Transcript NMR Training for Slightly Advanced Users

NMR Training
for Advanced Users
Huaping Mo
Summer, 2009
http://www.pinmrf.purdue.edu
http://people.pharmacy.purdue.edu/~hmo/index.htm
Overview
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Our facility
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Our capabilities
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800MHz, 600MHz, 500 MHz (2), 400 MHz(2) and 300MHz (4)
Observe almost all NMR active nuclei
Detect as low as 1 mM to as high as 100 M proton concentrations
VT from -80°C to above 100°C*
Many problems can be converted to and addressed by NMR observables!
Our expertise
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PINMRF: Purdue Interdepartmental Facility (http://www.pinmrf.purdue.edu)
Staff include John Harwood (director, D.Sc), Huaping Mo (associate director, Ph.D.), Jerry
Hirschinger (engineer) and other members.
Various 1D, 2D and 3D experiments
Novel pulse sequence development and simulations
Structural determinations
Quantitative analysis
NMR hardware trouble shooting and repair
Our track record
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Mo, Harwood et al. J. Magn. Reson., in press; Ye, Mo et al. Anal. Chem. 2009; Mo & Raftery
Anal. Chem. 2008; Bai, Mo, Shapiro, Bioorg Med Chem, 2008; Mo & Raftery J. Bio. NMR,
2008; Mo & Raftery J. Magn. Reson. 2008
Acknowledged in a number of publications (Thank you!)
Determined the structures for a series of novel natural products / metabolites
A schematic Diagram for NMR
RF
properties;
I(q)
pulse
calib/angle
q
excitation
t90
Mz
q
probe
tuning
network
raw FID
observation
receiving
efficiency
NMR signal:
transmitter
spectral
analysis
spectral
processing
R
Shims; chemical
shift; coupling;
line-shape;
relaxation;
exchange; h, c & V
pulse
sequence
ADC
FID
FT
receiver
g(RG)
receiver
gain
function
A = A0 * h * c * V *R *sin(q)*I(q)*g(RG)
© Huaping Mo, 2009
Insider scoops: a systematic
approach for NMR quantitation
•
Receiving efficiency (Mo et al. J. Magn. Reson. in press)
– conceptually, it is similar to extinction coefficient in UV spectroscopy in
characterizing how efficient a unit magnetization can be detected
– 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
•
Receiver gain function (to be submitted)
– how much gain is actually achieved by the receiver
•
Solvent signal offers a universal and robust concentration internal standard
(Mo & Raftery Anal. Chem. 2008)
– Normalized NMR signal size is strictly proportional to the concentration for a
given sample, regardless how concentrated or dilute the sample is
– Unit magnetization generates the same amount of total NMR response in the RF
coil, which is indifferent to chemical shift or line-shape
– No need to make additional internal or external standard
Common Misconceptions
• You need to prepare either an internal or external
concentration reference for quantitative NMR
• You need a chemical shift referencing compound in a
hetero-nuclear spectrum
• A compound has to be in a deuterated solvent to be
observed by NMR
• You need to separate the compound to find out if it is
right or how much is there
Outlines for this talk
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Basic preparations for NMR: safety, sample, lock, shim and tune
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Understanding NMR: excitation and observation
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RF pulse calibration
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Data acquisition: sweep width, carrier freq. and # of scans etc.
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NMR observables:
– Chemical shift, scalar couplings, NOE and relaxations
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Chemical shift referencing
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Introduction to basic 2D's
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Simulations for spin systems, pulses and sequences
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Basic operation demonstrations
Sample
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Basic requirements:
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Rule #1: for Bruker NMR spectrometers, the NMR
tube insert cannot exceed max depth (19mm or
20mm) from the center of the RF coil
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Proton observe: 1 uM or more
Cabron observe: 1 mg or more
Volume: 300 ul or more
Solvent: most solvents will do;10% deuterated solvent is
sufficient for locking
Spectrometers can be run without lock (deuterium).
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.
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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!
RF coil
~18mm
Coil Center
 20mm
Samples of smaller volumes
• Follow rule # 1 and then rule #2
• Shimming might be challenging due
to air/glass and air/solution
interfaces
– Bubbles should be avoided
• Consider Shigemi tubes
• Be careful with spinning
– Non-spinning is recommended for
volume ~ 300 ul or less
– 500 ul is sufficient for a regular tube
300ul
400ul
500ul
Sensitivity for smaller volumes
• For a regular tube, larger
volume helps shims/lineshape, but not sensitivity
directly
1.2
1
relative senstivity
• 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
0.8
0.6
0.4
0.2
0
200
400
600
volume (ul)
800
1000
Tune and match the probe
•
Why do we need tune and match
(wobb)?
•
Only higher fields (500, 600 and 800
HMz) in our facility need tuning
– Lower field probes have been tuned!
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•
Most of the time only proton 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 setting the numbers
to the pre-set values
RF reflection
– For best pulses and sensitivity
Carrier frequency
tune
match
Frequency
Significance of tuning/matching
• Shorter 90° pulse
– More efficient use of RF power
1.1
• Protects transmitter
• Better sensitivity
– Reciprocity: if excitation is efficient,
then detection is equally efficient
NMR signal size
– More uniform excitation in high power
0.9
0.7
• Potentially quantitative:
– NMR signal size is about inversely
proportional to the 90° pulse length
0.5
8
10
12
90 degree pulse length (ms)
14
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
Lock
• Lock depends on shims: bad shims make bad lock
– Initialize shims 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 auto-lock if multiple deuterium
signals exist)
Shims
• The goal of shimming 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) + lock field
• Shimming can be done either manually or by gradient, which can be
very efficient and consistent if done properly
• Sample spinning may improve shims
– However, spinning-side bands appear
• Recommendations:
– 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
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
Lock: avoid high lock power
Bad lock
Good lock
Lock power okay
Lock power too high
unstable and lower lock signal
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
– Small (<1%) or no spinning side-bands
Shim by line-shape
Plot made by G. Pearson, U. Iowa, 1991
z4 too small
z4 too big
make z4
smaller first
Understanding NMR
•
Modern NMR spectrum is an
emission spectrum
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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 xyplane magnetization
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
in
out
coarse attenuator
fine attenuator
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
Proton pulse calibration
• Most hard (highest power) 90° pulses are typically from 5 us to 20
us.
• High power pulse for proton (or other heteronuclei if sensitivity is
sufficient) is directly calibrated
– 360° method (not quite sensitive to radiation damping or relaxation)
– 180° method
90º
90º
q
First pulse with 2 us; 2 us increment
180º
360º
450º
270º
180º
270º
360º
NMR observables
• Chemical shifts
– Expressed in ppm; reflects chemical environment
• Scalar couplings
– Expressed in Hz; causing splitting / broadenings in 1D
– 2D or nD bond correlations
• NOEs / relaxation / line-shapes
– Reveals distance/conformation information
• Peak size
– Potentially useful in quantitative analysis
Chemical shifts
• Reflects chemical
environment:
– Ring current effect
• Outside: high ppm
• Inside: low ppm
– Effect of electron
withdrawing groups
• Donating: low ppm
• Withdrawing: high ppm
Chemical shifts of solvents/impurities
Gottlieb et al. JOC 1997
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
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
Scalar coupling (J)
1
1:1
1:2:1
1:3:3:1
AB system
“roofing”
JAB
JAB
dA
dB
Scalar coupling:
• proportional to gyromagnetic ratio
• through bond/electrons
• split into 2nI + 1 lines.
Scalar coupling: simulation helps!
pro-chiral!
8Hz
a
12Hz
b
8Hz
These are not
impurities!
ssb
Ha and Hb are not exactly equivalent, with
chemical shift difference of 0.025ppm
Observed at 300 MHz
simulated
Example:
satellites and spinning side-bands
6.6 Hz; 29Si satellites; 2.3% each
ssb: 20 Hz or
multiple of 20Hz
from center
~120 Hz; 13C satellites; 0.55% each
TMS
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-width offers an estimate of T2
Line-shape
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Full Width at Half Maximum is 1/(pT2*) Hz, with
T2* as apparent spin lattice relaxation time
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Magnetic inhomogeneity (shim) can increase
FWHM (2l) or distort the line-shape (reduce T2*)
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T1 > T2 > T2*
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Small molecules
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FWHM (2l)
1H:
T1 ~ T2 in the order of seconds
seconds to tens of seconds; even longer if no
proton attached (CO and quaternary)
13C:
-20
Large molecules
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-15
-10
-5
0
5
10
15
20
of f se t ( H z )
1H:
T1 ~ T2 hundreds of mini-seconds or shorter
13C: seconds or sub-seconds
Lorentzian: A(w)= l / (l2 + (w-w0)2)
2l=1/(pT2*)
integration
Lorentzian peak Integration
-n (2l)
0
n (2l)
n
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)
(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
Examples of (N)H resonance
14N
decoupling
800MHz
500MHz
300MHz
no 14N decoupling
Hz
NH4Cl in DMSO.
Triplet is due to 14N coupling (52 Hz)
Urea in water (6% D2O)
Direct observe: 31P, 13C or 15N
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19F, 31P
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13C
and 13C can be observed directly on all PINMRF
300 and 400 MHz instruments (please follow local
PINMRF instructions)
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 preamplifier
– BBO tuning is needed for both proton and observed nucleus
– Double check filters if re-cabled
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!
Dipolar coupling: NOE
• NOE depends on
correlation time
(molecule size) and
resonance frequency
NOE + 1
• NOE does not always
enhance the
observed signal
3
2
13C
1
31P
1H
0
-1
0
2
4
6
8
10
15N
-2
H
P
-3
C
N
-4
w htc
Molecule size
Temperature
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
Improving sensitivity: receiver gain
• Receiver gain needs to be
maximized which frequently
requires good water
suppression
• Excessive acquisition time
may end up with spending
time collecting noise and
down-grade signal-to-noise
ratio
S/N
• Avoiding excessive large
receiver gain (for signal
clipping)
sensitivity vs receiver gain
(arx300; chloform signal)
0
100
200
300
receiver gain
400
500
Improving sensitivity: Ernst angle
• Acquire more scans in a
given amount of time
cos a = exp(-Tc/T1)
• Increase concentration
and lower the solvent /
salt amount
sensitivity
• Use Ernst angle a for
excitation:
Tc/T1
Pulse angle (degrees)
Missing a carbonyl carbon
presumably due to insufficient
relaxation
R'
O
OR
About 5 mg in CD3OD. 2800 scans (~4 hrs)
OH
NH
OH
O
?
Missing a carbonyl
Solution: Use H2O
Why it works:
R'
O
OR
OH
NH
OH
O
• 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).
1D acquisition for very long hours
•
Helpful
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Split long experiments into smaller blocks and save
data regularly (multiple data can always be summed
if needed): multizg
Dissolve the compound in water (H2O) might be
helpful (shorter relaxation time)
Lower sample temperature may help
Not helpful
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Save several days’ data into one single FID
Use 300 ul or less volatile solvent
Chemical shift referencing
• 1H chemical shift can be readily
referenced by the solvent signal or
TSP/TMS
• Heteronuclei can be indirectly referenced,
by PROTON chemical shift!
– No need to have a separate internal or
external reference
From 1D to 2D
FT
1D
w1
t2
time domain
frequency domain
FT(t2)
2D
FT(t1)
w1
t1
t1
t2
time domains
w2
w2
frequency domains
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
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
2D NMR essentials: acquisition
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Proton tuning and matching
Calibration of proton (90 degree) pulse length
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Modest receiver gain
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•
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Larger td1 improves resolution in the indirect dimension
Rarely exceeds 512 (except occasionally in COSY
Detection method in the indirect dimension
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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)
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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)
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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
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
2D processing
•
Window functions
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Zero filling
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Typically double data points in each dimension
Phasing
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Allow FID to approach zero at the end of the acquisition time
Sine bell functions with some shifts are recommended most of the time
Indirect dimension 0th and 1st order corrections are recommended in the pulse
program. If not, use 0 for both to start with.
First data point is typically scaled by 1 or 0.5, depending on the pulseprogram
Direct dimension’s 1st order phase is rarely more than 50 degrees. 0th order can
be anywhere from 0 to 360 degrees
Phase in the 2D mode for best appearance
Referencing
–
–
Can be done by picking a known resonance in the spectrum
by (external) protons
HSQC: a Block Diagram
• Magnetization transfer pathway:
F1(H) -> F2(X) -> F2(X,t1) -> F1(H) -> F1(H,t2)
90
H
180
1/4J
1/4J
1/4J
1/4J
acq
180
90
t1/2
X
t1/2
dec
INEPT
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.
HMQC or HSQC
codeine
Magnitude HMQC (9 mins)
Phase sensitive HSQC (18 mins)
Easy set up and slightly higher sensitivity
Better resolution
adapted from acornnmr.com
HMQC and HSQC comparison
• HMQC
• HSQC
– Fewer pulses
– More tolerant to pulse
mis-calibrations
– More pulses
– Less tolerant to pulse
mis-calibrations
– Allows homonuclear
(proton) coupling in
the indirect dimension
– No homonuclear
(proton) coupling in
the indirect dimension
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
Pulse sequence:
the heart and soul of NMR
label
;zggpwg
;this is a bruker sequence
prosol relations=<triple>
#include <Avance.incl>
#include <Grad.incl>
"d12=20u"
1 ze
2 30m
d1
10u pl1:f1
p1 ph1
50u UNBLKGRAD
p16:gp1
d16 pl0:f1
(p11:sp1 ph2:r):f1
4u
d12 pl1:f1
(p2 ph3)
4u
d12 pl0:f1
(p11:sp1 ph2:r):f1
46u
p16:gp1
d16
4u BLKGRAD
go=2 ph31
30m mc #0 to 2 F0(zd)
exit
ph1=0 2
ph2=0 0 1 1 2 2 3 3
ph3=2 2 3 3 0 0 1 1
ph31=0 2 2 0
;comments for parameters…
Delay only; be very careful
with critical command in a
labeled line
Delay
90x
1H
180x
90-x
90-x
define f1 power level
90° pulse on f1
G
Gradient pulse
Shaped 90° pulse
Acq. and go to label 2
Write to disc. And go
to label 2
Phases
On-res: dephased by two gradients
Off-res: refocused by two gradients
Where Things are:
Bruker File Structure
•
•
•
•
•
•
•
•
•
User NMR data
Pulse programs
Gradient programs
Shaped pulses
decoupling
Frequency(f1) lists
Parameter sets
Shim sets
Macros
/u/data/username/nmr
/u/exp/stan/nmr/lists/pp
/u/exp/stan/nmr/lists/gp
/u/exp/stan/nmr/lists/wave
/u/exp/stan/nmr/lists/cpd
/u/exp/stan/nmr/lists/f1
/u/exp/stan/nmr/par
/u/exp/stan/nmr/lists/bsms
/u/exp/stan/nmr/mac
Gradients
• Homospoil gradients
– Size of duration may not matter much
– Stronger ones tend to clean up unwanted
magnetization better
• Gradient echoes:
– Exact ratios between multiple gradients must
follow
– Diffusion loss must be considered for small
molecules, especially during long echoes
• Log of signal size is proportional to -g2g2d2D
Simulations
• Can be easily performed for pulses, spinsystems or pulse sequences
• Save experimental time
• Enhance our understanding of NMR
• Most frequently used for shaped pulses
Shaped Pulse: What and Why
• What
– Narrow sense: amplitude modulation only, while
phase is constant
– Broad sense: amplitude and phase modulation
• Why
– To achieve perturbation over a certain frequency
range (uniform and selective)
• Narrow bandwidth: shaped pulse. e.g. Gaussian
• Wide bandwidth: adiabatic pulse
How is Shaped Pulse Different
• Composite pulse is typically a block of square pulses with constant
phases
– Pulse integration does not correlate with pulse angle
– Pulse calibration come from individual component
• Adiabatic pulse sweeps frequency (phase has strong time
dependence)
– Pulse integration does not correlate with pulse angle
– Pulse calibration depends on sweep range, and somewhat on
adiabaticity too
• Simple shaped pulse can be calibrated by integration
– Caveat: a 180° pulse is not necessarily twice of a 90° pulse
– Some shaped pulses are good for 180° inversions (z -> -z) while others
are good for 90° excitations (z -> x/y)
Shaped Pulse Examples
• Square pulse: simplest shaped pulse;
good for simple hard excitation
• Gaussian and Sinc: good selectivity;
for proton
Gauss
• Gaussian cascade: G4, G3, Q5 and
Q3; for carbon
–
–
–
–
G4 for excitation
G3 for inversion
Q5 for 90°
Q3 for 180°
Sinc1
G4
G4: four Gaussian lobes
Choosing Shaped Pulses
•
Define the goal
– excitation, inversion or refocusing
– length or power level
• Rule of thumb: bandwidth is ~ 1/P360 or RF strength (for square pulses)
– shape
•
Power requirement
– peak power may not exceed certain level
•
Length requirement
– Be aware of probe limit on length in case of high power
– While longer pulses tend to have better selectivity, relaxation / scalar coupling
may limit pulse length
•
Run pulse simulation and calculation
– Bandwidth needs to be first satisfied
– Simulated frequency profile is to have top-hat behavior
– Phase needs to be linear in the region of interest
Shaped Pulse Calculation
• Rule of thumb:
– 6db change in power results two fold change in pulse length
DdB = 20 log (P90/P90ref)
– e.g. 10us @0db => 20us @6db for the sample pulse angle
• For a shaped pulse with a imperfect linear amplifier,
DdB = 20 log (P90*shape_integ/P90hard*comp_ratio)
Modern spectrometers have comp_ratio close to 1
• Adiabatic pulses require different treatments
Example: Setting up a Sinc Pulse
• Within xwinnmr, launch shape
tool by typing “stdisp” or from
menu
• Within shape tool, choose
shapes -> sinc. Change lobe
number to 1 and click “OK”
• On the left is the amplitude
profile (sinc shape) and
(constant) phase is shown on
the right
1 means one sinc lobe
Example: a Sinc Pulse (cont’d)
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Within shape tool, choose analyze ->
integrate pulse. Make necessary updates.
In this particular case, we assume the
reference is 9.5 us @1.5db and you wish to
calculate for 1000us 90 degree pulse. Then
click OK
The power level is calculated as 35.8db
compared with the reference. Click “seen”
If satisfied, you can save this shaped pulse
under /u/exp/nmr/stan/lists/wave/.
Go back to xwinnmr->ased, and update the
sinc1 shaped pulse as pulse length of 1ms,
and power level to be 35.8 + 1.5 (since
reference 9.5us is @ 1.5db) = 37.3db
If needed, the shaped pulse power can be
fine tuned by gs, or a careful calibration
Pulse Simulation
• Within shape tool, choose
analyze -> simulate.
Update the length as
1000us and rotation angle
as 90 (for sinc1 we just set
up). Click “OK”.
• A new Bloch module will
show default (x,y) profile
for excitation. Click on z to
view z profile.
z
Pulse Simulation (cont’d)
• If you decide that the starting
magnetization is x, you can click
(in Bloch module) “calculate”>”excitation profile”. Change
initial Mx to 1 and Mz to 0. Click
“OK” and then the excitation
profile will be updated.
• If you wish to examine trajectory
(how a magnetization at a given
frequency responds to the sinc1
pulse), you can click “time
evolution”, and update initial
values etc (may not allow too
many steps). Click “OK”.
Demo
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Sample preparation; Shigemi tube
Lock and shim
Tune and match
Calibration of 90° pulse
Water suppression
Calculation / simulation of pulses
Set up 1D and 2D’s: mutizg; COSY and HSQC
Data processing: addition and subtraction
Data presentation: xwinplot
Backup slides
Safety
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Personal safety
– Cryogens: do not lean on or push magnets
– Cryoprobes: avoid contact with transfer line
– Magnetic and RF hazards
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Instrument safety
– Know the limits of instruments and be conservative
– Probe limits: avoid excessive long decoupling, hard pulses or their equivalents
– Double check pulse program and parameters for any non-standard new
experiment. Pay special attention to power switch statements in the
pulseprogram
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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
Xwinnmr: Spectra addition/subtraction
• Operations on processed data (spectra)
have no impact on raw data
• edc2: define 2nd dataset (to be compared)
and 3rd dataset (to save results into)
• dual: allow comparison