1. Chem-806 Identification of organic and inorganic compounds by advance NMR techniques Tool box 2D-NMR: Homonuclear 2D-NMR: Heteronuclear 3D-NMR

2. Parameter Consideration ReceptivityReceptivity –Spin Quantum Number –Resonance Frequency –Sensitivity –Natural abundance Frequency ShiftFrequency Shift –Absolute Frequency –Relative Chemical Shift Scale (Referencing) Relaxation – T 1 and T 2Relaxation – T 1 and T 2 –Definition –Mechanisms –Measurement –NOE

3. Multinuclear NMR Z Nuclei Spin I Frequency % natural abundance 1 1 H ½ 100.00 99.98 6 13 C ½ 25.15 1.108 7 14 N 1 7.23 99.935 15 N ½ 10.136 0. 365 15 N ½ 10.136 0. 365 9 19 F ½ 94.103 100 15 31 P ½ 40.43 100 At B o =2.35 Tesla When sampling a nuclei, following parameters shoud be considered: Sensitivity Natural abundance Relaxation time : T 1  recycle time, T 2  acquisition Time Influence of H-decoupler: {NOE and J} 0 = B0B0 

4. Sensitivity and Receptivity The sensitivity of a nuclei depends on: 1.Magnetic Field (   ) 2.Population excess (   ) 3.Magnetic field induced in receiver coil (   ) Sensitivity = k *  x 3 * I x (I x +1) e.g.  ( 13 C)/  ( 1 H) = 1/4  13 C less sensitive than proton (64 less) Receptivity R x = a x * Sensitivity Where a x = natural abundance e.g. Relative receptivity of 1 H and 13 C R = CH a C *  C 3 a H *  H 3 = 0.01 * (25) 3 1 * (100) 3 = 1.6 * 10 -4 R = 0.83 FH

5. T1 considerations Spin lattice relaxation time T 1  “lifetime” of First Order Rate process z y x 90 x z y x MoMoMoMo MyMyMyMy z y x z y x z y x MoMoMoMo 5 T 1 t..t Magnitude of T 1 is highly dependant on : 1.the type of nuclei 2.State of the sample T 1 governs the efficiency of the NMR experiment : recycle time For 1 H in solution T 1 can be 0.01 to 100 sec. For low  nuclei – spin ½ - relaxation can be much longer! Recovery of the magnetization along the Z axis

6. Relaxation (T 1 )

7. Dinitrobenzene: T 1 H2H2H2H2 H 4 /H 6 H5H5H5H518090 

8. Inversion recovery : 13 C 18090  D1= 5T 1  = 0.03 s  = 1.5 s  = 3 s  = 6 s  = 50 s T 1 =  null / ln2 = 1.443 *  null e.g. C2 => T 1 = 4.3 s (  null =3 )

9. Helping relaxation reducing relaxation timeaddition of paramagnetic relaxation reagentChromium III acetylacetonate Cr(acac) 3 One approach of reducing relaxation time is by the addition of paramagnetic relaxation reagent (Chromium III acetylacetonate => Cr(acac) 3 ) 1s delay, 30 o pulse Without Cr(acac) 3 With Cr(acac) 3

10. Intensity vs Pulse Interval MzMzt M(t) = M o (1-e -t/T1 ) PW=90 o D1 AQ NS z y x 90 x z y x MoMoMoMo MyMyMyMy t = pulse interval = D1 + AQ t 1 * T 1 M(t) M(t) 0.63 M 0 2 * T 1 0.86 M 0 3 * T 1 0.95 M 0 4 * T 1 0.98 M 0 5 * T 1 0.99 M 0 10 * T 1 0.99995 M 0

11. Optimum recycle delay (pulse interval) with 90` pulse Total experiment time  fixed During the experiment, t (D1+AQ) is repeated for NS PW=90 o D1 AQ NS t = pulse interval = D1 + AQ tSensitivity.1 T 1.3.2 T 1.41.5 T 1.56.75 T 1.61 1 T 1.63 1.26 T 1.64 1.5 T 1.63 2 T 1.55 Optimum delay

12. Optimum angle with D1 < T 1 PW<90 o D1 AQ NS D1 = 0 t = pulse interval = AQ z y x PW x MoMoMoMo M z y x Optimum angle  Ernst angle  = cos -1 e t/T1 t  T 1 (t=1) 100 T 1 90 o.01 10 T 1 90 o.1 10 T 1 90 o.1 2.5 T 1 86.3 o.4 2.5 T 1 86.3 o.4 1.5 T 1 77.1 o.67 1.5 T 1 77.1 o.67 1. T 1 68.4 o 1 1. T 1 68.4 o 1 0.5 T 1 52.7 o 2 0.5 T 1 52.7 o 2 0.25 T 1 38.8 o 4 0.25 T 1 38.8 o 4 0.1 T 1 25.2 o 10 0.1 T 1 25.2 o 10 0.01 T 1 8.1 o 100 0.01 T 1 8.1 o 100 Short T 1 Long T 1

13. Sensitivity curves for different pulse and different delays and relaxation time

14. Steady State

15. T 2 Consideration

16. T2T2

17. Refocusing of field inhomogeneity

18. Carr-Purcell-Meiboom-Gill

19. CPMG used to get rid of broad signals Polystyrene (50,000) + camphor 90180   = 1.5 ms 

20. SW and Memory size O1 SW spectral windowSWcarrier offsetO1 The spectral window (SW) and the carrier offset (O1) are chosen to match entire spectra (to avoid Fold-over and aliasing) SWDwell time (1/2SW). For a given SW, the time (Dwell time) between 2 data point is defined by the Nyquist theorem (1/2SW). total number of data pointTD Acquisition time AQ(DW*TD) The total number of data point (TD) acquired is related to the Acquisition time AQ (DW*TD) digital resolution depends on the window and on the number of points placed in that window The digital resolution depends on the window and on the number of points placed in that window Digital resolution = 2 * SW/ TD = 1/AQ TD = 2 * SW * AQ Sharp lines have long FID (long T 2 * ), broad peaks have short FID (short T 2 * ), AQ ~ 3 * T 2

21. Nyquist Theorem

22. DIGITALLIZATION Accuracy Speed Digitallization: Convert FID (Volt/Time) in Digital form Digitallization process is limited by: Carrier OffsetTransmitter Offset ReferenceRotating Frame Carrier Offset or Transmitter Offset or “O1” is the frequency of the irradiating field. It is also the “Reference” or “Rotating Frame” frequency WindowSpectral WidthSW The “Window” or “Spectral Width” also called “SW” define the range of frequencies that can be measured r.f.O1SW Dwell TimeDW The Sampling Rate => 2 Points/CycleDwell Time = DW= _1__ 2*SW

23. If Maximum Frequency to be sampled is f max = SW We must sample at a rate no less than 2 * SW sec. Digital Resolution The amount of memory limit the accuracy of the signal to be recorded # of memory# Points -> TD (time domain), For a given # of memory (# Points -> TD (time domain)), one obtain: NP (real)andNP (Imaginary) 2 2 D.R. Digital Resolution = D.R. =  f (Separation between 2 points) D.RSW NP D.R. = 2 * SW NP

24. Digitallization : resolution and Acquisition time

25. Example At 200 MHz At 200 MHzIf: SW=2000 Hz (10 ppm) TD = 16,000 points (16K) What is the Digital Resolution: D.R.0.25 Hz D.R. = 2*SW/TD = 4000 / 16,000 = 1 / 4 = 0.25 Hz Acquisition Time AQ What is the Acquisition Time AQ: AQ4 seconds AQ = TD * DW = TD / (2 * SW) = 4 seconds D.R.AQSWTD D.R. = 1 / AQ = 2 * SW / TD

26. C13-NMR : proton decoupling

27. Heteronuclear nOe positive  13 C For nuclei having positive  : (e.g. 13 C) Decoupling protonhigher signals due to nOe Decoupling proton can produce higher signals due to nOe. Enhancement is dependant on motiondistance between interacting nuclei motion and distance between interacting nuclei quaternary carbons much smaller protonated carbons As a consequence, quaternary carbons are much smaller than protonated carbons

28. Heteronuclear nOe positive Can yield higher positive signal for nuclei with positive  (e.g. 13 C) negative For nuclei with negative  (e.g. 29 Si, 15 N) can yield larger (negative) signals or for partial T 1DD can null the signal!!! e.g. 15 N { 1 H} Without NOE S = A 0 =1 NOE max  HHHH  N ~ -5 S =A 0 +NOE= -4 100% NOE 50% NOE NOE=-2.5 S =A 0 +NOE= -1.5 20% NOE S =A 0 +NOE= 0 NOE=-1

29. Refocusing : echo formation

30. Delay as a sequence building block J AX AA J 2 J 2 - AX Spin system A= 1 H, X= 13 C, J AX For each isochromats the distance they run in the xy frame during a delay t is: Distance = Frequency (cycles/sec) * delay (sec) Distance = 2  * (+/-) J/2 * t xxxx J= 10 Hz  t (1/2J) = 0.05 s J=100 Hz  t (1/2J) = 0.005 s J=140 Hz  t (1/2J) = 0.00357 s Delay Dist. t=0 0 t=1/4J  /4 (45 o ) t=1/2J  /2 (90 o ) t=1/J  (180 o )

31. APT experiment

32. APTPulse Sequence APT Pulse Sequence

33. Multiplet Modulation

34. APT : 8 msec delay

35. APT : 6 msec delay

36. INEPT experiment

37. INEPT

38. Comparing NOE Enhancement and INEPT Enhancement

39. INEPT sequence

40. Multiplet distortion in INEPT: 29 Si

41. Multiplet distortion in coupled INEPT: 13 C

43. 29 Si – INEPT coupled

44. Refocused INEPT

45. Multiplicity Modulation : HX

46. Multiplicity Modulation : XH 2

47. Multiplicity Modulation : XH 3

48. Decoupled INEPT: Menthol

49. Optimum delay in decoupled INEPT

50. Decoupled INEPT: compare normal and INEPT 29 Si

51. DEPT

52. Intensity vs pulse angle

53. Normal 13 C DEPT-45 DEPT-90 DEPT-90 DEPT-135 Menthol in Acetone-d 6

54. DEPT Ipsenol

55. DEPT Editing

56. ADEPT Editing

59. The BIRD pulse

60. Refocusing : echo formation

61. Field Gradient in NMR

62. Gradient Field replace phase Cycling Gradient Field: A Way to Speed up 2D & 3D NMR => replace phase Cycling If gradient g z is applied during t g Each isochromats accumulate phase:  =  *  f * t g n Coherences precess at n *  f Where n is the coherence order

63. GE-COSY: Gradient Enhance COSY

64. Homonuclear 2DJ-NMR

66. Different format in 2D-NMR

67. 2DJ stack plot

68. 2DJ- contour

69. 2DJ- expansion

70. 2DJ-ROTATE

71. 2DJ-rotate

72. NMR processing: apodization Window LB Line Broadning (LB) : Exponential Multiplication improves the signal to noise ratio (at the expense of resolution)

73. Processing : line broadening

75. NMR processing: apodization Window Line broadning : Exponential multiplication improves the signal to noise ratio (at the expense of resolution) Resolution Enhancement reverse exponential + Gaussian function also traf function, also sine function (for 2D – magnitude mode)

76. Processing : resolution enhancement

77. Processing : sine bell (phased mode)

78. Processing : sine bell (magnitude mode)

79. Processing : sine bell (Power mode)

80. Processing : Qsine (phased mode)

81. Processing : Qsine (magnitude mode)