Basic NMR Concepts

Chi-Fon Chang
NMR Core Facility
2002/12/23
 NMR Spectroscopy
Where is it?

1nm 10 102 103 104 105 106 107

(the wave) X-ray UV/VIS Infrared Microwave Radio Frequency

(the transition) Electronic Transition Vibration Rotation Nuclear
(spectrometer) X-ray UV/VIS Infrared/Raman NMR
Fluorescence
 Before using NMR
What’s N, M, and R?

Properties of the Nucleus

Nuclear spin
Nuclear magnetic moments

The Nucleus in a Magnetic Field

Precession and the Larmor frequency
Nuclear Zeeman effect & Boltzmann distribution

When the Nucleus Meet the right Magnet

Nuclear Magnetic Resonance
‹ Properties of the Nucleus

Nuclear spin
• Nuclear spin is the total nuclear angular momentum quantum number. This is
characterized by a quantum number I, which may be integral, half-integral or 0.
• Only nuclei with spin number I ≠ 0 can absorb/emit electromagnetic radiation.
The magnetic quantum number mI has values of –I, -I+1, …..+I .
( e.g. for I=3/2, mI=-3/2, -1/2, 1/2, 3/2 )

1. A nucleus with an even mass A and even charge Z Æ nuclear spin I is zero
Example: 12C, 16O, 32S Æ No NMR signal
2. A nucleus with an even mass A and odd charge Z Æ integer value I
Example: 2H, 10B, 14N Æ NMR detectable
3. A nucleus with odd mass A Æ I=n/2, where n is an odd integer
Example: 1H, 13C, 15N, 31P Æ NMR detectable
Nuclear magnetic moments
Magnetic moment µ is another important parameter for a nuclei
µ = γ I (h/2π)

I: spin number
h: Plank constant 6.626*10-34 joul-sec
γ: gyromagnetic ratio (property of a nuclei)

1H: I=1/2 , γ = 267.512 *106 rad T-1S-1

13C: I=1/2 , γ = 67.264*106
15N: I=1/2 , γ = 27.107*106
‹ The Nucleus in a Magnetic Field

Precession and the Larmor frequency

• The magnetic moment of a spinning nucleus processes with a characteristic
angular frequency called the Larmor frequency ω, which is a function of r and B0

Remember µ = γ I (h/2π) ? J

Angular momentum dJ/dt= µ x B0

Larmor frequency ω=rB0

Linear precession frequency v=ω/2π= rB0/2π

Example: At what field strength do 1H process at a frequency of 600.13MHz? What would be the
process frequency for 13C at the same field?
Nuclear Zeeman effect
• Zeeman effect: when an atom is placed in an external magnetic field, the
energy levels of the atom are split into several states.
• The energy of a give spin sate (Ei) is directly proportional to the value of mI
and the magnetic field strength B0
Spin State Energy EI=- µ. B0 =-mIB0 r(h/2π)
• Notice that, the difference in energy will always be an integer multiple of
B0r(h/2π). For a nucleus with I=1/2, the energy difference between two states is
∆E=E-1/2-E+1/2 = B0 r(h/2π)

m=–1/2

m=+1/2

The Zeeman splitting is proportional to the strength of the magnetic field

Boltzmann distribution
• Quantum mechanics tells us that, for net absorption of radiation to occur, there
must be more particles in the lower-energy state than in the higher one. If no net
absorption is possible, a condition called saturation.
• When it’s saturated, Boltzmann distribution comes to rescue:
Pm=-1/2 / Pm=+1/2 = e -∆E/kT
where P is the fraction of the particle population in each state,
T is the absolute temperature,
k is Boltzmann constant 1.381*10-28 JK-1

Example: At 298K, what fraction of 1H nuclei in 2.35 T field are in the upper and lower states?

(m=-1/2 : 0.4999959 ; m=1/2 : 0.5000041 )

• The difference in populations of the two states is only on the order of few parts per
million. However, this difference is sufficient to generate NMR signal.
• Anything that increases the population difference will give rise to a more intense
NMR signal.
‹ When the Nucleus Meet the Magnet
Nuclear Magnetic Resonance

•For a particle to absorb a photon of electromagnetic radiation, the particle must

first be in some sort of uniform periodic motion

v
• If the particle “uniformly periodic moves” (i.e. precession)
at vprecession, and absorb erengy. The energy is E=hvprecession

•For I=1/2 nuclei in B0 field, the energy gap between two spin states:
∆E=rhB0/2π

∆E =hvphoton

• The radiation frequency must exactly match the precession frequency

Ephoton=hvprecession=hvphoton=∆E=rhB0/2π
ÆThis is the so called “ Nuclear Magnetic RESONANCE”!!!!!!!!!
Nuclear Magnetic Resonance Spectrometer
How to generate signals?

B0:

B1:
( )
‹ Magnet B0 and irradiation energy B1
B0 ( the magnet of machine)
(1) Provide energy for the nuclei to spin
Ei=-miB0 (rh/2π)
Larmor frequency ω=rB0
(2) Induce energy level separation (Boltzmann distribution)
The stronger the magnetic field B0, the greater separation
between different nuclei in the spectra
∆v =v1-v2=(r1-r2)B0/2π
(3) The nuclei in both spin states are randomly oriented around the z axis.
M z=M, Mxy=0
( where M is the net
nuclear magnetization)
B1(the irradiation magnet, current induced)
(1) Induce energy for nuclei to absorb, but still spin at ω or vprecession
Ephoton=hvphoton=∆E=rhB0/2π=hvprecession

And now, the spin jump to the higher energy ( from m=1/2Æm= – 1/2)
m= –1/2

m= 1/2

(2) All of the individual nuclear magnetic moments become phase

coherent, and the net M process around the z axis at α angel
M z=Mcosα
Mxy=Msinα.
What happen before irradiation
• Before irradiation, the nuclei in both spin states are processing with
characteristic frequency, but they are completely out of phase, i.e., randomly
oriented around the z axis. The net nuclear magnetization M is aligned statically
along the z axis (M=Mz, Mxy=0)
What happen during irradiation
When irradiation begins, all of the individual nuclear magnetic moments become
phase coherent, and this phase coherence forces the net magnetization vector M
to process around the z axis. As such, M has a component in the x, y plan,
Mxy=Msinα. α is the tip angle which is determined by the power and duration of
the electromagnetic irradiation.

z
α

Mo x
x
B1 Mxy
y
y
ωo
α deg pulse 90 deg pulse
What happen after irradiation ceases
•After irradiation ceases, not only do the population of the states revert to a
Boltzmann distribution, but also the individual nuclear magnetic moments begin to
lose their phase coherence and return to a random arrangement around the z axis.
!)
•This process is called “relaxation process” ( )
•There are two types of relaxation process : T1(spin-lattice relaxation) & T2(spin-
spin relaxation)
T1 (the spin lattice relaxation)
• How long after immersion in a external field does it take for a collection of nuclei
to reach Boltzmann distribution is controlled by T1, the spin lattice relaxation time.
( )
•Lost of energy in system to surrounding (lattice) as heat
( )
•It’s a time dependence exponential decay process of Mz components
dMz/dt=-(Mz-Mz,eq)/T1
T2 (the spin –spin relaxation)
•This process for nuclei begin to lose their phase coherence and return to a random
arrangement around the z axis is called spin-spin relaxation.
( random )
•The decay of Mxy is at a rate controlled by the spin-spin relaxation time T2.
dMx/dt=-Mx/T2
dMy/dt=-My/T2

dephasing
‹ Collecting NMR signals
•The detection of NMR signal is on the xy plane. The oscillation of Mxy generate a
current in a coil , which is the NMR signal.
•Due to the “relaxation process”, the time dependent spectrum of nuclei can be
obtained. This time dependent spectrum is called “free induction decay” (FID)

Mxy

time
( relaxation )
( T1 &T2)
•In addition, most molecules examined by NMR have several sets of nuclei, each
with a different precession frequency.

0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
t1 sec

Time (sec)

•The FID (free induction decay) is then Fourier transform to frequency domain
to obtain each vpression ( chemical shift) for different nuclei.

frequency (Hz)
Fourier transformation (FT)

FT

FT
NMR signals
• We have immersed our collection of nuclei in a magnetic field, each is processing with
a characteristic frequency, To observe resonance, all we have to do is irradiate them
with electromagnetic radiation of the appropriate frequency.

•It’s easy to understand that different nucleus “type” will give different NMR signal.
( v =ω/2π= γB0/2π ? γ v !! )

•However, it is very important to know that for same “nucleus type”, but “different
nucleus” could generate different signal. This is also what make NMR useful and
interesting.
•Depending on the chemical environment, there are variations on the magnetic field
that the nuclei feels, even for the same type of nuclei.
•The main reason for this is, each nuclei could be surrounded by different electron
environment, which make the nuclei “feel” different net magnetic field , Beffect
•Electron surrounding each nucleus in a molecule serves to shield that nucleus
from the applied magnetic field. This shielding effect cause the ∆E difference,
thus, different v will be obtained in the spectrum
Beff=B0-Bi where Bi induced by cloud electron
Bi = σB0 where σ is the shielding constant
Beff=(1-σ) B0
vprecession= (rB0/2π) (1-σ)
σ=0 Æ naked nuclei
σ >0 Æ nuclei is shielded by electron cloud
σ <0 Æ electron around this nuclei is withdraw , i.e. deshielded

HO-CH2-CH3

low high
field field
ωo
 HO-CH2-CH3

low high ω0=rBeffect

field ωo field

Ref: Some figures copy from the web page by Guillermo Moyna, University of the Sciences in Philadelphia
•Example of 1D : 1H spectra, 13C spectra of Codeine C18H21NO3, MW= 299.4

1H

13C
•Homo nuclear 2D NMR : a series of 1D

F(t2) FT F(w2)
d0 DW
SW (f2) = 1/(2*DW)

d0+IN0
DW

F(t1) FT F(w1)

d0+2*IN0
DW SW (f1) = 1/(2*IN0)

d0+3*IN0
DW
……

incremented
Set up 1D/homo Nuclear 2D

(Bruker AV system)
 Before go into NMR LAB

1. What’s the nucleus? Æ Which Prohead to choose?

AV500 in IBMS : 5mm TXI-Z (1H/15N/13C , with Z gradient) only
AV600 in IBMS: 5mm QXI-Z (1H/15N/13C /31P) (not ready to use yet)
AV600 in CHEM: 5mm BBO and TXI-Z (1H/15N/13C, with Z gradient)
DRX600 in IBMS: 5mm TXI-XYZ (1H/15N/13C , with XYZ gradient) and others
5mm : 1H , 1H/19F , BBO, TXI(1H/15N/13C) , TXI-Z (1H/3C/31P)
8mm : TXI (1H/13C/15N) 8mm with Z gradient
10mm: 1H , 1H /19F , BBO

2. What’s the goal? Æ Which type of experiment you need?

3. Which parameter set to choose? Æ Experiment List
4. How to optimize condition? Æ Experiment Guide
Definition of some AQ Commands & parameters
edc,new edit current data set or generate a new data set
eda.ased edit AQ parameters (eda: shows all, ased: shows required only)
rga auto optimize rg value
zg zero memory, and start to collect FID (go)
go start to collect FID and add signals to the previous memory
stop stop the active job ( just one job)
kill kill active job ( can choose several jobs)

o1.o2,o3 center frequency of the spectrum for nuclear at f1 channel (ex: 1H),
f2 channel (ex:13C), and f3 channel (ex:15N)
sw spectrum width ( 1 sw : F1 dimension, 2 sw: F2 dimension…….)
td number of points for FID collection( 1 td: F1 dimension, 2 td: F2 dimension….)
d1 relaxation time ( usually > 5* T1)
ns number of scan
ds dummy scan
rg receiver gain ( usually use the value calculated by rga)
new :New data set edc: Edit Current data set
 eda: Edit AQ parameter 2D
1D
ased : shows required parameters only
Definition of some AQ Commands & parameters
edc,new edit current data set or generate a new data set
eda.ased edit AQ parameters (eda: shows all, ased: shows required only)
rga auto optimize rg value
zg zero memory, and start to collect FID (go)
go start to collect FID and add signals to the previous memory
stop stop the active job ( just one job)
kill kill active job ( can choose several jobs)

o1.o2,o3 center frequency of the spectrum for nuclear at f1 channel (ex: 1H),
f2 channel (ex:13C), and f3 channel (ex:15N)
sw spectrum width ( 1 sw : F1 dimension, 2 sw: F2 dimension…….)
td number of points for FID collection( 1 td: F1 dimension, 2 td: F2 dimension….)
d1 relaxation time ( usually > 5* T1)
ns number of scan
ds dummy scan
rg receiver gain ( usually use the value calculated by rga)
 ppm
2D/homo
1D

F1 dimension !!!
1.5
2.0

2.5
3.0

3.5
center (o1) at 4.5ppm
4.0
sw=6ppm
4.5
4.5 4.0 3.5 3.0 2.5 2.0 1.5ppm
1.5ppm (=o1-sw/2) to 7.5ppm(=o1+sw/2)
F2 dimension !!!
F2: H (f1 channel)
O1(f1 channel):3ppm
2 sw(F2 dimension): 5ppm
2D/hetero F1: H (f1 channel)
O1(f1 channel): 3ppm
F2: H (f1 channel) 1 sw(F1 diemnsion): 5ppm
O1(f1 channel):8ppm
2 sw(F2 dimension): 5ppm
F1: N (f3 channel)
O3(f3 channel): 120ppm
1 sw(F1 diemnsion): 40ppm
 In the NMR LAB

1. The best condition for sample? Æ Temperature, sample position

2. The best condition for NMR? Æ Wobble : Tune & Match

Tune
Match
RCVR
0%

N S Absorption

100%
Frequency

3. The best condition for field? Æ Lock and shim

( Simple Operation Guide for Bruker AV System)
4. Ready to go! ( LAB work sheet)
edhead :Edit probe head lock

edte :Edit temperature

edasp :Edit AQ Spectrometer
2002 NMR Training Course
Basic Operation for Bruker AV System

Basic Data processing for 1D/2D

using xwinnmr

Yong-Li Pan

12/23 (Mon) 11:00-12:00

37
 Topic
• 1D NMR data process
• 2D NMR data process

38
1D NMR spectrum
data process

39
 Data Acquisition on Console
• 1D:Time domain data: FID
• 2D:Time domain data: SER
• FTP: XFTP, WinSCP
data , XwinNMR,
aurelia , data process

40
 Procedure of 1D data process
• Getting the data set: Search
• Setup parameter of Fourier transform and execute
• Phase
– Biggest
– Cursor
– MC
• Use edp menu or key in window function
command to improve the resolution of spectrum
• Integration
• Plot
41
42
 Getting the data set
• Time domain data:
– /Disk/data/user/nmr/Filename/Expno/fid
• Frequency domain data:
– /Disk/data/user/nmr/Filename/Expno/pdata/pro
cno/1r
– /Disk/data/user/nmr/Filename/Expno/pdata/pro
cno/1i
1D data file path
Getting the data set
File Search
1D layout with fid (raw data)
 Data Process
process

Key in
General parameter for processing
(edp)
• The edp command opens a dialog box
which allows you to set the parameters
required by the various commands in the
Process menu. The parameters are described
along with the commands which make use
of them.
Setting the size of the real
spectrum

SI (>=1/2 TD)
 Process Command
• General parameter setup (edp)
• Line broadening factor (lb)
• Exponential multiply (em)
• Manual window adjust (winfunc)
• Real spectrum size (si)
• Fourier transform (ft)
 Process Command
• Autophase correction (apk)
• Alternative autophase corr. (apks)
• Manual phase correction (phase)
• Special processing
– Phase w. constants PHC0,1 (pk)
– Magnitude spectrum (mc)
– Power spectrum (ps)
– Special window function:
• Gaussian (gm), Trapezoidal ™, Sine (sine), Squared sine (qsin),
Sinc (sinc)
 Process Command
• Baeline correction
– Automatic full spectrum (abs)
 Spectrum calibration
• H2O: MRS, 4.7ppm
• TMS: , 0 ppm
• DSS: , 0 ppm

Use “SR” (edp )

 Window function

• Line broadening factor (lb)

• em, gm:window function
• key in lb =0.3, type em to execute
• MC: , spectrum

• Manual adjust window function:

window function ,
spectrum resolution
Manual adjustment of window function
 Fourier Transform Command
• ft
• Sequential operations:
– em + ft (ef)
– em + ft +pk (efp)
– gm +ft (gf)
– gm +ft +pk (gfp)
 Phase correction
• 1DZG: line width, shimming
– Phase , H2O biggest, phase
• 1DZGPR: one pulse with f1 presaturation,
, cursor peak,
phase
• PH0: first order
• PH1: second order
1D NMR spectrum scale adjust
 Baseline Correction
„ Automatic baseline correction
-abs

Spectrum integration

• integration button,
,
Manual integration
2D NMR Spectrum
data process
 Procedure of 2D data process
• Getting the data set: Search
• Setup parameter of Fourier transform and
execute (xfb)
• Phase
• Baseline Correction
• Setup contour level (edlev)
• Plot
 Getting the data set
• Time domain data:
– /Disk/data/user/nmr/Filename/Expno/ser
• Frequency domain data:
– /Disk/data/user/nmr/Filename/Expno/pdata/procno/2rr
– /Disk/data/user/nmr/Filename/Expno/pdata/procno/2ri
– /Disk/data/user/nmr/Filename/Expno/pdata/procno/2ir
– /Disk/data/user/nmr/Filename/Expno/pdata/procno/2ii
2d NMR data file
2d NMR data
2D FID (SER)
edp
edp
edp
 Fourier transform
• SI (>=1/2 TD)
• xfb (execute Fourier transform both)
2D NMR Phase (Row)
2D NMR Phase (column)
2D spectrum after phase
 Baseline Correction
• Automatic baseline correction
– abs2, abs1

Contour level (edlev)

 XWIN-PLOT
• XWIN-PLOT
– Windows menuÆplot-editor
•
• (title)
• Peak Picking
•
•
 Start XwinPlot
Plot editor

xwinplot
XwinPlot 1D
2D NMR Plot
Experiments Set up for hetero nuclear 2D/3D

Chi-Fon Chang
NMR Core Facility
2002/12/24
 Hetero Nuclear 2D/3D NMR
Why are they?

1D 1H

Aromatic & Amide Aliphatic

R1 R2

N Cα CΟ N Cα CΟ ………………..

H H H H
2D Homo Nuclear
1H-1H

R1 R2

N Cα CΟ N Cα CΟ ………………..

H H H H
 2D Hetero Nuclear
1H-15N

1H
15N

R1 R2

N Cα CΟ N Cα CΟ ………………..

H H H H
3D Hetero Nuclear
1H-15N-1H
1H 1H

1H

15N

R1 R2

N Cα CΟ N Cα CΟ ………………..

H H H H
 3D 15N-edit TOCSY-HSQC
15N 1H-all

1H-NH
 3D CBCA(CO)NH 13Ca & Cb
15N

1H-NH
 Hetero Nuclear 2D/3D NMR
What are they?

Example of 2D Hetero Nuclear NMR: HSQC or HMQC

The HSQC or HMQC experiment allows to trace out directly bonded 1H-X pairs
via the large 1JHX coupling constant
2D 1H-13C HSQC

Strychnine
Solvent: CDCl3
Example of 2D Hetero Nuclear NMR: HMBC

The HMBC experiment allows to trace out longa-range (typically two- and three-
bonds away) 1H-X pairs via the small nJHX coupling constant.
 2D 1H-15N HMBC

2D 1H-13C HMBC

2D 1H-13C HSQC
 Example of 3D Double Resonance NMR: HCCH-TOCSY (side chain assignment)

The 3D HCCH-TOCSY experiment is specifically designed to correlate side-

chain aliphatic proton and 13C resonances via 1J(CH) and 1J(CC) coupling
constants. The experiment provides nearly complete assignments of all
aliphatic 1H and 13C resonances.

1H-13C
.
Example of 3D Double Resonance NMR:NOESY-HS(M)QC (through space)

The 3D NOESY-HSQC or NOESY-HMQC experiment is specifically designed to

obtain X-edited NOESY spectra of X-labeled biomolecules from which
homonuclear 1H-1H NOEs can be clearly assigned even in overcrowded regions

3D 15N-edit NOESY-HSQC
1H-15N

1H-13C
3D 13C-edit NOESY-HSQC
Example of 3D Triple Resonance NMR: for Nucleic Acids

The 3D HCP experiment is a 1H-13C-31P triple-resonance experiment specifically

designed to assign the ribose H3'/C3', H4'/C4' on the 5' side and, H4'/C4' and
H5',H5''/C5' resonances on the 3' side of the intervening phosphorus in 13C-labeled
nucleic acids.

3D HCP
1H-13C-31P
The 3D HCN experiment allows to obtain sugar-to-base correlations from the
H1'(sugar) to N1/N9 (base) via C1'(sugar) (HsCsNb experiment)

3D HCN
1H-13C-15N

HsCsNb

or non-exchangeable base proton assignments from the H6/H8 (base) to N1/N9 (base)
via C6/C8 (base) (HbCbNb experiment) in 13C,15N-labeled nucleic acids.

1H-13C-15N

HbCbNb
Example of 3D Triple Resonance NMR: for protein/peptide sequential

1H-13C-15N

•Sequential Assignment
•Chemical Shifts of Cα , Cβ
•HN & NH chemical Shifts
 1H-13C-15N

•Sequential Assignment
•Chemical Shifts of CO
•HN & NH chemical Shifts
 Hetero Nuclear 2D/3D NMR
How to get spectra?

• What’s the sample? Æ Which Prohead to choose?

AV500 in IBMS : 5mm TXI-Z (1H/15N/13C , with Z gradient) only
AV600 in IBMS: 5mm QXI-Z (1H/15N/13C /31P) (not ready yet)
AV600 in CHEM: 5mm BBO and TXI-Z (1H/15N/13C, with Z gradient)
DRX600 in IBMS:
5mm : 1H , 1H/19F , BBO, TXI(1H/15N/13C) , TXI-Z (1H/3C/31P),
TXI-XYZ (1H/15N/13C , with XYZ gradient)
8mm : TXI (1H/13C/15N) 8mm with Z gradient
10mm: 1H , 1H /19F , BBO

• What’s the goal? Æ Which type of experiment you need?

• Which parameter set to choose? Æ Experiment List
• How to optimize condition? Æ For users: Experiment Guide
•Experiment Name: 3D HNCO
•Experiment Type: Using Echo/antiecho , f1: H, f2:C, f3:N, F1(CO), F2(N), F3(H)
•Standard Parameter Set: std_3D_HNCO
•Pulse Program: hncogp3d.2
•AQ parameters to check
1H pulses
pl1 (high power, ex: 0db), p1(90deg at pl1), p2(180deg at pl1)
pl19 (low power for dipsi2,pcpd1), p26(90deg at pl19), pcpd1(90deg ,ex: 40-50usec)
sp1 (shape pulse power for Sinc.1000) , p11(pulse length for sp1, ex: 2m)
Others
o1 (for 1H), o2 (for 13CO), o3 (for 15N)
1 sw, 1 td (for F1 dimension, ie: 13C)
2 sw, 2 td (for F2 dimension, ie:15N)
3 sw, 3 td (for F3 dimension, ie:1H)
d1, rg, ns(=8*n), ds ( 16)

Users need to adjust parameters in “red” ( meaning of the parameter in “green” )

• How to optimize condition? Æ For operators : pulse program
(1) hard pulse calibration for hetero nuclei
(2) Shape pulse calibration for hetero nuclei
(3) other “uniform” (sample independent) parameters set up
ex: delays, decouple program, gradient program, frequency jump..……

;hncogp3d.2
;avance-version (01/05/09)
;HNCO …………( skips)

…………( skips) ;d21: 1/(2J(NH) [5.5 msec]

; F1(H) -> F3(N) -> F2(C=O,t1) -> F3(N,t2) -> F1(H,t3) ;d23: 1/(4J(NCO) [12 msec]

; ;d26: 1/(4J'(NH) [2.3 msec]

;on/off resonance Ca and C=O pulses using shaped pulse …………( skips)

;phase sensitive (t1) ;cpds1: decoupling according to sequence defined by cpdprg1

;phase sensitive using Echo/Antiecho gradient selection (t2) ;cpd3: decoupling according to sequence defined by cpdprg3

…………( skips) ;pcpd1: f1 channel - 90 degree pulse for decoupling sequence

;sp1: f1 channel - shaped pulse 90 degree (H2O on resonance) ;pcpd3: f3 channel - 90 degree pulse for decoupling sequence

;sp2: f2 channel - shaped pulse 90 degree (C=O on resonance)

;sp3: f2 channel - shaped pulse 180 degree (C=O on resonance) ;use gradient ratio: gp 1 : gp 2 : gp 3 : gp 4 : gp 5

;sp5: f2 channel - shaped pulse 180 degree (Ca off resonance) ; 60 : -40 : 10 : 80 : 8.1

;sp8: f2 channel - shaped pulse 90 degree (C=O on resonance)

; for time reversed pulse
 In the NMR LAB

1. The best condition for sample? Æ Temperature, sample position

2. The best condition for NMR? Æ Wobble : Tune & Match

Tune
Match
RCVR
0%

N S Absorption

100%
Frequency

3. The best condition for field? Æ Lock and shim

4. Ready to go! ( LAB work sheet)
edhead :Edit probe head lock

edte :Edit temperature

edasp :Edit AQ Spectrometer
gradshim : gradient shimming
Wobble all 3 channels: 15NÆ 13C Æ 1H

Step 1.1: (15N first) edasp to change setting and connection

Step 1.2: type wobble to wobble 15N, type stop after tune and match
Wobble all 3 channels: 15NÆ 13C Æ 1H

Step 2.1: (13C ) edasp again to change setting and connection

Step 2.2: type wobble to wobble 13C,click any key on “HPPR” after 13C
tuning and matching are done.
Wobble all 3 channels: 15NÆ 13C Æ 1H

Step 3.1: click “Chn Ç“ on “HPPR” , then wait until wobble on 1H pop out
(this might take 10-20 sec, please be patient! )

1H LNA

XBB19F

2H

Step 3.2: type stop after tuning and matching are done for 1H.
Step 3.3: edasp to change setting and connection for the experiment
 After the LAB work

NMR data processing software

XWINNMR (process NMR data on IRIX 6.X & Linux )
License only available for older version
nmrPipe (process NMR data on IRIX6.X & Linux)

NMR data analysis software

AURELIA (analyze NMR data on IRIX 6.X & Linux )
License only available for older version
nmrDraw (analyze NMR data on IRIX 6.X & Linux )
nmrView (analyze NMR data on IRIX 6.X & Linux )
Sparky (analyze NMR data on Linux)
 Thank you!!

[email protected]
3D data processing and Display

Xwinnmr processing 3D data

Sparky display processed NMR spectra

NMR core tsunai yu

Strategies for protein NMR studies
 edp «—Display Processing parameter

Size of the raw data

(SI ≥ 1/2TD)

Spectral calibration

Window function
1800/3

Phase correction
 edp «—Display Processing parameter

Strip start
# of raw data points of
strip transform
Col: 350
Xwinnmr
 edp «—Display Processing parameter

Some expt. need to

set as “TRUE”
3D processing
•Phase 2D plane (F1-F3 plane and F2 F3 plane)
eg. HNCACB F3(1H), F2(15N), F1(13C)
rule: phase 1H-15N(HSQC) and 15N-13C plane

xfb«ˆ select direction (13or 23) :

enter slice number [1, …..,96]:
enter new PROCNO for 2D data:
Xwinnmr
Xwinnmr
Xwinnmr
Xwinnmr
Xwinnmr
Xwinnmr
 F3(H) F2(C) F1(N)
13 plane PHC0 49.3 150.252
PFC1 -5.8 -100.400
23 plane PHC0 X1 58.249
PHC1 X2 -180.6
If X1 13 plane PHC0 value, X2 13 plane PHC1 value
Use the HSQC plane phase value (eg.13 plane in this case)
3D processing
•Applying the 3D Fourier Transform
tf3 no (processed 3D data in F3 dimension without imaginary data)
tf2 no
tf1 no

•Baseline correction
tabs3 (automatic baseline correction in the F3 dimension )
tabs2
tabs1
Sparky display processed NMR spectra

Adventage
•Using a graphical interface (in PC, linux, and unix)
•Work with 2-4 dimensional spectra
•Suitabe display with other process program
•Output suitable for other structure determination program
•Free license
Start sparky
•Under unix the command to run Sparky is “sparky”. The installation
locaton is /usr/local/sparky/bin.
•Under windows(95, 98,or NT) double clicking sparky.bat in
c:\progranfiles\sparky\bin c:\progranfiles\sparky\bin

Open spectra
Converting processed data to UCSF format by bruk2ucsf,
pipe2ucsf,vnmr2ucsf………
eg. in PC
c:\progranfiles\sparky\bin> bruk2ucsf d:\data\nie.310\12\pdata\1\3rrr
d:\sparky\data\noe.ucsf