Its Time to Move On

After more than 28 years of serving the NMR Facility at the University of Ottawa, I have decided to retire at the end of 2020.  My position will be posted this fall.  What a fantastic job it is managing an NMR Facility!  I have learned so much about NMR over the years and have taken great pleasure in helping so many students.  I have particularly enjoyed writing this BLOG which was originally intended to answer the frequent questions asked of me by uOttawa students.  To my surprise and delight, it has been useful to so many more people than just those at the University of Ottawa.  Thank you for reading it.

It is now time for me to move on.  Much less of this:

 ....and much more of this:

Cheers.

NMR and Food Chemistry - Rhubarb

Many gardeners here in Canada are currently harvesting their rhubarb to make pies and other desserts.  Like cranberries, rhubarb is very sour due to the presence of organic acids.  The leaves of the rhubarb plant contain so much oxalic acid that they are toxic.  The stalks of the plant contain less oxalic acid and are not toxic (although I would argue that they taste awful).  Oxalic acid is used in many cleaning products, so if you are like me and hate the taste of rhubarb, you can still make use of it as a cleaning agent.  If you have a very dirty stainless steel pot, you can clean it by simply stewing rhubarb in it.  The figure below shows the ¹H and ¹³C NMR spectra of an aqueous extract of rhubarb stocks.  The major constituents are organic acids with smaller quantities of sugars.  Some of the ¹³C signals from oxalic, malic and citric acids are labelled.

NMR and the Liquid-Gas Interface

Most NMR spectra are recorded for liquid or solid phase samples.  Many chemists have not even considered measuring NMR spectra of gas phase samples.  Such spectra are indeed possible to record and the information available from such spectra has been studied and reviewed in detail.*  In our first high school science classes we learn that molecules in the gas phase diffuse much more quickly than those in the liquid phase and that there is an equilibrium between the liquid and gas phases.  These two elementary concepts can be demonstrated nicely with ¹H NMR spectroscopy.

A suitable sample was prepared by putting 1-2 µL of acetone in a standard 5 mm NMR tube.  A greased rubber plug was then forced into the tube such that it resided about 6 cm  above the bottom of the NMR tube.  This was done to limit the volume over which the vapour could diffuse to that of the active volume of the probe coil.  The tube was then sealed with a torch to prevent the loss of sample.  The sample contained a small amount of liquid in the bottom of the NMR tube and a mixture of acetone vapour and air above the liquid.  A sketch of the sample is shown in the figure below.

The 600 MHz ¹H data were collected in a cryoprobe at 298 K without a ²H lock.  The magnet was shimmed using the ¹H FID. The ¹H spectrum has two resonances, a broad one at ~2.2 ppm (Δν_1/2 = 30 Hz) and a narrower one (Δν_1/2 = 4 Hz) at ~3.8 ppm due to liquid and gaseous acetone, respectively.  The large 30 Hz line width for the liquid resonance is due to the magnetic susceptibility discontinuity boundary between the droplet of liquid with the glass and vapour interfaces.  There may also be broadening as the droplet resides near the edge of the homogeneous region of the magnetic field.  A DOSY spectrum, acquired with δ = 0.5 msec and Δ = 4.9 msec, illustrates the vastly different molecular diffusion rates between the liquid and gaseous phases of acetone.  An EXSY spectrum, acquired with a 2 second mixing time, clearly shows exchange peaks between the liquid and the gas phases, illustrating the liquid-gas equilibrium.

* C.J. Jameson. Chem. Rev. 91, 1375-95 (1991).

12C/13C Isotope Effects on 1H T1 Relaxation Times

What is the ¹H T₁ relaxation time of chloroform?  It seems like a simple enough question, but the answer is not so simple.  The relaxation rate for any proton is the sum of relaxation rates resulting from several different mechanisms (eg. homonuclear dipolar coupling, heteronuclear dipolar coupling, chemical shielding anisotropy, spin rotation etc...).  Each of these mechanisms of relaxation depends on dynamic effects and the extent to which those processes occur at the Larmor frequency.   Often, in proton-rich organic compounds, ¹H T₁ relaxation is dominated by the homonuclear dipolar coupling interaction.  For chloroform, with only a single proton, there can be no intra-molecular homonuclear ¹H dipolar interaction and the ¹H relaxation rate must depend on other mechanisms.  One of these mechanisms is the result of the heteronuclear dipolar coupling interaction.  For the ¹³C isotopologue of chloroform, one would expect a significant heteronuclear dipolar interaction between the directly bound ¹H and ¹³C.   This interaction is absent in the ¹²C isotopologue and one would therefore expect the T₁ relaxation time of ¹³CHCl₃ to be much shorter than that of ¹²CHCl₃.  This is illustrated in the figure below.

The ¹H T₁ relaxation times for both ¹²CHCl₃ and ¹³CHCl₃ were measured with the inversion recovery method for a degassed, dilute (1%) sample of chloroform in acetone-d6.  The inversion recovery delay was varied from from  1 sec. to 300 sec.  The recycle delay was 300 sec.  Relaxation is much more efficient for ¹³CHCl₃ compared to ¹²CHCl₃.  The T₁ for ¹³CHCl₃ is only 46% that of ¹²CHCl₃, indicating the significance of the heteronuclear ¹H - ¹³C dipolar coupling interaction as a relaxation mechanism.       

1H T1ρ Edited 13C CPMAS Spectra - Pharmaceutical Analysis

Active pharmaceutical ingredients (API's) are often mixed with other compounds (excipients) used to dilute, stabilize, sweeten, color, flavour, bind, coat (etc...) the medication.  Often the API is a crystalline compound while the excipients are noncrystalline or amorphous.  When analyzing pharmaceutical pills by solid-state ¹³C CPMAS NMR, one often wants to observe the active crystalline API and not the amorphous non-active excipients.  This can sometimes be accomplished by collecting spectra with differing recycle delays, as the excipients often have shorter ¹H T₁ relaxation times than the crystalline API.  An example of this is given here.  Crystalline API compounds often have much longer ¹H T_1ρ's than the amorphous excipient compounds.  One can modify the simple CPMAS pulse sequence to discriminate against the excipients with short ¹H T_1ρ's by introducing an additional ¹H spin locking pulse before the cross polarization during which the transverse ¹H magnetization of the excpient decays to zero while that of the API decays to a much smaller extent.  After the ¹H spin locking pulse, cross polarization is applied, transferring polarization from the remaining API ¹H magnetization to the API ¹³C.  This modification to the CPMAS experiment is shown in the figure below.

The figure below illustrates this technique applied to generic acetylsalicylic acid (ASA) tablets.

The spectrum in the bottom panel is a ¹³C CPMAS spectrum of a crushed generic ASA tablet with a 60 second recycle time and a 2 msec contact time.  One can see the nine ¹³C resonances of the ASA plus the broad excipient signals between 50 ppm and 110 ppm (highlighted in yellow).  The spectrum in the middle panel was acquired under identical conditions with an additional 10 watt, 12 msec ¹H spin locking pulse applied prior to cross polarization.  Clearly, the broader signals of the excipients (with short ¹H T_1ρ) are suppressed leaving only the resonances of the ASA.  The top panel is a weighted difference of the two spectra showing only the signals of the excipients.  Although a pharmaceutical example is used here, the technique is applicable generally to any mixture of solids with different ¹H T_1ρ's.

Where is my INEPT signal? - Proton Exchange Issues

INEPT and DEPT sequences are routinely used to enhance the NMR signals for low γ nuclides such as ¹⁵N or ¹³C.  The enhancement relies on polarization transfer between the protons J-coupled and the low γ nuclide.  The pulse sequences incorporate delays based on the reciprocal of the J-coupling constant between the protons and the low γ nuclide.  In the case of ¹⁵N INEPT, the enhancement for each scan can be as much as γ_H/γ_N (~ 10) compared to that from a conventional one-pulse sequence with inverse gated decoupling.  Furthermore, the recycle delay for the INEPT sequence depends on the the ¹H T₁ relaxation time rather than that of ¹⁵N.  ¹H T₁'s are typically an order of magnitude (or more) less than those of ¹⁵N so the recycle delays required for ¹⁵N INEPT spectra are at least ten (and possibly 100 times) shorter than those required for one-pulse data collection.  These two factors mean that the true time saving for a ¹⁵N INEPT measurement compared to a one-pulse ¹⁵N measurement can be on the order of 100 - 1000 times.  There are, however cases where ¹⁵N INEPT signals are attenuated or entirely nonexistent.  Attenuated ¹⁵N INEPT signals are observed when the protons (with short T₁) coupled to ¹⁵N exchange with those of water (longer T₁) on a time scale of seconds.*  The problem arises because of saturation transfer during the inverse gated decoupling used during the acquisition time. The partially saturated protons are unable to transfer as much polarization to the ¹⁵N as they would were they fully polarized. The problem can be reduced if a recycle delay much greater than the T₁ relaxation time of the water protons is employed.  If the protons bound to ¹⁵N undergo exchange with other labile protons at a rate fast with respect to the ¹H-¹⁵N J coupling interaction, polarization transfer from ¹H to ¹⁵N is not possible and a ¹⁵N INEPT signal cannot be observed.  This is demonstrated in the figure below.

Concentrated solutions of the methyl ester of anthranilic acid and anthranilic acid were prepared in DMSO-d₆.  The ¹⁵N NMR data were collected on a 600 MHz instrument with a cryoprobe.  The left-hand panel of the figure compares the ¹⁵N one-pulse spectrum with inverse gated decoupling (bottom) to the INEPT spectrum (top) for the methyl ester.  The spectra were collected with the same number of scans. For the methyl ester, the ¹⁵N bound protons do not exchange with any other labile protons.  The enhancement in the ¹⁵N INEPT spectrum is clear.  Similar spectra for anthranilic acid are shown on the right-hand side of the figure.  In anthranilic acid, the ¹⁵N bound -NH₂ protons undergo intramolecular exchange with the acid proton at a rate fast with respect to the one-bond ¹⁵N-¹H coupling constant (~90 Hz).  As a result, polarization transfer is not possible and no INEPT signal is observed.  The same is true for the meta- and para- isomers (data not shown). 

Thank you to Jin Hong for sharing her experience with collecting ¹⁵N INEPT data for anthranilic acid and Mojmir Suchy for kindly providing the samples.

* G.D. Henry and B.D. Sykes, J. Magn. Reson. B, 102, 193 (1993).

1H J-Resolved Spectroscopy to Evaluate 1H-1H and 1H-19F Coupling Constants

2D ¹H J-RESolved spectroscopy (JRES) is able to separate the ¹H chemical shift and J coupling interactions in the F2 and F1 domains of the 2D data, respectively. The F2 projection represents the pure-shift ¹H decoupled ¹H NMR spectrum while the individual F1 slices at each chemical shift reveal the ¹H - ¹H J coupling for each resonance.  When this technique is applied to a spin system with both homonuclear ¹H-¹H coupling and heteronuclear coupling, it has the ability to provide both the homonuclear and heteronuclear coupling constants.  This is demonstrated in the figure below for 2,3-difluoro pyridine which has both ¹H-¹H and ¹H-¹⁹F coupling.

The top trace in the figure is the ¹H NMR spectrum showing the complex resonances due to both the homonuclear and heteronuclear coupling.  The 2D JRES spectrum is highlighted in grey.  The ¹H-¹H coupling is shown in the F1 slices which were summed to produce the blue, red and green vertical traces in the figure for ¹H resonances A, C and B, respectively.  These traces are identical to the resonances in the separately collected ¹H spectrum with ¹⁹F decoupling shown in the bottom trace of the figure.  The F2 projection of the JRES spectrum is shown in the trace directly on top of the 2D spectrum, colour coded in yellow.  The F2 projection represents the ¹H decoupled ¹H spectrum showing only the ¹H- ¹⁹F coupling.  It can be compared to the separately collected PSYCHE pure-shift ¹H spectrum, colour coded in orange which is very nearly identical.  Clearly this very simple, often overlooked, technique can provide a great deal of both homonuclear and heteronuclear coupling information.