生物核磁共振波谱学
NMR in Biological Science
4,NMR EXPERIMENTS
THNMR
YAN
4.1 NMR HARDWARE
? Hardware Overview
? Magnet
? Field Lock
? Shim Coils
? Sample Probe
? RF Coils
? Gradient Coils
? Qadrature Detector *
? Digital Filtering *
? Safety
THNMR
YAN
plotter Computer
Pulse Programmer
A-D
Convertor
Audio
Filter
Detector
Conversion to
Audio Signal
RF Transmitter
and Amplifier Preamplifier
脉冲傅立叶变换核磁共振谱仪
To Sample
From
Sample
Magnet
Probe
THNMR
YAN
THNMR
YAN
THNMR
YAN
THNMR
YAN
脉冲傅立叶变换核磁共振谱仪
Normal rf pulses are square
shaped (in the time domain),
their excitation profile (in the
frequency domain) is given by
its FOURIER transform,the
sinc function (approximation
for b?180° ).
The excitation band width is
proportional to the reciprocal
of the pulse duration,pulses
must be short enough to keep
the "wiggles" outside the
range of interest.
Magnet
Most magnets are of the superconducting type,
THNMR
YAN
Why Magnet is so Important
? Boltzmann Statistics
? N-/N+ = e-E/kT = e-gB/kT,
? For 300 K one gets for 1H a population difference N--N+ of
B w DN
1.4T 60 MHz 9.9·10-6
11.7T 500 MHz 77 ·10-6
THNMR
YAN
Why Magnet is so Important
?Resolution ~ B0
?Sensitivity ~ (B0 )3/2
?Allignment ~ (B0 )2
?Relaxation ~ (complex)
THNMR
YAN
Nuclei (ppm)
A 1.89
B 2.00
C 2.08
Interaction J (Hz)
AB 4
BC 8
Field Lock
The field strength might vary
over time due to aging of the
magnet,movement of metal
objects near the magnet,and
temperature fluctuations.
?The field lock is a separate NMR
spectrometer within your spectrometer,
This spectrometer is typically tuned to
the 2D NMR resonance frequency.
?It constantly monitors the resonance
frequency of the 2D signal and makes
minor changes in the Bo magnetic field to
keep the resonance frequency constant,
?The 2D signal comes from the deuterium
solvent used to prepare the sample.
THNMR
YAN
Shim Coils
?Correct minor spatial inhomogeneities in the Bo magnetic
field,
?Inhomogeneities,caused by the magnet design,materials in
the probe,variations in the thickness of the sample tube,
sample permeability,and ferromagnetic materials around
the magnet.
Shim Coil
Z0 Z1 Z2 Z3 Z4 Z5
X XZ XZ2 XZ3 X2Y2 XY X3
Y YZ YZ2 X2Y2Z YZ3 Y3
XYZ
THNMR
YAN
Sample Probe
?Accepts the sample,sends RF energy into the sample,and
detects the signal emanating from the sample
?Contains the RF coil,sample spinner,temperature
controlling circuitry,and gradient coils.
Sample Temperature Bore
Sample Observe Coil
Matching Circuit
Preamplifier
Probe Side Arm
(connects to CCC through
Flexible transfer line)
THNMR
YAN
RF Coils
RF coils create the B1 field which rotates the net
magnetization in a pulse sequence,They also detect the
transverse magnetization as it precesses in the XY plane,
THNMR
YAN
where:
N = concentration of nuclear spins
wo = resonance frequency
Vs = volume of sample
Ts = sample temperature
h = coil filling factor
Qp = quality factor of probe
Tp = noise temperature of probe
TR = noise temperature of receiver
Sensitivity:
S
N
? K N w o
3 / 2
T S
h Q P V S
T P ? T R
Cryogenetic NMR Probe
THNMR
YAN
Cross-Section of Sample Area
Room Temperature Bore
5 mm Sample Tube
Observe Coil
25 K
300 K
Temperature Profile
THNMR
YAN
500 MHz S/N,4024:1!
(about 4590:1 with thin wall sample)
normal probe,about 900:1
5 mm 1H{13C/15N} PFG
Cryogenic Probe x 200
Safety
strong magnetic fields and cryogenic liquids
！！！ keep away from the magnet
? unauthorized entry
? all ferromagnetic items
? electronic circuitry,specifically pacemakers
? mechanical watches and some digital
watches
? credit cards and magnetic storage media
THNMR
YAN
4.2 Practical considerations
? Introduction
? Sample Preparation
? Sample Probe Tuning
? Determining a 90 Degree Pulse
? Field Shimming
? Phase Cycling
? Acquisition time and Truncation
? SNR Improvement
? Variable Temperature
? 1-D Hydrogen Spectra
? Integration
Sample Preparation
? deuterium lock solvent
? 5mm tubles,500 ml
? exact concentration
? well-resolved
? highly purified
? standard (reference)
? long-resistance
? N-15/C-13 labeled
THNMR
YAN
THNMR
YAN
Sample Probe Tuning
THNMR
YAN
Determining a 90 Degree Pulse
THNMR
YAN
Field Shimming
The purpose of shimming a magnet is to make the
magnetic field more homogeneous and to obtain
better spectral resolution,
Inhomogeneities
THNMR
YAN
THNMR
YAN
Acquisition Time and Truncation
a) not knowing f(t) from -? to ?,
but only from 0 to AQ (= length of acquisition time)
b) not acquiring a continuous,but a digitized FID
signal,with values known only for t = n DW
Spectral Width (SW) = 1/DW
digital spectral resolution = 1/AQ
Question,ideal acquisition time?
If too short ----> truncated
If too long ----> too much noise
THNMR
YAN
Truncation
Typical:
tmax > 3T2
THNMR
YAN
Spectra Width
Signals with absolute offset frequencies w out of the
Spectra Width (SW) will appear at wrong places in
the spectrum (folding)
Question,ideal SW?
If too narrow ---->?
If too wide ---->?
folding
THNMR
YAN
SNR Improvement
NS=1
NS=16
NS=8
NS=80
THNMR
YAN
LineWidth and T2
the linewidth depends on the decay rate of the FID:
lw1/2 = 1/pT2 (for the linewidth at half-height)
Since all signals are supposed to
decay exponentially in time,the
Lorentzian is the "natural" line
shape in the (frequency) spectrum
the faster the exponential decay,the
broader the Lorentzian.
THNMR
YAN
Typical 1D NMR Processing Steps
Sample preparation
Field lock
Probe tuning
Field shimming
Set 1D NMR parameters
Select pulse sequence (solvent suppression etc)
Time domain signal
Apodize
Zero-filling
Fourier transform
Phase correction
Baseline correction
Frequency domain function
Calibration,Integration,NMR spectra assignment
NMR Lock Solvents
? Acetone CD3COCD3
? Chloroform CDCl3
? Dichloro Methane CD2Cl2
? Methylnitrile CD3CN
? Benzene C6D6
? Water D2O
? Diethylether (DEE) (CD3CD2)2O
? Dimethylether (DME) (CD3)2O
? N,N-Dimethylformamide (DMF) (CD3)2NCDO
? Dimethyl Sulfoxide (DMSO) CD3SOCD3
? Ethanol CD3CD2OD
? Methanol CD3OD
? Tetrehydrofuran (THF) C4D8O
? Toluene C6D5CD3
? Pyridine C5D5N
? Cyclohexane C6H12 THNMR
YAN
THNMR
YAN
n = number of nuclei in the sample
NS = number of scans acquired
concentration
double sample concentration gives double
sensitivity,but to get the same result from
longer measuring time,one needs four times
the number of scans!
NMR in Biological Science
4,NMR EXPERIMENTS
THNMR
YAN
4.1 NMR HARDWARE
? Hardware Overview
? Magnet
? Field Lock
? Shim Coils
? Sample Probe
? RF Coils
? Gradient Coils
? Qadrature Detector *
? Digital Filtering *
? Safety
THNMR
YAN
plotter Computer
Pulse Programmer
A-D
Convertor
Audio
Filter
Detector
Conversion to
Audio Signal
RF Transmitter
and Amplifier Preamplifier
脉冲傅立叶变换核磁共振谱仪
To Sample
From
Sample
Magnet
Probe
THNMR
YAN
THNMR
YAN
THNMR
YAN
THNMR
YAN
脉冲傅立叶变换核磁共振谱仪
Normal rf pulses are square
shaped (in the time domain),
their excitation profile (in the
frequency domain) is given by
its FOURIER transform,the
sinc function (approximation
for b?180° ).
The excitation band width is
proportional to the reciprocal
of the pulse duration,pulses
must be short enough to keep
the "wiggles" outside the
range of interest.
Magnet
Most magnets are of the superconducting type,
THNMR
YAN
Why Magnet is so Important
? Boltzmann Statistics
? N-/N+ = e-E/kT = e-gB/kT,
? For 300 K one gets for 1H a population difference N--N+ of
B w DN
1.4T 60 MHz 9.9·10-6
11.7T 500 MHz 77 ·10-6
THNMR
YAN
Why Magnet is so Important
?Resolution ~ B0
?Sensitivity ~ (B0 )3/2
?Allignment ~ (B0 )2
?Relaxation ~ (complex)
THNMR
YAN
Nuclei (ppm)
A 1.89
B 2.00
C 2.08
Interaction J (Hz)
AB 4
BC 8
Field Lock
The field strength might vary
over time due to aging of the
magnet,movement of metal
objects near the magnet,and
temperature fluctuations.
?The field lock is a separate NMR
spectrometer within your spectrometer,
This spectrometer is typically tuned to
the 2D NMR resonance frequency.
?It constantly monitors the resonance
frequency of the 2D signal and makes
minor changes in the Bo magnetic field to
keep the resonance frequency constant,
?The 2D signal comes from the deuterium
solvent used to prepare the sample.
THNMR
YAN
Shim Coils
?Correct minor spatial inhomogeneities in the Bo magnetic
field,
?Inhomogeneities,caused by the magnet design,materials in
the probe,variations in the thickness of the sample tube,
sample permeability,and ferromagnetic materials around
the magnet.
Shim Coil
Z0 Z1 Z2 Z3 Z4 Z5
X XZ XZ2 XZ3 X2Y2 XY X3
Y YZ YZ2 X2Y2Z YZ3 Y3
XYZ
THNMR
YAN
Sample Probe
?Accepts the sample,sends RF energy into the sample,and
detects the signal emanating from the sample
?Contains the RF coil,sample spinner,temperature
controlling circuitry,and gradient coils.
Sample Temperature Bore
Sample Observe Coil
Matching Circuit
Preamplifier
Probe Side Arm
(connects to CCC through
Flexible transfer line)
THNMR
YAN
RF Coils
RF coils create the B1 field which rotates the net
magnetization in a pulse sequence,They also detect the
transverse magnetization as it precesses in the XY plane,
THNMR
YAN
where:
N = concentration of nuclear spins
wo = resonance frequency
Vs = volume of sample
Ts = sample temperature
h = coil filling factor
Qp = quality factor of probe
Tp = noise temperature of probe
TR = noise temperature of receiver
Sensitivity:
S
N
? K N w o
3 / 2
T S
h Q P V S
T P ? T R
Cryogenetic NMR Probe
THNMR
YAN
Cross-Section of Sample Area
Room Temperature Bore
5 mm Sample Tube
Observe Coil
25 K
300 K
Temperature Profile
THNMR
YAN
500 MHz S/N,4024:1!
(about 4590:1 with thin wall sample)
normal probe,about 900:1
5 mm 1H{13C/15N} PFG
Cryogenic Probe x 200
Safety
strong magnetic fields and cryogenic liquids
！！！ keep away from the magnet
? unauthorized entry
? all ferromagnetic items
? electronic circuitry,specifically pacemakers
? mechanical watches and some digital
watches
? credit cards and magnetic storage media
THNMR
YAN
4.2 Practical considerations
? Introduction
? Sample Preparation
? Sample Probe Tuning
? Determining a 90 Degree Pulse
? Field Shimming
? Phase Cycling
? Acquisition time and Truncation
? SNR Improvement
? Variable Temperature
? 1-D Hydrogen Spectra
? Integration
Sample Preparation
? deuterium lock solvent
? 5mm tubles,500 ml
? exact concentration
? well-resolved
? highly purified
? standard (reference)
? long-resistance
? N-15/C-13 labeled
THNMR
YAN
THNMR
YAN
Sample Probe Tuning
THNMR
YAN
Determining a 90 Degree Pulse
THNMR
YAN
Field Shimming
The purpose of shimming a magnet is to make the
magnetic field more homogeneous and to obtain
better spectral resolution,
Inhomogeneities
THNMR
YAN
THNMR
YAN
Acquisition Time and Truncation
a) not knowing f(t) from -? to ?,
but only from 0 to AQ (= length of acquisition time)
b) not acquiring a continuous,but a digitized FID
signal,with values known only for t = n DW
Spectral Width (SW) = 1/DW
digital spectral resolution = 1/AQ
Question,ideal acquisition time?
If too short ----> truncated
If too long ----> too much noise
THNMR
YAN
Truncation
Typical:
tmax > 3T2
THNMR
YAN
Spectra Width
Signals with absolute offset frequencies w out of the
Spectra Width (SW) will appear at wrong places in
the spectrum (folding)
Question,ideal SW?
If too narrow ---->?
If too wide ---->?
folding
THNMR
YAN
SNR Improvement
NS=1
NS=16
NS=8
NS=80
THNMR
YAN
LineWidth and T2
the linewidth depends on the decay rate of the FID:
lw1/2 = 1/pT2 (for the linewidth at half-height)
Since all signals are supposed to
decay exponentially in time,the
Lorentzian is the "natural" line
shape in the (frequency) spectrum
the faster the exponential decay,the
broader the Lorentzian.
THNMR
YAN
Typical 1D NMR Processing Steps
Sample preparation
Field lock
Probe tuning
Field shimming
Set 1D NMR parameters
Select pulse sequence (solvent suppression etc)
Time domain signal
Apodize
Zero-filling
Fourier transform
Phase correction
Baseline correction
Frequency domain function
Calibration,Integration,NMR spectra assignment
NMR Lock Solvents
? Acetone CD3COCD3
? Chloroform CDCl3
? Dichloro Methane CD2Cl2
? Methylnitrile CD3CN
? Benzene C6D6
? Water D2O
? Diethylether (DEE) (CD3CD2)2O
? Dimethylether (DME) (CD3)2O
? N,N-Dimethylformamide (DMF) (CD3)2NCDO
? Dimethyl Sulfoxide (DMSO) CD3SOCD3
? Ethanol CD3CD2OD
? Methanol CD3OD
? Tetrehydrofuran (THF) C4D8O
? Toluene C6D5CD3
? Pyridine C5D5N
? Cyclohexane C6H12 THNMR
YAN
THNMR
YAN
n = number of nuclei in the sample
NS = number of scans acquired
concentration
double sample concentration gives double
sensitivity,but to get the same result from
longer measuring time,one needs four times
the number of scans!
