University of Ottawa NMR Facility Web Site

Please feel free to make suggestions for future posts by emailing Glenn Facey.

Friday, April 22, 2016

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. 

Monday, April 11, 2016


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. γHC) 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.