NMR Training for Advanced Users

1. NMR Trainingfor Advanced Users Huaping Aug 18, 2008

2. Two Insider Scoops • Receiving efficiency • conceptually, it is similar to extinction coefficient in UV spectrospcopy; it characterizes how efficient a unit magnetization can produce a signal by a given NMR receiver • NMR signal size is proportional to receiving efficiency • Receiving efficiency can be pre-calibrated as a function of 90° degree pulse length • receiving efficiency is the same for all nuclei of the same type (indifferent to chemical shifts) in the same sample • Solvent signal offers a universal and robust concentration internal standard • Normalized NMR signal size is strictly proportional to concentration for a given sample, regardless how concentrated or dilute the sample is • Unit magnetization generates the same amount of total NMR response, which is indifferent to chemical shift or line-shape

3. Outlines • Basic preparations for NMR: safety, sample, lock, shim and tune • Understanding NMR: excitation and observation • RF pulse calibration • NMR observables: • Chemical shift, scalar couplings, NOE and relaxations • Introduction to basic 2D's • Simulations for spin systems, pulses and sequences

4. Safety • Personal safety • Cryogens: do not lean on or push magnets • Cryoprobes: avoid contact with transfer line • Magnetic and RF hazards • Instrument safety • Know the limits of instruments • Probe limits: avoid excessive long decoupling and long hard pulses or their equivalents • Be conservative • Double check pulse program and parameters for any non-standard new experiment • Data Safety • Back up data promptly and regularly • Data processing or manipulation has no impact on the raw (FID) data • Do not change parameters after data are acquired

5. Sample • Rule #1: for Bruker NMR spectrometers, the NMR tube insert cannot exceed max depth (19mm or 20mm) from the center of the RF coil • Longer insert than recommended may present problems for the probe, as well as cause frictions during spinning • Varian is more flexible in allowing longer insert • Rule #2: center of NMR sample should be as close as possible to the center of RF coil. • Normal sample needs to about 500 ul or slight more • Too much solvent is a waste! • Too little solvent may make shim difficult, but it does work! • 10% deuterated solvent is sufficient for locking RF coil 18mm Coil Center  20mm

6. Samples of smaller volumes • Follow rule # 1 and then rule #2 • Shimming might be challenging due to air/glass and air/solution interfaces • Consider Shigemi tubes • Be careful with spinning • Non-spinning is recommended for volume ~ 300 ul or less 300ul 400ul 500ul

7. Sensitivity for smaller volumes • Volume less than 300 ul may not offer additionally sensitivity improvement over that achieved by 300 ul, if the total amount of analyte is constant

8. Tune and match the probe • Only higher fields (500, 600 and 800 HMz) in our facility need tuning • Most of the time only proton channel requires tuning • Drx500-2 with BBO needs special attention • Proton always needs tuning • BB (used for 13C or 31P etc) channel needs tuning, by first dialing the numbers to the pre-set values Carrier frequency RF reflection tune match Frequency

9. Significance of tuning/matching • Shorter 90 degree pulse • More efficient use of RF power • Protects transmitter • More uniform excitation in high power • Better sensitivity • Reciprocity: if excitation is inefficient, then detection is equally inefficient • Potentially quantitative: • The product of NMR signal size is inversely proportional to the 90 degree pulse length

10. Recognizing Bruker probe types side view side view magnet 1H tuning/matching rods are labeled as yellow bottom view Do not touch those! Dials for broadband (BB) tuning/matching Tabulated values for BB tuning/matching BB Dialing stick BBO probe on drx500-2 TXI probe

11. Lock • Lock depends on shim: bad shim makes bad lock • Initialize shim by reading a set of good shims (i.e. rsh shims.txi) • Inheriting a shim set from previous users may present difficulties • Unusual samples (esp. small volumes) may need significant z1/z2 adjustments • Use “lock_solvent” or “lock” command • The default (bruker) chemical shift may appear as dramatically changed if the spectrometer assumes another solvent • Avoid excessive lock power • Lock signal may go up and down if lock power is too high due to saturation of deuterium signal • Apply sufficient lock power and gain so that lock does not drift to another resonance (this may happen by autolock if multiple deuterium signals exist)

12. Shim • The goal of shim is to make the total magnetic field within the active volume homogeneous (preferably <1Hz). Total magnetic field = static field (superconductor) + cryoshim (factory set) + RT shim (user adjust) • Shim can be done either manually or by gradient, which can be very efficient and consistent if done properly • Sample spinning may improve shim • However, spinning-side band appear • Recommendation: • Start from a known good shim set (by rsh on bruker or rts on varian). • Do not inherit shims from other users unless you know they’re good • Non-spinning and higher order (spinning) shims should not change dramatically from sample to sample for most applications

13. Lock: lock gain recommended not recommended higher lock gain Lower lock level due to lower lock gain may easily lose lock; change in lock level (during shimming) is less visible

14. Lock: avoid high lock power Bad lock Good lock Lock power okay Lock power too high unstable lock and lower level

15. Evaluate shims • Look for a sharp peak • No clear distortion • Full width at half height should be about 1 Hz or less for small molecules • Small (1% or smaller) or free of spinning side-bands • Check if peak distortions are individual or universal • Make sure that phasing is not causing peak distortions • Maximize the lock level • Higher lock level => better shim • Lock level does not drop significantly when spinning is turned off

16. Shim by line-shape Plot made by G. Pearson, U. Iowa, 1991 make z4 smaller first z4 too small z4 too big

17. Understanding NMR • Modern NMR spectrum is an emission spectrum • Equilibrium state • Magnetization is along +z axis • It is desired to have the largest +z magnetization prior to excitation • Excitation by a RF pulse • A projection of magnetization is made on xy plane • It is desired to have the largest xy plane project for observation • Observation • Precession of the projected xy- plane magnetization

18. in out coarse attenuator fine attenuator RF pulses • RF pulse manipulates spins • Important in excitation and decoupling • Defined by length, power and shape • RF power is expressed in decibels • Bruker • Power range: typically 0db (high power) to 120db (low power) • Varian: • Coarse power: typically 60db (high) to 0db (low); 1 db increment; absolute • Fine power: 4095 (high) to 0 (low); default is 4095; relative • e.g. 54.5db can be roughly achieved through setting coarse power to 55 and fine power to 3854

19. RF pulse calibration • Hard pulse (high power pulse) can be calibrated directly or indirectly • For best calibrations, pulses need to be on resonance (know the chemical shift or resonance frequency!) • Soft or shaped pulsed can be first calculated and then fine-tuned to optimum • Shapetool (by Bruker) or Pbox (by Varian) can be used for calculation and simulation • Be aware of possible minute phase shift (several degrees for soft pulses), which can be critical in water flip back or watergate

20. Proton pulse calibration • Most hard (highest power) 90° pulses are typically from 5 us to 20 us. • Direct observation for high power proton pulse calibration (or even for heteronuclei if sensitivity is sufficient) • 360° method (not quite sensitive to radiation damping or relaxation) • 180° method 90º First pulse with 2 us; 2 us increment 90º 180º 360º 450º 270º 180º 360º 270º

21. NMR observables • Chemical shifts • expressed in ppm • Scalar couplings • expressed in Hz • 2D or nD bond correlations • NOEs / relaxation / line-shapes • Peak size • Potentially useful in quantitative analysis

22. Chemical shifts • Reflects chemical environment: • Ring current effect • Outside of ring: high ppm • Inside: low ppm • Effect of electron withdrawing groups • Donating: low ppm • Withdrawing: high ppm

23. Chemical shifts of solvents/impurities Gottlieb et al. JOC 1997

24. Example: aliasing okay sw=16ppm aliased (from arx300) aliased from 0 ppm with phase distortion, because the peak is out of the “detection window” • Oversampled proton spectrum on higher fields (500 – 800 MHz) does not have the aliasing issue: peaks outside of sw will disappear

25. Spectral aliasing (cont’d) • In direct observe dimension, spectral aliasing is generally avoided by either increasing spectral width (sw) or moving center frequency (sfo1) • Sometimes the indirect detection dimension (in nD spectrum) may intentionally adopt aliasing to improve resolution in that dimension

26. 1 JAB JAB AB system “roofing” 1:1 dA dB 1:2:1 1:3:3:1 Scalar coupling

27. a b Scalar coupling: simulation helps! pro-chiral! 8Hz 12Hz 8Hz These are not impurities! Ha and Hb are not exactly equivalent, with chemical shift difference of 0.025ppm Observed (300 MHz) simulated

28. Example:satellites and spinning side-bands 6.6 Hz; 29Si satellites; 2.3% each spinning sideband; 20 Hz from center 120 Hz; 13C satellites; 0.55% each TMS

29. Dipolar coupling: NOE • NOE depends on correlation time (molecule size) and resonance frequency • NOE does not always enhance the observed signal 13C 31P 1H 15N Molecule size Temperature

30. NOE implication in Quantification • The observed nucleus should be free of interference from other nuclei • Pre-saturation in aqueous samples may not be appropriate for accurate quantification • Small molecules tend to gain signal size due to positive NOE from saturated water • Large molecules tend to lose signal size due to spin diffusion

31. Relaxation • T1 relaxation allows magnetization to recover back to +z axis • Nuclei with larger gyromagnetic ratios (resonance frequencies) tend to relax faster • 1H: 0.1 – 10 s (proteins have short T1’s) • 13C, 15N, 31P: much longer than 1H • Nuclei in a proton rich environment tend to relax faster • T2 relaxation contributes to the observed resonance line-shape • T2~T1 for small molecules • Line-shape offers an estimate of T2

32. Line-shape • Full Width at Half Maximum is 1/(pT2*) Hz, with T2* as apparent spin lattice relaxation time • Magnetic inhomogeneity (shim) can increase FWHM (2l) or distort the line-shape (reduce T2*) • T1 > T2 > T2* • Small molecules • 1H: T1 ~ T2 in the order of seconds • 13C: seconds to tens of seconds; even longer if no proton attached (CO and quaternary) • Large molecules • 1H: T1 ~ T2 hundreds of mini-seconds or shorter • 13C: seconds or sub-seconds FWHM (2) Lorentzian: A(w)=  / (2 + (-0)2) 2=1/(pT2*)

33. multiple chemical environments:chemical or conformational exchange • Fundamentally, chemical shift reflects chemical environment surrounding a nucleus’ • Multiple chemical environments may alter chemical shift or even cause significant peak broadening Fast exchange slow exchange Jin, Phy. Chem. Chem. Phys. (1999)

34. (N)H line-shape: influence of relaxation and scalar coupling In addition to chemical exchange, (N)H proton line-shape is also influenced by the coupled nucleus 14N JNH ~65 Hz Slow 14N relaxation (compared to JNH) medium14N relxation Fast relaxation this might be the very reason why CHCl3 proton appears as a singlet though JH-35Cl and JH-37Cl exist

35. Improving Sensitivity • More scans in a given amount of time • Use Ernst angle a for excitation: cos a = exp(-Tc/T1) • Increase concentration with less solvent / salt Tc/T1 sensitivity Pulse angle (degrees)

36. Improving sensitivity • Receiver gain needs to be maximized which requires good water suppression • Avoiding excessive large receiver gain (for signal clipping) • Excessive acquisition time end up with spending time collecting noise and down-grade signal to noise ratio

37. Missing a carbonyl carbonpresumably due to insufficient relaxation About 5 mg in CD3OD. 2800 scans (~4 hrs) ? Missing a carbonyl

38. Solution: Use H2O • Why it works: • Carbonyl 13C is reduced due to presence of a proton rich environment in H2O. • Potential intra-molecular hydrogen bond is weakened or broken, and decoupled from ring movement In H2O:D2O (1:1). 1400 scans (~2 hrs).

39. Direct observe: 31P, 13C or 15N • 19F, 31P and 13C can be observed directly on all PINMRF 300 and 400 MHz instruments (please follow local PINMRF instructions) • 13C can be observed on higher fields (500 MHz and above), without any cable change • Drx500-2 with BBO probe offers higher sensitivity for 31P, 13C, 15N and most other heteronuclei (19F excluded) • Observed nucleus needs to be cabled to x-broadband pre-amplifier • BBO tuning is needed for both proton and observed nucleus • Double check filters if re-cabled

40. Direct observe: 31P, 13C or 15N • Satellite peaks can frequently be indirectly observed in proton spectrum (so that we know the less sensitive heteronuclei are there to be observed directly!) • Decoupling of proton may improve signal by • Sharper peaks • NOE • Proton channel has to be tuned!

41. 1D acquisition for very long hours • helpful • Split long experiments into smaller blocks and save data regularly (multiple data can always be summed if needed) • Dissolve the compound in water (H2O) might be helpful (shorter relaxation time) • Lower sample temperature may help • Not helpful • Save several days’ data into one single FID • Use 300 ul or less volatile solvent

42. From 1D to 2D FT 1D t2 w1 time domain frequency domain FT(t2) FT(t1) 2D w1 t1 t1 t2 w2 w2 frequency domains time domains

43. 2D NMR • Correlate resonances through bond or space • COSY: coupling • Magnitude mode recommended. • 1 mg or less will do • Minutes to a couple of hours • TOCSY: coupling network • ~ 70 ms mixing time • 1 mg or less will do • An hour or longer • NOESY / ROESY: distance / NOE • Mixing time ranging from less than 100 ms (proteins) to 500 ms (small molecules) • 1 mg or more • Hours or longer • HSQC/HMQC: proton correlation to X, typically through one-bond scalar couplings (two or three bond correlation possible) • 1mg or less will do • An hour or longer • HMBC: proton correlation to X, through multiple bond scalar couplings • 1 mg or more • Hours or longer

44. 2D NMR • Resolve overlapping peaks • Resolution is provided largely through the indirect dimension • No need to have highest resolution in the direct detected dimension • Limit direct acquisition time to 100ms or less if heteronuclear decoupling is turned on • Lower decoupling power if longer acquisition time is needed • Change in experimental conditions may help

45. 2D NMR essentials: acquisition • Proton tuning and matching • Calibration of proton (90 degree) pulse length • Standard pulse lengths can be used if the solution is not highly ionic (< 50 mM NaCl equivalent) • All proton pulses are likely getting longer if the solution is ionic and/or the probe is not tuned • Modest receiver gain • rg about half of what rga gives or less • Carrier frequency (center of spectrum in Hz) and SW (sweep width) in both dimensions (avoid aliasing unless intended to) • Number of scans (NS) • The pulse program recommends NS (a integer times 1, 2, 4, 8 or 16) • Needs some dummy scans, especially with decoupling / tocsy • Number of increments in the indirect dimension (td1) • Larger td1 improves resolution in the indirect dimension • Rarely exceeds 512 (except occasionally in COSY • Detection method in the indirect dimension • Determined by the pulse program • Typically is either states (and/or TPPI) or echo-antiecho • Acquisition time (aq) less than 100 ms with decoupling • Modest gradients (cannot be more than the full power of 100% and typically less than 2 ms in duration) • Go through the pulse program if you really care

46. 2D processing • Window functions • Allow FID approach zero at the end of the acquisition time • Sine bell functions with some shifts are recommended most of the time • Zero filling • Typically double data points in each dimension • Phasing • Indirect dimension zeroth and 1st order corrections are recommended in the pulprogram. If not, use 0 for both • Direct dimension first order phase is rarely more than 50 degrees. Zeroth order can be anywhere from 0 to 360 degrees • Phase in the 2D mode to best appearance • Referencing • Can be done by picking a known resonance in the spectrum • Or referenced by (external) protons

47. Magnetization transfer pathway: F1(H) -> F2(X) -> F2(X,t1) -> F1(H) -> F1(H,t2) INEPT HSQC: a Block Diagram 90 180 1/4J 1/4J 1/4J H 1/4J acq 90 180 t1/2 t1/2 X dec States: =x and =y are acquired for same t1 and treated as a complex pair in Fourier transform. No need to change receiver phase TPPI: =x, y, -x and –y are acquired sequentially in t1, and receiver phase is incremented too. Real Fourier transform.

48. HMQC or HSQC codeine Magnitude HMQC (9 mins) Easy set up and slightly higher sensitivity Phase sensitive HSQC (18 mins) Better resolution adapted from acornnmr.com

49. HMQC Fewer pulses More tolerant to pulse mis-calibrations Allows homonuclear (proton) coupling in the indirect dimension HSQC More pulses Less tolerant to pulse mis-calibrations No homonuclear (proton) coupling in the indirect dimension HMQC and HSQC comparison

50. Data Presentation • Processed data can be readily viewed, manipulated and printed by xwinplot (wysiwyg) • Xwinplot can readily output .png, .jpg or .pdf files for publications or presentations • Files can be transferred through secure ftp