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Principles and selected applications of Diffusion-Ordered NMR Spectroscopy Stéphane Viel, Ph. D. Assistant Professor Aix-Marseille University Molecular.

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Presentation on theme: "Principles and selected applications of Diffusion-Ordered NMR Spectroscopy Stéphane Viel, Ph. D. Assistant Professor Aix-Marseille University Molecular."— Presentation transcript:

1 Principles and selected applications of Diffusion-Ordered NMR Spectroscopy Stéphane Viel, Ph. D. Assistant Professor Aix-Marseille University Molecular Sciences Institute II (UMR-6263) Chemometrics and Spectroscopy Laboratory Marseilles (France)

2 2 DOSY ? Diffusion Ordered NMR Spectroscopy Web of Science, 12 / 2007

3 3 DOSY ? Diffusion Ordered NMR Spectroscopy Web of Science, 12 / 2007

4 4 NMR and Diffusion… PGSE Pulsed Gradient Spin Echo 1965

5 5 NMR and Diffusion… DOSY Diffusion Ordered SpectroscopY 1992

6 6 NMR and Diffusion… DOSY Diffusion Ordered SpectroscopY 1992 PGSE Pulsed Gradient Spin Echo 1965

7 7 General outline Part 1: Theory about molecular mobility –Self-diffusion –Study of self-diffusion by NMR Principles of Pulsed Gradient Spin Echo (PGSE) Diffusion ordered NMR spectroscopy (DOSY) Part 2: Selected applications of DOSY

8 8 Self-diffusion Random translational motion of molecules or ions that arises from the thermal energy under conditions of thermodynamic equilibrium –No thermal gradient (convection) –No concentration gradient (mutual diffusion)

9 9 self-diffusion coefficient root mean square displacement Self-diffusion by Brown, 1828 time = t time = 0 « Random jostling of molecules which leads to their net displacement over time »

10 10 D self-diffusion coefficient k Boltzmann’s constant T absolute temperature f friction factor Self-diffusion coefficient D DD is related to the hydrodynamic volume of the diffusing particle through

11 11 Self-diffusion coefficient D DD is related to the hydrodynamic volume of the diffusing particle through D self-diffusion coefficient k Boltzmann’s constant T absolute temperature f friction factor Sphere 

12 12 For a sphere diffusing in an isotropic and continuous medium of viscosity  :  Stokes Einstein equation Diffusion Molecular Size

13 13 PGSEPGSEPulsed Gradient Spin Echo (PGSE) –Stejskal and Tanner, 1965 –Gradients of magnetic field (Pulsed) Study of self-diffusion by NMR Gradient Pulses OFF ON Time

14 14 1. Spatially label the nuclear spins using gradients of magnetic field. Study of self-diffusion by NMR 2. Monitor their displacement by measuring their spatial positions at 2 distinct times. Principle: 2 steps

15 15 Nuclear magnetogyric ratio Larmor frequency In NMR, each nuclear spin is identified by its Larmor precession frequency  0 B0B0

16 16 Magnetic field gradient Magnetic field gradient Spatially dependent magnetic field For a single and constant gradient oriented along the z direction

17 17 Magnetic field gradient Magnetic field gradient Spatially dependent magnetic field For a single and constant gradient oriented along the z direction Notion of effective gradient Coherence order

18 18 Phase shift of nuclear spins Assume that the magnetic field gradient is active during a time  A nuclear spin acquires a phase shift Static Field

19 19 Assume that the magnetic field gradient is active during a time  Phase shift of nuclear spins A nuclear spin acquires a phase shift Gradient

20 20 Phase shift of nuclear spins Assume that the magnetic field gradient is active during a time  A nuclear spin acquires a phase shift The spatial position of the nuclear spins is encoded into a phase shift Nuclear spin spatial labelling

21 21 Rotating frame In NMR, a common simplification consists in describing the evolution of the magnetization in a frame rotating at the Larmor frequency  0 For nuclear spins on resonance, the phase shift reduces to

22 22 Spin Echo or Hahn Echo (SE) Without magnetic field gradients Echo Signal

23 23 codingdecoding Spin Echo or Hahn Echo (SE) With magnetic field gradients

24 24 Spin Echo or Hahn Echo (SE) With magnetic field gradients Echo

25 25 Spin Echo or Hahn Echo (SE) With magnetic field gradients p = 1p = - 1

26 26 Spin Echo or Hahn Echo (SE) With magnetic field gradients Echo

27 27 Spin Echo or Hahn Echo (SE) With magnetic field gradients Attenuation factor 

28 28 I echo : Intensity at the echo with gradients I 0 : Intensity at the echo without gradients D: Self-diffusion coefficient  : gradient pulse duration  : Diffusion time q: gradient pulse area Attenuation factor 

29 29 How do we actually obtain D? Attenuation factor 

30 30 How do we actually obtain D? FITFIT D Attenuation factor 

31 31 codingdecoding Stimulated Echo (STE) With magnetic field gradients

32 32 BPP-STE-LED sequence Stimulated Echo (STE) with Bipolar gradient (BPP) pulses and longitudinal eddy current delay (LED)

33 33 The BPP-STE-LED sequence Stimulated Echo (STE): –T1 relaxation vs. T2 relaxation –No artefacts due to J modulation Bipolar gradient pulses (BPP): –Reduced eddy currents Longitudinal Eddy currents Delay (LED): –Less spectral distortions due to eddy currents

34 34 Stimulated Echo (STE) with Bipolar gradient (BPP) pulses and longitudinal eddy current delay (LED) The BPP-STE-LED sequence

35 35 The BPP-STE-LED sequence Stimulated Echo (STE): –T1 relaxation vs. T2 relaxation –No artefacts due to J modulation Bipolar gradient pulses (BPP): –Reduced eddy currents Longitudinal Eddy currents Delay (LED): –Less spectral distortions due to eddy currents

36 36 Stimulated Echo (STE) with Bipolar gradient (BPP) pulses and longitudinal eddy current delay (LED) The BPP-STE-LED sequence

37 37 The BPP-STE-LED sequence Stimulated Echo (STE): –T1 relaxation vs. T2 relaxation –No artefacts due to J modulation Bipolar gradient pulses (BPP): –Reduced eddy currents Longitudinal Eddy currents Delay (LED): –Less spectral distortions due to eddy currents

38 38 Stimulated Echo (STE) with Bipolar gradient (BPP) pulses and longitudinal eddy current delay (LED) The BPP-STE-LED sequence Echo Signal

39 39 Stimulated Echo (STE) with Bipolar gradient (BPP) pulses and longitudinal eddy current delay (LED) Séquence BPP-STE-LED

40 40 How can we use PGSE data? A B C NMR spectrum (frequency scale, ppm) D A > D C > D B DADA DADA DBDB DCDC DCDC ppm S I Z E

41 41 NMR spectrum (ppm scale) A BC D A > D C > D B DADA DADA DBDB DCDC DCDC A A B C ppmC SIZESIZE James & McDonald, 1978 Stilbs & Moseley, S I Z E

42 42 Size Resolved Spectrometry NMR spectrum (ppm scale) A BC D A > D C > D B B C C A A ppm Stilbs, 1981 S I Z E

43 43 ppm DADA DCDC DBDB D High Low A B C

44 44 ppm A A B C C DADA DCDC DBDB DOSY D A B C High Low

45 45 DOSYD iffusion O rdered NMR S pectroscop Y –Morris & Johnson, 1992 DOSY Antalek, B. Concepts in Magn. Reson 2002, 14,

46 46 DOSY Many processings available: - MaxEnt (Delsuc, M. –A.) - DECRA (Antalek, B.) - CORE (Stilbs, P.) - MCR (van Gorkom, L. C. M.) - MULVADO (Huo, R.) - iRRT (Mandelstham, V.) DOSYD iffusion O rdered NMR S pectroscop Y –Morris & Johnson, 1992 –Signal processing

47 47 DOSY Many processings available: - MaxEnt (Delsuc, M. –A.) - DECRA (Antalek, B.) - CORE (Stilbs, P.) - MCR (van Gorkom, L. C. M.) - MULVADO (Huo, R.) - iRRT (Mandelstham, V.) DOSYD iffusion O rdered NMR S pectroscop Y –Morris & Johnson, 1992 –Signal processing

48 48 DOSY map Adapted from Nilsson et al.

49 49 Distortions due to spectral overlap Adapted from Nilsson et al.

50 50 iRRT inverse Regularized Resolvent Transform Mixture of 2 isomers V. Mandelshtam A. J. Shaka Thureau, P.; Thévand, A.; Ancian, B.; Escavabaja, P.; Armstrong, G. S.; Mandelshtam, V. A., ChemPhysChem 2005, 6, 1 Armstrong, G. S.; Loening, N. M.; Curtis, J. E.; Shaka, A. J.; Mandelshtam, V. A., J. Magn. Reson. 2003, 163, 139

51 51 Part 1: Theory about molecular mobility –Self-diffusion –Study of self-diffusion by NMR Principles of Pulsed Gradient Spin Echo (PGSE) Diffusion ordered NMR spectroscopy (DOSY) Part 2: Selected applications of DOSY General outline

52 52 Chiral recognition Chiral recognition of dipeptides in a biomembrane model C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

53 53 Introduction The organization of biomembranes is based on molecular recognition phenomena (chiral recognition) To investigate the non covalent interactions involved in such systems, models are used C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126, we used Sodium N-doceanoyl-L-prolinate (SDP) Here Z E

54 54 Introduction (2) We studied by NMR the chiral recognition in SDP micelles of 2 dipeptides Ditryptophan (1) NMR techniques: 1 H, PGSE, ROESY +Molecular mechanic calculations Diphenylalanine (2) C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

55 55 1 H experiments: LL/DD couple Ditryptophan (1) +SDP micelles Diphenylalanine (2) +SDP micelles C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

56 56 1 H experiments: LD/DL couple Ditryptophan (1) +SDP micelles Diphenylalanine (2) +SDP micelles C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

57 57 PGSE experiments Monitor the D values of the dipeptides by PGSE experiments 2-site model: dipeptide in equilibrium between the bound (b) and free (f) phase SDP D + D Free State Bound State C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

58 58 PGSE experiments Monitor the D values of the dipeptides by PGSE experiments 2-site model: dipeptide in equilibrium between the bound (b) and free (f) phase SDP D + D C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

59 59 PGSE experiments Determine the partition coefficient of the dipeptides in the 2 phases C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

60 60 PGSE experiments Bound molar fractions x b and partition coefficients p 82 ± ± LL-2 82 ± ± DD ± ± DL ± ± LD ± ± LL ± ± DD ± ± DL ± ± LD-1 pXbXb Dipeptide: C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

61 61 82 ± ± LL-2 82 ± ± DD ± ± DL ± ± LD ± ± LL ± ± DD ± ± DL ± ± LD-1 pXbXb Dipeptide: PGSE experiments Bound molar fractions x b and partition coefficients p C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

62 62 82 ± ± LL-2 82 ± ± DD ± ± DL ± ± LD ± ± LL ± ± DD ± ± DL ± ± LD-1 pXbXb Dipeptide: PGSE experiments Bound molar fractions x b and partition coefficients p C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

63 63 Conformations of 1 isomers by NMR and Molecular mechanic calculations (1) Buffer 1 DL-1 + Buffer 1 LL-1 + Buffer C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

64 64 Conformations of 1 isomers by NMR and Molecular mechanic calculations (2) SDP micelles (LL/DD couple) 1 SDP LL-1 + SDP micelles 1 SDP DD-1 + SDP micelles C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

65 65 Conformations of 1 isomers by NMR and Molecular mechanic calculations (3) SDP micelles (DL/LD couple) 1 SDP DL-1 + SDP micelles 1 SDP LD-1 + SDP micelles C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

66 66 Binding modes of 1 isomers to SDP micelles LL/DD couple C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

67 67 Binding modes of 1 isomers to SDP micelles LD/DL couple C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc. 2004, 126,

68 68 Chemical exchange Determining chemical exchange rates in nucleobases P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006,

69 69 Hydrogen bonding in nucleic acids P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, DNA RNA Thymine –Adenine Uracil –

70 70 Effect of chemical exchange in DOSY Uridine P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, H2OH2O

71 71 Model Simple 2-site exchange P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, N-H +H 2 O HOH+N-H T = 50 msT = 200 ms T= 900 ms

72 72 Model Simple 2-site exchange P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, N-H +H 2 O HOH+N-H T = 50 msT = 200 ms T= 900 ms

73 73 Model Simple 2-site exchange P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, N-H +H 2 O HOH+N-H T = 50 msT = 200 ms T= 900 ms

74 74 Uracil exchange constants K a Simple 2-site exchange P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, N-H +H 2 O HOH+N-H H 1 k a = 8 s -1 H 3 k a = 18 s -1

75 75 Thymine exchange constants K a Simple 2-site exchange P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, N-H +H 2 O HOH+N-H H 1 k a = 5 s -1 H 3 k a = 7 s -1

76 76Self-aggregation Investigations of  complexes in solution S. Viel, L. Mannina, A. L. Segre Tetrahedron Lett. 2002, 43, C. Sanna, C. La Mesa, L. Mannina, P. Stano, S. Viel, A. L. Segre Langmuir 2006, 22,

77 77  stacking interactions are important in organic chemistry and for biological systems Here we consider 2 types of organic molecules bearing an aromatic ring and characterized by a: Introduction - low molecular weight (< 400 Da) Studied by: - NMR ( 1 H, PGSE, NOESY) - DLS - Physicochemical measurements - low H 2 O solubility S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

78 78 Molecules under study Class A Class B CH 3 H1D HHOCH 3 1C HHCH 3 1B HHH1AZYXName CH 2 CH 2 OCH 2 CH 2 CH 3 CH 2 CH 3 PRET CH 2 OCH 2 CH 3 CH 3 ACET CH(CH 3 )CH 2 OCH 3 CH 3 METOYXName S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

79 79 Monomeric resonances METOACETPRET 1 H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc < sol) 1 H experiments S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

80 80 METOACETPRET 1 H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc > sol) 1 H experiments Monomeric resonances Extra resonances S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

81 81 1 H experiments METOACETPRET 1 H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc > sol) Well resolved Upfield shifted S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

82 82 PGSE experiments (DOSY display) ACET PGSE on a dilute aqueous solution of ACET Much lower diffusion coefficient S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

83 83 PGSE experiments (nm)( m 2 s -1 )(mM)  PRET  METO  ACET Aggregate  PRET  METO  ACET Monomer RHRH D NMR Conc Hydrodynamic radii (Stokes Einstein, Sphere) S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

84 84 NOESY experiments ACET NOESY spectrum of a dilute aqueous solution of ACET 400 ms Color of cross peaks: Blue : Negative Green/Yellow : Positive S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

85 85 NOESY experiments ACET NOESY spectrum of a dilute aqueous solution of ACET 400 ms Color of cross peaks: Blue : Negative cross-peak Green/Yellow : Positive cross- peak Spin Diffusion S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

86 86 NOESY experiments ACET NOESY spectrum of a dilute aqueous solution of ACET 10 ms Color of cross peaks: Blue : Negative Green/Yellow : Positive S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

87 87 DLS experiments Hydrodynamic radii Hydrodynamic radii of the aggregates were also estimated by DLS (nm) ( m 2 s -1 ) (mM)  ACET  PRET  METO Aggregate RHRH DConc METO PRET ACET S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

88 88 Physico-chemical properties Surface Tension Activity Coeff Osmotic Coeff Rel. viscosity S. Viel et al. Tetrahedron Lett. 2002, 43, C. Sanna et al. Langmuir 2006, 22,

89 89 Diffusion-Ordered NMR Spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharides Molecular weight S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4,

90 90 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Introduction Polysaccharides constitute a major class of biomacromolecules and play key roles in biological recognition processes. Their structural elucidation relies mainly on NMR, but a complete characterization may also require the molecular weight (MW). Available techniques: Photonic Correlation Spectroscopy, Gel Permeation Chromatography Drawbacks: sample manipulation

91 91 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Diffusion and Mass Strictly, diffusion relates to molecular size. A calibration is hence required to establish the relationship between diffusion coefficient and molecular weight Pullulan (linear polysaccharide) 6 fractions (kDa): 5.8; 12; 28.3; 100; 180 and 853 Studied by PGSE experiments

92 92 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Diffusion and Mass 853 kDa5.8 kDa100 kDa

93 93 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Determination of Molecular Weight: Pullulan as a Model Sample MW (Da) D (m 2 /s)

94 94 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2002, 4, Determination of Molecular Weight: Calibration curve D (m 2 /s) MW (Da)

95 95 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2002, 4, Determination of Molecular Weight: Check with another polysaccharide D (m 2 /s) MW (Da)

96 96 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Determination of Molecular Weight: Check with oligosaccharides D (m 2 /s) MW (Da)

97 97 S. Viel, D. Capitani, L. Mannina, A. L. Segre Biomacromolecules 2003, 4, Determination of Molecular Weight: Check with saccharides D (m 2 /s) MW (Da)

98 98 Use of Pulsed Field Gradient Spin-Echo NMR as a tool in MALDI method development for polymer Mw determination Molecular Weight M. Mazarin, S. Viel, B. Allard-Breton, A. Thévand, L. Charles Anal. Chem. 2006, 78,

99 99Polymers M. Mazarin, S. Viel, B. Allard-Breton, A. Thévand, L. Charles Anal. Chem. 2006, 78, pMAM

100 D=f[PS] D 0 PS =f(Mw) Polymers PS CDCl 3 D = k Mw -a

101 101 PS : Comparison M w : SEC, NMR and MS

102 102 Analysis of mixtures (part I) Improved 3D DOSY-TOCSY experiment for mixture analysis S. Viel, S. Caldarelli Chem. Comm. 2008, in press

103 103 S. Viel, S. Caldarelli Chem. Comm. 2008, in press Introduction Overlapping signals severely complicate DOSY analysis A typical solution is the addition of another frequency dimension to spread the signals out Drawback: time consuming experiments due to the requirement of sampling the indirect frequency dimension

104 104 Speeding up 3D NMR experiments Various methodologies have been proposed to speed up 3D NMR experiments (FDM) S. Viel, S. Caldarelli Chem. Comm. 2008, in press

105 105 Speeding up 3D NMR experiments Various methodologies have been proposed to speed up 3D NMR experiments (FDM) One possibility is Hadamard (there are other ones S. Viel, S. Caldarelli Chem. Comm. 2008, in press ………...….….3D iRRT would be great!)

106 106 Speeding up 3D NMR experiments Various methodologies have been proposed to speed up 3D NMR experiments One possibility is Hadamard (there are other ones S. Viel, S. Caldarelli Chem. Comm. 2008, in press ………...….….3D iRRT would be great!) In Hadamard NMR spectroscopy, the evolution time in the indirect dimension of the 2D block is replaced by phase-encoded multisite selective excitation

107 107 Hadamard encoding S. Viel, S. Caldarelli Chem. Comm. 2008, in press Hadamard family matrices H Matrix dimension N: N = 2 k (k = 1, 2, 3…) +––+ ––++ –+– Pulse 1 Pulse 2 Pulse 3 Pulse 4 A B CD – HH HH M chemical sites –+ ++

108 108 Hadamard encoding S. Viel, S. Caldarelli Chem. Comm. 2008, in press Hadamard family matrices H Matrix dimension N: N = 2 k (k = 1, 2, 3…) +––+ ––++ –+– Pulse 1 Pulse 2 Pulse 3 Pulse 4 A B CD M chemical sites – HH HH –+ ++

109 109 Hadamard encoding S. Viel, S. Caldarelli Chem. Comm. 2008, in press Hadamard family matrices H Matrix dimension N: N = 2 k (k = 1, 2, 3…) +––+ ––++ –+– Pulse 1 Pulse 2 Pulse 3 Pulse 4 A B CD M chemical sites – HH HH –+ ++

110 110 Hadamard encoding S. Viel, S. Caldarelli Chem. Comm. 2008, in press Hadamard family matrices H Matrix dimension N: N = 2 k (k = 1, 2, 3…) +––+ ––++ –+– Pulse 1 Pulse 2 Pulse 3 Pulse 4 A B CD Signal B = + 1 – – 4 M chemical sites – HH HH –+ ++

111 111 Proposed pulse sequence S. Viel, S. Caldarelli Chem. Comm. 2008, in press Thrippleton, M. J.; Keeler, J., Angew. Chem. Int. Ed. 2003, 42, Cano, K. E.; Thrippleton, M.; Keeler, J.; Shaka, A. J., J. Magn. Reson. 2004, 167, ZQC filters

112 112 Proof of principle (1) S. Viel, S. Caldarelli Chem. Comm. 2008, in press TOCSY spectrum of a mixture of: - Methanol (M) - Ethanol (E) - Propanol (P) - Valine (V)

113 113 Proof of principle (2) S. Viel, S. Caldarelli Chem. Comm. 2008, in press M P E V

114 114 Effect of signal overlapping S. Viel, S. Caldarelli Chem. Comm. 2008, in press Propanol 2-Butanol

115 115 Effect of signal overlapping (2) S. Viel, S. Caldarelli Chem. Comm. 2008, in press Time saving factor:  64

116 116 Analysis of mixtures (part II) Enhanced diffusion-edited NMR spectroscopy of mixtures using chromatographic stationary phases S. Viel, F. Ziarelli, S. Caldarelli Proc. Natl. Acad. Sci. U. S. A. 2003, 100,

117 117 Can we selectively slow down the diffusion of some components of the mixture? S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Introduction PGSE experiments allow compounds to be discriminated according to differences in their effective size (mixture analysis) Corollary: similar sized compounds CANNOT be resolved by PGSE Chromatographic phases

118 118 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Principle A chromatographic phase interacts selectively with some of the mixture components (for instance: polarity/apolarity) Discrimination is achieved according to apparent diffusion rates (instead of free self-diffusion coefficients)

119 119 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Problem: spectral resolution! 1 H of Sol. + Stationary phase Conventional NMR HR High Resolution MAS Magic Angle Spinning: solid state technique

120 120 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Problem: spectral resolution! 1 H of Sol. + Stationary phase Conventional NMR HR High Resolution MAS Magic Angle Spinning: solid state technique HRMAS NMR

121 121 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, HRMAS HRMAS rotor HRMAS probe

122 122 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Example 1 Mixture 1: - Dichlorophenol - Ethanol - Heptane

123 123 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Example 1 Mixture 1: - Dichlorophenol - Ethanol - Heptane + SiO 2

124 124 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Example 2 Mixture 2: - Naphtalene - Dec-1-ene - Ethanol

125 125 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Example 2 Mixture 2: - Naphtalene - Dec-1-ene - Ethanol + C18

126 126 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Research directions Improve resolution of complex mixtures Characterize new chromatographic phases Investigate chromatographic phenomenon Discriminate stereoisomers

127 127 PFG MAS diffusion measurements Pulsed field gradient magic angle spinning NMR self-diffusion measurements in liquids S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190,

128 128 Gradients and MAS probes S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Courtesy of Bruker Instruments

129 129 Magic gradient S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Courtesy of Bruker Instruments

130 130 S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Courtesy of Bruker Instruments Stator Gradients Magic gradient

131 131 S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Gradient calibration: Profile Hahn echo on a H 2 O/D 2 O sample with gradient during acquisition Adapted from Hurd et al.

132 132 S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Gradient calibration: Profile 6% 95%

133 133 Gradient calibration: strength S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Rotor:

134 134 S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Rotor: V = 50  L V = 12  L G = 6.0 G cm -1 A -1 Gradient calibration: strength

135 135 Effect of spinning S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Water ACN 12  L

136 136 Effect of spinning S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Water ACN 50  L

137 137 Results: ACN 4 kHz S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190,  L12  L

138 138 S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, Results

139 139 Results S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli J. Magn. Reson. 2008, 190, PEO 116kDa D 2 O 4 kHzPEO 116kDa CDCl 3 3 kHz

140 140 S. Viel, F. Ziarelli, S. Caldarelli Proceedings of the National Academy of Sciences of the United States of America 2003, 100, Research directions Improve resolution of complex mixtures Characterize new chromatographic phases Investigate chromatographic phenomenon Discriminate stereoisomers

141 141 HPLC PFG MAS ODS phaseSilica gel G. Pagès et al. Anal. Chem. 2006, 78, G. Pagès et al. Angew. Chem. Int. Ed. 2006, 45, Mixture of: - Benzene - Naphthalene - Anthracene (ACN/H 2 O, 90/10)

142 142 Merci

143 143 Grazie

144 144 Thank you ! A B C

145 145 A B C


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