NMR Training
The goal of initial NMR training is to have a list (at least a mental list) of things you need or want to learn.
Learning NMR never ends, in the sense there will always be more that NMR can do for you in your work.
Think of the console training as going to office hours with questions before an assignment is due, rather than a
test to be passed.
NMR is “learn by doing”. It is a practical skill more than a theoretical one, although some understanding of
“what’s under the hood” of a modern (black box) spectrometer is essential. What has worked best in training
is to cycle repeatedly through the basic rote procedure of acquiring a simple 1D spectrum, filling in more
details as the procedure becomes familiar, the questions arise. This primer addresses:
Safety and local etiquette, necessary to get started, then
Gaps often encountered in intermediate users’ knowledge, and “urban legends”.
(Absolute beginners should ask for the tutorials of the chem4639 handouts).
When the defaults will not be what you want.
A menu of intermediate and advanced skills you can pick from, for your next goals.
Things to know before you begin operating an NMR
NMRs are expensive. They are delicate. Negligent use can be disastrous. Most damage is avoidable, some is not.
If you understand how the instrument works, you will also develop some feeling for why things should be done a
specific way. If, on the other hand, you do things your own way and disregard local procedures, you run the risk of
damaging the instrument. Repairs are often thousands of dollars, but the greater cost is if research and classes
cannot use the NMR for several weeks. If you are a beginner, relax: problems are rarely caused by attentive
beginners.
If you already know NMR, pay special attention: every NMR is different. The software can be set up in
many different ways for a different user base or for different hardware capabilities. The shortcuts or
procedures you learned at another site may not work here (may actually be dangerous, if not set up properly).
Each instrument has idiosyncracies to work around -- the calibrations are certainly different. Ignoring the local
etiquettes of how to leave the instrument ready, could effectively shut down the NMR. Your experience
hopefully puts you further along in understanding what underlies the steps of locking, shimming, acquiring and
processing, independently of the specific Varian or Bruker commands to do them. And so you may get up to
speed more quickly. But if you want access to this instrument, you must be willing to work within our user
community.
[ insert homer simpson picture ]
Basic Magnet Safety:
NMR magnets are always live and always at field. They cannot be
turned off like a light switch. Therefore, nothing ferromagnetic is allowed
near them (within 3 ft of the 400). At the top of the list are tools and other
heavy objects (wrenches, screwdrivers, metal chairs, and floor buffers).
These can dent or puncture the magnet dewar, or cause a quench: the
magnet is drawn to the object as strongly as the object is drawn to the
magnet, if the magnet hits the side of the dewar it can trigger a quench.
Also important are small object that can fly into the bore: paper clips,
staples, bobby pins, metal barrettes, costume jewelry, or wallet chains.
Items such as iPods, cell phones, and media storage devices can also be
damaged by the magnetic field. ATM and credit cards may become
unusable. Don’t get sloppy because the AS in AS400 means “actively
shielded”, so it has 1/5’th the fringe field of a common (unshielded)
magnet, because the next magnet you see won’t be. Please practice good
magnetic hygiene and leave all of these items in your lab or next to the
instrument’s computer when you approach the magnet.
Leave the door open when you are in the room. This is a good habit in general around cryogens, but
especially an NMR. The magnet contains a tremendous amount of energy that could be released as heat,
boiling off a lot of liquid helilum and displacing a swimming pool volume of air in a few seconds. Such a
quench happens rarely (about once per century per magnet), but if the magnet starts to blow like a steam
whistle, immediately follow your instinct to run away out the open door. In low oxygen you black out without
warning (you don’t get short of breath unless there’s high CO2).
Don’t waste time learning software: learn what the commands do.
It is possible to take spectra by memorizing commands, like cheatcodes to a video game, but that knowledge
will be quickly obsoleted by a new version of software. Put a little thought into what the data would look like
as a spreadsheet, and what each command does to the data. That knowledge is forever, and portable. What
happens if you learn on a Varian but they have a Bruker at your first job? Asking "Where’s the chloroform
lock?' or "what shims are responsive on this instrument" makes a good impression; In contrast, "what button
do I push", not so much.
Focus on learning your immediate need, but recognize you can keep learning NMR forever.
To be a competent chemist you need an appreciation (not necessarily fluency) of the things you don't know
that support your professional expertise. (Or produce the artifacts in your spectrum you must explain or
avoid.) Blaming your tools is unprofessional, merely reveals ignorance.
Pay attention, watch other students, ask questions, compare notes. .
There are also a lot of "urban legends" handed down in NMR. Like "lock power should be less than lock
gain".
There is a natural tendency to be shy about one’s lack of NMR knowledge, to avoid risking losing access to the
NMR, or perhaps to save face. The opposite is true. Asking naïve questions will get you an answer tailored to
your understanding and goals. Accounts only get cancelled if a user i) presents a problem or risk from not
knowing something; and ii) persistently resists learning it after being told how and why it is important.
Don’t be shy about having questions about NMR or software. I’ve been at this for twenty years, and I still
see new commands or capabilities of vnmr software I’ve never known were there. About half of the problems
that come to me require looking up or figuring out new things. Understanding what you are doing on the
console can get into a lot of interesting subjects: synthetic chemistry, signal processing, computer
programming, math, physics.
“No day without a tip”
This document outlines the steps of taking an NMR spectrum the way we do it here. it relies more on the
command line than on automation.
The basic steps are :
Making the sample
Inserting the sample
-- Making samples.
Use deuterated solvents.
Use a solvent with TMS. Chemical shifts are (by definition) with reference to TMS. Referencing to solvent
(e.g. chloroform at 7.26 ppm) is ok for a quickie, but it is not publishable. TMS
evaporates readily (it will disappear from samples in a week or so, even from a capped
tube. It is trivial to add TMS to a sample by pouring vapors from a bottle of TMS
over an open NMR tube. Non-deuterated solvents can be suppressed, but this is an
advanced technique – about 2 hr to learn and ½ hr each time you do it.
The sample volume should be 700 uL -- at least 4 (preferably 5 cm) in the NMR tube.
Making it smaller is hard to shim: you will get worse S/N and resolution. 3 cm
samples only make sense when spending 4 hr or more acquiring a severely masslimited sample, for expert at shimming, with time to spend a half-hour at it.
NMR tubes vary from $1 "disposable" tubes to $20 or more. Cheap tubes are fine for
most routine synthetic work, as long as they are straight enough, so they don’t scratch the probe when they
spin.
Expensive tubes can be shimmed to better linewidth: they have more uniform wall thickness. Cheap tubes
can't be shimmed as tight and may give spinning sidebands of a few percent. But they are fine for most work.
It is important that the tube not be curved, or it can scratch the probe when it spins. Wilmad HIP tubes are
fine. Even a tube that rolls true on a flat surface can be a problem. If in doubt, bring a tube to the NMR500
and I have a tester (a straight, tight sleeve: if the tube fits in the sleeve and spins smoothly, it is ok as far as the
instrument is concerned.) Advice on washing NMR tubes is given in an appendix.
-- Correct positioning of the NMR tube in the spinner.
Clean the tube thoroughly after inserting it into the spinner. Scrub it vigorously with an acetone-soaked
kimwipe, then dry off the dirty acetone with a dry section of the Kimwipe. There is a film of something on
most new NMR tubes that shows up in spectra acquired for 5 min or longer. The O-ring in the spinner was
put there by the devil to coat your sample with a mix of silicone oil, water bath fungus, skin flakes, sharpie ink,
cellotape adhesive. (Is chemistry the only profession where you wash your hands *before* you go to the
bathroom?). Even if you are taking a quick spectrum of a concentrated sample, keep that gunk out of the
probe. Plean clean all samples, habitually. No exceptions.
If the NMR tube slides too easily in the spinner, use the other spinner and email Roger that the O-ring needs
replacing. When the spinner drops into the magnet, the tube can keep going and bottom out in the probe.
Position the clean tube using the depth gauge.
Center the sample on the CL mark, but not lower than the maximum depth (bottom of the guide). The sample
must not only completely fill the detection coil observed volume (dotted box in guage) but the ends of the
sample should be at least 1 cm from the coil. The magnetic field bends in & out of the sample at the ends (the
sample/air interface) these cannot be shimmed. If the interface is 1 cm away, you can shim quickly with Z1C
and Z2C to under 1 Hz. If the interface is closer, you
need to shim Z1 and Z2 ten times at different Z3 and
Z4 settings to find the best combination.
If centering a large sample would put it deeper than
the max depth, then the ends will be far enough away
the asymmetry doesn't matter.
Please do not mess with the max depth adjustment of
the depth gauge. It should be 69 mm for the 4nucleus probe; 65 mm for the broadband probe
(backup probe).
-- NEVER DROP A SAMPLE INTO THE
MAGNET WITHOUT THE LIFT/EJECT AIR
FLOWING. Get in the habit of feeling the sample
float before letting go of it.
Turn the lift air on with software. (The manual
switch is only there if an sample must be removed
when the computer is down; it requires higher air
pressure and won't support a sample).
- Spin the sample at 20 Hz.
Spinning averages out poor X or Y shims, making it easier to get good Z shims on the first pass. It typically
sharpens peaks from 1.3 Hz (nonspinning) to 1.0 Hz (spinning), which is hardly noticeable in most spectra.
Spinning can introduce spinning sidebands around your peaks, and noise in 2D spectra. It is best turned off to
see small peaks near big solvent peaks, and for all 2D spectra.
- Locking
Locking does three things.
1) You make a fine adjustment of the magnet field (Z0) to tune your sample to the "tuning fork" in the RF
cabinet (spectrometer frequency), so our spectrum is in the spectrometer’s spectrum window (sw);
2) When the lock is turned on ("field frequency lock") the spectrometer will then auto-adjust Z0 to
compensate for trains, elevators, or magnet drift that would otherwise move the lines while the spectrum is
acquiring, and thereby blur your spectrum.
3) The lock level gives a signal to shim on.
The lock display on a Varian shows the "difference frequency" between the sample and the tuning fork. You
adjust Z0 to make this difference 0 (step function).
Each hump in the display is about 10 Hz of frequency, and is about one click of the 16's button of Z0. You
will see 2 humps if you are either 20 Hz too high (sharp) or 20 Hz too low (flat).
A complication is that the intensity of the trace is random. Try it: scan small values of Z0 (e.g. the 4's button)
you will see sometimes the humps are large, but often small or 0 (it depends on the relative phase of the tuning
fork and the signal from the ping'ed sample).
There is an urban legend that lock power should be lower than lock gain. What is true is the lock power must
not be too high. There is a specific number for each solvent (written in a chart near the spectrometer). If the
lock power is too high, it will saturate (suppress) the solvent peak instead of stimulate it. If this happens the
lock level will oscillate wildly, and will not reflect how good the shims are. The gain can be any number that
puts the lock level between 20 and 90%.
Another urban legend is you should shim to 70% lock level.
-- Shimming
Shimming means adjusting the magnetic field to be exactly the same at the top, middle, and bottom of the tube
- within one ppb (one part-per-billion). Note 1 ppm on your NMR spectrum would be one ppm of variation in
the magnetic field. )
Shims Determine Linewidth
The Z direction is the long axis of
the sample; X and Y are
transverse.
Shimming is an art. Explore it on
your own. Lesson 1 below should
get you decent shims in a couple
of minutes.
The field must be the same at the top, middle, and bottom of the tube.
The lineshape you get is the sum of all the volume elements
2 Hz marginal; 1 Hz good; 0.5 Hz limit
B(z)
ppm
ppm
type bestshim
to load a
good starting shim set.
Start with the 4's button of
Z1C.
if it goes 4 clicks, check Z2,
z
and go 2 clicks.
Good Homogeneity
When you go through the
maximum, step back to it using the 1's button.
Get to a point where any Z1 change or z2 change reduces the lock level.
Bad Homogeneity
Reduce lock gain if the lock level goes above 100% (and you can't see changes).
You can click about once per second for CDCl3 or D2O. With acetone or methanol, wait for the lock level to
stabilize before clicking again – about 3 sec.
The lock level is the height of the solvent deuterium line. The area is fixed, so the height depends on the
linewidth.
You should be able to get good (1 Hz) shims using the 4's button on Z1C and Z2C
The 64's button of Z1 is about equal to the 1's button of Z1C. If you start shimming on Z1, it may hit its limit
(of 2048) before you are shimmed.
The vnmr software supports a very wide range from fine to coarse, because it is the same software used for
many NMRs -- from microNMR up to imaging magnets. Additionally, on some spectrometers the shim values
range from -32K to +32K, others (like the 400) only from -2K to +2K.
Z1 to Z5 make corrections as a polynomial series ( f(x) = c + c1x + c2x2 + c3x3 + c4x4 + ...)
The bottom line in shimming is whether the lineshape of your H1 or C13 spectrum is good enough to see what
you need to see. The TMS line is usually sharp, and good to check your shims on. TMS can be shimmed to
0.7 Hz (but getting 0.7 Hz shims on TMS may be a waste of spectrometer time if others are waiting). Your
compound will have a natural linewidth from 1.2 to 1.5 Hz.
After a quick shimming, start acquiring, and check the TMS line. If yourshims aren’t good enough, abort
acquisition and shim some more.
The most common reasons for shimming problems are:
Bad starting shims (type bestshim and continue)
The sample is too small (dilute it to 4 cm)
The sample was not centered on the coil (reposition and re-insert)
A bad tube (throw it out and use a new one)
The sample precipitated, or didn’t dissolve.
You added solvent to solid in the NMR tube, and there is a high conc at the bottom (shake it up).
-- Tuning. We don’t tune the probe of the 400. Just be aware that on many spectrometers it is a good idea to
tune the probe with your sample inserted. It is mentioned in passing here so you won't look stupid if they do it
at your next job.
-- Set Up Acquisition
Typically click (MainMenu) > Setup > H1, CDCl3
(click other buttons as appropriate for different solvent, etc.)
You can also load a previous parameter set, just retrieve a spectrum, then type su ('setup") to load them from
the Sun Computer into the spectrometer (the RF cabinet).
Generally you acquire your proton 1D into exp1. If you will be taking a 2D, you copy your 1D paramters into
another experiment directory (exp2), join exp2, then setup the 2D.
If you load a parameter set for CDCl3, and your sample is in DMSO, some of your peaks may be outside of the
spectrum window. Either click setup again for the correct solvent, or type solvent='DMSO'
Set the number of scans, e.g. nt=16.
If your sample is dilute, increase the number of scans (transients).
10 times the number of scans takes 10 times longer but ives 3 times the S/N. Practical limits are:
H1 spectrum of 10 ug overnight
C31 spectrum of 10 mg overnight.
There is a tradeoff between sensitivity and resolution.
If your sample is dilute, you can get better sensitivity (S/N per hour) if you take small pulses quickly: d1=0,
np=16k, pw(30)
If you need accurate integrals increase time between scans:
d1=3, np=32K pw(60)
Type dps to see your current pulse sequence.
-- Start the acquisition
type ga.
-- Annotate the spectrum.
While waiting for acquisition, type gettext.
A minimal text file includes, file name (sample name, if different), your name and date, and location where
data is stored, e.g.
LM-Bz-ascorbate-top
in CDCl3, .05% TMS
John Smith, 23 August 2011
NMR400:smith
Any samples in your refrigerator or shelves without name and date are worthless and disposable, the same is
true of NMR data. If you don't record where data can be found, it doesn't exist, you never completed the
synthesis, and shouldn't get paid or recommended for a job.
3x the sample will acquire 10x faster;
10 times the sample will acquire 100x faster
Set the reference as soon as there are enough scans to see it.
It is possible to copy and paste the filename from the text file into the "save FID" dialog. It is a good habit to
save the data as soon as it is done (in case it is accidentally deltered, e.g. by another ga), and then to save it
again after processing (phase, set reference, set integrals, )
While data is acquiring, look at the FID (type df).
This is the signal recorded by the spectrometer. It is essentially an oscilloscope trace of a bell ringing, and
fading out over a second or so. A plot of amlitude vs. time. To see what frequencies are present, you need to
do a Fourier transform.
- Display and Plotting
The most noticeable skill on the spectrometer is your ease to zoom in and out of regions of the spectrum,
measure couplings and frequencies, and create plots that can make a point, sharply. This is something
everyone manages to learn well. In this draft I prioritize the things people miss and can’t move on without.
Things You Have Yet to Learn
Bilge Rat
Galley Slave
Deck Ape
Able-Bodied
How to take a simple 1D proton
How to lock on different solvents
How to shim Z1 and Z2 together (second-order optimization)
how to shim higher-order shims
Reduce noise with line broadening (what is an FID?)
Identify water and other solvents in spectra.
Plots with expansions or stacked plots
Export plots to powerpoint or word processor.
Use multiple exp’s (e.g. acquire in exp1, process in exp2)
Acquire multiple spectra overnight
Retrieve saved data
Predict Spectrum of a chemical structure
Back up your data (copy to another computer – ssh)
Off-line data processing (ACD, Mestre, spinworks)
Basic unix (list directory, change directory, copy files)
Acquire 13C spectrum.
Set parameters for sensitivity vs. accuracy
Measure a 90-degree pulse (prelim for all 2D).
Set gain. (describe signal path)
Basic 2D spectrum (e.g. gCOSY) – setup, process, plot
Ship’s Mate
Measure C13 90o pulses
Set decoupler power
2D proton-carbon spectra
Create and use shaped pulses
Use pulsed field gradients
-- When the Defaults Are Not What You Want
There will come a day when the defaults won't work for you. The default parameters are a compromise
between sensitivity and accuracy. One day you may have barely enough material to get a spectrum overnight,
and need to squeeze out another factor of 2 in sensitivity. Another day you may have a contaminant, and you
need more accurate integrals to distinguish the two mixed compounds.
For sensitivity set a shorter time between pulses, (and use smaller tip angles);
For quantitative accuracy, set a longer time between pulses.
This section very briefly describes the relevant of the parameters, and gives brief samples of the principal tools
for understanding them:
The pulse sequence,
The vector model
A 90 degree pulse calibration
At right is how we sketch the basic 1D pulse
and acquire sequence. The sample is pulsed to
stimulate emission of the signal, which is
acquired and recorded, until it dies out.
pw
pulse width
d1
at
relaxation delay
acquisition time
You can see your pulse sequence on the Varian
by typing dps.
time
This is repeated and the signal added:
Sample
Free Induction
Decay
(FID) Recorded
Relaxatio
n
Delay
time
Typical defaults would be:
a 45 degree pulse,
1 second acquisition time
1 second relaxation delay
What’s a 45-degree pulse? The angle refers to the vector model.
30o pulse
At equilibrium
Pulse Width
1 µsec
45o pulse
3
2
30o pulse
60o pulse
6
5
4
90o pulse
45o pulse
8
7
60o pulse
90o pulse
Figure: At top, the magnetization of the sample is tipped more, by longer pulses, up
to 90 degrees. The intensity of the spectrum (bottom) is proportional to the xy
component of the tipped magnetization.
For 1D NMR the pulse is not critical – very little difference is seen in the spectrum whether you used a 45
degree or 60 degree pulse. For 2D NMR, however, knowing the length of the 90-degree pulse is critical.
The default calibration is an approximate guess. For 2D NMR the 90 degree pulse is calibrated by taking a
series of spectra, with increasing pulse lengths, as shown below:
90o
0o
2 µsec
4
6
8
180o
10
13
16
360o
270o
19
21
24
27
30
A spectrum line when excited by pulses of indicated length. The 360-degree
pulse above is 32 usec, so the 90-degree pulse is 8 usec.
33
FID (free induction decay, “time domain”)
np
at =1 sec
=16K points
another tremendous degree of optimization and control comes in the
process of the Fourier Transform.
At left is the data recorded
ft
Fourier transform
Spectrum (“frequency domain”)
np
=16K points
The FID can be thought of as a sound recording of a bell ringing, and
dying out in a second or so. (Actually, a chorus of bells). The NMR
spectrum is plot of the frequencies present in the chorus, obtained by a
Fourier transform of the FID. There are some processing tricks in how
you do this Fourier transform that can improve your S/N significantly, or
can sharpen peaks if you need to resolve multiplets or measure coupling
constants.
The Fourier transform algorithm in the software requires that the number
of points must be a power of two. (If the number of data points acquired
is not a power of two, zeroes may be added to the end of the FID.
Typically it is 16 Kb or 32 Kb. )
The number of points in the spectrum is the same as the number of points in the FID. A surprising fact is
adding more points to the end of the FID (making it longer) increases the density of points in the spectrum,
rather than making the spectrum wider. We can use this if you want to see narrow lines or 2 Hz splittings:
zeroes may be added to the FID to get better digital resolution (more points on each peak) in the spectrum.
The above is a cartoon version of the first principles, to help you understand what the commands are doing.
You don’t need to understand them to get started – and probably can’t understand them until you get started.
But you will need to become a competent NMR spectroscopist if you want to become a competent chemist -to justify the structures of your compounds in publications.