FT-NMR

Fundamentals Nuclear spin Spin quantum number – ½ Nuclei with spin state ½ are like little bar magnets and align with a B field. Can align with (++) or against (+-) B Small energy gap between + and – spin alignment (NMR insensitive/Boltzman dist) Can probe difference with RW

(NMR insensitive/Boltzman dist) Small population difference between +1/2 and -1/2 state It is the small excess of nuclei in the -1/2 that produce NMR signal

Common NMR nuclei Protons, 1H 13C 15N 19F 31P Sensitivity depends on natural isotopic abundance and g DE = gћB0 , bigger magnet, greater sensitivity

Precession of nuclear dipoles z +1/2 M0; net magnetic moment From small excess of Nuclei in +1/2 state y M0 B0 from magnet x -1/2

FT pulse Radiofrequency generator A short, intense pulse generates a magnetic field in the x-y plane (excites all nuclei) M0 of the nuclei interacts with the magnetic field produced by the pulse. Tips M0 off axis Θ = gB1tp tp – length of pulse, 90 pulse

Vector Illustration of the pulse +1/2 M0 RF pulse B0 from magnet RF coil -1/2

Relaxation T1 spin-lattice (relaxing back to precessing about the z axis) T2 spin-spin (fanning out)

Induced current in coil After pulse, nuclei begin to precess in phase in the x-y plane Packet of nuclei induce current in RF coil Relaxation is measured by monitoring the induced coil → FID (→ FT) NMR spectrum

FID

Noise reduction and increasing resolution Apodization: Multiply the free-induction decay (FID) by a decreasing exponential function which mathematically suppresses the noise at long times. Other forms of apodization functions can be used to improve resolution or lineshape. Zero filling

Chemical Shift Shielding Electrons have spin, produce local B environments Protons in different electronic environments experience different B, different precessing frequencies, DE = hu Chemical shift proportional to size of magnet ppm {(s-s0)/s0}*106

Spin-Spin Coupling Adjacent nuclei have a 50/50 chance of being spin up (+1/2) or spin down (-1/2) Each produce a small magnetic field that is either with or against B0 1 adjacent proton CHOCH3 CH3 is a doublet at frequencies u0-ua, u0+ua (equal intensity), 1:1 CH is a quadruplet 1:3:3:1

Splitting Patterns J values Quadruplet                         Triplet         Multiplets 1 3 3 1 1 2 1 ¼ ½ ¼ ¾ 1 ½ ¾ ¾ 1 ½ ¾ ¼ ½ ¼ 1 2 1 3 6 3 3 6 3 1 2 1

13C NMR 13C frequency Different tuning folk Broadband Decoupling of 1H No spin-spin coupling NOE effect Assignments based on chemical shift Wider frequency range

Obtaining a 13C NMR Spectrum 1H Broadband decoupling Gives singlet 13C peaks, provided no F, P, or D present in the molecule) Continuous sequence of pulses at the 1H frequency causes a rapid reversal of spin orientation relative to the B0, causing coupling to 13C to disappear

Broadband Decoupling 1H channel 13C channel

H3C4-C3H=C2H-C1OOH solvent C-4 C-2 C-3 C-1 10 180

13C Chemical Shifts Reference is TMS, sets 0 ppm A range of 200 ppm Chemical shifts can be predicted Empirical correlations Ex. Alkanes di = -2.3 + 9.1na + 9.4nb – 2.5ng + 0.3nd + 0.1ne + Sij 2-methylbutane di = -2.3 + 9.1*1 + 9.4*2 – 2.5*1 - 1.1 = 22.0 (22.3)

Signal averaging 13C experiment generally take longer than 1H experiments because many more FIDs need to be acquired and averaged to obtain adequate sensitivity. NOE effect (enhancement/reduction in signal as a result of decoupling) N4 N4 13C 1H W2 N2 N2 N3 N3 W1 1H 13C N1 N1

NOE effect W2 (Enhancement) dominates in small molecules Relevant for all decoupling experiments

Other more complex 1D Experiments 1H NOE experiment Inversion Recovery Experiment; Determination of T1 J modulated Spin Echo INEPT Experiment DEPT Experiment

Targeted 1H Spin Decoupling Continuous irradiation at a frequency (n2) that corresponds to a specific proton in the molecule during the 1H NMR experiment All coupling associated with the protons corresponding to n2 disappears from the spectrum

1H targeted decoupling (NOE) n2 channel 1H channel

1 3 2 TMS n2

NOE- nuclear Overhauser effect Saturation of one spin system changes the equilibrium populations of another spin system NOE effect can be positive or negative. In small molecules it is usually positive

Selective Heteronuclear Decoupling Saturate at a specific frequency Multiplets collapse reveal connectivity

More Complex NMR Pulse Sequences J-Modulated Spin Echo experiment Cq and CH2 down and CH3 and CH up DEPT experiment Q= 45, 90, 135 CH3 [DEPT(90)], CH2 [DEPT(45)-DEPT(135)], CH [DEPT(45)+DEPT(135)-0.707DEPT(90)] 2D-NMR Het. 2D J resolved/Homo 2D J resolved 1H-1H COSY 1H/13C HETCOR

J-Modulated Spin Echo 13C channel 90x – t – 180x – t(echo) 1H channel _____________BBBBBBB t = 1/J(C-H) CH + CH3 (up) C(q) + CH2 down

Neuraminic acid

CH and CH3 Cq and CH2

DEPT 13C ch 90x–t –180x–t–FID 1H ch 90x–t–180x–t – fy –t-BBBBBB t = 1/2J(C-H) fy = 90, 45, 135 CH: DEPT(90) CH2: DEPT(45)- DEPT(135) CH3: DEPT(45) + DEPT(135) - 0.707DEPT(90)

DEPT DEPT(90) CH3 DEPT(45) – DEPT(135) CH2 DEPT(45)+DEPT(135)- 13C decoupled spectra

HET 2D J Resolved 13C ch 90x – t – 180y – t - FID 1H ch BBBBBB__________BBBBBBB t = 1/J(C-H) Gives J(H-C) values as a function of 13C chemical shift

Homo 2D J Resolved 1H ch 90x – t – 180x – t - FID t = 1/J(H-H) Gives J(H-H) values as a function of 1H chemical shift

H-H COSY 1H ch 90x – t1 – f- FID (t2) t = 1/J(H-H) Coupled protons give cross correlation peaks off the diagonal

HOOC(1)-C(2)H(NH2)-C(3)H2-C(4)H2-C(5)OOH

HETCOR Plots 13C chemical shifts as a function of 1H chemical shifts of the connected carbon/protons pairs.

F2 (13C NMR decoupled Spectrum) H(3) H(4) H(2) C(2) C(4) C(3)

Practical Aspects to “Running a sample” Deuterated solvent Air drop, sample height Lock-in the deuterated peak (B drift) Shimming the magnet; parallel magnetic field lines for limiting broadening of line width. Setting the parameter; nuclei, spectral range, FID time, number of scans, and apodization, ect.