an alphabetical listing of NMR terms
There are three things to define in pulsed NMR: when to do nothing, when to pulse, and when to record the signal (FID). These things are defined in the acquisition parameters list. In Topspin the AcquPars tab contains the acquisition parameters listing. There are two display modes in AcquPars: ased and eda. One is a short list of the most relevant parameters for the given pulse program (computer instructions for the NMR experiment including delays (when to do nothing) and when to transmit rf irradiation). The second (eda) gives the full listing of parameters. The corresponding text file for eda can be found on your computer under the folder for the relevent experiment number (expno) and is named acqus. Look under the letter "P" to find more information describing the basic acquisition parameters.
ased, acquisition parameters
edit assigned parameters
"ased" is a subset of the full acquisition parameters table (eda). When you read a parameter set (rpar) or choose an experiment with the "new" window or copy an old data set to a new parameter number, in each case a file called acqu, containing a list of relevant acquisition paramters is created inside the folder of the new experiment number.
If you navigate to the proper directory, you can view the acqu file with a text editor.
After acquiring an FID (so that a file called fid exists in the expno folder), a file called acqus will also be added. This file can also be opened with a text editor. It is a "second" acqu file that contains the parameters used to acquire the FID. These are the values printed when you plot the parameters with your spectrum.
atma or atmm
automatic tuning and matching in automatic (atma) or in manual (atmm) mode. A motor inside the probe and controlled by the computer adjusts the tuning (x-axis of the wobble curve) to get the frequency defined by the acquisition parameters of that specific experiment number. It also adjusts the wobble curve y-axis to match the impedence (V=IZ) of an external 50 ohm reference.
Tuning and matching are carried out under low power. When set correctly the reflected power of the pulses are minimized, meaning the energy reaches the nuclei instead of heating the probe.
To measure non-hydrogen nuclei in a broad-band probe, the probe must be tuned to the correct frequency first. When using the atma (or atmm) commands (with an atma probe, such as the BBFO or BBO on the Avance 400, 500 and 600 instruments), the computer will tell the probe's motor which nuclei are defined in the acquisition parameter set, and the motor will automatically adjust the capacitors in the self-resonating circuit to the ballpark frequency. Afterwards, either manual (atmm) or automatic (atma) fine-tuning of the circuit's frequency and match needs to take place.
reduction; to make smaller
The pulse power levels are given in dB of attenuation from the amplifier's full power output. At 120dB (maximum attenuation), the pulse occurs for the defined duration, but has no power. To increase the power output, one must decrease the attenuation.
The Z, Z2, Z3, Z4, Z5, Z6 shims.
button: adding a button to the topspin menu bar
right-click on the menu bar and choose to add a user-defined button.
You have a choice to have either text or a picture/graphic appear on your new button. It's simplest to start with text, you can change the text to a graphic later on. The three things needed are:
- what the button does when you click on it
(for example, rsh qnp)
- a description of the button, which appears when the mouse sits on the button
- the text on the button OR the file location for the graphic displayed on the button
modification: baseline correction
A correction that adds a constant to the y-axis in the time domain to center the FID if it has been shifted vertically by a dc-offset.
With Bruker software it is commonly set to "quad". If it is set to "no" then it is off.
It is sometimes useful to set bc_mod to qfil in order to smooth out artifacts in the center of the spectrum, particularly when doing on-resonance water suppression.
The degree of the affect is adjusted with bcfw (0.1 - 1 are common values).
The distribution of individual spins among the available energy levels (population bias) follows the Boltzmann equation:
--- = exp (- ∆E / kBT)
N is the total population of particles with spin
N- is the population in the higher energy spin state
N+ is the population in the lower energy spin state
The Bruker Smart Magnet control System is an auxillary keyboard (either physical or virtual) which allows user control over the following features:
The spectrum's x-axis normalized and referenced to TMS = 0
delta scale (ppm) = (frequency signal/frequency TMS) - (frequency TMS/frequency TMS)
chemical shift calibration
1) Standard automatic calibration
The Bruker Avance spectrometers use the parameter called "solvent" to reference the 1H chemical shift relative to the locked 2H signal. The offsets are defined in a table accessible via the edlock command and are stored in the 2H lock text file (../exp/stan/lists/2Hlock). You will usually get an accurate chemical shift calibration, excepting D2O.
The chemical shift of water (or heavy water) depends on the hydrogen (or even stronger deuterium) bonding. Water will be calibrated to 4.7 ppm with the automatic lock system; two spectra taken under different conditions (concentration, temperature, pH, or ionic strength), show the water signal at 4.7 ppm and a constant shift change in EVERY other peak. In reality, only the water resonance frequency changed.
(2) Internal calibration
Use the calibrate button (or type .cal) to reference chemical shifts relative to any peak, such as TMS, DSS (similar to TMS, but water soluble), solvent, or any other known peak.
(3) Universal referencing
For non-hydrogen nuclei, IUPAC guidelines explain how to calibrate any NMR sensitive nucleus relative to the 1H spectrum based on the ratio of the magnetogyric ratios. ALL nuclei are calibrated relative to the universal reference: the 1H signal of 1% TMS in CDCl3. In practice, calibrate the 1H spectrum, use the Chi value from the table to get the new spectral reference assuming the exact same field, IUPAC (2001).
Detailed instructions for Bruker spectrometers.
(4) External referencing
This method is popular for solid-state NMR. It is also an older method for solution NMR. The three listings given above are preferable for solution NMR (more exact, less error prone, and less time consuming). The tables in the IUPAC article give standard external references for all NMR sensitive nuclei.
Practical instructions are also available.
Cleaning NMR Tubes
The source of the following information came from the Wilmad website.
NMR tubes are not 'analytically clean' when delivered to you. If your NMR samples require scrupulously clean glass, follow the procedures below for Difficult Cleaning Problems to assure your sample purity is never jeopardized. Since NMR tubes are formed over a metal mandrel and certain organic lubricants are used, these cleaning steps will assure that any trace organic or inorganic residues from these procedures is removed.
Since the purpose of an NMR Sample Tube is to confine a liquid sample in a perfectly cylindrical volume within the spectrometer probe, the degree to which the tube accomplishes this determines the quality of the sample tube.
Improper cleaning can damage NMR tubes and reduce your apparent spectrometer performance. You should never use a brush or other abrasive materials to clean NMR tubes. Scratches on the inside surface of the tube allow a portion of the sample to extend beyond the perfect cylinder defined by the NMR tube. Because the portion of your sample which fills a scratch on the inner surface of a tube experiences a different magnetic field than the rest of the sample, lines will broaden and resolution will deteriorate when you use scratched tubes. And you'll see a reduction in apparent spectrometer performance, unless you reshim your spectrometer for each sample.
Proper cleaning of NMR tubes can be easy or difficult, depending on your sample. We'll start with simple cleaning situations and move to the harder cleaning problems. Because even difficult cleaning procedures end with a proper rinsing, explained under Simple Cleaning of NMR Tubes, you should be familiar with both cleaning procedures.
Simple Cleaning of NMR Tubes
Cleaning an NMR tube can be as simple as rinsing the tube with heavy water or a deuterated organic solvent, you can rinse them one at a time. Your main concerns, then, are what to do with the rinsate. And, if you're using Acetone, also preventing dermatitis that results when oils are removed from your skin by this potent solvent.
If you rinse a lot of tubes, there are apparatuses available that will make your job much simpler. Tube washers, listed in the Wilmad NMR Catalog as Solvent Jet Cleaners, provide an easy way to clean either one or five tubes at a time. Using a vacuum flask and aspirator, solvent recovery is simple. And your hands won't be so easily dried out by solvents, either.
A final rinse with Acetone is frequently used to remove the last organic contents from the tube. When your sample is to be dissolved in water or D2O, a final rinse with distilled water is usually adequate. You may want to take steps to remove traces of water from the surface of the tube. Follow the procedures for deuterium exchange, below.
Difficult Cleaning Problems
Tubes left with samples in them for a period of time frequently present a more challenging cleaning problem. Sample degradation or precipitation can cause material to adhere to the inner walls of the tube. Rinsing the tube doesn't always remove this adhered material. So Wilmad recommends using strong mineral acids such a concentrated or, in severe cases, fuming Nitric Acid soaks of 1-3 days, as needed. Nitric Acid can oxidize many organic chemicals and dissolves most inorganic materials, as well. Wilmad doesn't recommend using Chromic Acid, since residual Chromium can often adversely affect NMR experiments. Chromic Acid, while a stronger oxidizer, can leave paramagnetic Chromium VI behind, which can be removed only with repeated soaks with Nitric Acid.
Copious rinsing of NMR tubes washed in acids is required to assure removal of residual acids. A final rinse with distilled water or Acetone is also appropriate.
Tubes which contained polymeric samples can be even more difficult to clean. When the polymers are natural products, like proteins and polysaccharides, strong acid soaks will usually be sufficient. However, when dealing with synthetic polymers, the challenge is more severe, since many polymers are inert to acids or insoluble in organic solvents by design. Although polymers may not readily dissolve in solvents, it may be possible to soften them by soaking the tubes in a solvent that swells the polymer. Then a pipe cleaner might be sufficient to remove the softened material. It may take some experimentation to find the solvent combination that works best with your polymer system. Agitation in an Ultrasonic bath with an appropriate solvent can also help dislodge stubborn sample residues. However, you should take precautions to assure that NMR tubes don't touch, since contact and vibrations can fracture delicate thin wall tubes. Wilmad offers a special tube rack for use in its Ultrasonic bath that prevents such destructive contact between tubes.
Removing Water from NMR Tubes
Drying tubes at elevated temperatures can reshape and ruin precision NMR tubes. If you dry tubes in an oven, Wilmad recommends placing tubes on a perfectly flat tray at 125° C for only 30-45 minutes. Better is the use of a vacuum oven that will remove water at lower temperatures. In a flat position, tubes that do reshape could be out-of-round and may not fit the spinner turbine as well. But they'll not affect the spectrometer probe adversely.
Tubes placed in an oven in a beaker, flask, or tube rack can bend, increasing Camber (lack of straightness). Bent tubes may still fit the spinner turbine, but can damage or break the NMR probe insert, a costly repair with many probes.
Even drying at high temperatures doesn't remove water chemisorbed to the surface of the tube. Thus, the preferred method of water removal is chemical, not physical, treatment. In most cases, it is the protic content of water that must be avoided. So Wilmad recom-mends exchanging the protons of chemisorbed water with a deuterated solvent such as D2O prior to a short drying period in the oven. A bottle of D2O that isn't being used any longer is perfect for this purpose. When water chemically degrades your samples, then removal of water is essential. Here, reaction of the water with a hydride solution can be used, with caution. After rinsing the hydride solution, a final rinse with very dry Acetone can be used to remove rinse solvent prior to oven drying. Cap tubes promptly to avoid absorption of moisture when removing dry tubes from the oven.
University of Manitoba NMR Lab Hints for Users
The FID (an interferogram of all the frequencies contained in the spectrum, with each signal damped according to the its individual relaxation time constant) is recorded as signal intensity (induced voltage) as a function of time. The time points are digitized and recorded from the receiver according to two parameters defined by the user: the number of digitization time points (td) and the sampling rate (dw). If the analog-to-digital conversion is stopped (aq = td * dw) before the signal has fully decayed, then only part of the free induction decay is recorded. The resultant step function at the end of the exponential FID results in artifacts and distortions in the frequency domain (there are extra "wiggles" next to the resonance peaks at the baseline) that result from fourier transformation applied to a step function (try it yourself in EXCEL). To prevent "clippping" (truncation or apodization), increase td or decrease sw (sw = 1/(2dw)) in order to increase the acquisition time (aq).
Deuterium is a stable (non-radioactive) isotope of hydrogen, 2H. The nucleus contains one proton and one neutron. Thus it is heavier than the significantly more abundant hydrogen nucleus: 1H. When water is made from deuterium instead of hydrogen, its common name is "heavy water".
Deuterium bonds are stronger than hydrogen bonds.
There must be 2H nuclei in your NMR tube to use the 2H lock to prevent magnetic field drift and to allow the spectrometer to automatically calibrate the chemical shift (x-axis).
In addition, by replacing the strong and generally uninteresting 1H nuclei in the solvent with 2H nuclei, those proton signals will no longer appear in the proton NMR spectrum. Since deuteration is rarely 100.000%, you can usually observe a solvent peak (of significantly reduced intensity) due to residual protons.
edit acquisition parameters
A table of all acquisition parameters. Once a FID has been acquired two different parameter lists exist within that experiment number (expno) file. In one list the parameters can be freely changed, in case you were to use that experiment number to acquire another FID. (A short version of the same list can be viewed via "ased"). The second list contains the actual parameters that were used to acquire the stored FID file. To view the "second" list, use an "s" and a space before the parameter name (e.g. type: s ns) or click the big "S" icon in the acquisition parameters tab (AcquPars) of Topspin.
edit processing parameters
A table of all processing parameters. For example: defining FID baseline corrections (BC_mode, bcfw), zero-filling (SI), window functions (LB for sensitivitiy enhancements or both LB and GB for resolution enhanced experiments), and linear prediction.
Electron spin resonance (ESR) or electron paramagnetic resonance (EPR) spectroscopy is a technique for studying chemical species that have one or more unpaired electrons, such as organic and inorganic free radicals or inorganic complexes possessing a transition metal ion. The basic physical concepts of ESR are analogous to those of nuclear magnetic resonance (NMR), but electron spins are excited instead of nuclear spins. Since ESR cannot be performed on the majority of stable molecules which have all of their electrons paired, it is arguably less widely used than NMR. However, this limitation to paramagnetic species also means that the ESR technique is one of great specificity, since ordinary chemical solvents and matrices do not give rise to ESR spectra.
Continuous wave (CW) ESR spectra are recorded by putting a sample into a microwave (MW) irradiation field of constant frequency and sweeping the external magnetic field B0 until the resonance condition is fulfilled. In the experimental set-up, the MW field is built up in a resonator (typically a rectangular cavity), into which the sample tube is introduced. The recording of reflected MW power as a function of the magnetic field yields the CW ESR spectrum. Amplitude modulation of the magnetic field with a frequency of typically 100 kHz increases the signal-to-noise ratio (S/N) considerably and is responsible for the derivative shape of the spectra.
Bruker EPR theory and practice : http://www.bruker-biospin.com/cw.html)
exponential multiplication (em)
A window function which performs sensitivity enhancement.
The FID is multiplied by an exponential function.
This emphasizes the strong initial signal and downplays the noise.
This data manipulation occurs during the processing step.
Sensitivity is cosmetically increased at the expense of spectral resolution.
The line broadening (lb) processing parameter must be set greater than 0 (lb > 0).
One rule of thumb is to use an lb equal to half the line width at half-height in Hz.
defines the specific sequence of pulses and delays, the nuclei to be measured, the power levels of the pulses, length of the pulses, phase of the pulses.
In Bruker pulse programs, in the comments, the relevant literature references are also often given. Here is a partial table of experiments:
The free induction decay (FID) is the measured NMR signal. The y-axis is signal intensity (induced votage in the NMR coil). The x-axis is time. Analog-to-digital conversion (ADC) takes place at constant time intervals (dwell time). The signal oscillates at the frequency difference between the carrier frequency (on-resonance center frequency) and the actual precession rate of the nucleus being detected. The signal decays due to relaxation processes that return the signal to its equiliubrium (steady-state) condition in the external magnetic field.
A fourier transform of the FID results in an NMR spectrum, where the y-axis is signal intensity and the x-axis is resonance frequency.
Fourier transform (ft)
A mathematical operation to obtain the spectrum (frequency domain) from the FID (time domain). The algorithm deconvolutes the separate frequencies recorded in the time domain. The command FT will do a fourier transformation.
Gaussian broadening (gb)
A parameter of the Gaussian multiplication filter function (or window function) applied to an FID before Fourier transformation.
LB < 0 and GB > 0 used with a gaussian filter function.
Gaussian multiplication (gm)
To obtain "resolution enhanced spectroscopy," use gm before ft (or use gf) to improve resolution
at the expense of signal to noise. The observed splittings are mathematically emphasized.
go setup (gs mode)
a single transient is acquired and displayed every d1 seconds without phase cycling
until a stop command is given. Either the time domain or the frequency domain can be observed in real time.
Check that aq ~ 2 seconds.
Set d1 to zero.
Shim while observing either the FID or the spectrum (toggle at top of gs menu bar).
An intrinsic property of a nuclear spin. The ratio between its magnetic moment and angular momentum. The larger the gamma of a nucleus, the larger its resonance frequency and the higher its sensitivity.
the acquisition will halt after the next shot and the data will be saved.
If dummy scans are running, the experiment will halt after the first shot that records data.
homogeneous The same in all directions; the same throughout.
an au program that increases the experiment number by 1. If the next experiment number
does not yet exist, the title, acquisition and processing parameters will be copied into the newly created file.
the area of the signal is proportional to the number of nuclei resonating at that particular frequency in the sample and the value is calculated using the NMR software's integration routine.
using Topspin, automatic integration is initiated with the command abs. The same command also calls for automatic baseline correction, IF the processing parameter INTBC is set to yes.
to use the last scale for the current integration, right-click inside the integration mode and choose use last scal for calibration. if INTSCL is equal to -1 the current dataset is scaled relative to the reference, otherwise the current dataset is the reference scale.
through bond scalar dipolar interactions. Also called the indirect dipolar coupling.
It is a relatively small modulation frequency arising from the different energies associated with the different orientations of spins in the magnetic field.
The information on the orientation of the neighboring nuclei are transmitted via the influences on the preferred electron spin orientations via the Fermi contact term (non-zero proabability of finding an electron at the nucleus). The J-coupling magnitudes are influenced by geometry and substituent effects. The splitting patterns (multiplicities) are influenced by the number of neighboring nuclei.
empirical relations between J-couplings and dihedral angles.
Please click on the "Lab Policies" link from our training new users page.
line broadening, lb
a processing parameter used in conjunction with either exponential multiplication, lb > 0,
which emphasizes the first part of the FID with the strongest signal and results in enhanced spectral sensitivity (signal-to-noise ratio) at the expense of spectral resolution. Or with Gaussian multiplication, lb < 0 and 0 > gb > 1, which gives a resolution enhanced spectrum at the expense of the signal-to-noise ratio.
Lock prevents magnetic field drift.
The 2H Lock is a separate NMR experiment which runs in parallel to your main experiment. It is an electronic feedback loop whose role is to stabilize the static magnetic field, Bo.
If the magnetic field "drifts" during the experiment you will have broadened lines (less resolution and less signal intensity) since the observed nuclear Larmor frequency is directly proportional to the external magnetic field.
The better the shim, the stronger and sharper the 2H signal, and the easier it is to "lock" the 2H signal.
an easy to write auotmation routine.
To create a macro type: edmac newfilename.
In the text editor that automatically opens, type each regular keyboard command on a separate line, save and exit.
save and exit. To execute the macro, type: proc
An intrinsic property of a nuclear spin. The ratio between its magnetic moment and angular momentum. The larger the gamma of a nucleus, the larger its resonance frequency and the higher its sensitivity.
creates a new experiment file which needs a directory location defined according
to the standard hierarchy: directory/data/user/nmr/name/expno/
Typing "new" is identical to any of the following:
- type: rpar (choose the experiment from the list)
dx 2 (creates expno 2; note that the au program may need a slight modification)
wrpa 2 (puts a duplicate in expno 2)
Alternatively, from an open experiment you can type:
- re 2 (navigates to 2)
nuclear magnetic resonance is an analytical technique used to understand the structure and dynamics of molecules. For nearly each element in the periodic table there is an isotope (usually non-radioactive) that has a non-zero quantum mechanical property called "spin." These nuclear spins are observable by NMR. A familiar example would be the nucleus of a hydrogen atom, otherwise known as a proton, and found in many molecules.
NMR can be used to study nuclei in solids, semi-solids, solutions or gases. The NMR instrumentation consists of a computer, electronics, and a magnetic field. The computer controls radio-frequency pulses generated by the electronics and transmitted to a coil. The sample, in an appropriate glass tube or rotor, is placed into the coil at the center of the magnet where the field inhomogeneity is smallest. When the rf-pulse is in resonance with the nuclear spins of the sample, the spins are perturbed from equilibrium. Both the resonance frequency ("chemical shift") of the spins and the time to return to equilibrium (relaxation time constant) are fundamental NMR measurements. The chemical shift depends on the local electronic environment of the nucleus and is sensitive to the molecular structure. The relaxation time constant depends on dynamic fluctuations in the molecule. Connectivities and proximities of nuclei within a molecule are measured by NMR through spin-spin coupling constants, such as J-couplings (through-bond) and dipolar couplings (through-space).
From the 2003 Nobel Prize in Physiology or Medicine, October 2003 press release:
Atomic nuclei in a strong magnetic field rotate with a frequency that is dependent on the strength of the magnetic field. Their energy can be increased if they absorb radio waves with the same frequency (resonance). When the atomic nuclei return to their previous energy level, radio waves are emitted.
MORE about NMR . . . NMR tubes, cleaning
the offset of the carrier frequency relative to TMS = 0 Hz.
SF + o1 gives SFo1 which is the absolute carrier frequency.
o1p gives the value in ppm. This is the center of the spectrum in the frequency domain.
values you may change to optimize an NMR experiment.
examples include: pulse lengths, phases, frequencies, power levels, and shapes; delay times, digitization rate, acquisition time, the frequency window (spectral window), digital resolution, etc.
Basic experimental parameters
|name||parameter function||short description|
|zg||pulse program||A set of instructions to the spectrometer telling it when to wait, when to pulse, and when to acquire. The zg experiment is the single pulse-acquire routine shown above.|
|d1||first delay||The spectrometer will wait d1 seconds.|
|p1||pulse duration||The radio-frequency (rf)-pulse time in microseconds. For a square pulse, it is inversely proportional to the bandwidth of excitation.|
|pl1||pulse power level||The attenuation applied to the amplifier's output.|
|de||dead time||The time the spectrometer waits between the end of the final pulse and the start of detection.|
It allows for probe ring-down to end before beginning data collection.
|aq||acquisition||The total time that digitization of the "free induction decay" (FID) takes place.|
aq = dw * td = td / (2swh)
|td||time domain points||The number of points used to digitize the FID.|
|dw||dwell time||The time interval between the sampled points (td) of the FID.|
dw = 1 / (2swh)
|sw||spectral width||The frequency range of the acquired spectrum in ppm. If the units are in Hz, the parameter is swh.|
|ns||number of shots||The number of repetitions of the experiment. The more FID's added together, the higher the signal-to-noise ratio.|
|ds||dummy shots||The number of time that the full experiment is repeated, but the FID is not stored. It is used to establish an equilibrium between pulses and delays before storing data.|
|o1p||offset||The center frequency of the RF pulse (the carrier frequency) translated into ppm (the center of the spectrum after Fourier transform). The same value in absolute units (MHz) is called sfo1. Or in Hz, relative to TMS, is called o1.|
We have two QNP probes, each can measure 1H plus three other nuclei. And theree is a 2H lock channel, through which 2H NMR can also be performed.
qnp19F: 1H, 19F (QNP 1), 31P (QNP 2), 13C (QNP 3), 2H
qnp29Si: 1H, 31P (QNP 1), 13C (QNP 2), 29Si (QNP 3), 2H
The QNP number is the position of the "QNP wand" attached to the bottom of the probe and connected to a pneumatic unit that changes the position of the capacitor in the probe so that the (self-resonating) circuit will resonate at the same frequency used for irradiation, which depends on which nucleus is to be measured. The number defined by the parameter "QNP" in the pulse program must match that needed for the probe. The number appearing in the small window of the QNP wand must also match the probe requirement for that nucleus.
Ours our gradient probes, capable of pulsed linear field gradients of 50 G/cm. Our BBO probes use automatic tuning and matching which means there is a motor attached to the bottom of the probe which adjusts the tuning and matching via computer control (atma) or a computer interface (atmm) for user adjustment via the mouse.
This probe combines magic-angle spinning from the world of solid-state NMR spectroscopy with gradients and deuterium locking from liquid-state spectroscopy. This hybrid technology is ideal for high-resolution spectroscopy of materials that have partial averaging of orientation-dependent (anisotropic) interactions, such as membranes, viscous liquids, gels, and wet solids.
policies, NMR lab
Please click on the "Lab Policies" link from our training new users page.
The manipulations made to a FID (intensity vs. time) to obtain a spectrum (intensity vs frequency) with various cosmetic enhancements. The list of parameters to be applied to the FID can be found under the processing parameters tab of Topspin, or by typing: edp
The degree of rotation of the net magnetization vector caused by a pulse depends on the duration and power of the pulse.
tip angle = (p1)*(pl1, in units of kHz)
A 360° pulse rotates the spins back around to their starting positions.
At equibilbrium, the spins are along +z, which is 0°
A 90° pulse tilts the spins into the xy-plane, where the receiver detects the signal.
A "nutation" experiment is a single pulse experiment where either the pulse duration or power is varied in order to calibrate the duration and power needed to achieve a particular tip angle.
For example, after acquiring a single shot for a proton NMR experiment, using Bruker parlance, type: paropt
vary p1, starting from 6 microseconds, in 6 microsecond increments for 10 experiments. A sine wave will evolve, experiment-by-experiment, in process number 999. The highest amplitude appears at 90°, the first null at 180°, the lowest amlitude (negatively phased) at 270°, and the second null at 360°.
To fine tune the pulse calibration, adjust p1 in a single-shot experiment until you find an accurate 360° null.
Acquisition hints for quantitative integrations:
Measure the longest T1 and use 5*T1 for the recycle delay
Use a large sw to get a flat baseline
Center your data in the middle of the spectrum (o1)
Acquire a large number of dots (td)
Make sure the signal-to-noise ratio is high (ns)
Use an optimal aq time (FID going to zero at ~1/3 of the screen)
For decoupled measurements of X nuclei – AVOID NOE effects
Repeat for a few different independent measurements
A 2007 reference giving more details, and recommending 13C decoupling is:
J. Nat. Prod., 70 (4), 589 -595, 2007
Here is their list of “factors” for quantitative NMR (qNMR) as opposed to the “normal survey proton NMR.”
(1) Don't spin, do shim (really well)
(2) Remove the 13C satellites by decoupling
(3) Relaxation delays so that, in Bruker-speak, aq + d1 = 5*T1
(4) Spectral windows with about 2 ppm extra on each side of the data
(5) Transmitter positioning to center the data
(6) Pulse width selection to calculate the optimal pulse width according to the Ernst angle: cos(p1opt) = exp(-aq/T1), where aq < T1
Note: usually d1 = 0, but time is needed to minimize heating
(7) Acquisition time with enough points to i) meet the Nyquist frequency condition, and ii) get a digital resolution of ~ < 0.2 Hz/pt
(8) Select sufficient repetitions, number of shots, for good si:no ratios
(9) Receiver gain settings should be optimized: automatic is fine
(10) Steady state: use dummy scans (ds) to establish an equilibrium
(11) Set the 13C decoupling bandwidth and position with care
Processing hints for quantitative integrations:
Do not use a window function, just do a straight fourier transform
Be sure that the baseline is flat on both sides of the data
Include the 13C satellites in the integration
Adjust the slope and bias of the integration
Use the same limits of integration for all spectra
Use the same scale for all the integrations (“lastscal”)
Use deconvolution software, if lines are not resolved
When the coil of a superconducting magnet loses its superconduting properties, due to loss of the cryogenic liquid. The magnetic field decays and the magnetic energy is released as heat. The release of heat causes the cryogenic fluids to boil which causes pressure increases; this may produce small explosions. Quench may happen spontaneously and without warning signs.
There is a potential of suffocation from the released gases during a quench. If you observe a sudden release of vapor from the dewar openings it could be a sign of a quench. When a quench is suspected, one should quickly warn others present, leave the lab, and inform a Facility Manager. All doors remain unlocked from the inside and there is also an emergency door across from the offices which leads to the courtyard.
Quenches have happened in many laboratories when magnetic items brought too close to the magnet have been attracted to it and subsequently collided with it. The damage caused can easily reach tens of thousands of dollars. One must always be careful not to bring tools, magnetic chairs, dewars, glass cylinders, and other metallic objects too close to the magnet.
For everyone's safety, please report any occurance of an object hitting the magnet.
receiver gain, rg
the amplification of the signal - amplifies both the signal and the noise
analogous to the “volume control” of the radio: too loud is too noisy and too low is hard to hear
The signal emitted by the nuclear spins is first amplified by the preamplifier. rg is the maximum amount of preamplifier gain. It must be low enough not to saturate the signal during the following stages of amplification.
rg = 1 is the smallest value allowed. If the signal appears truncated with rg = 1, then decrease the pulse duration (p1).
A measure of the time required for spins to return to equilibrium after perturbation (induction). Spin-spin dephasing (transverse plane, xy-plane) is characterized by a T2 relaxation time constant. Spin-lattice relaxation (longitudinal axis, z-axis) is characterized by a T1 relaxation time constant.
automated rg setting
the rga command allows the software to optimize the value of rg used.
1. Sample Volume
For 5 mm outer diameter NMR tubes, we recommend a "three-fingers" high sample volume as optimal, equivalent to approximately 0.6 ml.
2. Sample Concentration
The relevant concentrations in NMR are the number of moles of a nucleus with a unique chemical shift. A good guess for a first time run is in the range of mM or a few mg's. The more concentrated the sample, the better the signal-to-noise ratio. If there is aggregation at high concentrations, the line widths will be broad and J-couplings may not be resolvable (easily fixed by dilution of the NMR sample).
To increase the signal-to-noise ratio by a factor of two, you either need twice as much sample or 22 the time.
Any sample that has not dissolved (is not in solution) will not give a high-resolution NMR signal and will probably degrade the quality of the spectrum obtained. If you have particulate material, filter the sample.
3. Sample Handling
Start with clean tubes (new tubes are not clean, rinse them first with deuterated solvent). The tube quality will affect the spectral resolution. Use high quality tubes for high fields. Prepare your samples in your own lab. Wipe the outside of the tube clean, before arriving to the NMR facility. Leave your gloves and lab coat behind. Before placing your NMR tube (with the spinner) into the magnet, once again wipe the outside of the tube and also the spinner.
A.E. Derome, Modern NMR Techniques for Chemistry Research, Pergamon Press, Oxford, 1987, pp. 36-37.
Rider University, NMR Collaborative Training Project
a system of coils used to improve the homogeneity of the static magnetic field, Bo.
Field inhomogeneity may cause broadening or deformation (even splitting) of lines. If all the lines of the spectrum are asymmetric or broadened in the same way or to the same extent, the reason is most likely poor static field homogeneity, which needs to be improved by better shimming.
Samples with different heights will require adjustments of the axial shims (along the z-direction).
Special samples (paramagnetic samples or use of an internal capillary tube) require adjustments of the radial shims (x,y, and x-y combinations), which is done without spinning the sample.
Small solid particles in the sample tube (precipitates, crystals, sediments, etc.) interfere with homogeneity adjustments and should be removed (e.g. filtering).
Bruker AV500 Spectrometer 1H spectra,
3% CHCl3, acetone-d6.
Spoiled shim current spectra displayed with double the intensity.
Ideal Lorenzian line shapes require a homogeneous magnetic field. The sample should be in the center of the magnet where the magnetic field homogeneity is optimal. To further improve the homogeneity - room temperature shim gradients can be used. The magnetic field interacts with its surroundings. A large metal object nearby will cause a perturbation in the field. For a constant environment, it is sufficient to create a decent shim file for each probe and to make minor adjustments to the shims for each new sample placed into the magnet.
Read a good shim file already created for you into the current settings. You still may need to fine tune the shimming to your particular sample correcting for slight changes in position, angle, solvent height, solution magnetic susceptibility and temperature. Note that FIELD can be considered the Z0 shim.
A simple and effective approach to making minor adjustments to the shim file for the current sample in the magnet is to shim on the locked 2H signal. If the lock signal moves diagonally across the lock display the lock power is too high and the deuterium signal is saturated. Turn down the lock power.
The final test for how well the magnetic field has been shimmed is the observed spectral resolution. A recommended approach is to acquire a single shot, fourier transform and evaluate the line widths and line shapes of the spectrum. Optimize the spectrometer parameters and increase ns to acquire a spectrum with a high signal-to-noise ratio.
To adjust the shims:
Press Z on the BSMS keyboard
using the control knob maximize the signal intensity on the screen
if you push Z again, the stored value of the shim gradient will be restored
if you push a different button, like STD BY, the current value of the shim gradient will be stored in the memory.
How quickly you can shim effectively is related to the relaxation time of the solvent.
Next push Z2 and use the knob to maximize the trace.
Repeat until no further changes.
Q: What happens to the lock experiment when you want to observe high-resolution 2H NMR as your main experiment?
A: You may:
(A) choose a different nucleus to use for the lock (commonly 19F), or
(B) acquire your spectrum without a lock, knowing that there's magnetic field drift.
Q: How do you shim if there is no deuterium lock signal?
A: Shim on the FID or on the spectral line.
GA Pearson, Shimming an NMR Magnet
VW Miner and WW Conover, Shimming Ain't Magic
C. Blake, The Lonely Struggle of the Long Distance Shimmer
Analytical techniques based on the interaction between electromagnetic irradiation and matter.
A plot of intensity versus energy. The intensity units are induced voltage in a current. The energy units are usually given divided by Plank's constant (h), namely frequency. The delta (ppm) scale is often used, where the frequency is referenced and normalized to TMS (1% TMS in CDCl3 is the universal chemical shift reference standard).
spin on/off (sample spinning)
Mechanical spinning of the sample along the z-axis averages the spatial differences in the magnetic field between x- and y-axes of the principle axis system of the NMR tube, thus averaging out inhomogeneities of the external magnetic field along x or y.
spinning should be above 15 Hz to be effective, but below 30 Hz to avoid a vortex in the solution. Inhomogeneities in the external field along x and y appear as spinning side-bands spaced symmetrically around the isotropic chemical shift at multiples of the spinning frequency.
Usually spinning is omitted for quantitative, 2D, gradient, or high-field NMR spectroscopy.
spin (nuclear spin)
An intrinsic property of a nucleus (or electron, or photon, . . .). Values of nuclear spin range from 0 to 6.
A few examples:
|1H, 19F, 31P||1/2|
|2H, 6Li, 14N||1|
In solution NMR, the spinner is a plastic holder for the glass NMR tube. It allows the tube to be properly positioned within the magnetic field. It also has black and white markings around its circumference which allow the tube to spin with a frequency regulation around its long axis (around z) when the spin-air is on. At relatively low external magnetic field strengths and for experiments that do not use gradient pulses, sample spinning (15 - 30 Hz) will average out magnetic field inhomogeneities between the x- and y-axes, so that x and y shimming will not be needed.
Spinning side-bands in solution NMR arise from field inhomogeneities that can be corrected using the X and Y shims.
If the sample is not spinning - you will not see spinning side-bands. With spinning of the sample, they appear symmetrically on both sides of the NMR resonance spaced at the spinning frequency. If the sample spin rate is set to 20 Hz, then the spinning side-bands will be spaced at plus and minus 20 Hz relative to the center band (or possibly at plus and minus 40 Hz depending on the source of the inhomogeneity).
How can you distinguish between a spinning side-band and a 113C satellite? For one, the 1JCH coupling has a different magnitude ~ 100 - 200 Hz, much higher than the sample spinning in solution NMR. An experimental method to diagnose spinning side-bands is to change the spinning speed (for example to 30 Hz). If the small, flanking peaks move to plus and minus 30 Hz distance from the NMR resonance, then you definitely have spinning side-bands. The way to remove them is to shim on the X and Y shim gradients (if your using the lock signal for shimming, then turn of the spinning while shimming X and Y).
The most common source for a magnetic field in modern NMR is generated by a superconducting magnet. Once charged, the current flows through a superconducting coil located in the inner dewar cooled with liquid He. The insignificant resistance at 4.2 K (liquid He temperature) means the current is essentially not dissipated. Maintenance of a superconducting magnet requires weekly refills of the outer dewars with liquid nitrogen (77 K) and less frequent refills of the inner dewars with liquid Helium (4.2 K).
spin-lattice relaxation time constant or longitudinal relaxation time constant
usually measured in solution using an inversion-recovery pulse sequence, such as t1ir1d or t1ir
spin-spin relaxation time constant or axial relaxation time constant
usually measured using a CPMG pulse sequence to tease out pure T2relaxation from T2* relaxation, which is equal to the inverse of the linewidth (Hz)
"indirect" dipole-dipole couplings, also called J-couplings, are interactions between spins that are mediated through-bonds via the electrons (Fermi contact term).
The J-coupling adds a small modulation frequency (0.1 - 102 Hz) onto the much larger chemical shift frequency (MHz).
Through-bond J-couplings are independent of field. In other words the coupling observed as splittings in the spectral lines will have the same value in Hz at every magnetic field.
interactions that don't require bonds between the nuclei in order to see the dipole-dipole interaction.
In solution: the internuclear dipole-dipole interaction has an r-6 dependence.
In solution, where anisotropic interactions (such as direct dipole-dipole coupling) are averaged out by rapid molecular tumbling, through-space interactions are measured via relaxation mechanisms using the nuclear Overhauser effect (NOE).
The FID (an interferogram of all the frequencies contained in the spectrum, with each signal damped according to the its individual relaxation time constant) is recorded as signal intensity (induced voltage) as a function of time. The time points are digitized and recorded from the receiver according to two parameters defined by the user: the number of digitization time points (td) and the sampling rate (dw). If the analog-to-digital conversion is stopped (aq = td * dw) before the signal has fully decayed, then only part of the free induction decay is recorded. The resultant step function at the end of the exponential FID results in artifacts and distortions in the time domain (there are extra “wiggles” next to the resonance peaks at the baseline) that result from fourier transformation applied to a step function (try it yourself in EXCEL). To prevent “clippping” (truncation or apodization), increase td or decrease sw (sw = 1/(2dw)) in order to increase the acquisition time (aq).
variable temperature, VT NMR
A general term that covers both high-temperature and low-temperature NMR experiments.
tuning the capacitors of the probe so the ciruit resonates at the frequency of interest
the impedence is matched to an external 50 ohm reference (V = IZ)
The wobble curve is a graphical display of forward vs. reflected (low) power into the probe.
a general term used for non-hydrogen nuclei
The separation of nuclear energy levels in a magnetic field into 2I+1 = m different spin states.
the bruker command to start an acquisition, first deleting any previous FID
in the current experiment number.
there is also a pulse program called “zg”
this codes for a single-pulse NMR experiment, also called “pulse-acquire” experiment.