How Big is my Particle

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How Big is my Particle

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1. How Big is my Particle? Adventures in Diffusion NMR

2. Biography B.S.: Caltech - Chemistry Prof. John H. Richards - DNA NMR Medical research, in vivo NMR spectroscopy & imaging; high school Chemistry, high school Physics Ph.D: University of Wisconsin, Madison - Biophysics Prof. Laura Kiessling & Prof. Laura Lerner - Carbohydrate NMR (also with Prof. John Markley) Quantum mechanical computations on biophysical systems (with Prof. Frank Weinhold) Postdoc 1: University of Chicago - Medicine Prof. Sanjeev Shroff - AFM of derivatized surfaces Postdoc 2: University of Chicago - Chemistry Prof. Ka Yee Lee - Lung surfactant peptide NMR, Langmuir films Technical Director, Biomolecular NMR Facility, University of Chicago

3. Web

4. Particle Size Measurement - Why? Aggregate size (monomer, dimer, trimer, etc.) MW Hydrodynamic radius Micelle characterization

5. Particle Size Measurement by NMR - How? Measure diffusion particle size a diffusion use rudimentary imaging technology to make signal intensity depend on diffusion constant

6. Diffusion & particle size Stokes-Einstein equation D = diffusion coefficient (cm2/sec) ? = viscosity ( g/(sec cm) ) rs = hydrodynamic radius (cm) kT = thermal energy ( g cm2/sec2 ) Measure D, calculate rs Assumes spherical particle, anisotropic diffusion

7. Particle Size Measurement - Why NMR? Other popular methods mass spectrometery - perfect for molecules, problems with noncovalent systems size exclusion gel chromatography - problems with noncovalent systems gel electrophoresis - limited applicability, problems with noncovalent systems light scattering - specialized apparatus, limits on MW range, no chemical info. Utility of NMR Relevant solution conditions No need for special sample prep or equipment* Good for noncovalent aggregates (monomer, dimer, etc.) Good for complex mixtures Large apparent MW range: 10 - 1,000,000 Can measure in different media

8. How do we measure D with NMR?

9. How do we measure D with NMR? Pulse sequence that makes signal intensity dependent on diffusion

10. What do the data look like?

11. DOSY Spectrum DOSY = “Diffusion-Ordered Spectroscopy” Representation = 2D X-axis = frequency, ppm Y-axis = diffusion, m2/sec

12. More points, better resolution

13. Other DOSYs

14. How Does it Work?

15. The Basics Examine a normal 1H 1D NMR peak Detected signal comes from all nuclei in detection region

16. The Basics Consider effects of missetting Z1 shim Nuclei in some parts of the sample spin at a different rates than others Capitalize on that spatial dependence

17. Shims & Gradients Z1 shim & Z gradient: extra Bo at top of sample = +++ extra Bo at center of sample = 0 extra Bo at bottom of sample = --- linear dependence Z2, Z3, Z4 ... polynomial dependence

18. Pulsed Gradient Consider effects of briefly switching on a powerful Z1 shim

19. Refocusing Gradient Reverse effects by applying Z1 gradient pulse in opposite direction exact same strength exact same duration

20. Group Exercise

21. Message Nuclei that move around between gradient pulses DON’T REFOCUS EFFICIENTLY weak signal Nuclei that stay in the same place DO REFOCUS EFFICIENTLY strong signal

22. Review data Series of 1D spectra vary either diffusion time or gradient strength (preferred) Intensities decay exponentially Diffusion constant derived from decay rate

23. Interpretation Depends on specific pulse sequence

24. Pulse sequences for diffusion measurement We used:

25. Pulse sequences for diffusion measurement First: “gradient spin echo”: Stejskal & Tanner (1965),

26. Pulse sequences for diffusion measurement “Stimulated Echo” Tanner, J. Chem. Phys. 52 (1970) 2523

27. Pulse sequences for diffusion measurement “Stimulated Echo, Bipolar Gradients” Wu, Chen & Johnson, J. Mag. Res. A 115 (1995) 260

28. Pulse sequences for diffusion measurement Ours on Bruker: “stebpgp1s” “stimulated echo, bipolar gradient”

29. Interpretation ? = gyromagnetic ratio g = gradient strength d = gradient duration

30. Application 1: Hydrodynamic Radius “Analysis of Molecular Square Size and Purity via Pulsed-Field Gradient NMR Spectroscopy” Otto et al., Inorganic Chemistry 41 (2002), 6172 “Characterization of Protein Unfolding by NMR Diffusion Measurements” Jones et al., J. Biomol. NMR 10 (1997) 199

31. Application 2: Diffusion Editing “NMR Spectroscopic Filtration of Polypeptides and Proteins in Complex Mixtures” Rajogopalan et al., J. Biomol. NMR 29 (2004), 505

32. Application 3: Micelle Characterization “A Diffusion-Ordered NMR Spectroscopy Study of the Solubilization of Arteminsinin by Octanoyl-6-O-ascorbic Acis Micelles” Bilia et al., J. Pharm. Sciences 91 (2002) 2265

33. Application 4: Nanoparticle Coverage “Surface Chemistry of Colloidal PbSe Nanocrystals” Moreels et al., J. Am. Chem. Soc. 130 (2008) 15081

34. Practical Concerns No need for high concentration 1 mM OK, = 1 mM takes more time Experiments take 30 min to overnight, depending on resolution, sample concentration, and sample T1 values Best done in a Shigemi tube (for optimal gradient field homogeneity) Particles 100+ KDa may require a specialized diffusion probe We have one for I400 Quantitation normally requires comparison to standard compounds

35. Summary Pulsed field gradients are used to make spectra whose peak intensities depend on particle/molecular diffusion rate Particle size calculated from measured diffusion rate Method is workable with standard hardware and little special training DOSY provides easy readout of diffusion behavior for a wide variety of soluble molecules, even those in complex mixtures DOSY is implemented on Bruker A600, capable on Varian M400, P500, I400

36. Reviews Price, Concepts in Magnetic Resonance 10 (1998) 197 Pelta, Magnetic Resonance in Chemistry 36 (1998) 706

37. Thanks

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