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Hyperpolarization

Nuclear spin energy levels Hyperpolarization of nuclear spins is the most powerful method of signal enhancement in NMR, in those cases where we can find ways to apply it. In hyperpolarization, a physical means is used to repopulate the Zeeman energy levels of the nuclear spin system before an NMR experiment. Because the NMR sensitivity is proportional to the population, this enhances the detectable signal by orders of magnitude.

Dissolution Dynamic Nuclear Polarization
picture 2Dissolution dynamic nuclear polarization (DNP) is a technology (J.H. Ardenkjaer-Larsen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 10158-10163) for providing hyperpolarization of various classes of molecules. It makes use of the high polarizability of electron spins. Even at a moderate magnetic field of several Tesla, and at a temperature of ~1 K, electron spins are almost completely polarized. At the same temperature, the polarization of nuclear spins is still small even at the highest magnetic fields available. In DNP, polarization is transferred from the electron spins of free radicals to nuclear spins by means of microwave irradiation.
To be able to utilize hyperpolarized molecules before their non-equilibrium spin states decay, we are developing rapid injection techniques. These techniques transfer a hyperpolarized sample into an NMR spectrometer for data acquisition. At the same time, they allow admixing of a second reactant in a stopped-flow type experiment. The injection and mixing process is achieved by using high-pressure gas or liquid for driving both sample components.

Real-time NMR
picture 2Using hyperpolarization, in a single scan we routinely achieve a signal gain of three to four orders of magnitude, when compared to conventional NMR. The availability of such a large signal in one instant permits the observation of rapid, transient processes in real-time, that would otherwise be too fast for NMR spectroscopy. We are using this capability of hyperpolarization enhanced NMR for the study of the mechanisms and kinetics of chemical and biochemical reactions, such as those catalyzed by enzymes. The figure demonstrates the time-resolved observation of a reaction on the example of trypsin catalyzed hydrolysis of benzoyl arginine ethyl ester. After rapid injection of the hyperpolarized substrate, the reaction was followed by 13C NMR in time intervals of 0.5 seconds. From the measured data points, the reaction rate can readily be determined. In addition to the measurement of enzyme kinetics of non-chromogenic substrates, or of multi-step reactions, this technique holds particular promise for the characterization of transient reaction intermediates. Identification of intermediates can readily be achieved through observation of chemical shift on the sub-second time scale, and has the potential of leading to a detailed understanding of the reaction mechanism.