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

Principles and selected applications of Principles and selected applications of Diffusion-Ordered NMR SpectroscopyDiffusion-Ordered NMR Spectroscopy

Stéphane Viel, Ph. D.Stéphane Viel, Ph. D.Assistant ProfessorAssistant Professor

Aix-Marseille UniversityAix-Marseille UniversityMolecular Sciences Institute II (UMR-6263)Molecular Sciences Institute II (UMR-6263)

Chemometrics and Spectroscopy LaboratoryChemometrics and Spectroscopy LaboratoryMarseilles (France)Marseilles (France)

2Year

1992 1996 2000 2004 2008

Nu

mb

er o

f p

ub

licat

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10

20

30

40

50

60

DOSY ?

Diffusion Ordered NMR Spectroscopy

Web of Science, 12 / 2007

3Year

1992 1996 2000 2004 2008

Nu

mb

er o

f p

ub

licat

ion

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10

20

30

40

50

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DOSY ?

Diffusion Ordered NMR Spectroscopy

Web of Science, 12 / 2007

4

NMR and Diffusion…NMR and Diffusion…

19501950

19541954

PGSEPulsed Gradient Spin Echo19651965

5


19811981

19871987

DOSYDiffusion Ordered SpectroscopY

19921992

6


DOSYDiffusion Ordered SpectroscopY

19921992

PGSEPulsed Gradient Spin Echo19651965

7

General outlineGeneral 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

Self-diffusionSelf-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

self-diffusion coefficient

root mean square displacement

Self-diffusion by Brown, 1828Self-diffusion by Brown, 1828

time = time = tt

stime = 0time = 0

• « Random jostling of molecules which leads to their net displacement over time »

tDns 2

10

•D self-diffusion coefficient

•k Boltzmann’s constant

•T absolute temperature

•f friction factorf

kTD

Self-diffusion coefficient Self-diffusion coefficient DD

• DD is related to the hydrodynamic volume of the diffusing particle through

11af 6

Self-diffusion coefficient Self-diffusion coefficient DD

• 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

f

kTD

12

• For a sphere diffusing in an isotropic and continuous medium of viscosity :

Stokes Einstein equationStokes Einstein equation

a

kTD

6

Diffusion Molecular Size

13

• PPulsed GGradient SSpin EEcho (PGSEPGSE)– Stejskal and Tanner, 1965– Gradients of magnetic field (Pulsed)

Study of self-diffusion by NMRStudy of self-diffusion by NMR

GradientPulses

OFF OFF OFF OFF

ON ON ON

Time

14

1. Spatially label the nuclear spins using

gradients of magnetic field.

Study of self-diffusion by NMRStudy of self-diffusion by NMR

2. Monitor their displacement by measuring

their spatial positions at 2 distinct times.

Principle: 2 steps

15

Nuclear magnetogyric ratio

Larmor frequencyLarmor frequency

In NMR, each nuclear spin is identified by its Larmor precession frequency 0

B0 00 B

16

zegg

Magnetic field gradientMagnetic field gradient

Magnetic field gradient

Spatially dependentmagnetic field

For a single and constant gradient oriented along the z direction

17

ze egg

Magnetic field gradientMagnetic field gradient

Magnetic field gradient

Spatially dependentmagnetic field

For a single and constant gradient oriented along the z direction

Notion of effective gradient

Coherenceorder

gpge

18

Phase shift of nuclear spinsPhase shift of nuclear spins• Assume that the magnetic fieldgradient is active during a time

• A nuclear spin acquires a phase shift

)()( 0 zgBz e

Static Field

19

• Assume that the magnetic fieldgradient is active during a time

Phase shift of nuclear spinsPhase shift of nuclear spins


)()( 0 zgBz e

Gradient

20

Phase shift of nuclear spinsPhase shift of nuclear spins• Assume that the magnetic fieldgradient is active during a time


The spatial position of the nuclear spins is encoded into a phase shift

Nuclear spin spatial labellingNuclear spin spatial labelling

)()( 0 zgBz e

21

zgz e)(

Rotating frameRotating 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

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

Echo

Signal

23coding decoding

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

24)( 1z )( 2z


Echo

252)( zgp 1)( zgp


p = 1 p = - 1

26 )( 21 zzg


Echo

27

Spin Echo or Hahn Echo (SE)Spin Echo or Hahn Echo (SE)

)( 21 zzg

With magnetic field gradients

Attenuation factor

28

3exp 2

0

qDI

Iecho

• Iecho: Intensity at the echo with gradients

• I0: Intensity at the echo without gradients

• D: Self-diffusion coefficient

• : gradient pulse duration

• : Diffusion time

• q: gradient pulse area

Attenuation factor Attenuation factor

gq

29Gradient strength g (G.cm-1)

0 10 20 30

No

rma

lize

d in

ten

sity

0,0

0,2

0,4

0,6

0,8

1,0

How do we actually obtain How do we actually obtain DD? ? A

ttenu

atio

n fa

ctor

3exp 2 gD

30

Gradient strength g (G.cm-1)

0 10 20 30

No

rma

lize

d in

ten

sity

0,0

0,2

0,4

0,6

0,8

1,0

How do we actually obtain How do we actually obtain DD? ?

FIT

D

Atte

nuat

ion

fact

or

3exp 2 gD

31coding decoding

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

32

BPP-STE-LED sequenceBPP-STE-LED sequence

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

33

The BPP-STE-LED sequenceThe 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



35





36



37





38



Echo Signal

39


SéquenceSéquenceBPP-STE-LEDBPP-STE-LED

40

How can we use PGSE data?How can we use PGSE data?

A B C

NMR spectrum (frequency scale, ppm)

DA > DC > DB

DADA

DB

DC

DC

ppm

S I Z ES I Z E

41

NMR spectrum (ppm scale)

A B C DA > DC > DB

DADA

DB

DC

DC

A AB Cppm C

SIZE

James & McDonald, 1978Stilbs & Moseley, 1978-80

S I Z ES I Z E

42

Size Resolved SpectrometrySize Resolved Spectrometry

NMR spectrum (ppm scale)

A B C DA > DC > DB

B CCA Appm

Stilbs, 1981

S I Z ES I Z E

43

ppm

DA

DC

DB

D

High

Low

AB

C

44

ppm A AB C C

DA

DC

DB

DOSY

D

AB

C

High

Low

45

• DDiffusion OOrdered NMR SSpectroscopYY– Morris & Johnson, 1992

DOSYDOSY

Antalek, B. Concepts in Magn. Reson 2002, 14, 225-258

46

DOSYDOSY

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.)

• DDiffusion OOrdered NMR SSpectroscopYY– Morris & Johnson, 1992– Signal processing

47

DOSYDOSY

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.)

• DDiffusion OOrdered NMR SSpectroscopYY– Morris & Johnson, 1992– Signal processing

48

DOSY mapDOSY map

Adapted from Nilsson et al.

49

Distortions due to spectral overlapDistortions due to spectral overlap

Adapted from Nilsson et al.

50

iRRTinverseRegularized ResolventTransform

Mixture of 2 isomers

V. MandelshtamA. 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

• 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 outlineGeneral outline

52

Chiral recognitionChiral 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, 13354-13362

53

IntroductionIntroduction• 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. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

we used Sodium N-doceanoyl-L-prolinate (SDP)

Here N

O

CO2

H

Na+-

C11H23

N

CO2

H

C11H23

O

Na+-

Z E

54

Introduction (2)Introduction (2)• We studied by NMR the chiral recognition

in SDP micelles of 2 dipeptides

Ditryptophan (1)-

N

N

N

O

H

CO2

NH3

H

H

+2

3a

45

6

77a

2'

3'a

4'5'

6'

7'7'a

''

NMR techniques: 1H, PGSE, ROESY+Molecular mechanic calculations

-

+N

O

H

CO2

NH3

Diphenylalanine (2)


55

11H experiments: H experiments: LLLL//DDDD couple couple

Ditryptophan (1)+SDP micelles

Diphenylalanine (2)

+SDP micelles


56

11H experiments: H experiments: LDLD//DLDL couple couple

Ditryptophan (1)

+SDP micelles

Diphenylalanine (2)

+SDP micelles


57

PGSE experimentsPGSE 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

SDPD + D

FreeState

BoundState


58


Monitor the D values of the dipeptides by PGSE experiments

2-site model: dipeptide in equilibrium between the bound (b) and free (f) phase

SDPD + D

fbbbobs DxDxD )1( C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

59


• Determine the partition coefficient of the dipeptides in the 2 phases

aqueous

micellar

DP

DPp

micellar

aqueous

b

b

V

V

x

xp

1C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

60

PGSE experimentsPGSE experimentsBound molar fractions xb and partition coefficients p

82 ± 120.69 ± 0.03LL-22

82 ± 120.69 ± 0.03DD-22

138 ± 250.79 ± 0.03DL-22

138 ± 250.79 ± 0.03LD-22

931 ± 1000.962 ± 0.004LL-11

860 ± 870.959 ± 0.004DD-11

1900 ± 4070.981 ± 0.004DL-11

1900 ± 4070.981 ± 0.004LD-11

pXbDipeptide:


61

82 ± 120.69 ± 0.03LL-22

82 ± 120.69 ± 0.03DD-22

138 ± 250.79 ± 0.03DL-22

138 ± 250.79 ± 0.03LD-22

931 ± 1000.962 ± 0.004LL-11

860 ± 870.959 ± 0.004DD-11

1900 ± 4070.981 ± 0.004DL-11

1900 ± 4070.981 ± 0.004LD-11

pXbDipeptide:



62

82 ± 120.69 ± 0.03LL-22

82 ± 120.69 ± 0.03DD-22

138 ± 250.79 ± 0.03DL-22

138 ± 250.79 ± 0.03LD-22

931 ± 1000.962 ± 0.004LL-11

860 ± 870.959 ± 0.004DD-11

1900 ± 4070.981 ± 0.004DL-11

1900 ± 4070.981 ± 0.004LD-11

pXbDipeptide:



63

Conformations of Conformations of 11 isomers by NMR and isomers by NMR and Molecular mechanic calculations (1)Molecular mechanic calculations (1)

Buffer

DL-1 1 + BufferLL-1 1 + Buffer


64


SDP micelles (LL/DD couple)

LL-1 1 + SDPSDP micelles DD-1 1 + SDPSDP micelles


65


SDP micelles (DL/LD couple)

DL-1 1 + SDPSDP micelles LD-1 1 + SDPSDP micelles


66

Binding modes of Binding modes of 11 isomers isomers to SDP micellesto SDP micelles

LL/DD couple


67

Binding modes of Binding modes of 11 isomers isomers to SDP micellesto SDP micelles

LD/DL couple


68

Chemical exchangeChemical exchange

Determining chemical exchange rates in nucleobases

P. Thureau, B. Ancian, S. Viel, A. ThévandChem. Comm. 2006, 200-202

P. Thureau, B. Ancian, S. Viel, A. ThévandChem. Comm. 2006, 1884-1886

69

Hydrogen bonding in nucleic acidsHydrogen bonding in nucleic acids

P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thévand Chem. Comm. 2006, 1884-1886

DNA

RNA

Thymine – Adenine

AdenineUracil –

70

Effect of chemical exchange in DOSYEffect of chemical exchange in DOSY

Uridine


O

OO

NN

OO

O

H

H

H H

H2O

71

ModelModel

Simple 2-site exchange


N-H +H2O HOH+N-H

T = 50 ms T = 200 ms T= 900 ms

72

ModelModel



N-H +H2O HOH+N-H

T = 50 ms T = 200 ms T= 900 ms

73

ModelModel



N-H +H2O HOH+N-H

T = 50 ms T = 200 ms T= 900 ms

74

Uracil exchange constants KUracil exchange constants Kaa



N-H +H2O HOH+N-H

H1 ka= 8 s-1

H3 ka= 18 s-1

75

Thymine exchange constants KThymine exchange constants Kaa



N-H +H2O HOH+N-H

H1 ka= 5 s-1

H3 ka= 7 s-1

76

Self-aggregationSelf-aggregation

Investigations of complexes in solution

S. Viel, L. Mannina, A. L. SegreTetrahedron Lett. 2002, 43, 2515-2519

C. Sanna, C. La Mesa, L. Mannina, P. Stano, S. Viel, A. L. SegreLangmuir 2006, 22, 6021-6031

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:

IntroductionIntroduction

- low molecular weight (< 400 Da)

Studied by: - NMR (1H, PGSE, NOESY)- DLS- Physicochemical measurements

- low H2O solubility

S. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

78

Molecules under studyMolecules under studyClass A

Class B

CH3CH3H1DHHOCH31CHHCH31BHHH1AZZYYXXNameName

CH2CH2OCH2CH2CH3CH2CH3PRETCH2OCH2CH3CH3ACET

CH(CH3)CH2OCH3CH3METOYYXXNameName

X

N

Y

O

Y

X

H

O

O

NO2

z


79

Monomeric resonances

1H spectra of dilute aqueous solutions of METOMETO, ACETACET and PRETPRET, (Conc < sol)

11H experimentsH experiments

6.97.07.17.27.37.4 ppm 0.91.01.11.2 ppm

a b


80

1H spectra of dilute aqueous solutions of METOMETO, ACETACET and PRETPRET, (Conc > sol)

11H experimentsH experiments

6.97.07.17.27.37.4 ppm 0.91.01.11.2 ppm

a b

Monomeric resonances

Extra resonances


81

11H experimentsH experiments1H spectra of dilute aqueous solutions of METOMETO, ACETACET and PRETPRET, (Conc > sol)

6.97.07.17.27.37.4 ppm 0.91.01.11.2 ppm

a b•Well resolved

•Upfield shifted


82

ppm

7 6 5 4 3 2 1 ppm

-9.0

-9.5

-10.0

-10.5

Aggregate

Monomer

Log D

(m s )2 -1

PGSE experiments (DOSY display)PGSE experiments (DOSY display)

PGSE on a dilute aqueous solution of ACETACET

Much lower diffusion coefficient


83


(nm)(10-11 m2 s-1)(mM)

101.943PRETPRET 151.346METOMETO 111.750ACETACET

Aggregate

0.45043PRETPRET 0.45146METOMETO 0.45350ACETACET

Monomer

RHDNMRConc

Hydrodynamic radiiHydrodynamic radii(Stokes Einstein,Sphere)


84

NOESY experimentsNOESY experimentsNOESY spectrum of a dilute aqueous solution of ACETACET

400 ms400 ms

Color of cross peaks:

Blue : NegativeGreen/Yellow : Positive


85


400 ms400 ms


Blue : Negative cross-peakGreen/Yellow : Positive cross-peak

Spin Diffusion


86


10 ms10 ms


Blue : NegativeGreen/Yellow : Positive


87

DLS experimentsDLS experiments

Hydrodynamic radiiHydrodynamic radii of the aggregates were also estimated by DLS

(nm)(10-13 m2 s-1)(mM)

3006.52ACETACET 2507.83PRETPRET 7002.813METOMETO

Aggregate

RHDConc

METOMETO

PRETPRET

ACETACET


88

Physico-chemical propertiesPhysico-chemical propertiesSurface Surface TensionTension

Activity Activity CoeffCoeff

Osmotic Osmotic CoeffCoeff

Rel. viscosityRel. viscosity


89

Diffusion-Ordered NMR Spectroscopy: a versatile tool for the molecular weight

determination of uncharged polysaccharides

Molecular weightMolecular weight

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847

90S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847

IntroductionIntroduction•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


Diffusion and MassDiffusion 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 853Studied by PGSE experiments


Diffusion and MassDiffusion and Mass

853 kDa5.8 kDa 100 kDa

93

100 1000 10000 100000 10000001E-11

1E-10

1E-9

Pullulan

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847

Determination of Molecular Weight:Determination of Molecular Weight:Pullulan as a Model SamplePullulan as a Model Sample

MW(Da)

D(m2/s)


Determination of Molecular Weight:Determination of Molecular Weight:Calibration curveCalibration curve

100 1000 10000 100000 10000001E-11

1E-10

1E-9

Pullulan Calibration Curve

D(m2/s)

MW(Da)


Determination of Molecular Weight:Determination of Molecular Weight:Check with another polysaccharideCheck with another polysaccharide

D(m2/s)

MW(Da)100 1000 10000 100000 1000000

1E-11

1E-10

1E-9

Calibration Curve Dextran


Determination of Molecular Weight:Determination of Molecular Weight:Check with oligosaccharidesCheck with oligosaccharides

D(m2/s)

MW(Da)100 1000 10000 100000 1000000

1E-11

1E-10

1E-9

Calibration Curve Dextran Cyclodextrins - Oligosaccharide


Determination of Molecular Weight:Determination of Molecular Weight:Check with saccharidesCheck with saccharides

D(m2/s)

MW(Da)100 1000 10000 100000 1000000

1E-11

1E-10

1E-9

Calibration Curve Dextran Cyclodextrins - Oligosaccharide Saccharides

98

Use of Pulsed Field Gradient Spin-Echo NMR as a tool in MALDI method

development for polymer Mw determination

Molecular WeightMolecular Weight

M. Mazarin, S. Viel, B. Allard-Breton, A. Thévand, L. CharlesAnal. Chem. 2006, 78, 2758-2764

99

PolymersPolymers

M. Mazarin, S. Viel, B. Allard-Breton, A. Thévand, L. CharlesAnal. Chem. 2006, 78, 2758-2764

pMAMpMAM

Concentration (mg.mL-1)

0.0 1.0 2.0 3.0 4.0

D (

m2 .s

-1)

0.0

2.0e-10

4.0e-10

6.0e-10

8.0e-10

1.0e-9

1.2e-9

1.4e-9

Mw 309 (D0 = 1.172e-9)

Mw 972 (D0 = 6.596e-10)

Mw 3460 (D0 = 3.405e-10)

Mw 9830 (D0 = 1.973e-10)

Mw 23800 (D0 = 1.189e-10)

Mw 74500 (D0 = 5.903e-11)

D=f[PS] D0PS=f(Mw)

Molecular weight Mw (Da)

10 100 1000 10000 100000 1000000

D0

(m2 .s

-1)

1e-11

1e-10

1e-9

1e-8

0.54122.721448e-8

Polymers Polymers PSPS

CDCl3

D = k Mw -a

101

PS : Comparison Mw : SEC, NMR and MS

12.012.1(Fluka)

83416 2038353974500PS 70000

-3.8-3.8(Fluka)

22890 492290723800PS 20000

-4.6-8.6(Fluka)

9375 1089869830PS 10000

0.3-5.2(Fluka)

3470 432783460PS 3000

-5.6-0.6(Fluka)

918 6966972PS 1000

40.5-8.1(Sigma-Aldrich)

434 10334309PS 400

MALDI-TOF-MSNMRSEC (provider)PS standards

102

Analysis of mixtures (part I)Analysis of mixtures (part I)

Improved 3D DOSY-TOCSY experiment for mixture analysis

S. Viel, S. CaldarelliChem. Comm. 2008, in press

103S. Viel, S. Caldarelli

Chem. Comm. 2008, in press


•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

Speeding up 3D NMR Speeding up 3D NMR experimentsexperiments

•Various methodologies have been proposed to speed up 3D NMR experiments (FDM)

S. Viel, S. Caldarelli


105


•Various methodologies have been proposed to speed up 3D NMR experiments (FDM)

•One possibility is Hadamard (there are other ones



………...….….3D iRRT would be great!)

106


•Various methodologies have been proposed to speed up 3D NMR experiments

•One possibility is Hadamard (there are other ones



………...….….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

Hadamard encodingHadamard encoding



Hadamard family matrices HMatrix dimension N:N = 2k (k = 1, 2, 3…)

+––+––++–+–+++++

Pulse 1

Pulse 2

Pulse 3

Pulse 4

A B C D

– HHHH

M chemical sites NM

–+++

108





+––+––++–+–+++++

Pulse 1

Pulse 2

Pulse 3

Pulse 4

A B C D

M chemical sites NM – HHHH

–+++

109





+––+––++–+–+++++

Pulse 1

Pulse 2

Pulse 3

Pulse 4

A B C D

M chemical sites NM – HHHH

–+++

110





+––+––++–+–+++++

Pulse 1

Pulse 2

Pulse 3

Pulse 4

A B C D

Signal B = + 1 – 2 + 3 – 4M chemical sites NM – HHHH

–+++

111

Proposed pulse sequenceProposed pulse sequence



Thrippleton, M. J.; Keeler, J., Angew. Chem. Int. Ed. 2003, 42, 3938-3941.

Cano, K. E.; Thrippleton, M.; Keeler, J.; Shaka, A. J., J. Magn. Reson. 2004, 167, 291-297.ZQC filters

112

Proof of principle (1)Proof of principle (1)



TOCSY spectrum of a mixture of:

- Methanol (M)- Ethanol (E)- Propanol (P)- Valine (V)

113

Proof of principle (2)Proof of principle (2)



M

P

E

V

114

Effect of signal overlappingEffect of signal overlapping



Propanol

OH

OH

2-Butanol

115

Effect of signal overlapping (2)Effect of signal overlapping (2)



OHOH

Time saving factor: 64

116

Analysis of mixtures (part II)Analysis of mixtures (part II)

Enhanced diffusion-edited NMR spectroscopy of mixtures using

chromatographic stationary phases

S. Viel, F. Ziarelli, S. CaldarelliProc. Natl. Acad. Sci. U. S. A. 2003, 100, 9696-9698

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, 9696-9698


•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

118S. Viel, F. Ziarelli, S. Caldarelli


PrinciplePrinciple

•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)



Problem: spectral resolution!Problem: spectral resolution!1H of Sol. + Stationary phase

Conventional NMRHHigh RResolutionMMagic AAngle SSpinning: solid state technique



Problem: spectral resolution!Problem: spectral resolution!1H of Sol. + Stationary phase

Conventional NMRHHigh RResolutionMMagic AAngle SSpinning: solid state technique

HRMAS NMR



HRMASHRMAS

HRMAS rotorHRMASprobe



Example 1Example 1

Mixture 1:Mixture 1:- Dichlorophenol- Ethanol- Heptane



Example 1Example 1

Mixture 1:Mixture 1:- Dichlorophenol- Ethanol- Heptane

+

SiO2



Example 2Example 2

Mixture 2:Mixture 2:- Naphtalene- Dec-1-ene - Ethanol



Example 2Example 2

Mixture 2:Mixture 2:- Naphtalene- Dec-1-ene - Ethanol

+

C18



Research directionsResearch directions

• Improve resolution of complex mixtures

• Characterize new chromatographic phases

• Investigate chromatographic phenomenon

• Discriminate stereoisomers

127

PFG MAS diffusion PFG MAS diffusion measurementsmeasurements

Pulsed field gradient magic angle spinning NMR self-diffusion

measurements in liquids

S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123

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Gradients and MAS probesGradients and MAS probes

S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli

J. Magn. Reson. 2008, 190, 113-123

Courtesy of Bruker Instruments

129

Magic gradientMagic gradient


J. Magn. Reson. 2008, 190, 113-123


130S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S. Caldarelli

J. Magn. Reson. 2008, 190, 113-123


Stator

Gradients

Magic gradientMagic gradient


J. Magn. Reson. 2008, 190, 113-123

Gradient calibration: Gradient calibration: ProfileProfileHahn echo on a H2O/D2O sample with gradient during acquisition

Adapted from Hurd et al.


J. Magn. Reson. 2008, 190, 113-123

Gradient calibration: Gradient calibration: ProfileProfile

6%

95%

133

Gradient calibration: strengthGradient calibration: strength


J. Magn. Reson. 2008, 190, 113-123

Rotor:


J. Magn. Reson. 2008, 190, 113-123

Rotor:

V = 50 LV = 12 L

G = 6.0 G cm-1 A-1

Gradient calibration: strengthGradient calibration: strength

135

Effect of spinningEffect of spinning


J. Magn. Reson. 2008, 190, 113-123

Water

Water ACN

ACN

12 L

136

Effect of spinningEffect of spinning


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Water

Water ACN

ACN

50 L

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Results: ACN 4 kHzResults: ACN 4 kHz


J. Magn. Reson. 2008, 190, 113-123

50 L12 L


J. Magn. Reson. 2008, 190, 113-123

ResultsResults

139

ResultsResults


J. Magn. Reson. 2008, 190, 113-123

PEO 116kDa D2O 4 kHz PEO 116kDa CDCl3 3 kHz



Research directionsResearch directions

• Improve resolution of complex mixtures

• Characterize new chromatographic phases

• Investigate chromatographic phenomenon

• Discriminate stereoisomers

141

HPLC

PFG MAS

ODS phase Silica gel

G. Pagès et al. Anal. Chem. 2006, 78, 561-566

G. Pagès et al. Angew. Chem. Int. Ed. 2006, 45, 5950-5953

Mixture of:

- Benzene- Naphthalene- Anthracene(ACN/H2O, 90/10)

142

Merci

143

Grazie

144

Thank you !

A

BC

145

A

BC

Documents

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