Stephane Viel Dosy

Principles and selected applications of Diffusion-Ordered NMR SpectroscopyStphane Viel, Ph. D.Assistant ProfessorAix-Marseille UniversityMolecular Sciences Institute II (UMR-6263)Chemometrics and Spectroscopy LaboratoryMarseilles (France)

DOSY ?Diffusion Ordered NMR SpectroscopyWeb of Science, 12 / 2007

NMR and DiffusionPGSEPulsed Gradient Spin Echo1965

NMR and DiffusionDOSYDiffusion Ordered SpectroscopY1992

NMR and DiffusionDOSYDiffusion Ordered SpectroscopY1992PGSEPulsed Gradient Spin Echo1965

General outlinePart 1: Theory about molecular mobility

Self-diffusionStudy of self-diffusion by NMRPrinciples of Pulsed Gradient Spin Echo (PGSE)Diffusion ordered NMR spectroscopy (DOSY)

Part 2: Selected applications of DOSY

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

Self-diffusion by Brown, 1828 Random jostling of molecules which leads to their net displacement over time

Self-diffusion coefficient DD is related to the hydrodynamic volume of the diffusing particle through

Self-diffusion coefficient DD is related to the hydrodynamic volume of the diffusing particle throughD self-diffusion coefficientk Boltzmanns constantT absolute temperaturef friction factorSphere

For a sphere diffusing in an isotropic and continuous medium of viscosity :Stokes Einstein equation

Pulsed Gradient Spin Echo (PGSE)Stejskal and Tanner, 1965Gradients of magnetic field (Pulsed)Study of self-diffusion by NMRGradientPulses

Study of self-diffusion by NMR1. Spatially label the nuclear spins using gradients of magnetic field.2. Monitor their displacement by measuring their spatial positions at 2 distinct times.Principle: 2 steps

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

Magnetic field gradientMagnetic field gradientFor a single and constant gradient oriented along the z direction

Magnetic field gradientMagnetic field gradientFor a single and constant gradient oriented along the z directionNotion of effective gradient

Phase shift of nuclear spinsAssume that the magnetic fieldgradient is active during a time A nuclear spin acquires a phase shift

Assume that the magnetic fieldgradient is active during a time Phase shift of nuclear spinsA nuclear spin acquires a phase shift

Phase shift of nuclear spinsAssume that the magnetic fieldgradient is active during a time A nuclear spin acquires a phase shift The spatial position of the nuclear spins is encoded into a phase shiftNuclear spin spatial labelling

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

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

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

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

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

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

Spin Echo or Hahn Echo (SE)With magnetic field gradientsAttenuation factor

Iecho: Intensity at the echo with gradientsI0: Intensity at the echo without gradients D: Self-diffusion coefficient : gradient pulse duration : Diffusion time q: gradient pulse areaAttenuation factor

How do we actually obtain D? Attenuation factor

Stimulated Echo (STE)With magnetic field gradients

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

The BPP-STE-LED sequenceStimulated Echo (STE): T1 relaxation vs. T2 relaxationNo artefacts due to J modulationBipolar gradient pulses (BPP):Reduced eddy currentsLongitudinal Eddy currents Delay (LED):Less spectral distortions due to eddy currents

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

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

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

How can we use PGSE data?NMR spectrum (frequency scale, ppm)DB

NMR spectrum (ppm scale)DBSIZEJames & McDonald, 1978Stilbs & Moseley, 1978-80

Size Resolved SpectrometryNMR spectrum (ppm scale)BCCStilbs, 1981

ppmDADCDBDHighLow

DADCDBDHighLow

Diffusion Ordered NMR SpectroscopYMorris & Johnson, 1992DOSYAntalek, B. Concepts in Magn. Reson 2002, 14, 225-258

DOSYDiffusion Ordered NMR SpectroscopYMorris & Johnson, 1992Signal processingMany processings available:- MaxEnt (Delsuc, M. A.)- DECRA (Antalek, B.)- CORE (Stilbs, P.)- MCR (van Gorkom, L. C. M.)- MULVADO (Huo, R.)- iRRT (Mandelstham, V.)

DOSY mapAdapted from Nilsson et al.

Distortions due to spectral overlapAdapted from Nilsson et al.

iRRT

inverseRegularized ResolventTransformMixture of 2 isomersV. MandelshtamA. J. ShakaThureau, P.; Thvand, A.; Ancian, B.; Escavabaja, P.; Armstrong, G. S.; Mandelshtam, V. A., ChemPhysChem 2005, 6, 1Armstrong, G. S.; Loening, N. M.; Curtis, J. E.; Shaka, A. J.; Mandelshtam, V. A., J. Magn. Reson. 2003, 163, 139

Part 1: Theory about molecular mobility

Self-diffusionStudy of self-diffusion by NMRPrinciples of Pulsed Gradient Spin Echo (PGSE)Diffusion ordered NMR spectroscopy (DOSY)

Part 2: Selected applications of DOSY General outline

Chiral recognitionChiral recognition of dipeptides in a biomembrane modelC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

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 usedC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362we used Sodium N-doceanoyl-L-prolinate (SDP)Here

Introduction (2)We studied by NMR the chiral recognition in SDP micelles of 2 dipeptidesNMR techniques: 1H, PGSE, ROESY+Molecular mechanic calculationsC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

1H experiments: LL/DD coupleDitryptophan (1)+SDP micellesDiphenylalanine (2)+SDP micellesC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

1H experiments: LD/DL coupleDitryptophan (1)+SDP micellesDiphenylalanine (2)+SDP micellesC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

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

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

PGSE experimentsDetermine the partition coefficient of the dipeptides in the 2 phasesC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

PGSE experimentsBound molar fractions xb and partition coefficients pC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Conformations of 1 isomers by NMR and Molecular mechanic calculations (1)BufferC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Conformations of 1 isomers by NMR and Molecular mechanic calculations (2)SDP micelles (LL/DD couple)C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Conformations of 1 isomers by NMR and Molecular mechanic calculations (3)SDP micelles (DL/LD couple)C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Binding modes of 1 isomers to SDP micellesLL/DD coupleC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Binding modes of 1 isomers to SDP micellesLD/DL coupleC. Bombelli, S. Borocci, F. Lupi, G. Mancini, L. Mannina, A. L. Segre, S. VielJ. Am. Chem. Soc. 2004, 126, 13354-13362

Chemical exchangeDetermining chemical exchange rates in nucleobasesP. Thureau, B. Ancian, S. Viel, A. ThvandChem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. ThvandChem. Comm. 2006, 1884-1886

Hydrogen bonding in nucleic acidsP. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 1884-1886DNARNAThymine AdenineAdenineUracil

Effect of chemical exchange in DOSYUridineP. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 1884-1886H2O

ModelSimple 2-site exchangeP. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 1884-1886T = 50 msT = 200 msT= 900 ms

Uracil exchange constants KaSimple 2-site exchangeP. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 1884-1886H1 ka= 8 s-1H3 ka= 18 s-1

Thymine exchange constants KaSimple 2-site exchangeP. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 200-202P. Thureau, B. Ancian, S. Viel, A. Thvand Chem. Comm. 2006, 1884-1886H1 ka= 5 s-1H3 ka= 7 s-1

Self-aggregationInvestigations of complexes in solutionS. Viel, L. Mannina, A. L. SegreTetrahedron Lett. 2002, 43, 2515-2519C. Sanna, C. La Mesa, L. Mannina, P. Stano, S. Viel, A. L. SegreLangmuir 2006, 22, 6021-6031

Introduction stacking interactions are important in organic chemistry and for biological systemsHere we consider 2 types of organic molecules bearing an aromatic ring and characterized by a: low molecular weight (< 400 Da)Studied by: - NMR (1H, PGSE, NOESY)- DLS- Physicochemical measurements low H2O solubilityS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

Molecules under studyClass AClass BS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

1H experimentsMonomeric resonances1H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc < sol)S. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

1H experiments1H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc > sol)Monomeric resonancesExtra resonancesS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

1H experiments1H spectra of dilute aqueous solutions of METO, ACET and PRET, (Conc > sol)Well resolvedUpfield shiftedS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

PGSE experiments (DOSY display)PGSE on a dilute aqueous solution of ACETMuch lower diffusion coefficientS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

PGSE experimentsHydrodynamic radii(Stokes Einstein,Sphere)S. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

NOESY experimentsNOESY spectrum of a dilute aqueous solution of ACET400 msColor of cross peaks:

Blue : NegativeGreen/Yellow : Positive

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


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



Blue : NegativeGreen/Yellow : Positive


DLS experimentsHydrodynamic radii of the aggregates were also estimated by DLSS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

Physico-chemical propertiesSurface TensionActivity CoeffOsmotic CoeffRel. viscosityS. Viel et al. Tetrahedron Lett. 2002, 43, 2515-2519C. Sanna et al. Langmuir 2006, 22, 6021-6031

Diffusion-Ordered NMR Spectroscopy: a versatile tool for the molecular weight determination of uncharged polysaccharidesMolecular weightS. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847

IntroductionS. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847Polysaccharides 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 ChromatographyDrawbacks: sample manipulation

Diffusion and MassS. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847Strictly, diffusion relates to molecular size. A calibration is hence required to establish the relationship between diffusion coefficient and molecular weightPullulan (linear polysaccharide)6 fractions (kDa): 5.8; 12; 28.3; 100; 180 and 853Studied by PGSE experiments

Diffusion and MassS. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847853 kDa5.8 kDa100 kDa

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847Determination of Molecular Weight: Pullulan as a Model SampleMW(Da)D(m2/s)

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2002, 4, 1843-1847Determination of Molecular Weight: Calibration curveD(m2/s)MW(Da)

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2002, 4, 1843-1847Determination of Molecular Weight: Check with another polysaccharideD(m2/s)MW(Da)

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847Determination of Molecular Weight: Check with oligosaccharidesD(m2/s)MW(Da)

S. Viel, D. Capitani, L. Mannina, A. L. SegreBiomacromolecules 2003, 4, 1843-1847Determination of Molecular Weight: Check with saccharidesD(m2/s)MW(Da)

Use of Pulsed Field Gradient Spin-Echo NMR as a tool in MALDI method development for polymer Mw determinationMolecular WeightM. Mazarin, S. Viel, B. Allard-Breton, A. Thvand, L. CharlesAnal. Chem. 2006, 78, 2758-2764

PolymersM. Mazarin, S. Viel, B. Allard-Breton, A. Thvand, L. CharlesAnal. Chem. 2006, 78, 2758-2764pMAM

D=f[PS]D0PS=f(Mw)Polymers PSCDCl3D = k Mw -a

PS : Comparison Mw : SEC, NMR and MS

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

IntroductionS. Viel, S. CaldarelliChem. Comm. 2008, in pressOverlapping signals severely complicate DOSY analysisA typical solution is the addition of another frequency dimension to spread the signals out

Speeding up 3D NMR experimentsVarious methodologies have been proposed to speed up 3D NMR experiments (FDM)S. Viel, S. CaldarelliChem. Comm. 2008, in press

Speeding up 3D NMR experimentsVarious methodologies have been proposed to speed up 3D NMR experiments (FDM)One possibility is Hadamard (there are other onesS. Viel, S. CaldarelliChem. Comm. 2008, in press.....3D iRRT would be great!)

Speeding up 3D NMR experimentsVarious methodologies have been proposed to speed up 3D NMR experimentsOne possibility is Hadamard (there are other onesS. Viel, S. CaldarelliChem. 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

Hadamard encodingS. Viel, S. CaldarelliChem. Comm. 2008, in pressHadamard family matrices HMatrix dimension N:N = 2k (k = 1, 2, 3)

Hadamard encodingS. Viel, S. CaldarelliChem. Comm. 2008, in pressHadamard family matrices HMatrix dimension N:N = 2k (k = 1, 2, 3)Signal B = + 1 2 + 3 4

Proposed pulse sequenceS. Viel, S. CaldarelliChem. Comm. 2008, in press

Proof of principle (1)S. Viel, S. CaldarelliChem. Comm. 2008, in pressTOCSY spectrum of a mixture of:

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

Proof of principle (2)S. Viel, S. CaldarelliChem. Comm. 2008, in pressMPEV

Effect of signal overlappingS. Viel, S. CaldarelliChem. Comm. 2008, in pressPropanol2-Butanol

Effect of signal overlapping (2)S. Viel, S. CaldarelliChem. Comm. 2008, in pressTime saving factor: 64

Analysis of mixtures (part II)Enhanced diffusion-edited NMR spectroscopy of mixtures using chromatographic stationary phasesS. Viel, F. Ziarelli, S. CaldarelliProc. Natl. Acad. Sci. U. S. A. 2003, 100, 9696-9698

IntroductionCan we selectively slow down the diffusion of some components of the mixture?S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698PGSE experiments allow compounds to be discriminated according to differences in their effective size (mixture analysis)Corollary: similar sized compounds CANNOT be resolved by PGSE

PrincipleS. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698A 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!S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-96981H of Sol. + Stationary phaseConventional NMRHigh ResolutionMagic Angle Spinning: solid state technique

Problem: spectral resolution!S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-96981H of Sol. + Stationary phaseConventional NMRHigh ResolutionMagic Angle Spinning: solid state techniqueHRMAS NMR

HRMASS. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698HRMAS rotorHRMASprobe

Example 1S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698Mixture 1:- Dichlorophenol- Ethanol- Heptane

Example 1S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698Mixture 1:- Dichlorophenol- Ethanol- Heptane+SiO2

Example 2S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698Mixture 2:- Naphtalene Dec-1-ene Ethanol

Example 2S. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698Mixture 2:- Naphtalene Dec-1-ene Ethanol+C18

Research directionsS. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698 Improve resolution of complex mixtures Characterize new chromatographic phases Investigate chromatographic phenomenon Discriminate stereoisomers

PFG MAS diffusion measurementsPulsed field gradient magic angle spinning NMR self-diffusion measurements in liquidsS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123

Gradients and MAS probesS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123Courtesy of Bruker Instruments

Magic gradientS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123Courtesy of Bruker Instruments

Gradient calibration: ProfileS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123Hahn echo on a H2O/D2O sample with gradient during acquisitionAdapted from Hurd et al.

Gradient calibration: ProfileS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-1236%95%

Gradient calibration: strengthS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123Rotor:

Gradient calibration: strengthS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123Rotor:V = 50 LV = 12 LG = 6.0 G cm-1 A-1

Effect of spinningS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123WaterWaterACNACN12 L

Effect of spinningS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123WaterWaterACNACN50 L

Results: ACN 4 kHzS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-12350 L12 L

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

ResultsS. Viel, F. Ziarelli, G. Pags, C. Carrara, S. CaldarelliJ. Magn. Reson. 2008, 190, 113-123PEO 116kDa D2O 4 kHz PEO 116kDa CDCl3 3 kHz

Research directionsS. Viel, F. Ziarelli, S. CaldarelliProceedings of the National Academy of Sciences of the United States of America 2003, 100, 9696-9698 Improve resolution of complex mixtures Characterize new chromatographic phases Investigate chromatographic phenomenon Discriminate stereoisomers

HPLCPFG MASG. Pags et al. Anal. Chem. 2006, 78, 561-566G. Pags et al. Angew. Chem. Int. Ed. 2006, 45, 5950-5953Mixture of:

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

Grazie

Thank you !

Good afternoon, my name is Stphane Viel and I am from the Aix Marseille University in France. First of all, I would like to thank the Irvine Chemistry dept, especially Pr. Rachel Martin, for giving me the opportunity to present our work, and also all of you for being here today. This presentation will describe the principles and a few selected applications of Diffusion Ordered NMR spectroscopy, commonly referred to as DOSY. But first of all, to introduce this talk I, would like you to focus first on the DOSY acronym, which involves the word diffusion, and indeed DOSY allows you to investigate diffusion processes. For curiosity, it is interesting to look at the number of publications that have actually mentioned the DOSY acronym since its first introduction in 1992, when Morris and Johnson set up the experiement.As you can see, there is almost a perfect exponential growth, suggesting the increase in popularity of this technique. However, diffusion has been studied by NMR well before 1992As a matter of fact, NMR and diffusion are very old friends, almost from the very beginning. Back in the early 50s, Hahn depicted the effect of diffusion on spin echoes, followed later on by Carr and Purcell. In 1965, Stejskal and Tanner published a very important paper which demonstrated the feasibility of measuring diffusion by using pulsed magnetic field gradients instead of steady gradients. This is the birth date of the so-called PGSE technique.1956 TorreyLater on, Stilbs introduced the concept of using the FT PGSE to investigate complex mixtures. He did a lot of work on diverse physicochemical systems, which he summarized in his review in 1987. And then, and I would say only then, came the moment Morris and Johnson published their paper on DOSYThe aim of this short introduction was to insist on the fact that the PGSE and DOSY techniques are very closely related, and one of the purposes of this presentation will be to clarify the small difference between them.Now, let us look at the outline that I will follow in this presentation, which will be organized in two parts. In the first part, I will give a little bit of theory, defining in particular a few concepts such as self-diffusion and explaining how self-diffusion can be measured by NMR. I will especially focus on the Pulsed Gradient Spin Echo technique and introduce also Diffusion Ordered NMR spectroscopy. Then, in a second part, I will present a few selected applications of the work we have been doingI would like to draw your attention to the fact that we are working at thermodynamic equilibrium, which means that we do not have:Now I will give a series of 3 simple equations, which in the end I will group to achieve the Stokes Einstein relation ship that relates the self diffusion coefficient to the molecular sizeFirst I recall the Larmor precession frequency, which is the product of the magnitude of the magnetic field with the nuclear magnetogyric ratioIn other words, the free precession has been removed I would like to define quickly the notion of echo. After the first 90 pulse, the magnetization is in the xy plane and we have an NMR signal that we could detect if we wanted. However, this signal will obviously not last for ever. In fact, due to the interactions of our nuclear spins with the surroundings, there is a progressive loss of coherence between the nuclear spins and the signal eventually dies. Now, by using the specific pulse sequence feature delay-180-delay, the nuclear spins can somehow get some coherence back and this in turn yields to an observable NMR signal. I will not explain this in details This NMR signal is called an echo and it is due to a refocussing of the magnetization.Note that we are detecting only molecular displacement in the direction along which the gradient is applied, here this is the z direction.Using the previous approximations it can be shown that the echo attenuation is related to the diffusion coefficient according to the following equation Here we suppose to deal with constant amplitude gradient pulses also called rectangular pulsesUsing the previous approximations it can be shown that the echo attenuation is related to the diffusion coefficient according to the following equation Here we suppose to deal with constant amplitude gradient pulses also called rectangular pulsesWe then fit the data and obtain the diffusion coefficient D. The fitting is one of the main limitations of the PGSE technique because, rigorously, to obtain D one must perform an Inverse Laplace Transform which an ill contidionned mathematical operationThe experiment does not provide the spectral assignment but it helps greatly in studying mixtures by individuating the different components.The correlation spots could also inform about the distribution of the particle size but we will not go any further on this and just focus on the chemical information the 2D plot may conveyThe first application I would like to present deals with Chiral recognition. In particular, in collaboration with Giovanna Mancini from the university of Rome, we investigated chiral recognition of dipeptides in a biomembrane model.

As you probaly know, cell membranes are organized according to molecular recognition phenomena, and in particular chiral recognition phenomena.The most widely used and simplest model to mimic the behavior of biomembranes are micelles. Here we have used as a model the micelles formed by this surfactant, which has 2 isomers, and, from now on, I will refer to these micelles as SDP micelles First of all, I am giving the results from the 1HNMR experiments, focusing on the homochiral couple of enantiomers: LL and DD. For 1+SDP, you can see that NMR spectra of both enantiomers are different. Note that here is reported only the aromatic region of the 1H spectrum. This observation suggests that the selective interaction between the micelles and the enantiomers lead to chemical shift non equivalence for the resonances of the enantiomers.

This can also be observed for the homo chiral couple diphenyalanineThe same observations hold for the heterochiral couple of enantiomers, as you can see first for ditryptophan and second for diphenyalanineFirst, looking at ditryptophan, compound 1, we can see that the p values of the heterochiral enantiomers LDDL (yellow box) are higher than the p values of the homochiral enantiomers LLDD (blue box). This observation is also true for diphenylalanine even though, in this case, the difference is closer to the experimental errors.

Another point I would like to stress is that the p values of all 1 isomers are higher than the p values of the 2 isomers, which suggests that ditryptophan has a higher affinity to SDP micelles than diphenyalalnine. Therefore, from now on, we leave out diphenyalanine and focus on ditryptophan.In particular, what we were interested in is studying the conformations of the enantiomers of 1 in the absence and presence of SDP micelles. To do so, we performed a series of NOESY and ROESY experiments, from which we could derive a set of distance restraints that we then used as restraints in molecular mechanic calculationsThe conformations are elongatedThe first application I would like to present deals with Chiral recognition. In particular, in collaboration with Giovanna Mancini from the university of Rome, we investigated chiral recognition of dipeptides in a biomembrane model.This result is in agreement with HOESY experiments that have shown that, in the first solvation shell of uracil, nitrogen N3 was surrounded by about twice as many water molecules as nitrogen N1.2 classes of molecules have been considered in this study. Here I am using the terms class A and B to refer to each group of molecules. For the sake of clarity, I will give the results for each group subsequently. In the end, I will focus on the main similarities and differences between both classes

Insist on the meaning of the acronymsYou can see two nice triplets. The small peaks upfield are due to impuritiesFor each resonance we can observe the corresponding new resonance which are upfield shifted with respect to the normal monomeric resonances: for instance we can see two rather well resolved triplets here. We systematically observed an upfield shift for all resonances in the spectrum including the resonances due to the aromatic protons. This indicates the presence of p-p stacking interactions; in fact, this is the only way to observe an upfield shift that is generalized to all the resonances in the spectrumThe Rh values were computed according to the Stokes Einstein equation by assuming spherical particles and infinite dilution. Before zooming to the aromatic spectral region, I would like to draw your attention to the fact we do not observe any exchange in this experimentThe blue/negative NOE cross peaks are in agreement with the molecular structure of the compound under investigation. They indicate true distance information. In contrast, the NOE cross peaks observed for the extra resonances are all green and yellow, meaning that they are all positive. They also indicate a specific feature that is sometimes called spin diffusion in NMR. Spin diffusion usually indicates that the molecule is in the slow tumbling regime, pretty much like in the case for instance of macromolecules such as for example proteins. What we need to do in such situation is to reduce the contact time in order to avoid any spin diffusion effectsHere we had to reduce the contact time down to 10 ms to avoid observing any spin diffusion effects and at this particular contact time we could detect a set of positive NOE cross peaksA = surface tension; B = Osmotic coefficient; C = Activity coefficient; D = Relative viscosityThe plot A is peculiar to amphiphilic systems; the break point indicates the non-critical multimer concentration NMC. For plots B and C, we can observe an ideal behavior below the NMC and above the observed decrease in the osmotic coefficient and the activity coefficient are in agreement with the aggregation taking place. For plot D, usually, aggregation processes increase the solution viscosity. Here, above the NMC, the viscosity only slightly increases, which may indicate the presence of small aggregates. Fitting the data gives rise to a rh value of 3 nm.in order to focus the experimental time on the signal-containing spectral regions.

in order to focus the experimental time on the signal-containing spectral regions.






And actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesAnd actually there is a group of people in Marseille who are working along those linesI would also like to thank you for your attention, and I will be glad to answer your questions HOWEVER, keep in mind that sometimes French people.get easily upset!!!

Stephane Viel Dosy

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