Basic Biomolecular Training Course

Experimental Biomolecular NMR Training Course

Day 1 Spectrometer Basics: magnet, probe, configuration, tuning, locking, shimming,

gradient shimming.

Day 1,2 1D spectra: determining H2O lineshape, calibrating 1H pulse widths, acquiring H2O
presaturation spectra.

Water suppression: calibrating water flip back pulses, acquiring flipback-watergate

1D spectra.

Day 2,3 Homonuclear 2D: Flipback, watergated NOESY spectra.

Heteronuclear 2D: Watergated and Sensitivity-Enhanced H-N HSQC.

Constant-Time, H-C HSQC.

Day 4 Heteronuclear 3D: NOESY-HSQC, HNCA


z
y
x
B0
A superconducting solenoid
is used to generate a
homogeneous magnetic
field B0 along the vertical
(z) axis. A sample containing
NMR active nuclei (e.g. 1H,
13C) is placed in the center
of the field.
Basic NMR Spectroscopy: the Magnet
n↓
γ B0
ν0 = 
2π
n↑
The nuclear spins
interact with B0 and,
based on their spin states,
distributed into
energy levels which are
separated by the Larmor
frequency ν0, proportional
to B0 and nuclear
gyromagnetic ratio (γ)
(for 1H, B0 = 11.7 T
⇒ ν0 = 500 MHz). For
+ve γ, the lower energy
"up" spins are in slight
excess over "down" spins,
at equilibrium.
z z
n↑ ν0
≡ ⇒
x y
n↓
Equivalently, the spins
may be regarded as
precessing around B0
with a frequency ν0, with
the n↑ and n↓ spins
pointing in opposite
directions along the
z axis, and the xy
components distributed
randomly.
Meq
x y
Meq ∝ (n↑ − n↓)
At equilibrium, this
results in the
development of a bulk
nuclear magnetization
(Meq) along the
z axis (parallel to B0),
proportional to the
excess of lower energy
spins.

z
B0
y
x z
B1 cos (2πν 0 t)
A transmitter generates
a radio frequency (rf)
field oscillating at
the Larmor ("resonance")
frequency in rf coils
surrounding the sample
in the probe. The direction
of the associated oscillating
magnetic field B1 is
perpendicular to B0.
Basic NMR Spectroscopy: the Transmitter
n↓ Meq
B1
≡
x y
B1 M0
n↑
The rf field induces This is equivalent to a
transitions between rotation of the bulk nuclear
the nuclear spin magnetization Meq from the
energy levels. z axis toward the xy plane.
Complete saturation of
spin populations corresponds
to a 900 rotation.
z
B0
x y
Mt
The transmitter is turned off,
and the magnetization vector
precesses freely in the xy
plane at an angular frequency
v0. A B1 field which induces
a 900 rotation is called a 900
pulse.
 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

The freely precessing magnetization vector M0

traces a spiral trajectory as it experiences "friction"
in the form of relaxation processes (T1,T2) which
return it to equilibrium (Mxy decays to zero,
Mz grows to Meq ).
In the xy plane, the footprint of the precessing
magnetization induces exponentially decaying
sinusoidal voltages in the rf coil, along the x and
y axes: Free Induction Decay (FID)
 Basic NMR Spectroscopy: the Digitizer & Computer
Real
ADC1

Computer
My(t) = A cos (2πν 0 t) e−t/T2
Imaginary

ADC2

Mx(t) = A sin (2πν 0 t) e−t/T2

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

Set the temperature

ensures rf pulses
are optimally absorbed Tune the probe
by the sample
interchangeable

corrects for magnetic Lock the field

field drift over time

ensures uniform
magnetic field over Shim - manual + gradient
sample volume
repeat until
lineshape's
OK
Analyze H2O or HDO lineshape

Determine 1H 900 pulsewidth

Determine H2O/HDO frequency

Acquire a 1H 1D spectrum
 Field-Frequency Locking

The spectrometer monitors the 2H signal from the deuterated solvent to

(a). Achieve spatial homogeneity of the B0 field over the sample volume (shimming).
(b). Smoothen out time-varying fluctuations in the B0 field (locking).

M0 sin (2πν 0 t) e−t/T2

Mt monitored for locking

y ν0
M0

Im (Dispersive)
x
A dedicated part of the spectrometer continuously
monitors the absorptive and dispersive lineshapes
of the 2H signal.

M0 cos (2πν 0 t) e−t/T2

monitored for shimming

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

The error voltage is not

Lock frequency B0 computed instantaneously,
variation
but after integrating the
lock signal over a period
Field Correction of time (> 1 s).
Tx

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

oscillations reduce as we approach

field not at resonance resonance

adjust z0

turn lock on

lock signal levels out at resonance

 NMR Probe
Characteristics of an NMR probe (tuned LC circuit) :

frequency ω = 1/√LC L = inductance,

quality factor Q = ωL/R C = capacitance,
impedance Z = R + i[ ωL − 1/(ωC)] R = resistance

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

These are adjusted using variable capacitors − tuning and matching:

Probe Base
L

decoupling tuning rodcoaxially

Cmatch
coil
matching rod mounted Ctune
observe
coil

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)

Configuration for 13C (or X) Observe , 1H Decouple

From Tx
(chan 1)

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

From Tx From Tx From Tx From Tx

xmtr xmtr xmtr xmtr

50 50
To
filter
filter Probe
To
probe Probe probe probe
probe probe probe
atten chan atten chan

1/4 wave 1/4 wave

length cable length cable
1H/19F 1H/19F
preamp preamp
40-400 MHz 40-400 MHz
BB preamp BB preamp

output tune output output tune output

output output

To Rx To Rx To Rx To Rx

H1 or F19 Observe X Nucleus Observe

 Tuning Configuration for H1
From Tx
From Tx

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

To Probe To Probe To Probe

To Rx
Ensure attenuation is 8 or 9 and
channel selection is 0. The display
is dark and LED is flashing red.
Press the + key to select
channel 1. The display turns on
and displays reflected power
(arbitrary units).
Adjust matching and tuning
capacitors to minimize reflected
power (< 10). DO NOT FORCE
TUNE/MATCH RODS IF
THEY ARE PEGGED OUT !!

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

To Rx Disconnect cable (LED stops Press the (−) key to select

flashing) and reconnect to channel 0. The display turns off
filter. but LED continues to blink.
 Spatial Magnetic Field Inhomogeneity

intense,narrow, symmetric

The signal height is the

sum of all these contributions. The resonance is broader
short,broad, distorted
If λ is small, the peak is intense than the intrinsic line-width
and narrow. Shimming line λ, and the line-shape
reflects the field
inhomogeneity profile

The spectrum
consists of contributions
from each of these
spin isochromats

Every part of the sample ∆v

resonates at the same frequency:
Different parts of the
ν(z) = γB0/(2π)
sample resonate over
a range of frequencies
The linewidth (λ) of each resonance
λ (∆ν) due to the field
is dictated solely by physico-
inhomogeneity:
chemical properties of the molecule
(relaxation, exchange, etc) ν(z) = γ B(z)/(2 π)

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

Field inhomogeneities along x and y directions also need

B(z) − B'(z) = B0
Ideally, a perfect B'(z) field will completely
cancel the field inhomogeneity and
yield a perfectly homogeneous field, B0.
to be shimmed out. Individual shims are not independent
and interact with one another. Higher order z shims are
"contaminated" by lower order shims.
B(x,y,z) = B(z) + c1 x + c2 y + c3 xz + c4 yz + c5 xy + …
There are 28 shims on our 500 MHz spectrometer
(type dgs in vnmr).

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−= −γb z3 ν0 = γB0 ν+= γb z3 ν+= γb z4 ν0 = γB0 ν+= γb z4

ν− ν0 ν+ ν0 ν+

b > 0, b < 0 b>0 b<0

Manual Shimming

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

ν(−z) = −γ G z ν(0) = γ B0 ν(+z)= γ G z

ν(−z) ν(0) ν(+z)

A gradient in Bo in the Z direction is achieved with an
antihelmholtz type of coil. Current in the two coils flow in
opposite directions creating a magnetic field gradient
between the two coils. The B field at the center
of one coil adds to the Bo field, while the B field at the
center of the other coil subtracts from the Bo field.

From: The Basics of NMR, J. Hornak

http://www.cis.rit.edu/htbooks/nmr/ ∆ν ∝ L (length of sample)
 Pulsed Field Gradient (PFG): A linear gradient G applied for a brief period of time (τ)

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).

The inhomogeneity can then be removed by subtracting a counteracting field B'(z) = B0 − b1 z − b2 z2 − b3 z3 + …

This forms the basis for Gradient Shimming.

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

B(z) Gz strong B(z) Gz strong

B0 B0
weak
z z

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')

tpwr too high

pw too long
gain too high

tpwr too low

pw too short
gain too low
 Step 2: obtain a shim map
The effect of varying different shims (z1, z2, etc) on these gradient profiles generate information regarding the
contribution of different shims to B(z). This is called a shim map:

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

white: field profile over sample length

red: best fit

φ(z)/τ ∝ Β(z)
ω(z) ∝ z

Final Iteration

magnetic field should be as

"flat" over the sample length
as possible
Acquiring Spectra
 Basic Startup Protocol

Set the temperature

ensures rf pulses
are optimally absorbed Tune the probe
by the sample
interchangeable

corrects for magnetic Lock the field

field drift over time

ensures uniform
magnetic field over Shim - manual + gradient
sample volume
repeat until
lineshape's
OK
Analyze H2O or HDO lineshape

Determine 1H 900 pulsewidth

Determine H2O/HDO frequency

Acquire a 1H 1D spectrum
 The RF Pulse B1

M0 νT = transmitter frequency (e.g. sfrq, dfrq)

z = frequency of rotating frame

B1 = amplitude of rf pulse (rf power ∝ B12)

M0
ν0 = ν T
x ≡ phase of rf pulse (axis of alignment of B1
vector in rotating frame)
y τP (pw)

B1 M0 = magnetization vector for a peak

(tpwr) on resonance (ν0 = νT)

B1 νT τP = duration of the rf pulse (pulse−width)

(sfrq)
x

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

recycle delay (d1) acquisition time

H2O Lineshape 1H 900 Pulse-Width H2O Frequency

1H carrier away from H O. 1H carrier on H O.

2 2 vary H2O presaturation frequency.

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

Set 1H carrier to H2O frequency pw

d1 at
Set power level

Array the pulsewidth (pw) to determine a null (zero signal)

- 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.

- A 1800 pulse requires long recycle (d1) delays between pulsewidths.

Best: determine a 3600 pulse: no radiation damping, short recycle delays. However, is affected by rf inhomogeneity.

radiation damping, signal distortion, long d1 measurement

window

270 360

10 90 180 2nd null

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

choose this one

intensity
presat frequency
End