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FT-NMR

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 .

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FT-NMR

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  1. FT-NMR

  2. 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

  3. (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

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

  5. 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

  6. 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

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

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

  9. 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

  10. FID

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Broadband Decoupling 1H channel 13C channel

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

  19. 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)

  20. 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 W2 1H N2 N2 N3 N3 W1 1H 13C N1 N1

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

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

  23. 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

  24. 1H targeted decoupling (NOE) n2channel 1H channel

  25. 1 3 2 TMS n2

  26. 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

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

  28. 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

  29. 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

  30. Neuraminic acid

  31. CH and CH3 Cq and CH2

  32. 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)

  33. DEPT DEPT(90) CH3 DEPT(45) – DEPT(135) CH2 DEPT(45)+DEPT(135)- 0.707DEPT(90) CH 13C decoupled spectra

  34. 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

  35. 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

  36. 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

  37. HOOC(1)-C(2)H(NH2)-C(3)H2-C(4)H2-C(5)OOH CH2(3) CH2(4) CH(2) CH2(3) CH2(4) CH(2)

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

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

  40. 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.

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