6. TUNING AND SHIMMING
Before any NMR experiment can be performed on a new
sample the probe must be tuned and shimmed to obtain the optimum performance
of the spectrometer. Both of these operations are critical if the user
is to obtain high quality NMR spectra. It is not worth spending weeks
preparing a sample if you do not spend the time to tune and shim the probe
correctly before carrying out your experiments.
The tuning process matches the resonant frequency of the coil with the
sample in place to the input frequency from the spectrometer. Each sample
will have a slightly different effect on the resonant frequency whilst
different solvents can have very large effects on the resonant frequency.
The shimmimg process adjusts the homogeneity of the magnetic field in
the vicinity of the sample. This is the most critical and perhaps the
most difficult part of setting up your experiment. To the new user it
can appear a time consuming and perplexing task however it is worth taking
the time and effort to learn how to shim because a poorly shimmed magnet
will never give a high quality spectrum.
6.1 SAMPLE VOLUME AND
DEPTH
- The magnets have all been shimmed on a sample volume
of 500 µl in a 5 mm NMR tube. You should not use a sample volume
greater than 500 µl. If you use a sample with a different volume
it will be MUCH harder to acheive good shimming within a reasonable
time.
- If you do not have sufficient volume for 500 µl of a 1-2 mM sample,
you may use a Shigemi tube. These require 320 µl of sample but should
only be used when your sample is limited as they are are much harder to
shim than the standard volume
- The sample should be in a Wilmad 5 mm NMR tube with the serial no 528
or 535 marked on the outside of the tube.
- The sample tube should be inserted into the correct spinner and the
bottom half of the tube should be wiped with a clean tissue to remove
dirt and grease.
- Now adjust the position of the tube so that the bottom is at the 62
mm mark in the Varian depth gauge. (Shigemi tubes will need to be lower).
Do not touch the bottom of the sample tube with your fingers.
NOTE If your sample is running above room temperature for a period
of days, some of the solvent will evaporate from the main body and condense
in the upper portion of the tube. This will significantly alter the magnetic
field homogeneity during the course of the experiment. If this is likely
to be the case for your sample you should discuss the possibility of using
a "Matched Susceptibility Plug" to prevent this from occuring.
6.2 REMOVING A PREVIOUS
SAMPLE
- Check the "Acquisition Status" window or the "Acquisition
Display Unit:".
- If the lock level is NOT zero this means there is a sample in the magnet.
If the reading is zero this normally means the probe is empty. However
proceed with caution there are times when the lock level will read zero
and there is still a sample in the probe.
- Click "Connect" in the Acquisition Window. The window should pop open
- Click on "Eject".
- At this point you should be able to hear the sound of air rushing through
the probe. If you cannot hear it WAIT for a few seconds. If you hear a
gradual rising tone like somebody whistling, it means there is another
sample in the probe and that this is being ejected. Wait until you see
the old sample emerge at the top of the magnet.
- Remove the previous sample and place it carefully in the sample holders
provided.
- Click "insert" in the Acquisition Window, the air flow should shut off.
6.3 SETTING THE
TEMPERATURE
6.3.1 Setting the FTS
Controller
The FTS controller consists of two parts: the main control
unit which has the LED display and touch pad for adjusting parameters
and the compressor unit which cools the input air into the probe if necessary.
- Check the temperature setting on the FTS control unit.
- The Set Point (SP) should be adjusted to 10[ring] C below the temperature
required for your sample. Adjust the setting by pressing the s and t arrows
on the control unit.
- If the temperature setting on the FTS is to be higher than 25[ring]
C, switch off the compressor.
- Wait for a few moments for the temperature to adjust, this should be
fairly rapid. If the temperature overshoots and continues to heat or cool
in the same direction press the "Run" button twice.
6.3.2 Setting the Sample
Temperature
- On the Command line type:
temp=XX where XX is the required temperature
su
- If the temperature was different to the previously set value you should
see the value on the display module change. As the temperature approaches
the required temperature the rate of change slows and it can take several
minutes to regulate the last 1-2 degrees, this is not unusual.
- If your sample is sensitive to high temperatures you should wait a few
minutes before putting your sample into the magnet. Otherwise you can
proceed immediately.
6.4 INSERTING YOUR SAMPLE
- Open the Acquisition Window and click on "eject"
- Wait until you can hear the sound of the rushing air.
- Place your sample gently into the top of the upper barrel. Make sure
that it is free from the sides.
- Click insert in the "Acquisition Window". The sample should now descend
into the probe. It will locate with an alarming "Clunk". If it does not
then click "eject", wait a few seconds and then click "insert".
- You should now wait 5-20 mins for your sample to equilibrate before
you tune and shim the probe. The time you will have to wait will depend
on the temperature of your sample before it went into the probe and the
temperature you have selected to run the experiments. Clearly if you have
taken your sample out of the fridge and are running at 60 [ring]C you
will have to wait the full 20 mins. (Go and get a coffee!!). If you are
running above room temperature it is advisable to let your sample sit
at room temperature for several minutes in order that the dissolved air
can come out of the sample.
6.5 TUNING THE 600s
Figure 6.1 shows a schematic layout of one of
the spectrometers and a more detailed diagram of the connections on the
preamp unit and the magnet/console interface
Figure 6.1
Layout of Preamplifiers and Tuning
Unit
6.5.1 Tuning Proton
- Make sure the temperature is set to the required
value.
- Check that the following parameters are set.
solvent = `D2O'' If you use a different solvent set this parameter accordingly
tn='H1'
- type "su" in the command window.
- Disconnect the cable (and any filters) from the Probe socket on the
1H preamp (Figure 6.1) and connect it to the Probe socket on the Tune
unit. A red light should start to flash.
- Move the cable from the Output socket of the 1H preamp to the Output
socket of the tuning unit.
- Press the channel selector button so that it reads 1. The green LCD
display will light up
- Set the Attenuation to 8, if the reading is off scale adjust the attenuation
to a lower value to get the reading on scale.
Figure 6.2
Location of the tuning rods on a Triple
Resonance HCN Probe
- On the probe find the rod labeled Proton (Figure 6.2),
it usually has a red label
- The rod has two movable parts, the lower smooth part adjusts the "Tune"
capacitor and the upper knurled part adjusts the "Match" Capacitor.
- Adjust the Tune capacitor so that the number on the display is a minimum
- Now turn the Match Capacitor a little in a clockwise direction (the
number may get bigger)
- Now readjust the Tune to minimize the reading.
- If the new reading is smaller than the previous value, turn the Match
in the same direction as before and repeat the tuning process.
- If the new reading is higher than the original reading, turn the Match
back in the opposite direction and readjust the Tune to minimize the display,
- Keep adjusting the Match and Tune in this way until the reading reaches
a minimum. It should be possible to get the meter reading down to 1-2
on all probes with the attenuation set to 8.
- When you have finished set the channel selector to 0 and move the cables
back to their original positions
6.5.2 Tuning the First
Decoupler Channel
Most modern NMR experiments use samples that are labeled
with C13 and/or N15 and it is necessary to apply pulses or decoupling
to the second channel. The larger spectral widths of C13 experiments place
a greater demand on the power requirements for applying RF pulses. The
first decoupler channel is routed through the Magnet-Console interface.
The presence of relays and filters within the interface leads to a loss
in 2-3 dB of power compared to the second decoupler channel which is routed
directly from the power amplifier into the probe. Consequently the spectrometers
are normally configured to apply N15 pulses on the first decoupler channel
and C13 pulses on the second decoupler channel. If you wish to change
this you can do so.
- Set tn='H1'
- Set dn='N15' or 'C13'
- type su
- Tune the Proton channel as above
- Connect the Cable from the B-Band Out socket on the back of the Magnet/Console
Interface to the "Probe" input of the tuning unit.
- Connect the output cable from the X-Preamplifier to the output of the
tune unit
- Set the channel selector switch to 2
- Adjust the 'C13' or 'N15' rods (as appropriate) as before
6.5.3 Tuning the Second
Decoupler Channel for C13
Tuning the second decoupler channel is similar to tuning
the first decoupler channel except that intially the cables are connected
differently:
- Set tn=`H1'
- Set dn=`N15'
- Set dn2=`C13'
- Type su
- Tune the Proton channel and the N15 channel as described in sections
6.5.1 and 6.5.2 above
- Locate the cable coming from the C13 channel on the probe. It should
be connected to a large cylindrical filter. The other end should be connected
to a thick black cable labeled 2ND DEC or DEC2
- Disconnect the thick cable and connect a one end of a thin cable to
the cylindrical filter and connect the other end to the Probe socket on
the tune unit
- Connect the Output from the X-Preamplifier to the Output of the Tune
unit
- Set the channel selector switch to 3 and tune as before
- When you have finished, reconnect the thick cable labeled DEC2. DO NOT
confuse it with the cable labelled DEC3. Replace all other cables in their
original positions
6.5.4 Tuning X Nuclei
for Direct Observe
- Repeat the above procedure except for the following
changes
- Set tn='N15', 'C13' or 'P31' etc.
- Set dn='H1'
- Set the channel selector to 1 and tune the X nucleus
- When you have finished connect the Probe cable to the Probe socket on
the X Preamp
- Tune the 1H channel as before except set the channel selector to 2
- Connect the 1H Probe Cable to the H-Band Out socket on the Magnet/Console
Interface
6.6 TUNING ON THE 300
AND 400
Tuning on the 300 and 400 is essentially the same as
the 600s except you only need to reconnect one cable to perform any tuning.
This is the cable that comes from the Probe and goes directly into the
back of the Magnet/Console Interface. You disconnect this cable and connect
it to the Probe input on the Tune interface which is located on the front
of the pre-amplifier housing and not the magnet leg.
The 300 and 400 are equipped with different hardware which can only produce
1H frequencies on the 2nd channel. Any other frequency must be produced
from the 1st transmitter. The spectrometer checks the setup for each experiment
and automatically switches the 1H to the second channel if necessary.
You must be aware of this change if you run experiments with 1H and X
nuclei set.
6.6.1 Tuning for 1H
Only Experiments
- Set tn and dn ='H1'
- Set the Channel Selector to 1
6.6.2 Tuning for any
Other Experiment
- For 1H observe/ X decouple experiments set tn='H1'
and dn='X'
- For X observe/ 1H decouple experiments set tn='X' and dn='1H'
- In both cases, connect the Proton cable to the tuning meter and set
the channel selector to 2 and tune the proton as before
- Then connect the X cable to the tuning meter and set the channel selector
to 1 and tune as before
6.7 LOCKING
The lock circuit is used to maintain a stable magnetic
field. It uses the deuterium signal of the solvent as a fixed reference
frequency. The lock circuitry detects changes in the magnetic field and
adjusts the field strength accordingly to maintain the deuterium signal
at the reference frequency. The exact frequency of the deuterium signal
is different for each solvent but the spectrometer uses a list of the
deuterium frequencies stored in one of the configuration files to adjust
its parameters to accommodate these differences.
- Check the setting of the "solvent" parameter. This is normally set to
D2O, however on some occasions other users will use different solvents
e.g. DMSO.
- Check that the temperature is set correctly in this experiment.
- Enter the command "su"
- Open the Acquisition window
- Click on the "Lock" Button
- In the pop-up window click the lock "OFF" button
- Change the "Lock Power": For D2O set the Lock Power to 25, for H2O set
the lock power to 36
- Adjust the "Lock Gain" so that you can see the lock signal
- Adjust the value of Z0 to get maximum intensity. The final signal should
have no modulation and only a small decay
- Click the lock "ON" button
- Adjust the "Lock Phase" to maximize the lock level
6.8 SHIMMING
6.8.1 Introduction
Shimming is the process of adjusting the magnetic field
to produce a homogeneous field throughout the sample volume. Good shimming
is essential for all experiments, particularly those that use selective
pulses and gradients for suppression of strong unwanted solvent resonances.
The number of shims on each magnet varies from 13 on the 300 to 39 on
the 600s. They can be classified into two major groups: The Axial shims
(also called the Z shims) are those that contain only Z components, i.e.
Z1, Z2, Z3, Z4 etc.. The Radial shims (also known as non-spinning shims)
are those that contain X and/or Y components, they may also contain Z
components e.g. X, Y, XZ, YZ, XZ2, YZ2 etc.
There are two methods for shimming your sample, the
first is the manual way by hand. The other is to use Gradient Shimming.
This latter method uses gradients to generate maps of the effects of each
shim on your sample and applies a mathematical correction to the shims.
It is a very powerful and rapid technique that can acheive amazing results
very quickly. The process for Gradient Shimming is discussed in detail
below.
The shims have initially been adjusted for the best
sample homogeneity by the Varian Engineers during the installation of
the machines. However, each time you put a sample in the magnet you may
have to adjust the following shims: Z1-Z5, X, Y, XZ, YZ. The Z shims are
the most sensitive with Z1 being the most sensitive, but Z4 and Z5 have
the most complex effects on the sample. The higher order shims are normally
adjusted using a sample of Chloroform in Acetone which has a line width
of less than 0.2 Hz. Protein samples typically have line widths 100 times
greater than this and it is difficult to observe any effects on the
6.8.2 Manually Shimmimg
Your Sample
In practice shims are normally adjusted in pairs.
Each pair of shims is adjusted in an iterative fashion to produce the
maximum signal possible before the next pair is adjusted. When all sets
have been adjusted the whole process is repeated. The following scheme
suggests one method for iterating the shims.
Step 1 - Adjust Z1 and Z2
Step 2 - Maximize Z3 and then readjust Z1 and Z2
- Repeat the above until there is no further
increase in the lock level
Step 3 - Move Z4 by 128 and repeat Step 1
- If the lock signal increases repeat the above
step
- If the lock level is lower adjust Z4 in the opposite direction and repeat
step 1
- Repeat the above steps until no there is no further increase in the
lock level
- Periodically check the adjustment of the lock phase as large changes
in Z4 can induce significant changes in the phase of the lock signal
Step 4 - Adjust X and Y
- Adjust
XZ and YZ and then readjust X and Y
- Then
repeat step 1-3
Step 5 - Repeat Steps 1 to 4 until there is no further
increase in the lock level.
If you have problems shimmimg your sample you can put
in a test sample of 99.99% D2O with the same sample volume as your sample
and shim on this to get a good lineshape. Then replace it with your own
sample making sure they are set at the same level in the depth guage.
You will have to adjust Z1, Z2 and Z3 but the remaining shims should be
very close to the required values. If their are significant differences
in the sample depth you will have to apply large corrections to the Z4
shim. This cannot be done on a 90% H2O sample and must be done
by using a D2O sample.
6.8.3 Storing and Retreiving
Shim Values
If it takes you a long time to shim your sample
and you intend to use this sample for other NMR experiments in the future
you can store the shim values for that sample. There are two ways to do
this but we recommend that you save the shims in a 1D data file. To do
this set up any of the 1D experiments outlined below and set nt=1, run
the single scan experiment. Now change to the shim directory under
vnmrsys and save the file with a meaningful name e.g. sry_m64i_122595_61mm.
To retreive these shim values load the file and then enter the following
commands:
- load=`y' su
NOTE The shim values are only valid for the machine
and probe for which they were adjusted. If you use a different machine
or a different probe you must reshim your sample. Furthermore your shim
values are only valid if the sample is exactly the same next time you
use it. Even then you will have to readjust the Z1-Z3 shims.
6.9 GRADIENT SHIMMING
Gradient shimming is the best way to acheive good line
shape. There are two distinct stages to the gradient shimming process
the first is to Generate a Shim Map for your sample. The second
is to perform the actual gradient shimming. In either case it is critical
to have well adjusted radial shims (X,YXZ and YZ etc...).
6.9.1 Generating Shim
Maps
The first step in generating a shim map is to optimise
Z1, Z2, X,Y XZ and YZ shims manually. This is critical if you want to
produce a good map on the first pass. You should also check the other
higher order radial shims. However if you do not see any significant change
in the lock level you should put them back to their initial settings.
Having made an initial adjustment to the shims we can
now set up the mapping parameters. Enter the command "gmapsys".
You should now get a new set of menu buttons on the command line.
There are X stages to Generating a Shim Map. The first
is to set up the parameters such as pulse width, offset and gradient
strengths, then set the optimal window for generating the shim map and
finally generate the shim map itself.
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