Transcript Bio-NMR - University of Kansas

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
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
Field homogeneity/lineshape
Radio frequency coils
Gradient coils
Temperature control
Probe design issues
Magnetic susceptibility considerations
Acoustic ringing
Background signals
Mechanical stability and robustness
Weight
Disassembly for cleaning and repair
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
Generally limited to two coils, often four
RF channels
“Triple” inverse probes for bio
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H on the inside
C and N (or C and P) outside
D either inside or outside
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
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
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
Number of nuclei present
Efficiency of the coil
Coil’s quality factor (Q)
Sample geometry
Ionic strength of the sample
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
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
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
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 runs - (min) to 63 (max) in
coarse steps of 1 dB, plus a fine power
adjustment in arbitrary DAC units
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Typical rectangular pulses 55-60
Typical presaturation 6
Bruker runs 120 (min) to –6 (max)
settable to at least 0.01 dB
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Typical rectangular pulses 0- -6
Typical presaturation 55
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 shape of a pulse in the time
domain and its profile in the frequency
domain are related by a Fourier
transform
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Exponential: Lorentzian
Rectangular: Sinc
Gaussian: Gaussian
Profiles of shaped 90 pulses
Profiles of shaped 180 pulses
Predefined power levels/shapes
for triple resonance
Nitrogen and deuterium:
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90 degree
decoupling
Proton:
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90 degree
Decoupling
Tocsy spinlock
Roesy spinlock
Flipback
Presat
Pulse shapes for carbon
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)
Duplicate lists for carbon as decoupler and direct observe
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
Our 400’s and new 500: 2
Existing 500: 3
600 and 800:4
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For proton observe experiments, channel 1
is proton
Channel 2 is carbon
Channel 3 is nitrogen
Channel 4 is deuterium
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
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
Probes come with a specification sheet
Newer Bruker probes have a memory
chip containing information on limits
Software power controls
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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
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
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
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
Varian pulse programs written in a C-like
language (possibly with a visual editor)
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Extensive use of flags to control optional features
Tends to minimize the number of programs
Bruker pulse programs written in a machinelike language
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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
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
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
Varian
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Global manuals on Help menu
Every BioPack sequence has a text manual
Bruker
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Global manuals on Help menu
NMR Guide and Encyclopedia
Terse but helpful hints in the comments
section of the pulse program
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
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
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
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
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