Measuring Proton NMR Spectra

Measuring Proton NMR Spectra
‫בס''ד‬
Measuring Proton NMR Spectra
by Roy Hoffman and Yair Ozery 20th September 2010
This guide is intended for routine use on the 400 MHz spectrometer
The sections that are adapted for use with the 200 MHz spectrometer are framed.
The sections that are adapted for use with the 500 MHz spectrometer are in a dotted
frame.
This guide is intended for use with the spectrometers of the Chemistry Institute of the
Hebrew University. See chapter 4 in order to adapt it for use in other laboratories.
1
Contents
1) Summary of instructions for measuring a proton NMR spectrum
4
2) General description of the equipment
5
3) Routine experiments
7
a) Logging on
7
b) Creating a work file
7
c) Specifying the probe
9
d) Inserting the sample
11
e) Check the temperature
13
f) Field-frequency lock
14
g) Tuning the probe
15
h) Shimming
18
i) Initial acquisition
20
j) Control of the one-dimensional spectrum display
22
k) Phase correction
23
l) Final acquisition
23
m) Baseline correction
24
n) Chemical shift calibration
24
o) Integration
24
p) Peak picking
25
q) Printing
25
r) Saving printouts to a file and sending them by email and fax
26
s) Exiting the program when finished work
27
4) Use of the guide in other laboratories
27
5) Less common probes
28
6) Measuring the pulse width
29
7) Temperature control and stabilization
30
8) Temperature calibration
38
9) Difficulties in locking and acquiring without lock
39
a) Improving lock stability
39
b) Manual locking
40
c) Normal values for the lock parameters
40
d) Acquisition without lock
41
10) Optimizing the spectral width and acquisition time
44
11) Apodization (window function) for increasing sensitivity or resolution 45
2
12) Parameter adjustment for optimizing sensitivity
46
13) Measuring the longitudinal relaxation time – T1
46
a) The inversion-recovery method
46
b) The DESPOT method
47
14) Chemical shifts of solvents for calibration purposes
47
15) Accurate quantitative acquisition
48
16) Manual peak picking
48
17) Fluorine decoupled proton acquisition
49
18) Proton acquisition decoupled from other nuclei
51
19) Transferring files from the spectrometers to other computers
52
20) Use of TOPSPIN on other computers
53
3
1. Summary of instructions for measuring a proton NMR spectrum
(Use the full guide for full details)
For all the instructions in the table there is an icon that appears in the menu at the top
of the program window. You can place the mouse cursor on it to be the pop-up help.
Clicking on the icon is equivalent to entering the command.
From the opening screen please enter the username and
password
Login:
Click on the icon to run the program
Topspin 2.1/1.3
Open a new file (not required) you can overwrite a previous file
but then all the old data will be lost
edc
Read the parameters for a proton spectrum
rpar 1_Proton
Lock the spectrometer to the solvent deuterium frequency
lock
Tune the frequency - you must do this when you start work or
change solvent, on the 400 you must use wobb and manual
tuning
atma / wobb
rga
Read the shim file. Each probe has its own shim file.
rsh
bbo/bbi/mas/hrmas
Automatic shimming is available only on the 500
topshim
Adjust the shimming (even if you used topshim on the 500)
according to this order: Press spin. On the 500 also click on 'on
axis' and correct Z and Z2. Cancel the spin and correct X, Xz, Y
and YZ, restart the spin and readjust Z and Z2. Sometimes it is
necessary to repeat the process several times to get good
shimming. Patience is recommended.
shimming
Initial scan with ds = 0 and ns = 1
zgfp
If the shimming is good please change ns as desired and ds to 2
Final acquisition
zgfp
Phase correction – the correction is done in two stages – in the
first the biggest signal is corrected by dragging the mouse on the
number 0 and the second stage is dragging the mouse on the
number 1. The phase is correct when the whole spectrum is
straight.
.ph
Calibration of the spectrum to TMS or a residual solvent signal.
Expand the region of the calibration signal, bring the line to the
peak and click. A new window appears with a chemical shift.
Enter the correct chemical shift and save.
.cal
Baseline correction: a menu appears – choose the second option.
.basl
Integration – click on the second left button on the menu bar in
the window that appears and select the signals by dragging the
mouse over them. Right clicking on a selected signal allows
.int
4
calibration.
Printing – click on the printer icon on the menu bar of press Ctrl
p. A new window with options appears, the second option is
recommended allowing the appearance of the plot to be edited.
plot
Logout from the computer
Lougout:
2. General description of the equipment
Figure 1. The spectrometer magnets from left to right: the 200, 400 and 500
MHz magnets
Figure 2. The console of the 400 MHz spectrometer with the doors closed on
the left and open on the right
5
Figure 3. The console of the 200 MHz spectrometer with the door closed on
the left and open on the right
Figure 4. The console of the 500 MHz spectrometer with the door closed on
the left and open on the right
Figure 5. The computer that controls the spectrometer
Figure 6. The control panel for the sample and magnet
6
3. Routine experiments
For a routine proton experiment
(http://chem.ch.huji.ac.il/nmr/techniques/1d/row1/h.html#BM1H) follow these
instruction. For proton NMR with fluorine decoupling see chapter 17 and for
decoupling from other nuclei see chapter 18.
a. Logging on
Log on to the computer using your username and password. The password will appear
as black circles for security reasons. Click on OK to enter.
If the computer is already logged on then log off as described in chapter 3r.
Figure 7. User logon window
Run the Topspin 1.3 (Topspin 2.1 on the 500 MHz spectrometer) program by double
or
clicking on the symbol
start>All Programs>Bruker>TOPSPIN>TOPSPIN1.3(2.1)>TOPSPIN1.3(2.1)
b. Creating a work file
The program will appear as in fig. 8. Below the title are menus. The next rows is a
toolbar for file management, editing, viewing options and acquisition commands. In
the last row there is a toolbar for controlling spectral appearance. On the left hand side
there is a list of files and on the right the spectrum will be displayed. The browser can
be used to open existing files (fig. 8).
7
Figure 8. The file browser
Alternatively you can click on PFolio (Last50 in Topspin 2.0) to see recent files (fig.
9).
Figure 9. File selection from recent files
To create a new file, enter edc (fig. 10) and enter the parameters: experiment name in
the field NAME, experiment number in EXPNO should be 1 (or a higher number if
there are already experiments under that name), processing number in PROCNO
8
should be 1, directory in DIR should be c:\bruker\topspin1.3 or c:\bruker\topspin ,
username in USER, name of the solvent in solvent, 1_Proton (for proton NMR) in
Experiment and the title in TITLE (The title can be changed later by clicking on the
TITLE tab on the spectrum window). Click on OK or press enter to create the file.
You can copy parameters from an existing file using edc by choosing Use current
paramters in the Experiment field.
On the 500 MHz spectrometer there is an extra field in the window Experimental
Dirs. Choose the directory C:/Bruker/TOPSPIN/exp/stan/nmr/par/user.
Figure 10. Creating a new file with edc
c. Specifying the probe
The probe that is in the magnet must be specified by entering edhead (fig. 11) and
choosing the relevant probe. Two probes are usually used on the 400 MHz
spectrometer. (See below regarding the probes for the 200 and 500 MHz
spectrometers.)
The connectors under the magnet of the probe 5 mm
Multinuclear Z3918/086 [09] that is known as BBO look like
this. It is used for carbon phosphorus and other nuclei but can
be used for proton NMR although about a third of the
sensitivity is lost compared with the BBI.
The connectors under the magnet of the probe 5 mm
Multinuclear inverse Z-grad Z8202/0051 [11] that is
known as BBI look like this. It is used for proton,
fluorine, most 2D-NMR and diffusion.
If the probe is not one of these two then see chapter 5.
On the 200 MHz spectrometer there are three probes. For the
routine purposes of measuring proton NMR, the
5 mm Multinuclear inverse Z03221/0022 [10] known as BBI
is used.
If the probe is not the BBI then see chapter 5.
9
On the 500 MHz spectrometer there are four probes. For routine purposes such as
proton NMR the 5 mm PABBO BB-1H/D Z-GRD Z800701/0114 [33] probe known
as BBO that looks like this
or the 5 mm PABBI 1H/D-BB Z-
GRD Z810701/0082 [34] probe known as BBI that looks like this
.
If the probe is not one of these two then see chapter 5.
After choosing the probe, click on Exit or press Return.
Figure 11. Window for specifying the probe
Read the shimming parameters by entering rsh bbi for the BBI probe or rsh bbo for
the BBO probe.
If using the BBO probe on the 400 MHz spectrometer for proton NMR change the
pulse width by entering p1 9. (The pulse width for the BBI is 5.6.)
If using the BBO probe on the 500 MHz spectrometer for proton NMR change the
pulse width by entering p1 11. (The pulse width for the BBI is 8.5.)
After clicking on Exit another window will appear. The connections should appear as
in fig. 12. Click on Save.
Figure 12. The connection window for the 500 MHz spectrometer (except of 19F
measurement and the CP-MAS probe)
10
If you need a quantitative spectrum see chapter 15 or if you want to optimize the
sensitivity see chapter 12.
d. Inserting the sample
The NMR tube is inserted into a spinner. The spinner is then inserted into a sample
gauge (fig. 13) and is then checked to see that the sample is in the region of the black
lines. The solvent height should be at least 4 cm from the bottom of the tube. The
bottom of the tube should be 2 cm below the coil center (fig. 14). If the sample depth
is less than 4 cm then the center of the solution should be placed at the coil center (fig.
15).
Figure 13. Sample depth
measurement
Figure 14. Sample
position with enough
sample depth
Figure 15. Placement of
a short sample
One can use the control panel (fig. 16 and for the 500 MHz spectrometer fig. 17) or
the bsmsdisp window (fig. 18) to insert the sample and for other actions mentioned
later such as locking and shimming.
If using the control panel, air is passed through the magnet by pressing on LIFT
ON/OFF. It is important to hear the rush of air to ensure that the sample may be
inserted safely (if no air is heard then do not insert the tube). If there is a sample in the
magnet it will be ejected. Before inserting a sample ensure that there is not already a
sample inside. Remove the previous sample and put the new sample in place. Press on
LIFT ON/OFF again and the sample sinks into the magnet.
11
Figure 16. The control panel for sample handling and magnet control as seen
from above
SPIN
LIFT
SWEEP
LOCK
Shims
Figure 17. The control panel of the 500 MHz spectrometer for sample handling
and magnet control as seen from above
12
in
If using bsmsdisp, enter bsmsdisp or, on the 500 MHz spectrometer, click on
order to open the window (fig. 18). Under the Main tab that opens automatically most
of the required actions are displayed. Air is passed through the magnet by clicking on
the LIFT button in the SAMPLE frame. It is important to hear the rush of air to
ensure that the sample may be inserted safely (if no air is heard then do not insert the
tube). If there is a sample in the magnet it will be ejected. Before inserting a sample
ensure that there is not already a sample inside. Remove the previous sample and put
the new sample in place. Click on LIFT again and the sample sinks into the magnet.
Do not spin the sample until the control panel or bsmsdisp shows that the sample is
inserted successfully (a green light appears). If the sample does not insert successfully
then reduce the air flow (see temperature control in chapter 7) eject and reinsert the
sample and increase the air flow to what it was after insertion is confirmed.
Figure 18. The bsmsdisp window for sample and magnet control
e. Check the temperature
Type edte and the temperature control window will appear. 298.0 K (24.85°C) is
considered to be room temperature. The temperature can be monitored using the
Monitoring tab (fig. 19).
On the 500 MHz spectrometer, the heater is shut off when leaving TOPSPIN and
must be reactived by clicking on Probe heater under the Main display tab (see ch. 7
fig. 47). It may be that the temperature is displayed in Celsius. You can change it to
Kelvin as described in ch. 7 figs. 48 and 49.
13
Figure 19. The edte window under the Monitoring tab for checking temperature
stability
If the temperature is not correct or not stable to within 0.1 degrees then the parameters
need to be adjusted – see ch. 7.
On the 200 MHz spectrometer the heater is usually left off for routine purposes. If you
want to control the temperature see ch. 7.
f. Field-frequency lock
Because the magnet field strength varies slightly affecting the frequency of resonance.
the resonance frequency of deuterium is locked by making slight adjustments to the
magnetic field. This is one of the reasons that deuterium substituted solvents are used.
In order to see the lock status click on
or enter lockdisp. A new window will
appear showing the lock signal (fig. 20).
14
Figure 20. The lock window (lockdisp) showing that the sample is locked
If the lock sweep signal appears in two colors clock on
so that it only appears in
one color. (The use of two colors can be misleading during shimming.) One can lock
by entering lock and then the solvent name (for example lock cdcl3). The sample then
usually locks automatically.
If the solvent has more than one type of deuterium of similar intensity such as THF-d8
or DMF-d7 or there is insufficient deuterium in the sample or the sample is not
homogeneous or isotropic enough then lock manually or do not lock as described in
chapter 9.
If there is high dynamic range, improving the lock stability as descried in ch. 9a may
improve the line-shape near the baseline of tall peaks.
g. Tuning the probe
Each time the solvent or probe is changed and at the start of your work you should
tune the probe.
On the 500 MHz spectrometer enter atma (or for better results atma exact) and wait a
minute or two for automatic tuning to finish. The computer will tell you when the
process is complete.
On the other spectrometers enter wobb and a window will appear like in fig. 21.
Likewise the preamplifier display next to the magnet will look like in fig. 22. If you
do not see the minimum (dip) you can sweep the T (tuning) or click on
at the top
of the wobb window and set a wider sweep width such as 20 MHz. It is recommended
to return the range to 4 MHz after finding the minium.
Bring the bottom of the signal to the center (figs. 23 and 24) with the T (tuning) screw
under the probe (fig. 25). Afterwards bring the signal down (figs. 26 and 27) with the
M (matching) screw (fig.25). Continue tuning and matching until the probe is tuned
(figs. 26 and 27).
15
Fig. 21. The wobb window showing the probe out of tune
Fig. 22. The probe out of tune as seen on the display on top of the preamplifiers
Fig. 23. The wobb window showing that the probe needs matching
16
Fig. 24. The display on top of the preamplifiers showing that the probe needs
matching
Fig. 25. The tuning and matching screws under the probe
Matching
Tuning
17
Fig. 26. The wobb window showing that the probe is tuned and matched
Fig. 27. The display on top of the preamplifiers showing that the probe is tuned
and matched
h. Shimming
On the control panel (fig. 16) and the bsmsdisp window (fig. 18) there are buttons for
the different shim functions for correcting magnetic field homogeneity.
(While shimming you can also do rga – see ch. 3i.)
If you have not read the shim file already the do it now by entering rsh probename
(rsh bbi or rsh bbo).
Check that the DIFF MODE button is not on.
The shims Z, Z2, X, and Y are adjusted with FINE on and the remainder of the shims
with FINE off.
The pure Z axis shims (Z, Z2, Z3) are adjusted while the sample is spinning (the SPIN
button is on) and the other shims are adjusted without spinning (SPIN button off).
On the control panel of the 500 MHz spectrometer (fig. 16) you need to press two
buttons to select each shim function. For a pure Z function (Z1, Z2, Z3) you must press
ONAXIS and the required functions. For functions without Z (X, Y, XY, X2-Y2)
18
press the function then Z0. For mixed functions press both function components for
example for XZ press X then Z1. To shim the 500 MHz spectrometer enter topshim
without sample spinning and wait about three minutes for the process to finish. For
the first sample you should also correct the non-spinning shims as explained below
and if there is a large change repeat topshim. You may be able to further improve the
shimming slightly by manually adjusting Z and Z2 with spinning. Sometimes,
particularly for non-homogenous samples or samples in non-standard tubes, topshim
will fail and you should shim manually as explained below.
On the control panel the adjustment is carried out by turning the wheel. In the
bsmsdisp window (fig. 18) under the Main tab in the SHIM frame there are buttons
for the main shim functions. Choose the shim function that you want and change it by
clicking on Step + and Step – in the VALUE frame. You can also enter the value
numerically under the word Actual. The higher the signal in the lockdisp window, the
better the shimming.
Start with the functions Z and Z2 (with spinning).
For the first sample that you do, adjust (without spinning) also functions X and Y then
XZ and YZ (if there is a large change return to X and Y), then XZ2 and YZ2 (if there
is a large change return to X, Y, XZ and YZ) and then XY and X2-Y2. Spin the
sample and readjust Z and Z2 and if the spectrum (see acquisition and phasing ch. 3i
to 3k) looks alright then you may adjust Z3 and return to adjust Z and Z2.
If using bsmsdisp then you must go to the Shim tab (fig, 28) to change XZ2 and YZ2.
Figure 28. The bsmsdisp window with the Shim tab for adjusting XZ2 and YZ2;
on the right is that for the 500 MHz spectrometer and of the left for the other
spectrometers
19
Afterwards, acquire and phase the spectrum as explained below (see acquisition and
phasing ch. 3i to 3k) and look at the signals (fig. 29). It is best to look at a singlet such
as the solvent of TMS and correct as necessary.
Figure 29. Signal distortion due to bad shimming
If the signals look like this increase Z2
If the signals look like this reduce Z2
If the signals look like this correct Z3
If the signals look like this correct Z and perhaps Z3
If the signals look like this correct X, Y, etc.
2 × the
spin rate
i. Initial acquisition
Adjust the sensitivity of the ADC by entering rga. (If you already did it while
shimming there is no need to do it again.)
If you just copied a previous proton file (e.g., by using Use current parameters in
Experiment under the command edc) and did not read new parameters (by specifying
1_Proton in Experiment under the command edc or by entering rpar 1_Proton all)
then enter ds 0 then ns 1.
Enter zgfp to run the spectrum.
Usually the spectral region and the acquisition is appropriate. Sometimes the fid may
be truncated and ringing will appear in the spectrum (fig. 61), that the signals may be
broad wasting time on acquiring noise or there may be signals outside the range.
Whenever any of these conditions are suspected the spectral range and acquisition
time need adjustemenr, see ch. 10.
Fig. 30 shows the acquisition window.
20
Figure 30. The acquisition window with an fid
After the acquisition a Fourier transform
(http://chem.ch.huji.ac.il/nmr/techniques/1d/1d.html) is carried out (fig. 31) that
converts the acquired signal into the spectrum (fig. 32).
Figure 31. The Fourier transform
Fourier transform
Frequency and chemical shift
Time since excitation pulse
21
Figure 32. A spectrum
j. Control of the one-dimensional spectrum display
The following toolbar is used for controlling how the spectrum is displayed.
From left to right: double the height, half the height, increase the height 8 times,
reduce the height 8 times, adjust the height interactively, display the full height,
display the full spectral width, display the who spectrum, contract the width, adjust
the width interactively, expand the width, define the region numerically, return to the
previous expansion, expand, keep the same region when reading another spectrum,
move half a screen left, adjust the horizontal position interactively, move half a screen
right, display the left end of the spectrum, display the right end of the spectrum, raise
the baseline to the center, adjust the vertical position interactively, lower the baseline
to the bottom.
The spectrum may be expanded by dragging the mouse while left-clicked. Releasing
the mouse key expands the spectrum (fig. 33).
22
Figure 33. Selecting a region by dragging the mouse
k. Phase correction
Enter .ph or click on
. The phasing window will open. The zero order phase
correction (at the point where there is a red vertical line) is done by left clicking on
and dragging the mouse up and down. First order phase correction (far from the
red vertical line) is done by left clicking
and dragging the mouse up and down.
When the phase correction is complete click on
correction (fig. 34).
The uncorrected
spectrum
to save (or on
to cancel) the
Figure 34. Phase correction
After zero order
phase correction by
left clicking on
and dragging the
mouse up and
down
After first order phase
correction by left
clicking on
and
dragging the mouse
up and down
l. Final acquisition
ns is the number of scans. If the sensitivity is good then 16 scans is enough. The less
sensitivity the more the scans needed: 32, 64, 128, etc. (see ch. 12).
23
ds is the number of dummy scans to allow the system to equilibrate. Set ds to 2.
Enter: ds 2
ns 16
zgfp
Correct the phase.
Check that the spectrum is alright.
At the processing stage one can improve the sensitivity or the resolution of the
spectrum but not both at the same time using a window function (apodization) – see
ch. 11. If there is still not enough sensitivity then other parameters may be adjusted –
see ch. 12.
m. Baseline correction
Enter bas or use the menu Procession > Baseline Correction…[bas]. A menu
window for baseline correction will open. Choose the option (second from the top)
Auto correct baseline using polynomial. Click on OK to save (or Cancel to abort).
n. Chemical shift calibration
Enter .cal or click on
. Bring the cursor (vertical red line) to the calibration peak
and left click. Enter the chemical shift and click on OK to save (or Cancel to abort).
The most common chemical shift references at room temperature are: TMS 0, CHCl3
7.261, DMSO-d5 2.504, HOD 4.81 and CD2HOD 3.312. Other chemical shifts are
given in table 2 ch. 14. See http://chem.ch.huji.ac.il/nmr/whatisnmr/chemshift.html.
Be careful not to confuse the reference signal with other overlapping signals. The
solvent and TMS usually have especially sharp signals.
o. Integration
Enter .int or click on
. The integration window (fig. 35) will open.
24
Figure 35. The integration window
and left dragging the cursor over the
You can add an integral by clicking on
regions of the spectrum that you want to integrate. To select an existing integral use
the
buttons. The right button selects and deselects all the integrals and the
other buttons select the integrals one by one.
deletes the selected integral(s).
splits and reconnects the selected integral. The integral window should look
something like in fig. 35 after manual integration.
Calibrate the integral intensity by right clicking on an integral of known intensity (of a
known number of protons). A menu will appear; select Calibrate current integral
and enter the intensity in the New value field.
The integrals in a regular proton spectrum are accurate to approximately ±10%. It is
possible to improve the accuracy to ±1% by acquiring a quantitative spectrum – see
ch. 15.
p. Peak picking
For the purposes of routine printing the peak picking is carried out automatically. See
ch. 16 to peak pick manually.
q. Printing
To print press ctrl-p, click on
or use the menu File > Print…[Ctrl P].
There are three print options:
1. Print active window (prnt) prints what appears in the spectrum window. Click on
OK to print (or Cancel to abort). Usually it prints without parameters and the print is
difficult to read.
2. Print with layout – start plot editor (plot) opens a plot editor (fig. 36) and you
can change the plot appearance. Choose the +/1D_H.xwp LAYOUT. Click on OK to
25
open the editor (or Cancel to abort). This is the preferred option even though it takes
more time.
Figure 36. Editing the plot using the TOPSPIN Plot Editor
Peak
Title
Toolbars
labels
Laboratory
logo
Editing
tools
Parameters
Spectrum
Scale
Integral
labels
The are many things that can be adjusted, the most important are as follows. Click on
the spectrum region far from the title. Small green squares appear. Click on 1D/2DEdit in the upper toolbar. An extra window appears. Use it to move and expand the
spectrum as necessary. Click on Close on that window when finished. Click on the
printer symbol on the upper toolbar to print. Choose the default printer (for our 400
and 500 MHz spectrometers this is Xerox Phaser 3117).
(For the 200 MHz spectrometer the default printer is HP LaserJet 5L.)
You can return to 1D/2D-Edit several times to print different regions of the spectrum.
When finished close the window and answer No to the question Save changes to
1D_H.xwp.
3. Plot with layout – plot directly (autoplot)
Save changes to 1D_H.xwp. Choose the +/1D_H.xwp LAYOUT. Click on OK to
open the editor (or Cancel to abort). This prints the region in the spectrum window at
the height in that window.
r. Saving printouts to a file and sending them by email and fax
Instead of printing on paper, one can prepare a printout file for sending by email or
fax or to save it on the computer. If this is a single plot then choose the printer Adobe
PDF (if you are using your own computer, check if Adobe PDF writer is installed),
Microsoft XPS Document Writer or Microsoft Document Image Writer from the
26
printer menu. If you want to insert a number of printouts into one document, chose the
Adobe PDF printer and choose a filename. If you want to save more than page, create
a file for each page and from within the Adobe PDF window, that appears after each
such 'printing', choose from the menu File -> Create PDF -> From Multiple Files…'
click on Browse, choose a filename, click on OK and repeat for each file. Click on
Save and choose a filename. You can send the file by email although it is not
recommended to send more than 30 pages at once. If you have a fax installed on the
computer (there is a fax in the 400 and 500 MHz spectrometers) you can send the
'printout' by fax. Open the file and print to fax. A fax window will appear. (The first
time that you use the fax, a window will appear for you to enter your personal details.)
Click on Next> then enter the name and phone number as dialed from the university
(there is no international line). Click on Next>, Next> and Finish to send it.
s. Exiting the program when finished work
When finishing work remove the sample (see ch. 3d) and close the window or enter
exit. A message will appear Close TOPSPIN This will terminate all possibly active
commands. Exit anyway? Click on OK or return. Leave your account: Start > Log
off. A message Are you sure you want to log off? will appear. Click on Logout.
4. Use of the guide in other laboratories
In order to prepare the parameters for acquisition, read the parameters for PROTON
(rpar PROTON all) change the parameters below and save under the name 1_Proton
(wpar 1_Proton all). Afterwards go into the new parameter directory and set all the
files in it to read only.
PL1
to the minimum permitted -6, -3 or 0
P1
length of the 90° pulse in μs
CY
12.5
NS
1
DS
0
SI
64k
Prepare another file from this one, modify it as below and save it as 1c_Protonfdec.
PL12 the fluorine pulse attenuation such that the 90° pulse is 100 μs
PL13 the fluorine pulse attenuation such that the 90° pulse is 100 μs
Enter edasp and change the parameters according to fig. 37.
27
Figure 37. The edasp setup for proton with fluorine decoupling
Copy the file 1D_H.xwp to 1D_Hold.xwp and cancel the readonly property from
1D_H.xwp. Go into the plot editor and change the file as you wish as explained in the
book "Topspin plotting" from Bruker. Reinstate the readonly property on 1D_H.xwp.
Prepare the following macros:
zgft =
zg
ft
zgfp =
zg
fp
zgef =
zg
ef
zgefp =
zg
efp
5. Less common probes
In addition to the commonly used probes there are four extra probes for special
applications.
The 5 mm Dual 19F/1H Z3752/0007 [04] probe is used for
measuring fluorine with proton decoupling and proton with
fluorine decoupling. With this probe change p1 to 6.9.
28
The 5 mm SEX 3He-BB Z3488/0109 [10] probe that was once
the BBI probe of the 300 MHz spectrometer is now used only for
measuring 3He.
The 10 mm QNP 1H/15N/13C/31P Z8222/0001 [20] probe is
used for measuring 15N, 13C and 31P in 10 mm tubes for samples
that are too insoluble to measure in 5 mm tubes.
The 10 mm Multinuclear low freq. Z00411/0004 [22] probe is
used for measuring low frequency nuclei such as 57Fe without
lock.
The 5 mm Dual 13C/1H Z3225/0347 probe is used for carbon
spectra but can be used for proton NMR with half the sensitivity
of te BBI probe. If using the dual probe on the 200 MHz
spectrometer change the proton pulse p1 to 10.4. See ch. 3c.
The 10 mm Multinuclear Z01400/992 probe is used only for
other nuclei such as phosphorus. It is not recommended to use
the BBO probe of the 200 MHz spectrometer for proton but if
using it set p1 to 20. See ch. 3c.
On the 500 MHz spectrometer there is a CP-MAS probe that looks like this
and an HR-MAS probe that looks like this
put a regular NMR tube into these probes.
. Do not
6. Measuring the pulse width
Acquire a regular spectrum and correct the phase.
Enter pulprog zg.
Set p1 to close to the 360° pulse: 20 for the BBI and 34 for the BBO.
On the 200 MHz spectrometer the 360° pulse for the BBI is close to 20, dual 39 and
BBO 90.
On the 500 MHz spectrometer the 360° pulse for the BBI is close to 34 and the BBO
44.
Acquire a provisional spectrum by entering zgfp.
If the spectrum is positive reduce p1 and if it is negative increase p1 until you find a
value of p1 that yields a spectrum close to zero intensity.
The value of p1 for a φ° pulse is (p360° - 0.8) × φ / 360 + 0.8
29
For a regular pulse of 90°: p1 = p360°/ 4 + 0.6
On the 500 MHz spectrometer the value of p1 for a φ° pulse is
(p360° - 0.16) × φ / 360 + 0.16
For a regular pulse of 90°: p1 = p360°/ 4 + 0.12
For a fluorine decoupled proton spectrum you can calibrate the fluorine decoupling
pulses. Open a file for measuring fluorine as described in the guide "Measuring NMR
spectra of carbon and other non-proton nuclei" ch. 1 and calibrate the decoupler pulse
as described there in ch. 6.
7. Temperature control and stabilization
On the temperature unit that is in the console on the right hand side at the top, check
that there is an airflow of about 270 L/h and that the HEATER is on (fig. 38).
Figure 38. The temperature unit on the 400 MHz spectrometer
Flow guage
Heater button Cooler button
Read the value at the
bottom of the ball
On the 500 MHz spectrometer all the controls are in the software so there is no need
to physically touch the temperature unit.
Use a ceramic spinner for temperatures over 310 K but do not use a ceramic spinner
for cooling. The ceramic spinner is fragile and expensive. Do not drop it.
In the edte window under the Main display tab (fig. 46) you can change the
temperature by clicking on Change… and entering the new temperature in the
Sample temp. field. The actual measured temperature appears in Target temp. You
can use any temperature up to 453 K as long as the solvent does not boil.
At room temperature and above, air is passed to the probe from under the magnet via
a black pipe shown in fig. 39 and for the 500 MHz spectrometer shown in fig. 40
Figure 39. Air connection (black tube) for room temperature and above
30
Figure 40. Air connection for the 500 MHz spectrometer (black tube) for room
temperature and above
In order to cool or to, stabilize a temperature close to that of the room better than
normally required, use the cooling unit. Do not use it without special permission.
To cool, fill the Dewar with liquid nitrogen, insert the transfer tube with its O-ring
and close it tightly. Turn of the HEATER and release the air hose clip (fig. 41).
Figure 41. Opening the air hose clip
Position the cooling pipe against the probe opening using the clamp attached to the
magnet leg (fig. 42) and check that it is in exactly the right place. The air opening is
very fragile so the pipe has to be positioned accurately.
Figure 42. Clamp for the cooling pipe under the magnet
Connect the pipe to the probe and attach the straps (fig. 43).
31
Figure 43. Connection of the cooling pipe to the probe
Press the LN2 button on the temperature unit then turn off the air flow. Adjust the
nitrogen flow using the buttons either side of the LN2 button according to fig. 47.
When you finish working with cooling stop the cooling (pres the LN2 button) then
turn the air flow on.
Before touching the cooling pipe heat the joint between the pipe and the probe with a
hairdryer until it is totally thawed. The connection is very fragile and trying to
disconnect it without heating may break it. After the joint has thawed disconnect it
gently and connect the air pipe.
On the 500 MHz spectrometer the cooling connect is different and is opened by
pussig the plastic sheath (fig. 44) and connecting it to the probe (fig. 45). On finishing
work with cooling disconnect the cooling pipe with heating from a hairdryer, connect
the air pipe and start the air flow and heating.
Figure 44. Connection of the cooling pipe on the 500 MHz spectrometer
Make a
gap here
Push
Figure 45. The cooling pipe connected to the probe of the 500 MHz spectrometer
32
Figure 46. The Main display tab of the edte window for temperature control
Figure 47. The Main display tab of the edte window for temperature control on
the 500 MHz spectrometer
On the 500 MHz spectrometer the temperature unit is entirely controlled from the edte
window (fig. 47). Check that the Probe Heater is On and that the Gas flow is 270
l/h. To cool, fill the Dewar, connect it to the probe and set Cooling to On and Gas
flow to 0 L/h.
On the 500 MHz spectrometer it is possible to display the temperature in Celsius in
the range -9.99°C to 99.99°C that allows an extra decimal place of precision. Under
the Config. tab, change the Display unit to Celsius and the Decimal Precision to
99.99. In the frame Target limits click on change… and select a minimum of -9.99
and a maximum of 99.99. The resulting window should look like in fig. 48.
33
Fig. 48 The Config. tab of the edte window in Celsius mode
If you want to work outside this temperature range or to display the temperature in
Kelvin, change the Display unit to Kelvin and the Decimal Precision to 999.9. In
the frame Target limits click on change… and select a minimum of 123.15 and a
maximum of 453.15. The resulting window should look like in fig. 49.
Fig. 49 The Config. tab of the edte window in Kelvin mode
If the temperature does not stabilize, set these parameters manually. They are correct
for room temperature. For other temperatures see fig. 50.
Proportional Band: 60
Integral Time: 72
Derivative Time: 18
After making these changes click on Apply PID changes. There is no need to change
these parameters for small changes in temperature. When cooling it is best to use a
higher nitrogen flow rate than that in fig. 50 and then to reduce it. If the temperature
34
does not stabilize to ±0.1 K, use self-tune (fig. 51) and wait several minutes while the
unit calibrates itself. If the temperature still does not stabilize then there is a leak or
insufficient gas flow.
Figure 50. Parameters for temperature control on the 400 MHz spectrometer
Proportional band
Cooling
Heating
Integral time
60
60
40
40
20
20
0
0
200
300
400
200
300
Temperature /K
Temperature /K
Derivative time
Cooling/%
400
50
15
40
30
10
20
5
10
0
0
200
300
400
200
Temperature /K
300
400
Temperature /K
-1
Max heating
Gas flow/L h
20
300
200
10
100
0
0
200
300
200
400
300
Temperature /K
Temperature /K
35
400
Figure 51. The Self-tune tab of the edte window for calibrating the temperature
unit
On the 500 MHz spectrometer the parameters for room temperature are:
Proportional Band: 74.5
Integral Time: 232
Derivative Time: 38
When cooling or heating use the values in fig. 52 (just like fig. 50 for the 400 MHz
spectrometer.
36
Figure 52. Parameters for temperature control on the 500 MHz spectrometer
Proportional band
Integral time
Cooling
Heating
80
With
decoupler
200
60
40
100
20
0
0
200
300
400
200
300
Temperature /K
Temperature /K
Derivative time
Cooling/%
40
40
30
30
20
20
10
10
400
0
0
200
300
400
200
Gas flow/L h
300
400
Temperature /K
Temperature /K
-1
Max heating
60
600
40
400
20
200
0
200
300
200
400
300
400
Temperature /K
Temperature /K
On the 200 MHz spectrometer the heater and the temperature controller are not used
for routine purposes.
If despite this you want to control the temperature. Turn the heater on in the
temperature controller (fig. 53) that is in middle of the console. The unit is accurate to
one degree.
Figure 53. The temperature controller of the 200 MHz spectrometer
Heater button
Flow gauge
37
The software control is similar to that of the 400 MHz spectrometer with the
following differences. Unter the Main display tab of edte there is control of the air
flow. 650 L/h is recommended. The manual paramters for Self-tune are:
‫ בלשונית‬.‫ מגהרץ עם השינויים האלה‬400-‫הפיקוד עליו בתוכנה דומה לזה של ספקטרומטר ה‬
Main display ‫ של‬edte .‫ ליטר לשעה‬650 ‫ מומלץ‬.‫ניתן לפקח על מהירות זרימת האוויר‬
-‫הפרמטרים הידניים ל‬self-tune:‫המומלצים הם‬
Proportional Band: 2
Integral Time: 7
Derivative Time: 1
When the temperature is close to room temperature it is accurate to the nearest
degree. If you want to be more accurate you can calibrate the temperature as
described in ch. 8.
8. Temperature calibration
When the temperature is close to room temperature it is accurate to about one
degree. The accuracy becomes worse the further the temperature is from room
temperature. If you want better accuracy you can calibrate the temperature using
methanol for room temperature and below or glycol for above room temperature.
You can also do the calibration after the acquisition.
Cancel the lock – click on LOCK on the control panel or Lock in the LOCK
frame of the bsmsdisp window. Wait a few seconds until the SWEEP light comes
on then cancel the sweep by clocking on SWEEP on the control panel or On-Off
in the SWEEP frame of the bsmsdisp window.
Read the methanol file by entering re meoh.
If the file does not exist an error will appear: Data set does not exist and you will
need to create it using edc. In the menu that appears (fig. 10) set the NAME to
meoh, EXPNO to 1, PROCNO to 1 and Experiment to 1_Proton. Click on
SAVE and the window will disappear. Enter p1 60 then rg 64. The file is then
ready.
Off-tune the probe by three turns of the TUNING screw (T, fig. 24). It is a good
idea to remember the direction that you turned the screw so that you can correct it
later.
On the 500 MHz spectrometer enter atmm wait for wobb to start then click six
times on <<<. To return the tuning afterwards enter atma. On leaving atmm it will
ask you if you want to save. Click on Cancel.
Acquire the NMR spectrum of methanol or glycol by entering zgfp. Correct the
phase as described in ch. 3k. Measure the chemical shift difference (Δδ) between
the two main peaks (fig. 54). The measure of temperature stability is that the result
of repeated acquisitions is the same to within 0.001 ppm. So repeat the
measurement until such stability is obtained. This usually takes 10 to 15 minutes.
If the temperature does not stabilize, check the temperature controller (ch. 7).
38
Calculate the temperature using the relevant equation like the one for methanol in
fig. 54 or use the program (fig. 55): Start > NMRThermometer >
NMRThermometer.
T(MeOH)/K = 409 – 36.54Δδ - 21.85(Δδ)2
T(Glycol)/K = 465.77 – 100.91Δδ - 0.39(Δδ)2
ppm
3.29912
4.86625
ppm
Figure 54. Temperature calibration spectrum
T(MeOH)/K = 409 – 36.54Δδ - 21.85(Δδ)2
Δδ=1.56713
T = 298.08 K
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
Figure 55. Program for calculating the temperature
9. Difficulties in locking and acquiring without lock
It is possible that the lock will fail when there are multiple solvents, the signal is weak
or there are multiple signals in the deuterium spectrum such as with THF-d8 and
DMF-d7 when you will need to release the lock by pressing on LOCK ON/OFF on
the control panel and relock manually.
a. Improving lock stability
High dynamic range spectra are especially sensitive to lock stability. The lock
stability may be improved by adjusting the lock parameters. If the lock goes up and
down in a wavy manner, reduce the LOCK POWER by 6 dB. If the lock is stable,
note the lock level, increase the LOCK POWER by 6 dB, note down the LOCK
GAIN and reduce it until the lock returns to it s previous level. If the reduction in
39
LOCK GAIN is significantly less than 6 dB then return the LOCK POWER to what
it was. Continue until the optimum LOCK POWER is found. Enter loopadj2 and
wait for it to finish. This makes the lock more stable.
b. Manual locking
Finding the field: on the control panel (fig. 16, 17 ) click on the FIELD button.
The current value of the FIELD appears in the small window. Using the wheel,
search for the signal. When you see a lock signal that is reminiscent of a butterfly,
bring it to the center. If you use bsmsdisp (fig. 18) open the Lock tab (fig. 56).
There you can change the value of Field. Sometimes two 'butterflies' appear
because the solvent (such as CD3OD) has two different types of deuterium. Then
it is preferable to choose the larger signal and if they are of the same intensity, the
right-most signal.
Figure 56. The Lock tab of the bsmsdisp window for advanced lock functions
Click on lock in the LOCK frame and the 'butterfly' should disappear and become a
flat line.
Click on LOCK GAIN on the control panel or Gain in the LOCK frame of the
bsmsdisp window to bring the lock level up or down to near, but not completely to,
the top of the lock window.
c. Normal values for the lock parameters
The lock parameter values are adjusted by clicking on the relevant button and
changing the value.
FIELD in the LOCK frame is the position of the magnetic field and changes between
solvents. For example for CDCl3 on the 400 MHz spectrometer the field is about 2750
and on the 500 MHz spectrometer it is about 1000.
40
SWEEP AMP on the control panel or Ampl in the LOCK frame of the bsmsdisp
window is used for controlling the width of the signal. It is usually 2.0 but may be
increased to 10 in order to search for a signal or reduced to 0.1 to separate very
similar deuterium signals such as in DMF-d7 and nitrobenzene-d5.
SWEEP RATE on the control panel or Rate in the SWEEP frame of the bsmsdisp
window is usually 0.15.
LOCK PHASE on the control panel or Phase in the LOCK frame of the bsmsdisp
window corrects the symmetry of the signal. This is usually about 280 on the
400MHz spectrometer and 50 on the 500 MHz spectrometer. If the two 'wings' of the
'butterfly' then the phase must be corrected until their heights match.
LOCK POWER on the control panel or Power in the LOCK frame of the bsmsdisp
window depends on the solvent. The more deuterium in the solvent molecules the
lower the value. There are solvents where the signal after lock are more likely to be
unstable and wobbling like a wave. This phenomenon is called saturation. In such a
case, reduce the value of the lock power until the signal stabilizes then reduce further
by five units. (Solvents that are more likely to give saturated signals are methanol-d4,
acetone-d6 and acetonitrile-d3.)
LOCK GAIN on the control panel or Gain in the LOCK frame of the bsmsdisp
window controls the height of the lock signal and can be varied according to need.
Usually it is increased so that the signal is in the upper part of the window.
d. Acquisition without lock
If the sample contains less than 2% of deuterated solvent or the sample is
ansotropic (common in liquid crystals) it is impossible to lock the field. For a
regular (isotropic) sample, as long as it does not adversely affect the measurement,
you can add 10% deuterated solvent containing lots of deuterium such as acetoned6, benzene-d6 or even D2O. If this is not possible but at least 2% of solvent can be
added without disturbing the experiment and there is a BB channel on the probe,
then tune the BB channel to deuterium using the sliders as explained in the guide
"Measuring NMR spectra of carbon and other non-proton nuclei" and then
connect the deuterium cable (2H) to the BB channel of the probe. (When finishing
work please return the cables to their normal configuration: 2H to 2H and BB to
BB. Tune the BB channel away from deuterium. This way, the next experiment
can be run with normal locking.)
If there is no possibility of locking the field then the acquisition is slightly
different from normal. It is preferable to take a similar but deuterated sample and
do all the preparations for acquisition: locking, tuning and shimming. If this is not
possible then enter the lock command and select a deuterated solvent most similar
to the sample and after the lock fails, cancel the lock. Click on LOCK on the
control panel or Lock in the LOCK frame of the bsmsdisp window. Wait a few
seconds till the SWEEP light comes on. Cancel the SWEEP on the control panel
or On-Off in the SWEEP frame in the bsmsdisp window. Enter locnuc off.
Tune the probe and do rga. Use gs to do the shimming. This is done interactively
by observing the fid and spectrum. First acquire an initial spectrum and correct the
phase then enter gs (fig. 57).
41
Figure 57. fid with poor homogeneity that requires shimming
You can correct the Z shims with spinning and the shims containing X and Y
components without spinning in order to improve the fid so that its decay is slow
and monoexponential (fig.58). If the spectrum contains a strong multiplet then
ringing will appear in the fid rather than a monoexponential.
Figure 58. fid in a homogeneous field with good shimming
In order to correct shim Z2 it is easier to use the real-time spectrum. Click on
and an unphased spectrum will appear. Click on
and the window in fig. 59
will appear. Change the Window function: to none and the Phase correction
mode: to pk. The spectrum will appear like in fig. 60.
42
Figure 59. Parameter window for real-time spectrum display
Figure 60. Real-time spectrum with poor homogeneity that requires
shimming
43
Figure 61. Real-time spectrum in a homogeneous field with good shimming
Correct the shape of the signal according to fig. 29 which in the case of fig. 60
means correcting Z2 such that the spectrum appears like in fig. 61. Return to the
and continue shimming until both the fid and
fid display by clicking on
spectrum look alright. Enter stop to leave the gs mode. The final acquisition and
all the subsequent steps are identical to those with lock. To return to acquisition
with lock you must read new parameters and enter ii or locnuc 2h. Afterwards
click on SWEEP on the control panel or On-Off in the SWEEP frame in the
bsmsdisp window.
10. Optimizing the spectral width and acquisition time
The default spectral width is between -4 and 16 ppm. In rare cases signals may
appear beyond this range. Signals may occur at up to 22 ppm in aldehydes,
carboxylic enols and around macrocycles. There may be signals at low chemical
shifts down to -20 ppm in metal hydrides in organometals and protons within or
over macrocycles. Paramagnetic signals may appear tens or hundreds of ppm from
the normal region. On the other hand it is possible to reduce the range if all the
signals appear in a small region and there is a need to cure fid truncation (fig. 59).
If it is suspected that there are signals outside the default region then it needs to be
expanded. Change o1p to the chemical shift at the center of the region such that:
o1p = (δmax + δmin) / 2
and sw to the spectral width such that:
sw = δmax - δmin
Alternatively you can display the required region in the spectrum window and
click on
. Once the spectral region has been selected it is possible to adjust the
acquisition time by changing td and si. Carry out one scan and look at the fid and
the spectrum. If the fid decays into the noise in the first half of the acquisition you
can reduce td and si. If the fid is truncated or there is ringing in the spectrum (fig.
62) then you should increase td and si.
44
Figure 62. The effect of truncation of the fid on the spectrum
Truncated fid
Fourier transform
ringing
Fourier transform
The value of td must be a power of two and si should be double td. You can use the
letter 'k' instead of '× 1024'. Therefore if td is 64k that means that it is equal to 65536
and si should be set to 128k or 131072. Look at the fid in the acquisition window or
under the fid tab of the spectrum window. It the fid does not decay into the noise at
the end of the acquisition then double td and si. On the other hand if the fid decays
into the noise before halfway through the acquisition then halve the td and si. Do not
increase the td to more than 1024k or reduce it below 256.
11. Apodization (window function) for increasing sensitivity or
resolution
To increase sensitivity set lb to the number of Hz that you want to broaden the signal.
Choose a value less than or equal to the line-width without a window function. Up to
the line-width, the larger the lb the greater the sensitivity but the worse the resolution.
Enter efp to obtain the result. See http://chem.ch.huji.ac.il/nmr/techniques/1d/1d.html.
To improve the resolution set lb to a negative value but not of greater magnitude that
the line-width without a window function. Set gb to a positive number up to 0.5. Up
to these limits the greater the magnitude of lb and the greater gb the better the
resolution but the worse the sensitivity. Enter gfp to obtain the result.
Commands that include the window functions, Fourier transform and acquisition
appear in table 1. The regular command for acquiring and processing a proton
spectrum is zgfp but it is best to be familiar with the other commands in table 1 in
order to use window functions.
Table 1. Fourier transform commands
Command
Meaning
zg
Aquisition
45
em
Sensitivity enhancment
ft
Fourier transform
pk
Phase correct
zgft
zg;ft
zgfp
zg;ft;pk
zgef
zg;em;ft
zgefp
zg;em;ft;pk
12. Parameter adjustment for optimizing sensitivity
If the sensitivity is too low despite a large number of scans (ns) it is possible to
increase the sensitivity a little. Measure the pulse width (see ch. 6) and the relaxation
time (T1) (see ch. 13) of the signals of a similar compound under similar conditions.
Enter pulprog zg. Change p1 so that the pulse width I 68° such that p1
=0.189 × p360° + 0.65 and set d1to T1 – aq or 1.44d7 – aq.
On the 500 MHz spectrometer this is p1 = 0.189 × p360° + 0.13
For example if d7 = 4 s then T1 = 5.76 s. If aq = 3.972 s then
d1 = (1.44 × 4 – 3.972) s = (5.76 – 3.972) s ≈ 1.79 s
If p360° = 20 then p1 = 0.189 × 20 + 0.65 = 4.43
13. Measuring the longitudinal relaxation time – T1
These measurement techniques for longitudinal relaxations (T1) are accurate enough
for accurate integration and optimizing sensitivity. These methods do not provide very
accurate measurements of T1. For more accurate measurements see the quide
“Measuring relaxation” and
http://chem.ch.huji.ac.il/nmr/techniques/other/t1t2/t1t2.html#t1.
a. The inversion-recovery method
The inversion-recovery method is suitable for relaxations times up to approximately
10 s if there is enough sensitivity to see the spectrum in one pulse.
Calibrate the pulse width and set p1 to 90°, see ch. 6.
Enter ds 0 and ns 1.
Acquire a regular spectrum and phase correct.
Enter pulprog t1ir1d.
Put the initial guess for T1ln2 (0.69 T1) into d7. If you do not have a guess use 1 s.
Acquire a spectrum by entering zgfp.
If the signal of interest is negative increase d7 and if it is positive decrease d7.
Wait at lesat 5 times T1 (7.2 d7) between acquisitions.
Repeat the experiment until you find a value of d7 that gives near zero intensity for
the signal of interest. Usually the signal of interest is that with the longest relaxation
but is not a solvent signal.
Calculate the relaxation time: T1 = d7/ln2 = 1.44d7
46
b. The DESPOT method
When T1 is longer than 10 s or the sensitivity is low it is preferable to use DESPOT.
Set pulprog to zg. Note down the values of aq and d1. Change ds to int[5T1max/(aq +
d1)]+1. Acquire the spectrum with a pulse width of 90° (p1 = p360° / 4 + 0.6 and on the
500, p1 = p360° / 4 + 0.12). Open another file with the same parameters and change the
pulse width to 45° (p1 = p360° / 8 + 0.7 and on the 500, p1 = p360° / 8 + 0.14). Run both
spectra.
From the second spectrum click on
to enter multi spectrum display. If other
spectra appear, select them in the left window and cancel them by clicking on
.
Add the first spectrum by opening it in the usual manner and select it in the left
window. Match the heights of the signal of interest by dragging with the mouse up
and down on
. Note down the value that appear at the upper left next to Scale:.
Calculate T1 as follows.
T1 = (aq + d1)/ln[1/(1-scale/√2)]
Click on
to return to the normal spectrum window.
14. Chemical shifts of solvents for calibration purposes
See http://chem.ch.huji.ac.il/nmr/whatisnmr/chemshift.html.
Table 2. Proton chemical shifts of deuterated solvents relative to internal TMS at
298 (25.15°C).
Solvent name
Solvent formula
Chemical shift
Acetic acid-d4
CD3COOD
1.899, *10.60
Acetone-d6
(CD3)2CO
2.053
Acetonitrile-d3
CD3CN
1.940
Benzene-d6
C6D6
7.16
Chloroform-d
CDCl3
7.261
Deuterium chloride (1M) in D2O
DCl
*5.17
Deuterium oxide
D2 O
*4.81
Dichloromethane-d2
CD2Cl2
5.279
DMF-d7
(CD3)2NCH
2.744, 2.915, 8.025
DMSO-d6
(CD3)2SO
2.504
Formic acid-d2
DCOOD
8.309, *10.241
Methanol-d4
CD3OD
3.312, *4.877
Nitrobenzene-d5
C6D5NO2
7.503, 7.671, 8.120
Sodium deuteroxide (1M) in D2O
NaOD
*4.97
THF-d8
C4D8O
1.721, 3.574
Toluene-d8
C7D8
2.08, 6.97, 7.01, 7.10
Trifluoroacetic acid-d
CF3COOD
*10.98
47
*The chemical shift of this signal is very temperature dependent and therefore not
accurate for the purposes of chemical shift calibration.
15. Accurate quantitative acquisition
It is possible to achieve integration to an accuracy of 1% by quantitative acquisition.
First measure the longitudinal relaxation, T1 (see ch. 13).
Change ds and ns according to requirements and enter pulprog zg. Enter aq and note
down the value. Change (usually increase) d1 to 5T1 – aq (or 7.21d7 – aq) but not less
than 0.03 s. For example, if d7 is 4 s then T1 is 5.76 s. If aq is 3.972 s then
d1 = (7.21 × 4 – 3.972) s = (5 × 5.76 – 3.972) s ≈ 24.8 s
Enter rga and wait for it to complete. This may take a minute or more.
Reacquire the spectrum which may take longer than usual.
16. Manual peak picking
The command pp does an automatic peak pick. Once can change the parameters of the
automatic peak pick: MI, MAXI and PC. The automatic peak pick chooses the peaks
between the heights MI and MAXI where the height relative to the local minima is
greater than PC times the noise. The noise level is determined automatically. One can
increase PC to reduce the number of peaks or reduce PC to increase the number of
peaks.
One can change the peak picking manually as follows. Enter .pp or click on
. The
peak picking window will open. Click on
and drag the mouse over the spectrum
(fig. 60). If you want to restrict the number of signals stop the selected region above
the baseline.
cancels the selected peaks.
cancels one peak. When finished click on
shows the peak picking window.
allows individual peak picking.
to save (or on
48
to cancel). Figure 63
Figure 63. Manual peak picking in selected regions
17. Fluorine decoupled proton acquisition
A fluorine decoupled proton spectrum is acquired just like a regular proton spectrum
but with the following differences.
You cannot acquire a fluorine decoupled proton spectrum on the 200 MHz
spectrometer.
You cannot acquire a fluorine decoupled proton spectrum on the 500 MHz
spectrometer.
Use only the 5 mm Dual 19F/1H Z3752/0007 [04] probe (see ch. 5), Put the 2H stop
filter in the X channel (fig. 64) and read the shims by entering rsh dualf.
49
Figure 64. The 2H stop filter connected to the X channel of the preamplifiers
Set p1 to 6.9 and run a regular proton spectrum (see ch. 3). Afterwards, run a fluorine
spectrum (see the guide "Measuring NMR spectra of carbon and other non-proton nuclei")
with the parameter p1 set to 13. In the fluorine spectrum click on
the center of the signals and note the frequency.
, and put the cursor at
Tuning the proton and fluorine is carried out using adjustment screws (fig. 65).
Figure 65. Adjustment screws for tuning the dual proton fluorine probe
Blue
screws for
tuning
fluorine
Yellow
screws for
tuning
proton
50
Create a file with the parameters 1c_Protonfdec in the Experiment field (see ch. 3b).
Change the parameter sfo2 to the frequency that you noted down. Run and process the
spectrum like a regular proton spectrum.
18. Proton acquisition decoupled from other nuclei
It is possible to decouple other nuclei such as phosphorus from a proton spectrum. It
is preferable to use a BBI probe but a BBO probe can be used.
Only on the 500 MHz spectrometer and only on the BBO probe can decoupling from
rhodium be carried out.
Run a regular proton spectrum as described in ch. 3. If the chemical shift range of the
coupled nucleus is unknown then run a spectrum of the coupled nucleus (see the guide
"Measuring NMR spectra of carbon and other non-proton nuclei") or if there is
insufficient sensitivity run a heteronuclear correlation (see the guide "Measuring 2D
NMR spectra"). If using the 1D spectrum of the coupled nucleus click on
in that
spectrum, place the cursor at the center of the signals and note down the frequency.
Put the 2H stop filter in the BB channel (fig. 64).
Table 3. Parameters for decoupling other nuclei from proton
Decoupled
nucleus
Parameter
name
pl12 BBI
400
pl12 BBO
400
pl12 BBI
500
pl12 BBO
500
Phosphorus, 31P
1e_Protonpdec
10.1
19.6
11.0
17.7
Rhodium, 103Rh
1f_Protonrhdec
13.8
Open a file with the correct parameters according to table 3. If you have not tuned the
BB channel then do it now according to the instructions in the guide "Measuring
NMR spectra of carbon and other non-proton nuclei" ch. 1b. If you measured the
frequency of the center of the signals of the decoupled nucleus from the 1D spectrum
then set sfo2 to this value. Otherwise set sfo2 to sf × ΞX(1 + 10-6δX) where ΞX for the
coupled nucleus is as found in "Measuring NMR spectra of carbon and other nonproton nuclei" table 3 and δX is the chemical shift in ppm of the center of the signals
of the coupled nucleus. For example if phosphorus is the coupled nucleus and the
chemical shift of the center of the spectrum is 20 ppm:
sfo2 = sf × ΞX(1 + 10-6δX)
= 400.13 × 0.40480742(1 + 10-6 × 20)
= 161.9788325
If using a BBO probe (except for rhodium decoupling) you must change the value of
pl12 according to table 3.
On the 500 MHz spectrometer when decoupling at a temperature close to room
temperature, increase the gas flow to 535 L/h and change the parameters
(Proportional band, Integral time and Derivative time) according to the dashed
lines in fig. 49. Even so, the temperature may not stabilize and you will need to raise
the temperature by as much as five degrees or attach the cooling unit.
Run and process the spectrum like a regular proton spectrum.
51
19. Transferring files from the spectrometers to other computers
Request an account on the backup server of the laboratory that is called
nmrdisk.ch.huji.ac.il.
The files on the 200 and 400 MHz spectrometers are in the directory
c:\Bruker\TOPSPIN1.3\data\username\nmr and on the 500 MHz spectrometer in the
directory c:\Bruker\TOPSPIN\data\username\nmr. The files are backed up every night
to /Public/spectrometername/Bruker… (where spectrometername is the200, the400
and the500) on the server nmrdisk.ch.huji.ac.il.
On your personal computer you can install TOPSPIN as described in chapter 20. The
default directory for NMR files on your computer is
c:\Bruker\TOPSPIN\data\username\nmr but you can use any directory in the form
homedirectory\data\username\nmr and transfer your NMR files to there.
In order to transfer files to your computer you need to use an ftp client: either the
command line based client that comes with Windows or for greater convenience a
GUI ftp client such as FileZilla (download from http://filezilla-project.org/). To
install FileZilla from their website click on Download FileZilla Client, choose your
operating system and follow instructions.
In order to run the program click on
. A window will open like in fig. 66.
Figure 66. The FileZilla Client program
Directory name
on the remote
(server)
computer
List of actions
Directory name
on your (client)
computer
Explorer bar on
your (client)
computer
Explorer bar on
the remote
(server)
computer
The files in the
above directory
on your (client)
computer
The files in the
above directory
on the remote
(server)
Current progress
and logs
The first time the program is used, enter nmrdisk.ch.huji.ac.il in the Host: field, your
username is the Username: field, your password in the Passowrd: field and 21 in the
Port: field. On subsequent occasions, click the down arrow next to the Quickconnect
button and select the nmrdisk entry..
52
You can open directories on your computer (top left window) and on the server (top
right window) and drag files and directories from one to the other (fig. 66).
Likewise the software is already installed on the spectrometers and you can transfer
files to the server so that you can process them elsewhere on the same day. If you
want to transfer files before they are automatically transferred every night then you
can transfer them to your personal directory on the server: /username.
20. Use of TOPSPIN on other computers
There are floating licenses that you can use for TOPSPIN from any computer with
Windows XP Pro SP2 or Linux RHEL3 or RHEL4 operating systems that is
connected to the University network. If you want to use the software outside the
University then you need to request permission to connect to the University network.
For details see http://ca.huji.ac.il/services/internet/connect/home.shtml. Your
computer must have at least a 1 GHz CPU, 512 MByte of RAM, a 64 MByte graphics
card, a screen resolution of 1280 × 1024, DVD drive and a three-button mouse.
Check that you have the SSH Secure Shell program installed on your computer (see
ch. 18). Install it if it is not there but do not run it yet.
Install the TOPSPIN2.1 program from the DVD. Log in as an administrator (root on
Linux). Insert the TOPSPIN2.1 DVD. After a few seconds an install shield will
appear. Click on Next> and a Adobe Acrobat Professional – [rellet.pdf] window
will appear. Close the window by clicking on the X at the top right then click on
Next>. If you have a previous version of TOPSPIN installed, it will ask you to
uninstall the previous version. If there is no previous version skip to "continue
here…"
Click on:
Yes
Next>
Next>
Next>
OK
Next>
OK
Next>
OK
Next>
OK
Next>
OK
OK
OK
Continue here if the was no previous version of TOPSPIN to uninstall.
53
Click on:
Next>
Yes
Next>
A Password Input window will appear. Choose and enter a password in both places
on the window and click on Next. Wait several minutes (about 10 minutes with a 3.4
GHz single processor and 16× DVD). At the end a message will appear TOPSPIN
Setup Installation Complete. Click on Finish.
If this is an upgrade from a working copy TOPSPIN 2.0 then skip to the next
paragraph. Otherwise, copy (see ch. 18) the license file license.dat from the directory
/shares/internal on nmrdisk.ch.huji.ac.il to the directory c:\flexlm\Bruker\licenses on
your Windows computer (or the directory /user/local/flexlm/Bruker/licenses on
Linux). After the installation run TOPSPIN (in Linux you must log on as a user other
than root). A LICENSE window will appear. Accept the condition by clicking on I
Accept.
A Configuration check window will appear. Click on Expinstall and enter the
password that you chose earlier.
Click on:
OK
Next>
Next>
Choose the basic frequency (such as 400.13 or 500.2). Specify the printer/plotter.
Specify the page size as A4/Letter. Click on Next> then Finish then Close. After a
few seconds a message will appear at the bottom expinstall: Done and then you can
use the program.
Once the installation is complete you can transfer your files to your computer (see ch.
18) and use TOPSPIN from your computer. The number of licenses are limited so
please leave the program when you finish using it.
54
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