Bio-NMR

1. Bio-NMR June 30-July 1, 2008

2. Overall goals • Be able to set up a sample for quality data collection • Be able to use vendor-supplied parameter sets and pulse programs • Be able to run the instrument safely • Improve understanding of how the instrument works

3. Probe design • Special purpose probes emphasize a few aspects of performance at the expense of others • General purpose probes are compromises • Everything gets more difficult at high field • Everything gets more difficult in a cryoprobe

4. Probe capabilities/performance • Field homogeneity/lineshape • Radio frequency coils • Gradient coils • Temperature control

5. Probe design issues • Magnetic susceptibility considerations • Acoustic ringing • Background signals • Mechanical stability and robustness • Weight • Disassembly for cleaning and repair

6. Practical RF coils • Helmholtz shape • Limitations on the uniformity of excitation inside the coil, restriction of field outside the coil, degree of inversion • Coil’s magnetic susceptibility must be masked

7. Coil layout • Generally limited to two coils, often four RF channels • “Triple” inverse probes for bio • H on the inside • C and N (or C and P) outside • D either inside or outside

8. Electrical performance • Tuning is adjustment of circuit resonant frequency to NMR frequency • Matching is impedance matching of the circuit to the pulse amp output and preamplifier input • Dielectric mostly changes tuning, ionic strength mostly changes matching • Ions in solution degrade the electrical performance

9. Gradient coils • Gradients used for coherence selection, artifact control, shimming, diffusion • Reproducibility of gradient pulses is of highest importance • Linearity is not perfect • Z is most important, x and y useful • For shimming, x and y room temp shim coils can be ramped

10. Temperature control • Thermocouple cannot be placed inside RF coils • Tall, thin tubes maximize temperature gradients; very complex behavior • Watch out for cancelling a temperature gradient with a homogeneity gradient • Convection cells in the tube are really bad • Temperature limits fixed by hardware

11. Factors influencing S/N • Number of nuclei present • Efficiency of the coil • Coil’s quality factor (Q) • Sample geometry • Ionic strength of the sample

12. Power handling • Risk from high power is excessive voltage • Risk from long pulses/decoupling is coil/sample heating • Cooling is critical during decoupling

13. Ways of describing an RF pulse • Voltage V • Power, W (P=V2/R) • Decibels, dB • Duration, us/ms/s • Phase/phase cycle • Purpose • Tip angle, degrees (proportional to time x voltage x profile) • Field strength, Hz (gammaH1/2pi)

14. Decibel scale for voltage and power • Log scale • Can be either relative or absolute • dB=20 log(Vin/Vout) (proportional to pw) • dB=10 log(Pin/Pout) • Change 90 degree pulse width by 10x =20 dB • Change 90 degree pulse width by 2x = 6dB

15. Effects of pulses at various power levels • Excitation (full power, usually 90) • Inversion (full power, 180) • Refocusing (full power, 180) • 13C, 15N decoupling (down 10-12 dB) • Spin lock (down 15-20 dB) • 1H decoupling (down 18-20 dB) • Water flipback (down 30-35 dB) • Water presaturation (down 55-75 dB)

16. Varian and Bruker dB scales • Varian runs - (min) to 63 (max) in coarse steps of 1 dB, plus a fine power adjustment in arbitrary DAC units • Typical rectangular pulses 55-60 • Typical presaturation 6 • Bruker runs 120 (min) to –6 (max) settable to at least 0.01 dB • Typical rectangular pulses 0- -6 • Typical presaturation 55

17. Pulse shaping • Software allows creating pulses of arbitrary shape • Pulses can be optimized for a particular property (at the expense of others) • Pulses can be selective for certain frequencies (a.k.a. structural type), or made very broadband • Calibrating pulses at one power/shape can be used to predict calibration for other powers and shapes

18. The most important thing to remember about what shaped pulses do! • The shape of a pulse in the time domain and its profile in the frequency domain are related by a Fourier transform • Exponential: Lorentzian • Rectangular: Sinc • Gaussian: Gaussian

19. Profiles of shaped 90 pulses

20. Profiles of shaped 180 pulses

21. Predefined power levels/shapes for triple resonance • Nitrogen and deuterium: • 90 degree • decoupling • Proton: • 90 degree • Decoupling • Tocsy spinlock • Roesy spinlock • Flipback • Presat

22. 90 degree rectangle Decoupling Adiabatic inversion Adiabatic refocusing Alpha or carbonyl selective 90 (also “time reversed”) Even more selective 90 Alpha and carbonyl 90 (also “time reversed”) Alpha/carbonyl 180 Alpha or carbonyl decoupling Alpha and carbonyl decoupling Adiabatic decoupling (two power levels) Pulse shapes for carbon Duplicate lists for carbon as decoupler and direct observe

23. Shaped gradient pulses • Original Bruker scheme: sine pulses, very soft • Original Varian scheme: rectangular pulses, very hard • New schemes: Bruker=smoothed square; Varian=WURST; very similar in practice

24. RF channel configurations • Our 400’s and new 500: 2 • Existing 500: 3 • 600 and 800:4 • For proton observe experiments, channel 1 is proton • Channel 2 is carbon • Channel 3 is nitrogen • Channel 4 is deuterium

25. Spectrometer evolution • 20th century spectrometers: frequency synthesizer sources, lots of components needed to adjust the gating, amplitude, and phase of pulses • 21st century spectrometers: direct digital synthesis, far fewer components, each running their own software (with their own bugs)

26. RF amplifier considerations • All modern spectrometers use linear amplifiers • Gain typically 60 dB • Maximum power 100-500 watts • Long linear range, compression near top end of power output • Software incorporates some type of correction for nonlinearities

27. Safe power handling • Probes come with a specification sheet • Newer Bruker probes have a memory chip containing information on limits • Software power controls • Current ones do not cover every scenario • Cryoprobes handle power much more efficiently, but can accept less; overall, pulse widths are longer than in conventional probes

28. Streamlining setup of many experiments • A pulse sequence using a consistent nomenclature for pulses and delays • A parameter set that contains all the information specific to the experiment, e.g. sweep widths, delay lengths, gradient powers • Calibration of local probes and amplifiers in a probe file • Merge the three and go

29. The Varian way—BioPack • Pulse and delay names managed by George Gray at Varian • Parameters accessed through drop down menus • A probe file called HCN is managed out of the gHNCO parameter set • An extensive set of auto-calibration and auto-acquisition tools

30. The Bruker way—rpar, getprosol • Rpar = read global experiment parameters (and pulse sequence) • Getprosol = read probe and solvent specific parameters • Probe file managed through edprosol • Every pulse sequence references a relations file

31. Pulse programming issues • Varian pulse programs written in a C-like language (possibly with a visual editor) • Extensive use of flags to control optional features • Tends to minimize the number of programs • Bruker pulse programs written in a machine-like language • Most features are hard coded in the sequence • Tends to maximize the number of programs • Bruker-supplied programs follow a specific naming convention

32. Vendor-specific differences • Varian favors fewer gradient pulses, Bruker favors more • Varian favors States-TPPI scheme for phase sensitive detection, Bruker favors echo-antiecho • Bruker programs mostly written by one person, Wolfgang Bermel; Varian programs come from many sources; several customer labs contribute

33. Sample specific parameters • The proton 90 degree pulse in an inverse probe is very sensitive to the composition of the sample because proton is on the inner coil • The carbon and nitrogen pulses are much less sensitive, because they share the outer coil

34. Accessing help and documentation • Varian • Global manuals on Help menu • Every BioPack sequence has a text manual • Bruker • Global manuals on Help menu • NMR Guide and Encyclopedia • Terse but helpful hints in the comments section of the pulse program

44. Power limits • High power is limited such that proton pw90 >6, carbon >15, nitrogen >35 in Varian probe • Proton >8, carbon >16, nitrogen >40 in Bruker probe • Decoupling power limits are reduced as at/aq is increased

45. Duty cycle considerations • Maximum duty cycle is 8% with conventional garp decoupling at ~10 watts • To a first approximation, duty cycle is at/(d1 + at) • “Canned” parameters will be safe with d1=1 and at~0.08 sec • Modified parameters may not be safe • Refer to the document “Cp800.pdf”

46. Lower power decoupling • Varian prefers adiabatic decoupling on nitrogen and carbon channels at about 60% of the average power of garp decoupling • Bruker parameterizes many experiments for adiabatic carbon decoupling, some fast (“BEST”) nitrogen experiments for garp4 decoupling at 25% of normal power (pulse width apx. doubled, power down 6 dB) • Garp4 works for carbon too and we can use simultaneous garp4 decoupling for both nitrogen and carbon

47. Temperature limits and sample positioning • Cold probes have a limited temperature range (Bruker 10-60, Varian 0-50) • Actual temperature is a few degrees colder than thermocouple reading • Temperature gradients a bigger problem than in warm probes • Bruker probes have a solid, and FRAGILE, bottom

48. Radiation damping and water suppression • What works in warm probes at low field may not work, or make things worse, in cold probes at high field • Recalibrate flipback pulses • Zero out any trim pulses in hsqc type experiments