Documente Academic
Documente Profesional
Documente Cultură
Day 1,2 1D spectra: determining H2O lineshape, calibrating 1H pulse widths, acquiring H2O
presaturation spectra.
A superconducting solenoid The nuclear spins Equivalently, the spins At equilibrium, this
is used to generate a interact with B0 and, may be regarded as results in the
homogeneous magnetic based on their spin states, precessing around B0 development of a bulk
field B0 along the vertical distributed into with a frequency ν0, with nuclear magnetization
(z) axis. A sample containing energy levels which are the n↑ and n↓ spins (Meq) along the
NMR active nuclei (e.g. 1H, separated by the Larmor pointing in opposite z axis (parallel to B0),
13C) is placed in the center frequency ν0, proportional directions along the proportional to the
of the field. to B0 and nuclear z axis, and the xy excess of lower energy
gyromagnetic ratio (γ) components distributed spins.
(for 1H, B0 = 11.7 T randomly.
⇒ ν0 = 500 MHz). For
+ve γ, the lower energy
"up" spins are in slight
excess over "down" spins,
at equilibrium.
Basic NMR Spectroscopy: the Transmitter
z
B0
y
x z z
B0
n↓ Meq
B1
≡
B1 cos (2πν 0 t)
x y x y
B1 M0
n↑ Mt
A transmitter generates The rf field induces This is equivalent to a The transmitter is turned off,
a radio frequency (rf) transitions between rotation of the bulk nuclear and the magnetization vector
field oscillating at the nuclear spin magnetization Meq from the precesses freely in the xy
the Larmor ("resonance") energy levels. z axis toward the xy plane. plane at an angular frequency
frequency in rf coils Complete saturation of v0. A B1 field which induces
surrounding the sample spin populations corresponds a 900 rotation is called a 900
in the probe. The direction to a 900 rotation. pulse.
of the associated oscillating
magnetic field B1 is
perpendicular to B0.
Basic NMR Spectroscopy: the Receiver
z Mxy(∞) = 0
sin (2πν 0 t) e−t/T2
Mz(∞) = Meq
y
Mt
x
M0 y
cos (2πν 0 t) e−t/T2
Computer
My(t) = A cos (2πν 0 t) e−t/T2
Imaginary
ADC2
The NMR signal comprising of two FIDs representing x and y components of the precessing magnetization
is fed into two Analog to Digital Converters (ADCs), where they are sampled at equal time intervals.
The digitized data is transferred to a computer and stored as the "real" and "imaginary" components of a
complex time domain signal.
The combined data is subjected to a complex Fourier Transform (FT) to yield a frequency domain spectrum.
FT
ν0
ν0
Mxy(t) = My(t) + i Mx(t) = A e−i(2πν 0 t) e−t/T2
Re (Absorptive) Im (Dispersive)
Re Im
Basic Elements of an NMR Spectrometer
Probe + Sample
z
y
x
RF Transmitter
+ Pulse Programmer
Computer
Receiver
B0 Magnet + Digitizer
Preamplifier
Basic Startup Protocol
ensures rf pulses
are optimally absorbed Tune the probe
by the sample
interchangeable
ensures uniform
magnetic field over Shim - manual + gradient
sample volume
repeat until
lineshape's
OK
Analyze H2O or HDO lineshape
Acquire a 1H 1D spectrum
Field-Frequency Locking
(a). Achieve spatial homogeneity of the B0 field over the sample volume (shimming).
(b). Smoothen out time-varying fluctuations in the B0 field (locking).
y ν0
M0
Im (Dispersive)
x
A dedicated part of the spectrometer continuously
monitors the absorptive and dispersive lineshapes
of the 2H signal.
ν0
Re (Absorptive)
Field Drift Correction by Feedback Loop
B = B 0 + ∆B
V V
+∆V
ν0
ν0
−∆V
B = B 0 − ∆B
Positive error voltage +∆V at lock resonance Negative error voltage −∆V at lock resonance
frequency: negative field drift: increase frequency: positive field drift: decrease
current to compensate B by +∆B current to compensate B by − ∆B
Rx V
B0(t)
misadjusted lock phase
t ∆B
Proper locking (and shimming) requires the lock phase to be properly adjusted
so that the resonance frequency of the dispersion mode signal and the peak height
of the absorption mode signal - for shimming- are registered correctly.
The Locking Procedure
lock off
adjust z0
turn lock on
The probe is an LC circuit which must be tuned to the resonance frequency of the
nucleus being studied (impedance = R when ω = 1/√LC) and,
The impedance of the rf coil + sample must be matched to that of the source
(spectrometer) electronics. V2 Rcoil
Pcoil =
(Rcoil + Rsource)2
Probe Base
L
Probe Sensitivity
Schematic of an NMR Probe
Coil Length related to
(a). length of homogeneous region of B0.
From: Protein NMR Spectroscopy,
(b). length of RT shims active region.
Principles and Practice, (c). RF homogeneity.
J. Cavanagh, W.J. Fairbrother, Coil Filling Factor η (increases with η).
A.G. Palmer III, N.J. Skelton, Probe Q (increases with Q).
Associated Press, San Diego, 1996, page 98. Dielectric constant of sample (decreases with
increasing dielectric - reduces Q, tuning/matching is difficult).
Tuning using a Directional Coupler
L
D.C.
Transmitter
Cmatch
Frequency
Ctune
tuning
frequency
or Graphical
Reflected Power
Display
Power Meter
extent of tuning
Adjust tuning and matching capacitors extent of matching
(minimize separation)
to minimize reflected power in the (maximize dip)
power meter.
These adjustments are usually independent if you are close to optimal tuning/matching. Otherwise,
they are likely to interact with each other.
Q = ωR/∆ω
∆ω
V ωR = resonance frequency
∆ω = width corresponding
VR to V= VR/√2
ωR Low Q, High R
High Q, Low R Insensitive to changes in sample conditions,
High sensitivity around center frequency or changes in center frequency.
and changes in sample conditions, e.g.
Low ω, or lossy samples: R = heat, due to
dielectric constant.
high salt/conducting conditions
Configuration for 1H Observe 13C,15N (or X) Decouple
From Tx
(chan 1)
C13
xmtr To
H1
Probe Probe
a N15
probe probe filter A
1H/19F
preamp
b
13C filter
tune
B
15N filter
output output
To Rx From Tx
From Tx (dec 1,
(dec 2, chan 2)
chan 3)
To
Probe xmtr
C13
H1 probe
Probe filter probe
N15
1/4 wave
length
cable
BB preamp
tune
output
1H filter
15N filter
output
To Rx
From Tx From Tx
(dec 1, (dec 2,
chan 2) chan 3)
Preamplifier Housing Configuration
To Rx To Rx To Rx To Rx
xmtr To
xmtr To A Probe H1
a
Probe
AProbe H1
probe Probe
a Probe probe filter
probe probe filter
1H/19F
1H/19F preamp
preamp
b Disconnect cable from A
b tune
B
tune
B Cable connections for output output
(filter) and connect it to
output output H1 observe operations a (port labeled probe)
To Rx on the tuning interface.
To Rx 1
From Tx
xmtr To
A Probe H1
a
Disconnect cable from B Probe
probe probe filter
on H1 preamp (labeled
output) and connect it 1H/19F
preamp
to b on tuning interface b
(labeled tune output). B
tune
output output
To Rx
2
Procedure for Tuning H1/F19
From Tx
100 001
xmtr
a
probe probe - - a - - a - - a
c LED c LED c LED t
1H/19F
t t h
h h
preamp a 0 9 et a 1 9 et a 1 9 et
b n a n n a n n a n
B probe probe probe
tune + + + + + +
output output
From Tx
To
xmtr
Probe
a - - a - - a
probe probe c LED c LED
filter A t t
Disconnect tune out cable h h
1H/19F from b and reconnect to a 0 9 et a 0 9 et
preamp H1 preamp output (B). n a n a
n n
probe probe
b + + + +
tune
B
output output
intense,narrow, symmetric
The spectrum
consists of contributions
from each of these
spin isochromats
ν0 ν0
Idealized, perfectly Inhomogeneity of the
homogeneous static field static field along the
profile along the z axis: B0 B0 z axis, B(z).
B(z)
B(z)
B(z) = B0
z z
Shimming
z2
z z4
B(z) = B0 + b1 z + b2 z2 + b3 z3 + …
z3
The field inhomogeneity is expressed
as a polynomial in z (actually z6
z5
spherical harmonics, or angular
parts of atomic orbitals).
B'(z) = − b1 z − b2 z2 − b3 z3 − … Shimming
lock level
The user (or software) manipulates
current in shim coils surrounding
the sample to generate a counteracting Improvement in field homogeneity is assessed by observing
magnetic field B'(z). Adjusting a shim an increase in the height of the absorption mode display
z2, for example, modulates the b2z2 term. lock signal (lock level) during the shimming procedure
Shimming is a complex and often tedious process, but is key to obtaining good spectra.
Shimming
Lower order z shims (z, z2) affect the central part of the sample; higher order shims (z3−z6) affect the edges of the sample.
z2
z z4
z3
Odd order shims distort the lineshape symmetrically; Even order shims distort the lineshape asymmetrically
z3 z4
+b z3 +b z4
B0
−b z3 B0
ν− ν0 ν+ ν0 ν+
adjust shims to
maximize lock level
Pulsed Field Gradients
A gradient is a linear variation of the static field imposed on the sample, through gradient coils in the probe,
and strictly under user control, as opposed to variations in B0 field due to field inhomogeneity.
A field gradient is defined through it's slope as a function of distance. A z-gradient (Gz) is defined as:
B(z) = Gz z
As a result, each part of the sample tube resonates at a different frequency depending upon it's position along
the z axis (length of the tube):
ν(z) = γ G z
A spectrum measured in the presence of a field gradient establishes a correlation between frequency and position
z
Gz
B(z) B0
−G z
Gz
Gz Gz Gz
B0 B0 B0 B0 B0
The maximum gradient strengths are ~ G = 60−70 Gauss/cm. Most gradient probes are z-axis only.
The rapid turning on and off of the gradients lead to eddy currents in the probe and magnet
bore tube. These disturbances take several milliseconds to subside, during which observation of spectra is
impossible. Gradient probes contain actively shielded gradient coils which do not produce significant
eddy current outside the sample volume, and the gradient stabilization time is typically 50−250 µs.
Using special pulse sequences, PFGs can be used to map the magnetic field inhomogeneity along the z axis
i.e., determine the coefficients bn in B(z) = B0 + b1 z + b2 z2 + b3 z3 + … in a short period of time (few minutes).
A strong signal (usually solvent) is required for grad shimming: 1H for H2O solvent, 2H for D2O solvent.
Basic Principles of Gradient Shimming
B(z) Gz strong
B0
weak
Record spectra in presence of strong, user controlled gradient Gz, to amplify effects of relatively weak static field inhomogeneity B(z).
B(z) = 0 Gz = 0 B(z) Gz = 0
B0 B0
z z
10-50 Hz
perfectly homogeneous linewidth, in absence of any broadened line, in presence of static field homogeneity, linewidth
field gradients is tens of Hz.
~ 10 kHz
broadened line, in presence of a strong z-gradient and in presence of a strong z-gradient and static field homogeneity,
no static field homogeneity, linewidth is several kHz. effect of B0 inhomogeneity is amplified.
Gradient Shimming Basics
Step 1
Step 1: obtain a gradient profile points to remember
gradients not on
(pfgon='nnn')
z2
z1 z5
z6
z3
field
z4
sample length
This shim map shows that there is a large z2, z4 and z5 contribution to B(z) and relatively minor z1, z3 and z6 contributions
Based on these shim maps, the gradient shimming program can calculate how much to alter different shims in order to
remove their contributions from B(z). This is usually a quick, iterative procedure.
Step 3: carry out iterative gradient shimming
First Iteration
φ(z)/τ ∝ Β(z)
ω(z) ∝ z
Final Iteration
ensures rf pulses
are optimally absorbed Tune the probe
by the sample
interchangeable
ensures uniform
magnetic field over Shim - manual + gradient
sample volume
repeat until
lineshape's
OK
Analyze H2O or HDO lineshape
Acquire a 1H 1D spectrum
The RF Pulse B1
A radio frequency pulse at a transmitter frequency νT and amplitude (rf field strength) B1 may be represented
as a vector of length B1 in a reference frame rotating at νT.
The phase of the B1 field coincides with the x axis of the rotating frame.
The B1 field acts on the magnetization vector (M0) of nuclear spins with resonance frequency ν0.
If the transmitter frequency is on resonance, i.e. νT = ν0, M0 "sees" B1 as the only effective magnetic field for
the duration of the pulse (τP) (M0 does not have to be aligned along the z axis).
The RF pulse rotates the nuclear magnetization from the z axis towards the xy plane
z
B1 z τP z
γ
1
ω
M0
/2
M0 M0
1=
θ=
ω
θ=π
y
y τP θ y pw90 θ
τP = τ90
B1
B1 B1
x
x x
A 900 pulse (pw90) rotates M0 into the xy plane, giving rise to maximum signal.
A 3600 pulse (pw360) rotates M0 back to the z axis, yielding minimum signal.
H2O Lineshape, 1H 900 Pulse-Width, H2O Frequency
are determined using variations of the same pulse sequence
(t2pul on Varian, zg on Bruker)
pw
short pw (1-2 µs), low power. vary (array) pw. pw close to pw90.
long acquisition time (1-2 s). short acquisition time (0.25-0.5 s). short acquisition time (0.25-0.5 s).
no window functions before FT. window function before FT. window function before FT.
short recycle delay (d1) 1-2 s. long d1 for D2O samples (5 s). short recycle delay (d1) 1-2 s.
low receiver gain in H2O. low receiver gain in H2O. high receiver gain.
1H Pulse Width Determination
Load parameters
- nulls are easier to detect than maxima. A 1800 pw is the first null but results in signal distortion
and ADC overflow because of radiation damping in H2O samples.
Best: determine a 3600 pulse: no radiation damping, short recycle delays. However, is affected by rf inhomogeneity.
270 360
signal intensity
1st null
pulse-width
360 null
Determining the H2O Frequency
In H2O samples, the H2O line is too broad (> 30 Hz) to allow proper peak identification using the cursor
pw90
A better way is to presaturate the H2O signal prior to detection and array the saturation frequency. at
presat
The H2O frequency corresponds to a minimum in the H2O signal intensity (maximum saturation). 1−1.5 s
intensity
presat frequency
End