NMR SPECTROSCOPY JM Franconi 1

MR SPECTROCOPY Principle of FT NMR spectroscopy In a spectrum, each signal represent the resonance frequency of a nucleus in a specific environment Bo is fixed a RF pulse irradiate a broad range of frequency RF irradiation sampling FT t FID spectrum f

MR SPECTROCOPY Non selective RF pulse In spectroscopy the RF pulse used is < 100 microsecond RF rectangular sinc g.B1 > 2pF F frequency range

RF pulse Magnetisation evolution MR SPECTROCOPY RF pulse Magnetisation evolution During RF pulse M rotate Around x direction with an angle: Bo z Q = g. t . B1 q Mz M y O Mxy x

MR SPECTROCOPY spectrum parameter chemical shift f f1 f2 f3 f4 A same nucleus can have different resonance frequency

chemical shift phenomenon MR SPECTROCOPY chemical shift phenomenon Bo Bo Bo1 H2 Bo2 H1 O C Bo2 Bo1

chemical shift measurement MR SPECTROCOPY chemical shift measurement u i u ref reference f paramagnetic low field high electronégativity Diamagnetic High field Low electronégativity

MR SPECTROCOPY J coupling J coupling is an interaction between close nuclei This interaction is independant of Bo field It does not exit on one isolated molecule JAB fA fB f

MR SPECTROCOPY J scalar coupling H1 C C 50%(+1/2) H2 50%(-1/2) f1+dBH2 H1 is influenced by the 2 H2 sites Jcoupling is transported by chemical bond C C 50%(+1/2) H2 50%(-1/2) f1 f1+dBH2 f1- dBH2

MR SPECTROCOPY J dipolar coupling Coupling is also possible directly through space r H1 H2 1/r6

The Nuclei magneticaly equivalent have the same chemical MR SPECTROCOPY J coupling The Nuclei magneticaly equivalent have the same chemical shift. They do not show any coupling effect Ha C fHa = fHb = fHc C Hb Hc

The componants relative intensity are given by a binomial MR SPECTROCOPY J coupling Rules for multiplicity determination A I nucleus coupled to n spin S give a signal with: 2.n.S + 1 componants The componants relative intensity are given by a binomial Distribution.

MR SPECTROCOPY Signal area NMR spectroscopy is a quantitative method f

Concept of digital resolution MR SPECTROCOPY spectrum quality DR Concept of digital resolution AT = N.DW DW t f At R = 1/AT Digital resolution FT Frequency domain Time domain T2* TR=1/pT2* t f True NMR resolution CONDITION: TR > DR

MR SPECTROCOPY Sampling rules Application of Nquist theorem DW f1 f2 f2 f1 f2 F=1/Dw 2. F. Aq = N

The gyromagnetic ratio and the natural isotopic abundance MR SPECTROCOPY Sensitivity The gyromagnetic ratio and the natural isotopic abundance determine the NMR signal available - the nucleus sensitivity is proportional to Bo2 and g3

concept of population and coherence MR SPECTROCOPY concept of population and coherence Mz Mz MR signal E2 RF E2 E1 E1 population Cohérence=transition

Polarisation transfer (NOE) MR SPECTROCOPY Polarisation transfer (NOE) C +1/2 -1/2-1/2 Transition C13 C -1/2+1/2 +1/2 Long distance +1/2 H Transition H1 Niveau C13 +1/2-1/2 -1/2 +1/2+1/2 H Niveau H1 2 independant level of energy system of 4 levels of energy NO COUPLING COUPLING

Polarisation transfer (NOE) MR SPECTROCOPY Polarisation transfer (NOE) dH = 4.dC f f +5 -dH-dC +dH-dC C13 transition -dH+dC H1 transition dH-dC -dH-dC -3 -dH+dC dH+dC POLARISATION H1 TRANSITION SATURATION

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SEQUENCE SPIN ECHO SEQUENCE 90° 180° te/2

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SEQUENCE DEMI TOUR!

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SEQUENCE ECHO = coherence recovery

SEQUENCE SPIN ECHO 90 o 180 o FID ECHO DE SPIN T2 T2* Mxy t

CONCEPT OF PULSE SEQUENCE SPIN ECHO SEQUENCE 180° 90° TR TE RF GS GP DAC on GR CONCEPT OF PULSE SEQUENCE

NMR SPECTROSCOPY APPLICATION Chemical structure determination (protein, ADN,…) Quantitative measurement (isotopic enhancement determination) food and beverage quality control Spectroscopy in vivo (metabolism caracterisation)