin. In the absence of external magnetic field the nuclei are randomly oriented. When the external field is applied, nuclei align themselves parallel to external magnetic field. When energy is applied in the form of radio-frequency wave transition of proton from lower energy to higher energy state occurs and nmr signal is thus recorded.

H1 - NMR 

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When Proton(hydrogen) is studied, then it is called as PROTON MAGNETIC RESONANCE(PMR). Any proton/nuclues with odd mass number spins on its own axis.when external magnetic field is applied, nuclues spins and c\a magnetic moment is created giving rise to frequency called “precisional frequency”. when Applied frequency = precisional frequency, nmr signal is recorded. Transition of proton from lower energy to higher energy takes place.

NUCLEAR & ELECTRON SPIN

All nuclei are assigned a spin quantum number, I, on the basis of thenumber of protons and neutrons in the nucleus, the values being zero,half-integral of integral. The number of permitted orientations in Space.  hence quantized energy levels, or spin states, that can be adopted by nucleus subjected to an externally applied magnetic field is given as (2I + 1). Thus, for I = 1⁄2, two orientations, or energy levels, are possible. The following should be noted. ● A spinning nucleus, being charged, generates a magnetic moment vector, or dipole, along its axis of rotation. 

The NMR Spectrometer

Basic Theory NMR  

Only nuclei with spin number (I) ≠ 0 can absorb/emit electromagnetic radiation.    

Even atomic mass & number ⇒ I = 0 (12C, 16O) Even atomic mass & odd number ⇒ I = whole integer (14N, 2H, 10B) Odd atomic mass ⇒ I = half integer (1H, 13C, 15N, 31P)

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The spin states of the nucleus (m) are quantized:

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Properly, m is called the magnetic quantum number

Basic Theory NMR 

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For 1H, 13C, 15N, 31P (biologically relevant nuclei with I = 1/2):

magnetic moment (m),

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It is a vector quantity that gives the direction and magnitude (or strength) of the ‘nuclear magnet’ h is the Planck constant g is the gyromagnetic ratio, and it depends on the nature of each nuclei. 

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The energy of a spin in a magnetic field will depend on the magnetic field, which we call Bo, and m:

Basic Theory NMR When the Bo field is applied, spins have to possible energy limits. In one we are in favor of the field, and in the other one we are against it. The energy is the dot product of the corresponding vectors: ENERGY, EE==--µµ . . BBo o

Population ratio ∆ E/ RT NNα //NNβ ==ee ∆ E/ RT α β

Basic Theory NMR Net magnetization

If we decompose the µ vectors in z and <xy>, we get: z

• We’ll have a slight excess of spins aligned with Bo, but at any angle with respect to z. The distribution is proportional to Nα / Nβ .

x y

Bo

z

z z

= Mo

y

x

y x

=

“0”

The net magnetization is aligned with Bo, and this is what we use in NMR.

Bo

Chemical Shift • Measured in parts per million. • Ratio of shift downfield from TMS (Hz) to total spectrometer frequency (Hz). • Same value for 60, 100, or 300 MHz machine. • Called the delta scale. Frequency of signal –frequency of reference x 106 Chemical shift, δ = Spectrometer frequency

Protons in a Molecule Shielding effect

e.g.

CH3 - CH2 - CH2 - OH 0.92 1.57 3.58

• CHEMICAL SHIFT: • Depends on carbon group attached: onacarbonyl (aldehyde): O

H

C

9- 10ppm

αtocarbonyl (aldehydes&ketones): O

C/H

C

C

H

1.6- 3ppm

C/H αtoC=C(allylic): C/H C

C

C

H

1.6- 3ppm

H

1.6- 3ppm

C/H αtoaromaticring(benzylic): C/H C C/H

Depends in hybridization C-C: means must be C or H, not O, N, or X

C/H C/H

H

C

0.2 to 2 ppm

C/H

C=C:

C/H C

C

H

4.5 to 7 ppm

C

H

1.6 to 3 ppm

H

6.8 to 8 ppm

= C=C: C

Aromatic:

Depends on anisotropy Acetylenic Protons, δ 2.5

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NMR Signals The number of signals shows how many different kinds of protons are present.  The location of the signals shows how shielded or deshielded the proton is.  The intensity of the signal shows the number of protons of that type.  Signal splitting shows the number of protons on adjacent atoms. 

NMR Spectra

Carboxylic Acid Proton, δ 10+

Intensity of Signals

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

EG:1,1,2-Tribromoethane Nonequivalent protons on adjacent carbons.

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Spin-Spin Splitting Triplet: 2 Adjacent Protons

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

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. 

Coupling Constants Complex Splitting a H

H C

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C

c

Hb

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).

Carbon-13 nmr: C 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 onefourth of that of 1H.  Signals are weak, getting lost in noise.  Hundreds of spectra are taken, averaged. 

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Combined 13 C and 1H Spectra

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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. FT

FT

FT

Basics of 2D NMR Experiment

General Scheme for 2D NMR

COSY Spectrum

H-3 —> H-5 —> H-10 —> OH H-10 -> H-9 H-3 —> H-16 H-16 —> H-11

2D NMR

WHY 2D NMR? 

As you can see, 1D protein spectra are far too complex for interpretation as most of the signals overlap heavily. By the introduction of additional spectral dimensions these spectra are simplified and some extra information is obtained. The invention of multidimensional spectra was the major leap in NMR spectroscopy apart from the introduction of FT-NMR. Consequently, both techniques were acknowledged by a nobel prize.

Anatomy of a 2D experiment:

2D NMR: 

The construction of a 2D experiment is simple: In addition to preparation and detection which are already known from 1D experiments the 2D experiment has an indirect evolution time t1 and a mixing sequence. This scheme can be viewed as:

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Do something with the nulcei (preparation), let them precess freely (evolution), do something else (mixing), and detect the result (detection, of course). After preparation the spins can precess freely for a given time t1. During this time the magnetization is labelled with the chemical shift of the first nucleus. During the mixing time magnetization is then transferred from the first nucleus to a second one. Mixing sequences utilize two mechanisms for magnetization transfer: scalar coupling or dipolar interaction (NOE). Data are acquired at the end of the experiment (detection, often called direct evolution time); during this time the magnetization is labelled with the chemical shift of the second nucleus.

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NMR APPLICATIONS:     

Structural (chemical) elucidation Natural product chemistry. Organic chemistry. Analytical tool of choice for synthetic chemists. Study of dynamic processes

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Reaction kinetics.

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Study of equilibrium (chemical or structural). Structural (three-dimensional) studies 

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Proteins. DNA/RNA. Protein complexes with DNA/RNA.

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Polysaccharides Drug design

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Structure Activity Relationships (SAR) by NMR Medicine - Magnetic Resonance Imaging (MRI)

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