Principles and selected applications of Diffusion-Ordered NMR Spectroscopy

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
DOSY ?

• Diffusion Ordered NMR Spectroscopy

Web of Science, 12 / 2007
3
DOSY ?

• Diffusion Ordered NMR Spectroscopy

Web of Science, 12 / 2007
4
NMR and Diffusion
PGSE Pulsed Gradient Spin Echo
1965
5
NMR and Diffusion
DOSY Diffusion Ordered SpectroscopY
1992
6
NMR and Diffusion
PGSE Pulsed Gradient Spin Echo
1965
DOSY Diffusion Ordered SpectroscopY
1992
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
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
Self-diffusion by Brown, 1828

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

10
Self-diffusion coefficient D

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

11
Self-diffusion coefficient D

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

• D self-diffusion coefficient
• k Boltzmanns constant
• T absolute temperature
• f friction factor

Sphere
12
Stokes Einstein equation

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

13
Study of self-diffusion by NMR

• Pulsed Gradient Spin Echo (PGSE)
• Stejskal and Tanner, 1965
• Gradients of magnetic field (Pulsed)

Gradient Pulses
14
Study of self-diffusion by NMR
Principle 2 steps
1. Spatially label the nuclear spins using
gradients of magnetic field.
2. Monitor their displacement by measuring their
spatial positions at 2 distinct times.
15
Larmor frequency

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

16
Magnetic field gradient

• Magnetic field
• gradient

For a single and constant gradient oriented along
the z direction
17
Magnetic field gradient

• Magnetic field
• gradient

For a single and constant gradient oriented along
the z direction
Notion of effective gradient
18
Phase shift of nuclear spins

• Assume that the magnetic field
• gradient is active during a time ?

• A nuclear spin acquires a phase shift

19
Phase shift of nuclear spins

• Assume that the magnetic field
• gradient is active during a time ?

• A nuclear spin acquires a phase shift

20
Phase shift of nuclear spins
Nuclear spin spatial labelling

• 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
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
Spin Echo or Hahn Echo (SE)

• Without magnetic field gradients

Echo
23
Spin Echo or Hahn Echo (SE)

• With magnetic field gradients

24
Spin Echo or Hahn Echo (SE)

• With magnetic field gradients

Echo
25
Spin Echo or Hahn Echo (SE)

• With magnetic field gradients

26
Spin Echo or Hahn Echo (SE)

• With magnetic field gradients

Echo
27
Spin Echo or Hahn Echo (SE)

• With magnetic field gradients

Attenuation factor ?
28
Attenuation factor ?

• 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

29
How do we actually obtain D?
Attenuation factor ?
30
How do we actually obtain D?
Attenuation factor ?
31
Stimulated Echo (STE)

• With magnetic field gradients

32
BPP-STE-LED sequence

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

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
The BPP-STE-LED sequence

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

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
The BPP-STE-LED sequence

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

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
The BPP-STE-LED sequence

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

Echo
Signal
39
SéquenceBPP-STE-LED

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

40
How can we use PGSE data?
NMR spectrum (frequency scale, ppm)
DB
41
James McDonald, 1978 Stilbs Moseley, 1978-80
NMR spectrum (ppm scale)
DB
S I Z E
42
Size Resolved Spectrometry
NMR spectrum (ppm scale)
Stilbs, 1981
B
C
C
43
ppm
Low
DB
D
DC
DA
High
44
Low
DB
D
DC
DA
High
45
DOSY

• Diffusion Ordered NMR SpectroscopY
• Morris Johnson, 1992

Antalek, B. Concepts in Magn. Reson 2002, 14,
225-258
46
DOSY

• Diffusion Ordered NMR SpectroscopY
• Morris Johnson, 1992
• Signal processing

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.)
47
DOSY

• Diffusion Ordered NMR SpectroscopY
• Morris Johnson, 1992
• Signal processing

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.)
48
DOSY map
Adapted from Nilsson et al.
49
Distortions due to spectral overlap
Adapted from Nilsson et al.
50
iRRTinverseRegularized ResolventTransform
Mixture of 2 isomers
V. Mandelshtam A. J. Shaka
Armstrong, G. S. Loening, N. M. Curtis, J. E.
Shaka, A. J. Mandelshtam, V. A., J. Magn. Reson.
2003, 163, 139
Thureau, P. Thévand, A. Ancian, B. Escavabaja,
P. Armstrong, G. S. Mandelshtam, V. A.,
ChemPhysChem 2005, 6, 1
51
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

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, 13354-13362
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

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

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

54
Introduction (2)

• We studied by NMR the chiral recognition in SDP
  micelles of 2 dipeptides

NMR techniques 1H, PGSE, ROESY Molecular
mechanic calculations
C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L.
Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc.
2004, 126, 13354-13362
55
1H 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, 13354-13362
56
1H 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, 13354-13362
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

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, 13354-13362
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

C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L.
Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc.
2004, 126, 13354-13362
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, 13354-13362
60
PGSE experiments

• Bound molar fractions xb 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, 13354-13362
61
PGSE experiments

• Bound molar fractions xb 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, 13354-13362
62
PGSE experiments

• Bound molar fractions xb 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, 13354-13362
63
Conformations of 1 isomers by NMR and Molecular
mechanic calculations (1)

• Buffer

C. Bombelli, S. Borocci, F. Lupi, G. Mancini, L.
Mannina, A. L. Segre, S. Viel J. Am. Chem. Soc.
2004, 126, 13354-13362
64
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. Viel J. Am. Chem. Soc.
2004, 126, 13354-13362
65
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. Viel J. Am. Chem. Soc.
2004, 126, 13354-13362
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, 13354-13362
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, 13354-13362
68
Chemical exchange

• Determining chemical exchange rates in nucleobases

P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 1884-1886
69
Hydrogen bonding in nucleic acids
Thymine
Adenine
DNA
Adenine
Uracil
RNA
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
70
Effect of chemical exchange in DOSY
Uridine
H2O
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
71
Model
Simple 2-site exchange
T 50 ms
T 200 ms
T 900 ms
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
72
Model
Simple 2-site exchange
T 50 ms
T 200 ms
T 900 ms
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
73
Model
Simple 2-site exchange
T 50 ms
T 200 ms
T 900 ms
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
74
Uracil exchange constants Ka
Simple 2-site exchange
H1 ka 8 s-1 H3 ka 18 s-1
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
75
Thymine exchange constants Ka
Simple 2-site exchange
H1 ka 5 s-1 H3 ka 7 s-1
P. Thureau, B. Ancian, S. Viel, A. Thévand Chem.
Comm. 2006, 200-202 P. Thureau, B. Ancian, S.
Viel, A. Thévand Chem. Comm. 2006, 1884-1886
76
Self-aggregation

• Investigations of ??? complexes in solution

S. Viel, L. Mannina, A. L. Segre Tetrahedron
Lett. 2002, 43, 2515-2519
C. Sanna, C. La Mesa, L. Mannina, P. Stano, S.
Viel, A. L. Segre Langmuir 2006, 22, 6021-6031
77
Introduction

• ??? 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

• low molecular weight (lt 400 Da)

• low H2O solubility

Studied by - NMR (1H, PGSE, NOESY) -
DLS - Physicochemical measurements
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
78
Molecules under study
Class A
Class B
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
79
1H experiments
1H spectra of dilute aqueous solutions of METO,
ACET and PRET, (Conc lt sol)
Monomeric resonances
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
80
1H experiments
1H spectra of dilute aqueous solutions of METO,
ACET and PRET, (Conc gt sol)
Monomeric resonances
Extra resonances
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
81
1H experiments
1H spectra of dilute aqueous solutions of METO,
ACET and PRET, (Conc gt sol)

• Well resolved
• Upfield shifted

S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
82
PGSE experiments (DOSY display)
PGSE on a dilute aqueous solution of ACET
Much lower diffusion coefficient
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
83
PGSE experiments
Hydrodynamic radii (Stokes Einstein, Sphere)
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
84
NOESY experiments
NOESY spectrum of a dilute aqueous solution of
ACET 400 ms
Color of cross peaks Blue Negative Green/Yello
w Positive
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
85
NOESY experiments
NOESY spectrum of a dilute aqueous solution of
ACET 400 ms
Color of cross peaks Blue Negative
cross-peak Green/Yellow Positive cross-peak
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
86
NOESY experiments
NOESY spectrum of a dilute aqueous solution of
ACET 10 ms
Color of cross peaks Blue Negative Green/Yello
w Positive
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
87
DLS experiments
Hydrodynamic radii of the aggregates were also
estimated by DLS
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
88
Physico-chemical properties
Surface Tension
Osmotic Coeff
Activity Coeff
Rel. viscosity
S. Viel et al. Tetrahedron Lett. 2002, 43,
2515-2519 C. Sanna et al. Langmuir 2006, 22,
6021-6031
89
Molecular weight

• Diffusion-Ordered NMR Spectroscopy a versatile
  tool for the molecular weight determination of
  uncharged polysaccharides

S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
90
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
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
91
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
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
92
Diffusion and Mass
853 kDa
5.8 kDa
100 kDa
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
93
Determination of Molecular WeightPullulan as a
Model Sample
D (m2/s)
MW (Da)
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
94
Determination of Molecular WeightCalibration
curve
D (m2/s)
MW (Da)
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2002, 4, 1843-1847
95
Determination of Molecular WeightCheck with
another polysaccharide
D (m2/s)
MW (Da)
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2002, 4, 1843-1847
96
Determination of Molecular WeightCheck with
oligosaccharides
D (m2/s)
MW (Da)
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
97
Determination of Molecular WeightCheck with
saccharides
D (m2/s)
MW (Da)
S. Viel, D. Capitani, L. Mannina, A. L.
Segre Biomacromolecules 2003, 4, 1843-1847
98
Molecular Weight

• Use of Pulsed Field Gradient Spin-Echo NMR as a
  tool in MALDI method development for polymer Mw
  determination

M. Mazarin, S. Viel, B. Allard-Breton, A.
Thévand, L. Charles Anal. Chem. 2006, 78,
2758-2764
99
Polymers
pMAM
M. Mazarin, S. Viel, B. Allard-Breton, A.
Thévand, L. Charles Anal. Chem. 2006, 78,
2758-2764
100
Polymers PS
D0PSf(Mw)
DfPS
CDCl3
D k Mw -a
101
PS Comparison Mw SEC, NMR and MS
102
Analysis of mixtures (part I)

• Improved 3D DOSY-TOCSY experiment for mixture
  analysis

S. Viel, S. Caldarelli Chem. Comm. 2008, in press
103
Introduction

• Overlapping signals severely complicate DOSY
  analysis
• A typical solution is the addition of another
  frequency dimension to spread the signals out

S. Viel, S. Caldarelli Chem. Comm. 2008, in press
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
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

...
.
.3D iRRT would be great!)
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
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

...
.
.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

S. Viel, S. Caldarelli Chem. Comm. 2008, in press
107
Hadamard encoding
Hadamard family matrices H Matrix dimension N N
2k (k 1, 2, 3)
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
108
Hadamard encoding
Hadamard family matrices H Matrix dimension N N
2k (k 1, 2, 3)
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
109
Hadamard encoding
Hadamard family matrices H Matrix dimension N N
2k (k 1, 2, 3)
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
110
Hadamard encoding
Hadamard family matrices H Matrix dimension N N
2k (k 1, 2, 3)
Signal B 1 2 3 4
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
111
Proposed pulse sequence
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
112
Proof of principle (1)
TOCSY spectrum of a mixture of - Methanol (M) -
Ethanol (E) - Propanol (P) - Valine (V)
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
113
Proof of principle (2)
M
E
P
V
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
114
Effect of signal overlapping
Propanol
2-Butanol
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
115
Effect of signal overlapping (2)
Time saving factor ? 64
S. Viel, S. Caldarelli Chem. Comm. 2008, in press
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, 9696-9698
117
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

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
118
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)

S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
119
Problem spectral resolution!
1H of Sol. Stationary phase

• High Resolution
• Magic Angle Spinning
• solid state technique

Conventional NMR
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
120
Problem spectral resolution!
1H of Sol. Stationary phase

• High Resolution
• Magic Angle Spinning
• solid state technique

Conventional NMR
HRMAS NMR
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
121
HRMAS
HRMAS probe
HRMAS rotor
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
122
Example 1
Mixture 1 - Dichlorophenol - Ethanol - Heptane
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
123
Example 1
Mixture 1 - Dichlorophenol - Ethanol - Heptane
SiO2
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
124
Example 2

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

S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
125
Example 2

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

C18
S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
126
Research directions

• Improve resolution of complex mixtures
• Characterize new chromatographic phases
• Investigate chromatographic phenomenon
• Discriminate stereoisomers

S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
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, 113-123
128
Gradients and MAS probes
Courtesy of Bruker Instruments
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
129
Magic gradient
Courtesy of Bruker Instruments
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
130
Magic gradient
Courtesy of Bruker Instruments
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
131
Gradient calibration Profile
Hahn echo on a H2O/D2O sample with gradient
during acquisition
Adapted from Hurd et al.
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
132
Gradient calibration Profile
6
95
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
133
Gradient calibration strength
Rotor
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
134
Gradient calibration strength
Rotor
V 12 ?L
V 50 ?L
G 6.0 G cm-1 A-1
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
135
Effect of spinning
12 ?L
Water
ACN
ACN
Water
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
136
Effect of spinning
Water
ACN
50 ?L
ACN
Water
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
137
Results ACN 4 kHz
50 ?L
12 ?L
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
138
Results
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
139
Results
PEO 116kDa D2O 4 kHz
PEO 116kDa CDCl3 3 kHz
S. Viel, F. Ziarelli, G. Pagès, C. Carrara, S.
Caldarelli J. Magn. Reson. 2008, 190, 113-123
140
Research directions

• Improve resolution of complex mixtures
• Characterize new chromatographic phases
• Investigate chromatographic phenomenon
• Discriminate stereoisomers

S. Viel, F. Ziarelli, S. Caldarelli Proceedings
of the National Academy of Sciences of the United
States of America 2003, 100, 9696-9698
141
Mixture of - Benzene - Naphthalene -
Anthracene (ACN/H2O, 90/10)
HPLC
PFG MAS
G. Pagès et al. Anal. Chem. 2006, 78, 561-566 G.
Pagès et al. Angew. Chem. Int. Ed. 2006, 45,
5950-5953
142
Merci
143
Grazie
144
Thank you !
145
(No Transcript)