Excitation Profiles for Shaped Pulses

Shaped pulses are very commonly used for selective excitation and nonselective inversion in a large number of NMR pulse sequences. The frequency domain excitation profile of a radio frequency pulse is the Fourier transform of the time dependent pulse shape and determines the width, uniformity and phase of the frequency spectrum excited. Since time and frequency are reciprocals of one another, short rf pulses have very wide excitation profiles and long rf pulses have very narrow selective excitation profiles. In a previous BLOG post the excitation profiles of rectangular pulses of varying duration were determined experimentally. The Fourier transform of a rectangular pulse is a sinc function which is observed to be the experimentally determined excitation profile. Short high power rectangular pulses are very desirable for uniform excitation over wide frequency ranges as the entire NMR spectrum of interest usually occupies only a very small central portion of the central lobe of the sinc shaped excitation profile which, to a first approximation, is flat over the observed spectral width. Rectangular pulses are often not desirable when narrow excitation profiles are required as the excitation is not uniform over the desired region and the ripples of the sinc excitation profile cause nodes of excitation and negative peaks.

The Fourier transform of a sinc function is a box function, so if a long low power sinc shaped rf pulse is used one obtains a narrow flat box shaped excitation profile. This is indeed the case as can be seen in the bottom trace of the figure below. The trace is composed of a series of ¹H NMR spectra of H₂O/D₂O where a truncated, 200 msec, 10 cycle sinc shaped monochromatic rf pulse was applied with varying rf frequency offsets. One can see that the excitation profile is, to a good approximation, a narrow flat box function. The deviation from a flat box function is the result of the truncation of the sinc pulse. It should be noted that the phase of the resonances is not constant across the excitation profile. Sinc pulses are not frequently used for selective excitation because of the phase problem and the fact that very long, minimally truncated pulses must be used. A frequently used alternative to the sinc pulse for selective excitation is the Gaussian shaped pulse. The Fourier transform of a Gaussian is a Gaussian and one therefore will obtain a narrow Gaussian shaped excitation profile when a long low power Gaussian shaped pulse is used. This is shown in the top trace of the figure below. This trace is similar to the bottom trace except a 20 msec Gaussian shaped pulse (with truncation at 1 % of the total height) was used rather than a 200 msec sinc pulse. Although the excitation is not flat, the phase is constant across the excitation profile and the total duration of the pulse is 10 times shorter than the sinc pulse.

First-Order Phase Errors

The phase of a signal in an NMR spectrum is described here and is determined by the axis on which the magnetization vector resides after the observe pulse relative to the receiver. The phase of the spectrum is typically corrected such that the peak in the real spectrum is entirely in absorption mode while that in the imaginary spectrum is entirely in dispersion mode. The correction in phase is referred to as the zero-order phase correction. A zero-order phase correction applies to all peaks in the spectrum regardless of their offset, Ω, from resonance. There are also first-order phase errors where the phase error for a resonance is linearly dependant on Ω and the duration of the pulse, t_p. The further a peak is away from the center of the spectrum for a given pulse duration, the larger the first-order phase error. Similarly, the longer the duration of the pulse for a peak of given offset, the larger the first-order phase error. These errors arise from the fact that a 90° pulse of fixed duration is only a true 90° pulse for a peak on-resonance. For off-resonance peaks, the pulse will not only be slightly less than 90° but also produce a small amount of magnetization along the axis of the pulse. For example, a 90°_x pulse applied to an equilibrium magnetization vector will produce only –y magnetization for an on-resonance signal but for an off-resonance signal, it will also leave a small amount of residual z magnetization as well as produce some x magnetization. It is the x magnetization that gives rise to the first order phase error. The effect of the pulse duration for a peak of given offset frequency is demonstrated in the figure below. The sample was a mixture of chloroform and acetone. The transmitter was set such that the chloroform signal was on-resonance. Six 90° pulses were calibrated at different power levels based on the on-resonance chloroform signal, ranging from 10 µsec in the lower trace to 320 µsec in the upper trace. For all spectra, the same zero-order phase correction was applied such that the chloroform signal was in-phase. No first-order phase correction was applied. One can see that the phase error in the off-resonance acetone signal increases as a function of the pulse duration. It should also be noted that the overall intensity of the acetone signal decreases with respect to the chloroform signal as a function of the pulse duration due to the width of the excitation profile decreasing as a function of the pulse duration.

Agilent's MR BLOG - SPINSIGHTS

Recently Agilent Technologies has started a MR BLOG called "SPINSIGHTS". The posts to this BLOG are written by Agilent employees and discuss the advances of Agilent's MR products as well as educational posts on both MRI and NMR spectroscopy. It is a very interesting read and promises to be a very useful resource. Thanks Agilent - keep it up!

"The MRI Photocopier"

A very interesting note* has recently been published in Concepts in Magnetic Resonance Part A by Joseph Hornak (generous provider of the two excellent free online text books, The Basics of NMR and The Basics of MRI ). In this work, it is demonstrated that an MRI scanner can be used to reproduce an image of printed text. The technique is based on the fact that the toner used to print text contains ferromagnetic particles and when placed in the uniform magnetic field of an MRI magnet, the magnetic field near the ferromagnetic toner is distorted. The "sample" is prepared by placing the printed page face up on a polycarbonate plate and covering it with a very thin sheet of polyethylene. The polyethylene is covered with water (doped with CuSO4 as a relaxation agent). The "sample" is placed in the magnet such that the plane of the page is perpendicular to the magnetic field. The text is "scanned" by taking a 2D proton MR image of a slice of the water near the surface of the polyethylene. The distortion in the magnetic field due to the ferromagnetic toner renders the text visible in the 2D magnetic resonance image. This is demonstrated in the figure below (from the reference) where a 2.5 mm thick slice above the polyethylene was imaged. The text was printed in #36 Arial font. The author speculates that this technique may be applied to the analysis of paintings.
* Joseph P. Hornak, "Magnetic Resonance Imaging of Printed Text", Concepts in Magnetic Resonance Part A. 36A, 347 (2010).

Sample Slice Selection in NMR Spectroscopy

In MRI, field gradients are used routinely for slice selection while imaging a sample. Gradients are also finding applications in high resolution NMR spectroscopy employing fast data collection techniques using parallel data acquisition for multiple slices of the same sample. A good example of this can be found in Carlos' BLOG (and the references therein). The slice selection is accomplished by turning on a linear field gradient across the sample while applying an excitation pulse. While the gradient is on, the frequencies of each of the NMR resonances is spread out according to length of the sample and the strength of the field gradient across the sample. A particular individual slice of the sample can be selected by either modifying the strength of the gradient while using a semi-selective pulse of a given excitation frequency or by modifying the offset frequency of the semi-selective pulse for a constant gradient strength. Both the gradient strength and the excitation profile of the pulse determine the thickness of the slice selected. When the gradient is turned off and the receiver turned on, the FID representing only the spectrum of the selected slice of the sample is collected. An educational example is shown in the figure below. A sample of toluene and H₂O/D₂O was prepared. As these two liquids are immiscible, the sample is layered with the less dense toluene on top and the more dense water on the bottom. The bottom trace in the figure shows a conventional ¹H NMR spectrum. Since the pulse used to collect the spectrum was a hard 90° pulse with a wide excitation profile, one can see both the toluene and the water in the spectrum. For the middle and upper traces, a field gradient of 48 G/cm was turned on while a 100 µs 90° Gaussian excitation pulse was applied. The only difference between the middle and upper traces is the offset frequency used in the excitation pulse. The middle trace represents the spectrum of a slice of the water in the bottom layer of the sample and the upper trace represents the spectrum of a slice of the toluene in the top layer of the sample.

NMR and Candy Canes

The Phase of an NMR Spectrum

Most students know that the phase of an NMR spectrum has to do with the degree to which the NMR resonances are above or below the baseline of the spectrum (i.e. the amount of absorption and dispersion character). Most students have also learned that the phase of a periodic time domain function depends only on the value of the function at time zero. Thus, the only difference between a cosine and a sine function is where the function starts at time zero. A sine is said to be 90° out of phase with respect to a cosine. Many students do not understand the connection between the phase of their NMR spectrum and the phase of the periodic time domain function giving rise to their spectrum by way of a Fourier transform. The figure below is an attempt to make the connection.

The left-hand portion of the figure shows equilibrium magnetization vectors being rotated by radio frequency pulses. 90° pulses along the x, y, -x and -y axes rotate the z magnetization vector to the -y, x, y and -x axes of the rotating frame, respectively according to the right-hand screw rule. After the pulse, the magnetization vector rotates in the rotating frame of reference at a frequency equal to the difference between the transmitter frequency and the frequency of the NMR resonance. In the figure, the magnetization is assumed to be rotating anti-clockwise representing an NMR resonance with a positive frequency with respect to the transmitter frequency. The NMR spectrometer measures the time dependent voltages on two of the four orthogonal axes of the rotating frame separated by 90° (quadrature detection). The time dependent voltages are proportional to the amount of magnetization on the axis as a function of time. One of the two time dependent voltages is called the "real" signal and the other is called the "imaginary" signal. Together these two signals make up the complex free induction decay (FID) which is Fourier transformed to produce the NMR spectrum. In the figure, the -y(t) voltage is the real FID and the x(t) voltage is the imaginary FID. The figure shows representations of the real and imaginary FIDs after the delivery of pulses along each of the orthogonal axes. Note that the phases of the real and imaginary FIDs depend on the pulse delivered. For example, after a 90°_x pulse, the magnetization resides on the -y axis. The real FID (along the -y axis) starts at a maximum (cosine) and the imaginary FID (along the x axis) starts at zero (sine) and increases as the magnetization vector rotates anti-clockwise. The Fourier transform of the complex FID produces a real spectrum (typically the one displayed to the user) and an imaginary spectrum (typically not displayed to the user). Note that the degree of absorption vs. dispersion character in the spectrum depends on the phase of the FID signals. If an NMR spectrum is not in phase, perhaps due to a receiver dead time problem, it can be corrected after the collection of the data by calculating the phase angles needed to put the real spectrum enitirely in absorption mode and the imaginary spectrum entirely in dispersion mode.

New Bench Top 1H NMR Spectrometer

A very interesting product has just been put on the market from a company called picoSpin. - a 7 lb bench top 45 MHz ¹H NMR spectrometer at a very tempting price. From the information available on their website (including a YouTube video), it looks as if this instrument would be an excellent teaching tool in a university laboratory.








This is not meant as a product endorsement.

NMR and Food Chemistry - Coffee

Many of us enjoy a hot delicious cup of coffee. Many (not including myself) will even spend a great deal of money and a great deal of their time in a long queue to get it! The coffee snobs will preach about the differences between a freshly brewed cup of java and the lowly cup of instant coffee. Is there really a difference? Spectrum A in the figure below shows the ¹³C CPMAS NMR spectrum of ground coffee. Among other things, the spectrum consists of resonances from the woody fibers, polysaccharide gums, tannins, alkaloids and aromatic oils. Spectrum B is the spectrum of used coffee grounds which were subsequently dried. The spectrum is due to the woody fibers and other insoluble material. Spectrum C is the difference between spectrum A and B and represents all the "goodness" of a fresh brewed cup of coffee. Among other things, it represents all of the alkaloids, tannins and aromatic oils. Spectrum D is the ¹³C CPMAS NMR spectrum of instant coffee which is manufactured by freeze drying brewed coffee. The spectrum is very similar to the difference spectrum, C. This indicates either that the difference between a freshly brewed cup of coffee and a cup of instant coffee lies in very low concentration constituents or that the coffee snobs are wasting their money and time in long queues. I think both are true.

13C NMR of "Perdeuterated" Solvents

When one acquires a ¹³C NMR spectrum of a sample, the deuterated solvent is observed with resonances characterized by the J coupling pattern of the deuterons attached to the carbon atoms. (CD = 1:1:1 triplet, CD₂ = 1:2:3:2:1 quintet, CD₃ = 1:3:6:7:6:3:1 septet). Often, however; small peaks are observed near the main solvent resonances. These are due to other isotopomers of the solvent. An example of this is shown in the bottom spectrum of the figure below for the high frequency resonance of "THF-d₈". The spectrum consists mainly of the expected 1:2:3:2:1 quintet however, there is a small peak present on the high frequency side of the quintet marked by the arrow. It is due to one of the components of the C1 resonance from THF-1,2,2,3,3,4,4-d₇. The supplier of the solvent claims that the isotopic purity is 99.5 atom % D. If the deuteration is uniform, approximately 0.5 % of the molecules will contain a single proton. Half of these molecules will be mono-protonated on the high frequency carbons. If a DEPT spectrum is acquired on the same sample, the non-protonated carbons are suppressed leaving only the mono-protonated carbons. This is shown in the top spectrum of the figure, which shows a 1:1:1 triplet from CHD. The isotope shift between CHD and CD₂ is 0.354 ppm. Other examples can be found here and here.

Deuterium Decoupling

Bruker NMR spectrometers equipped with a ²H TX board and ²H amplifier can be setup to carry out ²H observation and decoupling experiments while still maintaining a ²H lock. Two channel systems can use the ²H transmitter for ²H, ¹H[²H], ²H[¹H] and X[²H] measurements. In addition to these, a three channel system can be set up for X[²H, ¹H] measurements. The first figure below shows the ¹³C and ¹³C[²H] spectra collected for a mixture of deuterated solvents using two channels of an AVANCE spectrometer. The second figure shows the ¹³C, ¹³C[¹H], ¹³C[²H] and ¹³C[¹H, ²H] spectra for the standard ¹³C sensitivity sample (40% para-dioxane and 60% deuterobenzene) collected on a three channel Bruker AVANCE spectrometer.

HSQC - TOCSY

There are many different NMR techniques used to probe molecular structure. Two very useful methods are the HSQC (or HMQC) sequence used to obtain heteronuclear coupling correlations and the TOCSY sequence used to look at extended homonuclear coupling correlations. These are among the most widely used pulse sequences by chemists to help elucidate the molecular structure of organic molecules. The sequences can also be combined into a single HSQC-TOCSY sequence which is simply an HSQC followed by a TOCSY spin lock. For the case of ¹H and ¹³C in organic molecules, the HSQC-TOCSY spectrum provides ¹H TOCSY subspectra of the isolated spin systems in the rows at each of the ¹³C frequencies. Overlap of ¹H resonances which may have appeared in a simple ¹H TOCSY spectrum can be resolved in the HSQC-TOCSY spectrum provided there is enough shift dispersion in the ¹³C spectrum. An example of this is shown in the figure below. The top panel shows the HSQC-TOCSY spectrum for 3-heptanone collected on a 300 MHz instrument. One can see the two spin systems on either side of the carbonyl group, color coded in yellow and pink. The second panel shows an expansion of the region within the red square of the first panel. Here we can separate the overlapping quartet and triplet for the methylene protons on either side of the carbonyl group.

Temperature Calibration - An Alternative Method

It is well known that the actual temperature of a sample in an NMR probe is not necessarily the same as that read from the variable temperature unit on the spectrometer. This is because the thermocouple used by the variable temperature unit is below the sample tube and not in the center of the rf coil where the NMR measurements are made. One normally must make a calibration plot for the actual temperature vs. the set temperature. For temperatures above room temperature this can be done by employing the known temperature dependent chemical shift difference between the two proton resonances of ethylene glycol (see this link). At temperatures below room temperature, the same measurement can be made for the known temperature dependent chemical shift difference between the two proton resonances of methanol. The actual temperature is determined from the chemical shift difference and plotted against the temperature read from the variable temperature unit. One potential problem with this method is that the resistance of the magnet shim coils change slightly with temperature affecting the shim currents and the NMR line shapes of the resonances. This makes it difficult to measure a precise chemical shift difference. In order to obtain reliable results, the magnet must be reshimmed at each temperature.

I have used is a very simple alternative method for calibrating the temperature of the sample compared to that of the variable temperature unit. This is illustrated in the figure below.A "sample" is prepared by pushing a NONMAGNETIC thermocouple through an NMR tube cap. The depth of the thermocouple is adjusted such that when the cap is put on the NMR tube, the tip of the thermocouple sits in the center of the rf coil. The NMR tube should contain a suitable liquid filled to the correct depth. The tube is placed in the spinner and set to the proper depth with a depth gauge. While holding onto the thermocouple, the sample is lowered into the magnet until it sits correctly in the NMR probe. The thermocouple is connected to a digital thermometer (some of these devices can use a second thermocouple in an ice water bath as a reference). The desired temperature is set on the variable temperature unit. When the temperature on both the variable temperature unit and digital thermometer have stabilized (~ 10 minutes), the values from each are recorded. This is repeated for temperatures over the desired temperature range and a calibration plot is constructed. Shimming is not an issue. Note that no NMR measurements are made and that the sample tube is not spinning.

Solid State NMR of Half Integer Quadrupolar Nuclei

Many students who do liquid state NMR or solid state NMR of spin I=1/2 nuclei have very little appreciation for the information content and complexity of the solid state NMR spectra of spin I = n/2 quadrupolar nuclei (n= 3, 5, 7....). In part, I think this may be due to the mathematics involved with explaining the important effects. With this post, I attempt to describe the NMR spectrum of an I = 5/2 nucleus in the soild state without resorting to mathematics. I hope that this post helps to boost the understanding and appreciation for the NMR spectra of these important nuclei.

When one collects the NMR spectrum a spin I = n/2 nucleus in solution, one normally observes a single NMR resonance line for each species as the (n+1) Zeeman energy levels are equally spaced and the nucleus is undergoing fast isotropic motion. The width of the resonance depends upon the efficiency of the relaxation (usually dominated by the quadrupolar interaction). The more efficient the relaxation, the broader the resonance. In many cases, the width of the resonance may be comparable to the complete chemical shift range for the nucleus and in some cases it may be so broad as to make its observation impractical or impossible. For these reasons I = n/2 quadrupolar nuclei generate less than their share of interest in liquid state NMR compared to spin I = 1/2 nuclei like ¹H, ¹³C, ³¹P ..... etc.

In the solid state, the situation is much different as there is rarely fast isotropic motion. When the quadrupolar interaction is much less than the Zeeman interaction, the Zeeman energy level diagram is affected by both a first order and a second order quadrupolar perturbation as shown in the figure below for an I = 5/2 nucleus. The second order perturbation is much smaller than the first order.The energy levels are no longer equally spaced. Furthermore, the position of the resonances resulting from the transitions depends on the orientation of the nucleus with respect to the magnetic field. Since all orientations are represented equally in a powder sample, each transition gives a powder spectrum. This is illustrated on the right-hand side of the figure. Note that the m = 1/2 and m = -1/2 energy levels are affected equally to first order and that the central (m = 1/2 - m = -1/2) transition is affected only by the second order perturbation. As a result the line width of the resonance from the central transition is much narrower than the other transitions. (In the figure, the central transition is clipped significantly.). Unlike the first order interaction, the second order interaction is field dependent. The width of the central transition depends inversely on the strength of the magnetic field.

Magic angle spinning is a technique used by solid state NMR spectroscopists to obtain high resolution NMR spectra of solids. Magic angle spinning at infinite speed completely averages the first order quadrupolar interaction but only partially averages out the second order interaction. The energy level diagram for a spin I = 5/2 nucleus spinning infinately fast at the magic angle is shown in the figure below along with a simulated spectrum. The spectrum consists of a central transition, CT, and two satellite transitions, ST1 and ST2. Note that along with a lineshape due to the orientational dependence of the nucleus in the magnetic field, there is also an isotropic quadrupolar shift. The central and satallite transitions are not at the same frequency. This effect is completely separate from the chemical shift.
In practice, we cannot spin at infinite speed, however, we can often spin at a rate fast with respect to the width of the central transition. If the quadrupolar coupling constant is small enough, the central transition will be observed and affected only by the second order interaction. The satellite transitions, affected by both the first and second order interaction, are observed as a manifold of spinning sidebands. The intensity of the centerbands for the satellite transitions is greatly attenuated as the overall intensity is spread among all of the sidebands. Often the centerbands for the satellite transitions are so small in comparison to that of the central transition that they are not observed. This is illustrated with simulations in the lower portion of the figure below. For comparison, the upper portion shows similar simulated spectra without magic angle spinning. The spectra highlighted in yellow are expansions of the central portion of the spectrum. These simulations are also supported by observations.

Heat Dissipation in Bruker AVANCE Spectrometers

It is very important that an NMR spectrometer operate within a fixed temperature range. To control the temperature, many units within the console are equipped with cooling fans which must be kept in good working order. Failure to do so will shorten the life of the spectrometer and cause instability or malfunctions. In the Bruker AVANCE series of spectrometers, the SGU (Signal Generation Unit) boards are particularly sensitive to temperature. Each SGU houses a DDS (Direct Digital Synthesizer) containing numerically controlled oscillators which regulate rf frequencies and amplitudes. The DDS is normally temperature regulated at 55° C to insure stability. Both the SGU board temperature and DDS temperature can be measured either with the "UniTool" (AVANCE and AVANCE II) or web based tools (AVANCE III).

After years of service, two of our Bruker AVANCE spectrometers were running very warm. Using "UniTool" to check the temperature of the SGUs revealed that the board temperatures averaged 58° C! The DDS temperatures were all >55° C and not regulated. The air filters in the console doors were removed and cleaned and all eight fans of the AQS unit, housing the SGU's, were replaced on each spectrometer. After a day for the instruments to come to a thermal steady state, the SGU and DDS temperatures were again measured. The SGU board temperatures averaged 49°C. The DDS units in three of the five SGU's were now regulated properly at 55°C. The other two were still > 55°C and unregulated. In an attempt to lower the temperature further, the backs of the spectrometers were removed and again the instruments were allowed to reach a thermal steady state over a 24 hour period. The SGU and DDS temperatures were measured again. This time the SGU board temperatures averaged 38°C and all five DDS units were regulated properly at 55°C. The SGU temperatures in our AVANCE II and III spectrometers (with backs on) did not exceed 40°C and the DDS temperatures were regulated properly.

Although this may not be recommended by Bruker, I now run our AVANCE spectrometers with the back panels removed.

19F - 13C HMQC

If one has an NMR spectrometer with hardware capable of synthesizing and amplifying the frequency of ¹⁹F from the ¹H channel and a broadband NMR probe whose ¹H channel can tune down to ¹⁹F, then one is able to do ¹⁹F - ¹³C HMQC experiments. The figure below shows an example of a ¹⁹F - ¹³C HMQC spectrum collected on a Bruker AVANCE 500 NMR spectrometer using a 5 mm broadband probe. The fluorine spectrum is plotted on the top and the ¹³C[¹⁹F] spectrum is plotted on the side. The panel on the left shows the spectrum optimized for one-bond coupling, while that on the right shows the spectrum optimized for two-bond coupling. Note that the protonated carbons are doublets as proton decoupling is not possible in this configuration. The large signal, off scale in the ¹³C[¹⁹F] spectrum is due to the solvent (benzene-d₆).

E.COSY and the Relative Signs of Coupling Constants

Spin-spin coupling constants can have values greater than or less than zero. The absolute sign of the coupling constants cannot be discerned from the simple examination of a ¹H NMR spectrum. The E.COSY¹ (Exclusive COrrelation SpectroscopY) technique is one method which can be used to determine the relative signs of coupling constants. E.COSY is a phase sensitive COSY variant which produces off-diagonal signals showing only the active coupling (i.e. the coupling directly responsible for the cross-peak) as 2x2 antiphase square tetrads displaced in both the F2 and F1 domains by an amount equal to the passive coupling constants (i.e. the couplings not directly responsible for the cross peak). The slope of a line drawn through the cross-peaks is used to determine the relative signs of the passive coupling constants. The sign of the slope depends on whether the signs of the passive couplings are the same or whether they differ. The figure below shows the gradient E.COSY spectrum² (using the Bruker pulse program,"ecosygpph" ) for the ABX three-spin system in phenylalanine.The cross-peaks highlighted in yellow in the top panel of the figure are expanded in the bottom panel. Black contours are positive and red contours are negative. The panel on the bottom left shows the cross-peaks for AX and BX. In the case of the AX cross-peak, the line drawn through the cross peak has a positive slope indicating that the passive couplings J_AB and J_BX are of opposite sign. The line drawn through the BX cross peak also has a positive slope indicating that the passive couplings J_AB and J_AX are of opposite sign. From this we can deduce that J_AX and J_BX are of the same sign. The panel on the bottom right shows the cross-peak for AB. In this case, the line drawn through the cross peak has a negative slope confirming that the passive couplings J_AX and J_BX are of the same sign. In conclusion, the geminal and vicinal coupling constants are of opposite sign.

1. C. Griesinger, O.W. Sorensen & R.R. Ernst, J. Magn. Reson. 75, ; 474 - 492 (1987).

2. The ecosygpph pulse program produces spectra similar to those described in reference 1 as "complimentary" E.COSY spectra. The slope of the lines through the cross peaks in "complementary" E.COSY spectra are of opposite sign to those obtained from the E.COSY spectra described in reference 1.

Job Opening: NMR Technician, University of Ottawa

The Faculty of Science of the University of Ottawa is seeking a technician for its very active Nuclear Magnetic Resonance (NMR) facility. This rapidly growing NMR facility serves more than 25 active research groups and consists of 7 NMR instruments ranging in field from 200 to 500 MHz. The facility conducts a wide range of modern NMR experiments on both solids and liquids as well as MRI. As the NMR technician, you must hold at least a B.Sc. degree in chemistry or a related field (M.Sc. preferred) with at least two years experience in either one or both high resolution or solid state NMR spectroscopy. You must be enthusiastic, highly motivated and work well both independently and in groups. Extensive computer experience is mandatory. As you will be interacting daily with students and faculty, you must have excellent interpersonal skills as well as excellent oral and written communication skills. Reporting to the NMR facility manager, you will collect routine NMR data as a service, assist students and answer questions, train new users of the equipment, help with equipment maintenance (including cryogen fills), maintain a data archive, keep accurate time and usage records, and assist the NMR facility manager in the general operation of the facility. French/ English bilingualism is a definite asset. The position is open for one year starting August 15, 2010 and will be renewable, availability of funds permitting. The annual salary range for this position is $38,862 - $51,504. For consideration, please email your CV and cover letter to the Human Resources Service mycareer@uottawa.ca and refer to competition number TSCI9583NEP. Please also have three letters of recommendation emailed or sent to:

Dr. Glenn A. Facey
NMR Facility Manager,
Department of Chemistry, University of Ottawa
10 Marie Curie, Ottawa, Ontario, K1N 6N5
CANADA
ph. (613) 562-5800 ext 6077
fax. (613) 562-5170
email. gafacey@uottawa.ca

The deadline for applications is Friday June 25 at 5:00 pm.

More information is available at the following web site:

http://www.hr.uottawa.ca/careers/support/competition/cont/sci/TSCI9583NEP.php

Retrieving Empty Spinners From Magnets

Despite my best efforts in teaching students how to properly use NMR spectrometers, invariably a new user will, on occasion, drop a sample spinner into a magnet without a sample. This is usually done in our undergraduate laboratory by inexperienced students who have finished collecting their data and feel a need to return the empty spinner to the magnet. The problem is that ejecting the empty spinner with the eject air is not possible as the air passes through the spinner. In the past I have removed empty rotors from magnets on Varian spectrometers by removing the upper barrel of the magnet. On Bruker spectrometers, I have removed the probe and inserted a semi-rigid NONMAGNETIC plastic hose in the bottom of the magnet and pushed the spinner to the top of the upper barrel where it can be removed by a helper. In either case, the entire operation took between 5 minutes (Varian) and 30 minutes (Bruker) depending on how much reshimming was required. To avoid this tedious chore, I had our machine shop make a plastic tool for empty rotor removal shown below.

This tool consists of a pole with a diameter slightly less than that of the spinner. On the end of the pole is a tapered prong with a diameter of 4 mm at the tip and 6 mm at the base. This tool is inserted in the top of the magnet and gently lowered until the prong gets wedged into the empty spinner. The spinner is removed with the tool. The entire operation takes less than 10 seconds and no reshimming is required.

Gradient Spin Echoes for Selective Excitation

Shaped excitation pulses can replace the non-selective hard pulses typically used in a one-pulse measurement to achieve selective excitation. Another method of achieving selective excitation is the gradient spin echo using a selective 180° pulse. This technique is demonstrated in the figure below. A non-selective hard 90°_x pulse is first given followed by a pair of identical pulsed field gradients sandwiching a soft selective 180° pulse about the y axis. The hard 90° pulse rotates all spin vectors onto the -y axis. During the first gradient pulse the spin vectors dephase and evolve according to their offset frequencies. The soft 180°_y pulse flips a single resonance 180° about the y axis leaving all other resonances untouched. During the second gradient pulse, the "selected" resonance is rephased and its offset frequency evolution is refocused. The unselected resonances dephase more and continue to evolve according to their offset frequencies. The receiver is then turned on to collect the FID of the "selected" resonance, all others are dephased and therefore suppressed. This is demonstrated in the figure below which shows ¹H NMR spectra for a mixture of methylence chloride and acetone. The bottom trace shows a standard one-pulse measurement. The middle and top traces show results from a selective gradient spin echo measurement with the selective 180° pulse set for methylene chloride and acetone, respectively.

Background Suppression in Liquids

High resolution NMR probes for liquids may contain parts near the coil consiting of the nuclei being observed. The parts give rise to background signals which can severely affect the NMR data. When observing¹¹B, there is a background signal from boron containing parts near the coil and also the borosilicate glass in the NMR tube containing the sample.

Cory and Ritchey* introduced a very simple, clever method to suppress background signals in 1988. Their method uses a composite pulse, consisting of a 90° and two 180° pulses with appropriate phase cycling, in place of a conventional 90° pulse. The phase cycled composite pulse is essentially a 90° pulse for all spins inside the coil and 0° for all spins outside of the coil. An example of its implementation is shown in the figure below. The bottom traces show the ¹¹B [¹H] NMR spectra for a dilute sample of NaBH₄ and a "real" synthetic sample on the left and right, respectively. One can see an enormous background signal from both the NMR probe and the NMR tube. In the case of the "real" synthetic sample, the information from the spectrum is difficult or impossible to recover. The top traces show similar spectra acquired using the composite pulse. The only background signal remaining is that from the portion of the NMR tube inside the coil. This pulse sequence (without proton decoupling) is in the Bruker pulse program library called "zgbs". It is not exclusive to ¹¹B.

D.G. Cory and W.M. Ritchey. Journal of Magnetic Resonance, 80, 128 (1988).

Hahn Echo for 11B Background Suppression in Solids

Solids NMR probes often contain boron rich parts near the coil in which the sample resides. Boron nitride, in particular, is a material very commonly used. This can be very problematic if one wishes to collect ¹¹B NMR data, in that a strong background signal may be observed. Even though these parts are not directly inside the coil with the sample, they do experience a small amount of rf from the coil and the coil does detect a ¹¹B NMR signal from them. One simple way to avoid this problem is to use a Hahn echo to observe the ¹¹B spectrum. Unlike the sample inside the coil, which experiences the 90° and 180° pulses required for the Hahn echo, the boron rich parts outside of the coil experience pulses of much smaller flip angles and therefore the echo signal from them is much reduced. This is illustrated in the figure below, which shows ¹¹B NMR spectra collected in a 4 mm MAS probe without magic angle spinning. In the lower trace a simple one-pulse measurement was made with high power ¹H decoupling. The spectrum contains a very large background signal and it is very difficult to extract any useful information from the data. The spectrum in the upper trace was collected with a Hahn echo with high power ¹H decoupling during the acquisition. The background signal is completely removed revealing a beautiful, information-rich line shape for the central transition resulting from the second order quadrupolar and chemical shielding anisotropy interactions.

Thank you to Joseph Weiss of David Bryce's laboratory for providing the sample and collecting the data. See more of Joseph's beautiful ¹¹B spectra here:

Joseph W.E. Weiss and David L. Bryce J. Phys. Chem. 114 (2010) ASAP.
http://dx.doi.org/10.1021/jp101416k

Dummy Scans

Dummy scans (DS, Bruker) or steady state scans (SS, Varian) are scans taken in an NMR acquisition before the receiver is turned on and data are collected. Each dummy scan contains all of the rf pulses, delays and gradients used in the pulse program; the only difference is that the receiver is not turned on to collect data. Dummy scans are typically used to ensure that a spin system is in a steady state before data are collected. For example, One may collect a spectrum using a relaxation delay short with respect to the T₁'s of some of the resonances in the spectrum. The first scan may find the system at equilibrium, as the sample may have sat in the magnet for a few minutes while the probe was being tuned and matched or the magnet was being shimmed. The second and subsequent scans will find the system not at equilibrium, as the system has been perturbed by the rf pulses of the preceding scan(s) and not allowed to fully relax. The data from the first and seccond scan are therefore not the same. After several scans, although not at equilibrium, the system will be in a steady state before each additional scan and therefore each subsequent scan will collect similar data. An example of the use of dummy scans is shown in the figure below. The bottom trace shows a single scan ¹³C NMR spectrum of a concentrated solution of menthol in CDCl3 after the sample had sat in the magnet for a minute or so. The top trace shows a similar single scan acquisition preceded by 16 dummy scans. The relaxation delay was set to 1 second and the acquisition time for the FID was 1 second. 90° pulses were used in both spectra. One can see that some of the resonances in the top spectrum are attenuated in comparison to the bottom spectrum. The ¹³C signal from the CDCl₃ (which has a long T₁) is missing in the spectrum acquired with dummy scans. The dummy scans have selectively presaturated the solvent.

Household Dust (Bunnies)

Many people will be celebrating Easter this coming weekend. Children will be searching for Easter eggs left by the Easter Bunny. A word of caution though: Not all bunnies are cute!

Household dust bunnies seem to magically reproduce and grow. They must be collected regularly either with a broom or a vacuum cleaner and disposed of. What exactly is household dust? A bit of internet searching reveals that household dust is composed largely of fibers from clothing, dead human skin and the bodies of dust mites - a very disgusting mixture indeed. The top trace in the figure below is the ¹³C CPMAS NMR spectrum of a sample of household dust. The middle trace is a similar spectrum of clothing fibers from a sample of dryer lint. The difference spectrum (in the bottom trace) is consistent with a complex mixture of proteins and largely represents the spectrum of dead human skin and the bodies of dust mites.

Free and Inexpensive NMR Processing Software for Students

Processing NMR data has just become more affordable for students. Our friends at Advanced Chemistry Development have recently decided to make their NMR processing software free of charge to academics. As a student, you can put this software on your personal laptop or PC and process your NMR data at home or anywhere your travels take you. You can register and download the software here. They have even started a BLOG dealing with use of the software. I think I speak for all students when I say "Thanks guys!!"

Also, our friends at Bruker Biospin have recently introduced an inexpensive personal student license for their TOPSPIN software which can be purchased online.
There are other excellent NMR processing software options available. I mention specifically those above as the University of Ottawa currently holds network licenses for them and our students are most familiar with them.

Watergate vs Presaturation

Biochemists and protein chemists are often interested in observing the NH protons in their samples. Since the NH protons usually undergo slow chemical exchange with water, it is desirable to run the samples in H₂O rather than D₂O so the NH protons will not exchange with the deuterium in the solvent which would make them invisible in the ¹H NMR spectrum. In practice, a mixture of 10% D₂O and 90% H₂O is used as a solvent so that a deuterium lock can be established and used while running the spectrum. The very high concentration of water compared to the very low concentration of solute necessitates the use of solvent suppression methods.

Both presaturation and WATERGATE are efficient techniques used to suppress strong water signals from proton NMR spectra, however, there are differences between the two methods of which the user must be aware. Presaturation employs a selective, long, low power pulse to saturate the water resonance. This pulse is usually several seconds in duration during which exchange can occur between the unsaturated NH protons and the saturated water protons. If this exchange occurs, the NH protons become partially saturated to an extent related to the rate of chemical exchange between the NH and the water. The intensity of the NH protons in the spectrum is non-quantitative. WATERGATE, on the other hand, uses a pair of gradients surrounding a composite pulse which in effect inverts all but the water signal. The duration of the composite pulse is about 4 orders of magnitude shorter than a presaturation pulse, so exchange between the NH protons and the solvent occurs to a much lesser extent during the WATERGATE sequence compared to presaturation. As a result, the NH region is much less attenuated and more quantitative in a spectrum collected using WATERGATE compared to a similar spectrum run with presaturation. This is illustrated in the figure below which shows the NH region of the ¹H spectrum of a small disaccharide substituted peptide.One can see that some of the NH's are greatly attenuated in the spectrum acquired with presaturation compared to the spectrum run using WATERGATE. The more attenuated the signal, the faster the chemical exchange for that particular NH with water.

The WATERGATE suppression sequence is very similar to the gradient spin echo sequences used to measure diffusion constants and DOSY spectra. As a result, when WATERGATE suppression is used, one expects diffusion losses for small molecules (which diffuse quickly) compared to large molecules (which diffuse slowly). This effect is demonstrated in the figure below which shows the NH/aromatic region of the ¹H spectrum of a partially degraded ¹⁵N labelled 10 kDa protein.Here, one can see the same exchange losses pointed out in the previous figure for the presaturation spectrum compared to the WATERGATE spectrum. In addition, one can see that the intensity of a very sharp peak marked in yellow (likely due to a CH proton from free histidine) is less intense in the WATERGATE spectrum compared to the presaturation spectrum. This loss is due to the fast diffusion of the small free amino acid compared to the very large protein. In conclusion, one must be aware of the differences between the two solvent suppression methods if quantitative results are being sought.

I would like to thank Roger Tam from Robert Ben's Laboratory and Allison Sherratt from Natalie Goto's Laboratory for kindly providing the samples of the peptide and protein, respectively.

Fast 90 Degree Pulse Determination

Almost all NMR measurements rely on the correct calibration of 90° pulses. This is traditionally done by collecting a series of spectra as a function of pulse duration, finding a null for the 180° or 360° pulse and calculating the 90° pulse by simple division by 2 or 4 in the case of the 180° and 360° nulls, respectively. This determination, although trivial, can be very time consuming. Wu and Otting* have presented a much faster method of determining a 90° pulse based on measuring the nutation of a magnetization vector directly. Continuous nutation is depicted in the figure below. Here, the sample is subjected to continuous irradiation about the x axis. While being irradiated, the magnetization vector rotates in the z-y plane at a nutation frequency proportional to the pulse power. The magnetization on the -y axis is defined by a sine function. Fourier transformation of this magnetization gives an antiphase doublet centered at zero whose splitting Δν is twice the nutation frequency. The reciprocal of the nutation frequency is the time it takes the magnetization vector to rotate one complete cycle in the z-y plane and therefore the time it takes to rotate by one quarter of a cycle (i.e. the 90° pulse duration) is defined as 1/(2 Δν). The problem with continuous irradiation is that the sample must be irradiated at the same time magnetization is being detected. To eliminate this problem, a scheme similar to homonuclear decoupling is used where the radiation is turned off long enough to sample a data point. This is depicted in the figure below.Here each dwell period is divided up into a period for irradiation and a period for detection. The duty cycle for the irradiation is the fraction of time for which the sample is being irradiated. The magnetization is sampled when the power is off. As in the case for continuous irradiation, the magnetization vector still rotates in the z-y plane however, the rotation is slower as it is scaled according to the duty cycle. The duration of the 90° pulse is d/(2 Δν), where d is the duty cycle for irradiation. An example of this is shown in the figure below. The nutation spectrum was measured for HDO using a duty cycle, d = 0.10 and a power level of 12 dB (Bruker). Since the response of the amplifiers is linear, the 90° pulses at higher power levels can be calculated. Each decrease by 6 dB cuts the duration of the 90° pulse in half. In this case the 90° pulse at 0 dB was calculated to be 10.93 µsec at 0 dB based on the measured 90° pulse of 43.71 µsec at 12 dB. This pulse agrees to within a couple of percent of that measured by the more traditional method however, the measurement took only a few seconds. You can use a program called "pulsecal" on newer Bruker spectrometers to do this in complete automation.

--

* Peter S.C. Wu and Gottfried Otting J. Mag. Res. 176, 115 (2005).

Faster Relaxation Time Measurements in Solids

T₁ relaxation times are typically measured with the inversion recovery technique. In this method the magnetization is inverted and its recovery is monitored as a function of time. For nuclei with long T₁'s, the measurements are very time consuming as a recycle delay of at least five times T₁ must be used between scans. Typical T₁'s for ¹³C in the solid state range from several seconds to tens of minutes, so their direct measurement via the inversion recovery method could be prohibitively long.

High resolution ¹³C solid state NMR spectra of solids are routinely measured with cross polarization and magic angle spinning (CPMAS) in order to take advantage of the signal enhancement due to magnetization transfer between the abundant protons and isotopically dilute ¹³C nuclei. Additionally, the recycle delay needed for this measurement depends on the T_1ρ of the protons rather than the T₁ of the ¹³C. Proton T_1ρ's are typically shorter than ¹³C T₁'s by at least an order of magnitude, so many more scans can be collected per unit data collection time compared to a direct one-pulse measurement.

One might think that ¹³C T₁'s can simply be measured with cross polarization using a simple inversion recovery scheme by applying a 90° pulse to the ¹³C spins immediately following the contact pulse and then following their recovery over time. This method would have both the advantages of signal enhancement due to CP and more scans per unit time. The problem however, is that the enhanced magnetization of the inverted spins relaxes back to its unenhanced Boltzmann value and not its enhanced value. So, in order to measure the T₁, the direct ¹³C magnetization would have to be measured first (without CP) which would be very time consuming. This difficulty can be eliminated with the pulse sequence introduced by Torchia in 1978^* shown in the figure below.
This sequence uses a simple two step phase cycle to subtract out the effect of the direct ¹³C Boltzmann magnetization. The first part of the sequence uses a (90°_-y) pulse to return the CP enhanced magnetization to the z axis. The decay of the enhanced magnetization down to its Boltzmann value is followed using a (90°_x) pulse with detection of signals on the -y axis. The second part of the sequence uses a (90°_y) pulse to put the CP enhanced magnetization on the -z axis. The recovery of the enhanced inverted magnetization back to its equilibrium Boltzmann value is followed using a (90°_x) pulse with detection of signals on the y axis. The addition of the first and second parts of the experiment by way of the phase cycle allows for a simple calculation of the ¹³C T₁ with both the advantages of CP enhancement and the ability to collect more scans per unit time. An illustration of this method is shown in the figure below where the ¹³C T₁'s of glycine were measured. (The small peak in the spectrum is a spinning sideband of the carbonyl carbon)

* D.A. Torchia, J. Mag. Res. 30, 613, (1978).

NMR WIKI

An excellent resource for NMR users has been gaining more and more popularity on the web over the last few years. The brainchild of Evgeny Fadeev (Director of the Biomolecular Spectroscopy Facility at the University of California Irvine), NMR Wiki is an information sharing site offering a question and answer forum, job postings, a pulse sequence library, course material, history, information on meetings etc.... I think this is a fantastic place to learn more about NMR and Evgeny is to be commended for his efforts.

The Scale on an NMR Spectrum

Some people new to NMR spectroscopy have trouble with the meaning on the scales of their NMR spectra. Usually the data are plotted with a chemical shift scale (δ). This scale increases to the left and is usually reported in units of parts per million (ppm). The chemical shift of a resonance in a sample, δ_sample , in ppm is defined as follows:where ν_sample is the absolute frequency of the sample resonance and ν_reference is the absolute frequency of an agreed upon reference compound. For ¹H, ¹³C and ²⁹Si NMR, tetramethylsilane (TMS) is the agreed upon reference standard. When the scale is plotted in this manner, the peak positions are relative to that of the standard compound. This scale is particularly useful, as it independent of a single absolute frequency and therefore does not depend on the magnetic field strength, which varies from laboratory to laboratory. Spectra recorded using magnets of unequal field strength can be compared more easily.

Scales are also plotted in frequency units (Hz), usually with the reference compound assigned a value of 0 Hz. This scale also increases to the left and is usually of use when coupling constants (which are independent of field) are being measured.

Although absolute electronic shielding values are inconvenient to measure, one may hear people refer to one resonance being "more shielded" or "less shielded" than another, The shielding constant, σ, for a particular nucleus in a particular environment can be expressed by rearranging the Larmor equation.The shielding scale increases to the right.

In the age of continuous wave NMR spectrometers, NMR spectra were measured by irradiating the sample with continuous wave radiation and sweeping the magnetic field from a low value (on the left) to a high value (on the right). The scales were sometimes plotted in magnetic field units and one still hears about one peak being described as "upfield" or "downfield" from another. These terms have little relevance in FT NMR and should be avoided.

Some of the older literature reported ¹H NMR peak positions on a τ scale. Here, τ was equal to (10 ppm - δ ) and the scale increased to the right. This is no longer in use and has caused considerable confusion. It should be avoided.