University of Ottawa NMR Facility Blog: 2015

Monday, December 21, 2015

NMR of the Christmas Tree

One of my fondest memories as a child is the colorful lights and especially the smell of a decorated Christmas tree. The hot incandescent lights used years ago would heat up the tree evaporating the fragrant compounds in the needles producing the very memorable and wonderful smell of Christmas. Although modern artificial Christmas trees and cool LED lights have made the holiday season safer with respect to fires, they have taken much of the magic out of Christmas. Among many other compounds, it is pinene, bornyl acetate and citronellol that contribute to the Christmas smell of evergreen needles.

We can use NMR spectroscopy to look for these compounds and perhaps recover a bit of the Christmas magic. The bottom panel of the figure below shows the ¹³C CPMAS spectrum of spruce needles. One can easily identify the signals from cellulose in the CPMAS spectrum of the needles while some of the smaller peaks can be attributed to fragrant compounds. Many of the fragrant compounds in the needles are likely to be in a liquid-like state and not cross polarize very well. These will either be absent or under-represented in the CPMAS spectrum. The top panel of the figure shows the ¹H - ¹³C HSQC spectrum of a benzene-d₆ extract prepared from crushed spruce needles. The top and left-side projections are the high resolution ¹H and ¹³C NMR spectra, respectively. This sample is expected to contain all of the benzene soluble compounds. The spectrum is free of cellulose resonances and shows a mixture of fragrant compounds.

These data don't recover the childhood magic of Christmas but they do bring a little bit of joy to this NMR spectroscopist.

Merry Christmas

Wednesday, November 18, 2015

NMR Signals in Tuning Curves

The quality of the tuning and matching of an NMR probe on a Bruker NMR spectrometer can be monitored by using the wobble (or "wobb") routine in the TOPSPIN software. This routine sweeps the frequency using low power and displays a plot corresponding roughly to the absorbance vs frequency for the probe electronics. If the probe has very high sensitivity (eg: a cryoprobe) and contains a sample rich in protons (eg: water) then one is able to observe the proton spectrum in the frequency swept wobble curve. This is demonstrated in the figure below which shows wobble curves for a 600 MHz cryoprobe containing a sample of water.

The curves show the tuning profile of the probe with an anomaly at the proton frequency. The anomaly is the proton signal of the water in the NMR probe. The top panel is the wobble curve for the probe in a well-shimmed magnet and the bottom panel is the same curve after the magnet shims had been grossly maladjusted. One can see that the anomaly is much more broad in the wobble curve collected on a poorly shimmed magnet as one would expect the signal from the water to be much broader in an inhomogeneous magnetic field.

Thank you to Stan Woodman of Bruker Canada for pointing this phenomenon out to me.

Wednesday, June 24, 2015

Radiation Damping and Pulse Calibration

Radiation damping causes broadening in the NMR resonances of very strong signals (such as the ¹H signal of pure water) as a result of currents induced in the coil from the strong transverse magnetization. Radiation damping can also produce asymmetry and phase irregularities in the affected resonances. These problems make pulse calibration by the standard nutation curve problematic when very strong signals are used for the calibration. The left-hand panel (black) of the figure below shows the standard ¹H nutation curves for 0.1% H₂O in D₂O (bottom) and 80% H₂O in D₂O (top). In both cases, single-scan spectra with a recycle delay of 30 sec were collected and plotted horizontally. The pulse was varied from 1 µsec to 24 µsec in steps of 1 µsec. In the case of 0.1% H₂O in D₂O, the nutation curve is well behaved and one is easily able to read off the 90°, 180°, 270° and 360° pulse durations. In the case of 80% H₂O in D₂O, where radiation damping is a problem, the nutation curve is not well behaved. There are asymmetry and phase distortion problems which make it impossible to determine the 90° pulse, based on maximum signal height, with any accuracy. Nor is it possible to determine a reliable 180° based on the first minimum. The spectra show very little distortion in the vicinity of the second minimum so the 360° pulse can be used reliably to determine the 90° pulse. The right-hand panel of the figure (red) shows the integrals of the corresponding nutation spectra. The integrals for both samples behave similarly. It is clear that even in the case of severe radiation damping, one is able to determine a well behaved nutation curve from the integrals.

Friday, May 8, 2015

TROSY

The chemical shift resolution and sensitivity of NMR generally benefit from an increase in magnetic field strength. As a result, large sums of money are spent on magnets with higher and higher fields. The boost in sensitivity means that smaller and smaller quantities of sample are needed and measurements can be completed in shorter periods of time. There are particular cases however, where an increase in magnetic field can lead to a loss of sensitivity and resolution. This is the case for ¹⁵N decoupled ¹H spectra of the ¹H-¹⁵N spin pairs in very large ¹⁵N labelled proteins. The very long correlation times of the protein combined with the high resonance frequencies associated with high field strength lead to very short T₂ relaxation times and therefore broader lines. The broad lines account for a significant loss in resolution and sensitivity. One might then wonder why protein structural chemists spend so much money on very high field magnets. What follows is one possible answer to this question.

The two main relaxation mechanisms for the ¹H and ¹⁵N in proteins are dipolar coupling and chemical shielding anisotropy. These two mechanisms are also cross correlated with one another. The cross correlation term is of different sign for each of the two peaks in a ¹H-¹⁵N doublet resulting in one of the peaks of the doublet having a shorter T₂ (and broader line) than the other one. If ¹⁵N decoupling is applied, one sees a single resonance with a line width determined by the average of the two components of the doublet. This is illustrated in the figure below for a ¹H-¹⁵N spin pair in a small and large molecule at high field . The same is true in the ¹⁵N-¹H doublets in the ¹⁵N spectra of ¹⁵N-¹H spin pairs.

At very high fields, One of the lines in the ¹H-¹⁵N doublet is very sharp and the other very broad. If, in the ¹H spectrum of a protein, we could eliminate all of the broad doublet components leaving only the sharp ones, we would have a high resolution ¹H spectrum. Further, if we could combine such a measurement with an HSQC, we would have a high resolution ¹H-¹⁵N HSQC at high field. The combination of these two measurements is called transverse relaxation optimized spectroscopy (TROSY). TROSY data collection employs an HSQC measurement with neither ¹H nor ¹⁵N decoupling elements (as described in a previous post) as well as other elements which suppress the broad lines of the doublets and retain the sharp lines. The results of this are illustrated in the figure below for small and large proteins at high field.

Clearly, it is not advantageous to use the TROSY technique on small proteins rather than the conventional HSQC. For large proteins at high field however, there is a significant sensitivity and resolution advantage compared to a conventional HSQC. It should be noted that the TROSY cross peaks are shifted by ½ ¹J_HN in both the F2 and F1 domains. The figure below shows a superposition of a conventional HSQC (black) and a TROSY (blue) for a protein at 500 MHz.

One can clearly see the ½ ¹J_HN shift in the F2 and F1 domains of the TROSY compared to the HSQC. In this case, the conventional HSQC gives higher sensitivity than the TROSY.

Thank you to Adam Damry of Professor Roberto Chica’s research group at the University of Ottawa for providing the sample of ¹⁵N labelled protein.

Wednesday, May 6, 2015

Decoupling in 2D HSQC Spectra

HMQC and HSQC NMR data are commonly used to correlate the chemical shifts of protons and ¹³C (or ¹⁵N) across one chemical bond via the J coupling interaction. The data are ¹H detected, with the ¹H chemical shift in the horizontal F2 domain and the ¹³C (or ¹⁵N) chemical shift in the vertical F1 domain. In the case of ¹H and ¹³C, the technique depends on protons bonded to ¹³C. ¹H–¹²C spin pairs provide no coupling information and are suppressed by the method. If one is to observe the ¹H signal of a ¹H-¹³C spin pair, one expects to observe a doublet with splitting ¹J_H-C (i.e. the ¹³C satellites). Likewise, if one is to observe the ¹³C signal of a ¹H-¹³C spin pair, one expects to observe a doublet with the same splitting. 2D HSQC spectra are normally presented with both ¹H and ¹³C decoupling yielding a simplified ¹H-¹³C chemical shift correlation map over one chemical bond. The figure below shows one of the most commonly used gradient HSQC pulse sequences. The ¹H and ¹³C decoupling elements of the sequence are highlighted in yellow and pink, respectively.

During the evolution time, t1, the ¹³C chemical shift and ¹H-¹³C coupling evolve. The ¹H 180° pulse (color coded in yellow) in the center of the evolution time refocuses the coupling and as a result decouples protons in the F1 (¹³C) domain of the spectrum. ¹³C is broadband decoupled from the F2 (¹H) domain by applying a GARP pulse train (color coded in pink) at the ¹³C frequency during the collection of the FID. One can turn each of these elements “on” or “off” for data collection. The figure below shows the ¹H-¹³C gradient HSQC spectrum of benzene with all possible combinations of ¹H and/or ¹³C decoupling.

In the top left panel both ¹H and ¹³C decoupling are turned “on” and one observes a singlet in both the F2 (¹H) and F1 (¹³C) domains. In the top right panel, the ¹H decoupling element is “on” while the ¹³C decoupling element is “off”. The result is a ¹H-¹³C doublet in the F2 (¹H) domain and a singlet in the F1 (¹³C) domain. In the bottom left panel, the ¹H decoupling element is “off” while the ¹³C decoupling element is “on”. The result is a ¹³C-¹H doublet in the F1 (¹³C) domain and a singlet in the F2 (¹H) domain. In the bottom right panel, both the ¹H and ¹³C decoupling elements are “off”. The result is a ¹H-¹³C doublet in both the F2 (¹H) and F1 (¹³C) domains.

Tuesday, April 28, 2015

Dead Time and Phase

The phase of an NMR peak depends on the sine/cosine character of the free induction decay (FID) when the receiver is turned on. Positive and negative cosine FIDs will yield positive and negative in-phase peaks, respectively whereas positive or negative sine FIDs will yield peaks 90° out of phase. FIDs that are neither a pure sine nor pure cosine with yield peaks which are out of phase to an extent dependent on the cosine/sine character of the FID. In a perfect world, the receiver is gated on immediately after a perfect 90° pulse and all FID’s are cosines producing positive in-phase NMR peaks. In the real world however, there are problems with acoustic ringing, pulse breakthrough imperfect pulses of finite duration and finite electronic switching times. These problems produce a dead time between the end of the pulse and time at which the receiver is gated on during which data cannot be collected. As a result the FID may not be a perfect cosine function and a phase correction will need to be applied after Fourier transform. The first two figures below illustrate this point for a single off-resonance NMR signal as the dead time is increased. The first figure shows the FID’s as a function of increasing dead time from bottom to top while the second figure shows the Fourier transformed spectra without phase correction as a function of dead time from left to right.

When there is more than one signal, the FID is an interferogram representing the sum of all time domain signals, each with a different frequency. Since each component has a different frequency, its phase is affected to a different extent as a result of the dead time. Higher frequency time domain components (i.e. those representing peaks further off-resonance) are affected more than lower frequency components (i.e. those representing peaks closer to being on-resonance). This is illustrated in the figure below for the ¹H NMR data for p-xylene. The left-hand portion of top panel of the figure shows the FID containing both methyl and aromatic components while the right-hand portion of the top panel shows an expansion of the initial portion of the same FID. The bottom panel of the figure shows a stacked plot of the NMR spectra collected as a function of dead time. One can see that the phase of the aromatic peak furthest off-resonance is affected to a greater extent by an increased dead time than the methyl peak closer to resonance.

The last figure also shows a stacked plot of the ¹H NMR spectra of p-xylene as a function of dead time.

In this case, the methyl signal was set on-resonance. One notices immediately that the phase of the on-resonance methyl peak is unaffected by an increase in the dead time whereas that of the off-resonance aromatic peak is severely affected. The on-resonance methyl peak is not affected by an increase in dead time as its time domain signal is a simple exponential with no sine/cosine oscillations. A loss of the beginning of a simple exponential FID due to the dead time still leaves a simple exponential and thus the phase is not affected.

Thank you to Dr. Michael Lumsden of the NMR Facility of Dalhousie University for suggesting the subject of this post.

Friday, April 17, 2015

The Information Content of an FID

The free induction decay (FID) is a function representing the decay of transverse magnetization as a function of time after the application of a pulse (or pulse sequence). The NMR spectrum is obtained by Fourier transforming the FID. All of the information in the NMR spectrum (line width, intensity, phase, line shape ....) is contained in the FID. Knowledge of what part of the FID represents a particular property of an NMR spectrum allows a user to process the raw time domain data in a way that maximizes the quality and information content of the processed frequency domain spectrum. The figure below shows the real component of the complex FID for a sample in which there is a single resonance. The FID is labelled in terms of its information content.

The shape of the envelope of the overall decay defines the line shape of the NMR resonance. Exponential decays yield Lorentzian line shapes. The frequency in the FID represents the offset of the resonance from the carrier frequency or in other words, how far the resonance is from the center of the spectrum. The higher the frequency, the further off-resonance the peak. The FID for an on-resonance peak is a simple decaying exponential with no oscillation frequency present. The first point of the FID contains the integrated intensity and phase information. Since time and frequency are reciprocals of one another, the duration of the decay before it disappears into the noise determines the sharpness of the NMR resonances and ultimately the resolution in cases where there is more than one resonance in the spectrum. The longer the decay - the narrower the lines. The early portion of the FID (short time) defines the broad features (wide frequency distributions) whereas the the latter portion of the FID (long time) defines the sharp features (narrow frequency distributions). Also, since the FID is a decaying function, the early portion has a higher signal-to-noise ratio than the latter portion. With this knowledge its is very easy to understand how best to apply apodization functions to the raw data for the purpose line broadening and resolution enhancement. Processing techniques such as zero filling, forward linear prediction, backward linear prediction and signal-to-noise optimization as well as spectral artifacts such as truncation errors, baseline roll and receiver saturation errors are easily understood with knowledge of the information content in the FID.

Wednesday, April 1, 2015

NMR of Edible Oils

NMR spectroscopy is one of the most informative techniques for the study of structure, composition and dynamics of matter. One of the many thousands of applications of NMR spectroscopy is in the study of edible oils. Plant and animal oils are composed of complex mixtures of fatty acid tri-esters of glycerol. The fatty acid moieties are generally straight chains of 16 - 24 carbons in length with various degrees of unsaturation. In natural oils the double bonds are all cis-. Fatty acids with trans- double bonds are usually the result of food processing. The double bonds in polyunsaturated fatty acids are generally separated by single methylene groups. The end methyl carbon of each fatty acid chain in the glycerol tri-esters is referred to as the omega position. Omega-3 fatty acids are those with a double bond on the third carbon from the omega methyl position. The most common omega-3 fatty acid in plant oils is α-linolenic acid (ALA), a C₁₈ acid with three cis- double bonds in the 9-, 12- and 15- positions. Two of the most common omega-3 fatty acids in marine oils are eicosapentaenoic acid (EPA), a C₂₀ acid with five cis-double bonds in the 5-, 8-, 11-, 14-, and 17- positions and docosahexaenoic acid (DHA), a C₂₂ acid with six cis- double bonds in the 4-, 7-, 10-, 13-, 16- and 19- positions. The human body benefits from EPA and DHA which are only inefficiently synthesized from ALA in the human body. Also, it is recommended that consumption of saturated oils should be limited and that polyunsaturated oils are a better alternative. With these concerns, the study of edible oils has become important. One might expect that the complexity of the mixtures constituting the natural edible oils would limit the usefulness of NMR as a method of study however; the spectra contain a great deal of information as can be seen in the figures below. The ¹³C NMR spectra of 4 plant oils and 1 commercial “wild fish” oil are shown in the first figure.

The olefinic carbons are color-coded in yellow and give an indication of the degree of unsaturation in the oils. Clearly, the coconut oil is saturated and the fish oil contains the highest degree of unsaturation. The ¹H NMR spectra of the same oils are shown in the second figure.

The resonances color-coded in yellow are those of the protons on olefinic carbons and are a direct indication of the degree of unsaturation. The resonances color-coded in pink are methylene protons on carbons adjacent to two olefinic carbons and represent the degree of polyunsaturation. The resonances color-coded in blue are methylene protons attached to carbons adjacent to both methylene carbons and olefinic carbons. The methyl resonances are at the lowest chemical shift. Those color-coded in green are from the omega-3 fatty acid moieties. Qualitatively, from the data, it is obvious that the coconut oil is saturated and the fish oil contains the most omega-3 polyunsaturated fatty acid moieties.

Tuesday, March 31, 2015

Cross Polarization Based Mixture Resolution in Solids

One of the most common techniques used to collect solid-state NMR data for spin I = ½ nuclides is a combination of cross polarization (CP) and magic angle spinning (MAS). CPMAS provides high sensitivity from the CP and high chemical shift resolution from the MAS. Furthermore, the scan repetition rate depends on the shorter relaxation time of the protons rather than the longer relaxation time of the spin I = ½ nuclide therefore, more scans can be collected per unit time. It must be remembered however, that the success of the CP technique depends on the dipolar coupling interaction between proximate protons and the nucleus being observed. In the absence of dipolar coupled protons, CP signals are not observed. For this reason, it is sometimes necessary to use a conventional one-pulse method (Bloch decay) which can be used to observe the spin I = ½ nuclide, albeit with lower sensitivity, whether protons are present or not. When a sample consists of a mixture with some protonated components and some components without protons, then it may be advantageous to collect both a CPMAS and a Block decay spectrum. When both methods are used, the data can be used to resolve the spectrum of the mixture into subspectra; the protonated components in one spectrum and those components without protons in another. An example of this is shown with the ¹³C NMR data for a common antacid tablet in the figure below.

The two most abundant carbon containing components of an antacid tablet are calcium carbonate and sucrose. Spectrum (a) is the CPMAS spectrum. It consists only of the resonances of sucrose (color coded in yellow) since calcium carbonate (color coded in pink) contains no protons. Spectrum (b) is the Bloch decay spectrum with high power proton decoupling. It consists of both the resonances of sucrose and calcium carbonate. Spectrum (c) is a linear combination of (a) and (b) and represents, primarily, the spectrum of calcium carbonate. Another interesting example of CP based mixture analysis is given here using Christmas shortbread cookies as an example.

Thursday, March 26, 2015

Mixture Resolution in ¹³C CPMAS NMR

The recycle delay necessary to get the highest signal-to-noise ratio in a multi-scan ¹³C CP MAS NMR spectrum depends on the relaxation properties of the protons in the sample. The protons in pure solid samples normally belong to a single homogeneous dipolar coupled network. As a result, all of the protons in the coupled network have a common T₁ relaxation time. One would expect the same behavior for a mixture of compounds only if the components were mixed at the molecular level. If the compounds are not mixed at the molecular level, the sample consists of domains of pure materials, each of which has a common proton T₁. If the proton T₁'s of the domains are significantly different, then one has a means of discriminating between the domains and hence the compounds of the mixture with ¹³C CPMAS NMR data. The figure below illustrates this principle for a tablet of vitamin C ground into a powder. The vitamin C tablet consists primarily of ascorbic acid for which the structure is shown in the figure. The other major solid organic additives are hypromellose (hydroxypropyl methylcellulose), stearic acid (n-C₁₇H₃₅COOH), magnesium stearate and carnauba wax (a complex mixture of C₂₆ to C₃₀ acids, esters and alcohols). When the tablet is ground up, the powder consists of ascorbic acid domains, stearic acid domains, magnesium stearate domains and carnauba wax domains.

¹³C CPMAS NMR spectra were acquired with a 30 second and a 2 second recycle delay and are shown in (a) and (b), respectively. One can see that relative intensity of the components in the mixture depends on the recycle delay. The proton T₁ of the ascorbic acid is obviously longer than that of the other components of the mixture. The spectra in (c) - (e) are linear combinations of (a) and (b). The linear combination in spectrum (c) was created such that the ascorbic acid resonances were nulled. The resulting spectrum is that of only the organic additives. The hypromellose resonances are in the 50 ppm to 110 ppm range. The aliphatic resonances of the stearic acid, magnesium stearate and carnauba wax overlap in the 10 ppm to 50 ppm range and appear to have similar proton T₁'s. The linear combination in spectrum (d) was created such that the aliphatic stearic and wax resonances were nulled. The resulting spectrum is that of ascorbic acid and the inverted spectrum of the hypromellose. The linear combination in spectrum (e) was created such that the hypromellose resonances were nulled. The resulting spectrum is that of the ascorbic acid with the stearic acid, magnesium stearate and carnauba wax additives. This combination allows observation of the ascorbic acid with no overlapping resonances from the additives.

Wednesday, March 25, 2015

¹³C NMR of Vitamin C - Solids vs. Liquids NMR

In ¹³C[¹H] NMR spectra of liquids, one observes a single resonance for each symmetrically nonequivalent carbon atom. The same is true of the ¹³C CPMAS spectra of solids. The difference is that the molecular symmetry is the determining factor in liquids NMR whereas the crystallographic symmetry is the determining factor in solids NMR. As a result, solids NMR can give different spectra for different solid polymorphs and multiple resonances due to multiple nonequivalent molecules in the asymmetric unit of the crystal structure. The figure below compares the solids ¹³C CPMAS and liquids ¹³C[¹H] NMR spectra of a vitamin C (ascorbic acid) tablet.

The structure is provided in the figure and the assignment for all of the carbon atoms is color coded. In the liquids spectrum, one observes resonances for each carbon in the molecule. In the ¹³C CPMAS spectrum, one observes that three of the six ¹³C resonances are doubled. This doubling is consistent with there being two nonequivalent ascorbic acid molecules in the asymmetric unit of the crystal. Presumably, the other three resonance are doubled as well but there is insufficient resolution in the spectrum for this observation. The broad features in the spectrum are due to additives in the vitamin C tablet which include, hypromellose, stearic acid and carnauba wax.