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

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bio nmr


June 30-July 1, 2008

overall goals
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
probe design
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
probe capabilities performance
Probe capabilities/performance
  • Field homogeneity/lineshape
  • Radio frequency coils
  • Gradient coils
  • Temperature control
probe design issues
Probe design issues
  • Magnetic susceptibility considerations
  • Acoustic ringing
  • Background signals
  • Mechanical stability and robustness
  • Weight
  • Disassembly for cleaning and repair
practical rf coils
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
coil layout
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
electrical performance
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
gradient coils
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
temperature control
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
factors influencing s n
Factors influencing S/N
  • Number of nuclei present
  • Efficiency of the coil
  • Coil’s quality factor (Q)
  • Sample geometry
  • Ionic strength of the sample
power handling
Power handling
  • Risk from high power is excessive voltage
  • Risk from long pulses/decoupling is coil/sample heating
  • Cooling is critical during decoupling
ways of describing an rf pulse
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)
decibel scale for voltage and power
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

effects of pulses at various power levels
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)
varian and bruker db scales
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
pulse shaping
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
the most important thing to remember about what shaped pulses do
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
predefined power levels shapes for triple resonance
Predefined power levels/shapes for triple resonance
  • Nitrogen and deuterium:
    • 90 degree
    • decoupling
  • Proton:
    • 90 degree
    • Decoupling
    • Tocsy spinlock
    • Roesy spinlock
    • Flipback
    • Presat
pulse shapes for carbon
90 degree rectangle


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

shaped gradient pulses
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
rf channel configurations
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
spectrometer evolution
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)
rf amplifier considerations
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
safe power handling
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
streamlining setup of many experiments
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
the varian way biopack
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
the bruker way rpar getprosol
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
pulse programming issues
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
vendor specific differences
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
sample specific parameters
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
accessing help and documentation
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
power limits
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
duty cycle considerations
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”
lower power decoupling
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
temperature limits and sample positioning
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
radiation damping and water suppression
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