NMR and Food Chemistry - Maple Syrup

Maple syrup is arguably one of the tastiest traditional Canadian condiments.  In honor of Canada Day (July 1), it is appropriate to take a look at this delicious golden treat.  The bottom trace of the figure below shows the 600 MHz ¹H NMR spectrum of pure Quebec maple syrup dissolved in D₂O.

The spectrum is overwhelmingly dominated by sucrose.  Clearly, nature gives us the maple flavor with very low concentration components.  The top trace is a similar spectrum of "table" syrup which has a taste somewhat similar to maple syrup.  The spectrum is much more complicated than that of pure maple syrup.  In order to mimic the flavor of pure maple syrup, the food chemists resort to a complex mixture of sugars and artificial flavors.  Canada keeps it simple and of course better!  Happy Canada Day.

Non-uniform Sampling (NUS)

Collecting 2D or 3D NMR data can be very time consuming. The indirect dimension of a 2D experiment is sampled linearly via the t1 increments in the pulse sequence.  An FID must be collected for every single linearly spaced t1 increment. In the interest in collecting 2D or 3D NMR data in a more time efficient manner, a great deal of effort is made towards faster data collection techniques.  While some of these methods are based on spatial selectivity, others are based on sparse sampling techniques in the indirect dimensions of nD NMR sequences.  One such sparse sampling method, given the name non-uniform sampling (NUS), samples a sub-set of the indirect dimension in a random (or weighted random) manner and then predicts the uncollected data based on the data sampled, in much the same way data are predicted in the forward and backward linear prediction methods.  The reconstructed data is then used for the indirect Fourier transforms.  A comparison of the conventional and non-uniform data sampling methods is illustrated in the figure below.

Collecting only a fraction of FID's reduces the experiment time by the same fraction.  The figure below shows a superposition of partial 600MHz ¹H-¹³C HSQC spectra of a D₂O solution of sucrose.

All of the spectra were collected with 2 scans per increment using a 1.5 second recycle time.  The lower spectrum in black was collected conventionally with 256 increments in 15 minutes. The middle spectrum in blue was collected conventionally with 64 increments in 3.75 minutes. The top spectrum in purple was collected using NUS with 25% of 256 increments (i.e. 64 increments) collected in 3.75 minutes.  A comparison of the two conventionally collected data sets shows the expected loss in F1 resolution with the 4-fold reduction in experiment time by reducing the number of increments by a factor of 4. The bottom (black) conventional spectrum and the top (purple) NUS spectrum are however virtually indistinguishable despite the 4-fold reduction in experiment time for the NUS spectrum.  NUS is a very valuable technique for reducing experiment times without sacrificing resolution.

CEST - Chemical Exchange Saturation Transfer

Chemical Exchange Saturation Transfer (CEST) is a technique where one resonance, in slow exchange with a second resonance, is saturated with a selective low power pulse followed by a hard non-selective 90° pulse.  The intensity of the second resonance is then diminished due to the transfer of saturation from the first resonance as the result of  chemical exchange.  The figure below demonstrates this for a 25 mM solution of salicylic acid in H₂O/D₂O buffered at pH 7.

The left-hand panel of the figure is a stacked plot of extracted spectra collected in a pseudo 2D acquisition as a function of saturation frequency.  The saturation frequency was varied from an initial value of 20 ppm to a final value of -20 ppm in steps of 0.2 ppm.  The spectra are plotted such that only the water resonance is on scale.  One can see that the intensity of the water resonance dips when a saturation frequency of ~14 ppm is applied, corresponding to the resonance frequency of the –COOH and –OH protons of the salicylic acid (which appear to be in fast or intermediate exchange with one another).  The water resonance of course also dips to zero when a saturation frequency of ~4.7 ppm is used, corresponding to a simple presaturation of the water.  The right-hand panel of the figure is a plot of the integral of the water resonance as a function of saturation frequency, showing again a dip at ~14 ppm.

CEST is used in MRI to provide image contrast where a chemical exchange agent is introduced and images are collected with and without saturation of the exchange agent.  The difference provides an image enhanced by the presence of the chemical exchange agent.

Thank you to Dr. Mojmir Suchy of Prof. Adam Shuhendler’s group at the University of Ottawa for arousing my interest in the use of CEST for MRI and preparing the sample used in this post. 

INEPT

The sensitivity of a low γ, spin I = ½ nucleus is determined by the difference in populations between the low energy and high energy states, governed by the Boltzmann distribution. If the low γ, spin I = ½ nucleus is coupled to a proton the energy level diagram is more complicated than simply two levels and is shown in the figure below where a ¹³C-¹H spin pair is used as an example.

The populations of the states involved in the ¹³C transitions and hence the sensitivity of the ¹³C signal can be altered by inverting the H1 or H2 ¹H transitions with 180° pulses. This is illustrated in the figure below.

In the left panel, the H1 transition of a ¹³C-¹H spin pair is inverted (i.e. the populations of the two energy levels of the H1 transition are swapped). This also affects the populations of the energy levels involved in the C1 and C2 ¹³C transitions. After inversion of the H1 ¹H transition, the intensities of the C1 and C2 ¹³C transitions have changed from their equilibrium value of 1:1 to an enhanced value of 5:-3. If the H2 transition is inverted (right-hand panel), the C1:C2 intensity ratio is -3:5. In both cases the sensitivity of the ¹³C doublet has been enhanced compared to its equilibrium value. This enhancement is called INEPT (Insensitive Nuclei Enhanced by Polarization Transfer) and is one of the most common sensitivity enhancement techniques used in NMR pulse sequences. The simplest implementation of INEPT is shown in the figure below along with the vector diagrams.

Phase cycling can be employed to obtain a -4:4 anti-symmetric doublet, rather than doublets with components of unequal magnitude. This is represented in the figure below.

A refocusing element can be added to the end of the sequence to refocus the anti-symmetric doublets and data can be collected with proton decoupling.

The result is a singlet with 4 times  (i.e. γ_H/γ_C) the intensity of the singlet one would expect under equilibrium conditions without an NOE.  For ¹⁵N, one obtains a sensitivity gain of ~10. The results of these implementations of INEPT are compared to the equilibrium situation in the figure below.

INEPT has the additional advantage that its repetition rate is determined by the ¹H T₁ rather than the ¹³C T₁.  This is a tremendous additional sensitivity improvement when multiple scans are collected because the ¹H T₁ is often shorter than the ¹³C T₁ by an order of magnitude.  One can collect approximately ten times as many scans per unit time.  This advantage is even more significant for ¹⁵N.  Reverse INEPT is used in the collection of ¹H data for  carbon-proton pairs to suppress the protons bound to ¹²C. 

Solid-State 13C NMR of Chicken Eggshells

¹³C CP/MAS and direct single pulse ¹³C MAS NMR with high power decoupling can give very different results for a wide variety of materials.  ¹³C CP/MAS NMR relies on the transfer of magnetization from protons to ¹³C via the dipolar coupling mechanism whereas the direct single pulse method does not.  An interesting material to demonstrate this principle is the shell of a chicken egg.  Chicken eggshell is a complex bio-mineral consisting largely (~ 95%) of the calcite polymorph of calcium carbonate as well as proteins and lipids.  The ¹³C CP/MAS and ¹³C single pulse MAS NMR spectra of a sample of dry ground eggshell, from which the membranes had been removed, are shown in the lower and upper traces in the figure below, respectively.

The ¹³C CP/MAS spectrum in the lower trace has very broad resonances in the aliphatic region of the spectrum due to the proteins and lipids.  The carbonyl region of the spectrum  consists of two resonances; one at ~173 ppm due to the carbonyl carbons of the amino acid residues of the proteins and a resonance at  ~169 ppm which has been shown^1,2 to originate from bicarbonate ions (HCO₃)^-.  Although ~95% the eggshell consists of CaCO₃, the carbonate resonance is not present in the ¹³C CP/MAS spectrum as there are no proximate protons for cross polarization.  In contrast, the single pulse ¹³C MAS spectrum in the top trace shows only the ¹³C resonance from the carbonate ions which has a coincident chemical shift with that of the bicarbonate ions.  All of the other ¹³C resonances are buried in the noise as they are in much lower concentration. These spectra are an excellent example of how one can obtain different information from ¹³C CP/MAS and single pulse ¹³C MAS spectra.

1. D.M. Pisklak, L. Szcleszczuk, I. Wawer, Journal of Agricultural and Food Chemistry, 60, 12254 (2012).
2. J. Feng, Y.J. Lee, R.J. Reeder, B.L. Phillips, American Mineralogist, 91, 957 (2006).

Ultra-Fast 1H COSY

It cannot be disputed that the introduction of routine 2D NMR spectroscopy in the 1980's revolutionized the way in which NMR measurements are made. Now, with literally thousands of 2D methods available, the quantity of accessible information has dramatically increased. One cannot imagine a modern NMR lab without a 2D NMR toolbox.  One of the main drawbacks to traditional 2D NMR spectroscopy has always been the time required to collect the data.  Data collection can take anywhere from a few minutes to tens of hours.  Many 1D FIDs (typically more than 128) must be acquired as a function of evolution time to construct the 2D data matrix.  The measurement of each of these signals may require multiple scans as a result of necessary phase cycling between which a relaxation delay must be employed.  Once all of the data have been collected, each of the FID's is Fourier transformed followed by a second Fourier transform with respect to the evolution time.  Typical data collection and processing are illustrated here. The introduction of pulsed field gradients for coherence selection has reduced the time required to collect 2D spectra by reducing or  eliminating the need for phase cycling however, one still has to collect many FID's as a function of evolution time.  Even when multiple scans are not required for sensitivity, data collection can take minutes to hours.

Ultra-fast 2D measurements, employing an entirely different method of data collection, were introduced in 2002 and subsequently improved.  In this method, z-field gradients combined with linearly swept chirp pulses are used to phase encode spins linearly along the z axis of the sample according to specific evolution times.  The dephasing depends on both the position along the z axis of the sample and the resonance frequency of each spin.  After this encoding scheme is applied, each slice element of the sample has experienced a different evolution time as a function of its position in the sample.  After a conventional mixing period dictated by the type of 2D measurement, the site specific, spatially phase encoded spins must be read.  This is accomplished by applying a series of bipolar gradient pulse pairs while the receiver is collecting data.  During each gradient pulse (lasting typically 250 μsec) echos are collected.  The position of each echo during a single gradient pulse is related in a one-to-one fashion to the frequency of each of the spins in the sample thus mimicking a mini NMR spectrum whose frequency axis is replaced by a linearly related time axis.  The "spectra" collected during the negative gradient pulses are mirror images to those collected during the positive gradient pulses and must be reversed during data processing.  A series of typically 128 bipolar gradients are applied with the receiver open thus all of the data are acquired in a single scan.  Each "spectrum" collected is a function of the z slice position in the sample, which in turn is linearly related to the evolution time. The collection of "spectra" represent the ultra-fast domain and is Fourier transformed point by point as a function of evolution time (or z position).  The entire data collection sequence takes approximately 100 msec.

The left panel of the figure below shows a conventional 300 MHz gradient enhanced COSY-45 spectrum for a concentrated sample of menthol in CDCl₃ collected in 4.5 minutes.  The panel on the right shows a 300 MHz ultra-fast COSY spectrum of the same sample collected in only 100 msec - a time saving factor of 2700!  Both spectra were collected on a Bruker AVANCE II 300 NMR spectrometer equipped with a standard BBOF probe.  Both data sets were symmetrized.  Although the ultra-fast data set has noticeably lower resolution and sensitivity, one can see that it is very similar to the conventional COSY.

There are, of course, a number of drawbacks to the ultra-fast scheme including low sensitivity, limited resolution and limited accessible spectral widths.  Some of these drawbacks can be overcome  with the use of cryoprobes and strong pulsed field gradients. Molecular diffusion over the course of the measurement may also cause problems.  Despite the drawbacks however, the method is extremely well suited to time studies of chemical reactions where conventional 2D data collection would simply take too long.

The references below are a good place to start in order to find out more about this technique.  There is also a very well documented setup procedure available on the Bruker User Library, provided by Patrick Giraudeau, including pulse sequences and processing scripts.

Annual Rev. Anal. Chem. 7, 129-161 (2014).
Mag. Res. Chem. 53, 986-994 (2015).
J. Am. Chem. Soc. 125, 9204–17 (2003).
J. Am. Chem. Soc. 125, 12345–50 (2003).