Nitrogen-15 nuclear magnetic resonance spectroscopy

Nitrogen-15 nuclear magnetic resonance spectroscopy is a version of nuclear magnetic resonance spectroscopy that examines samples containing the ¹⁵N nucleus. ¹⁵N NMR differs in several ways from the more common ¹³C and ¹H NMR. To circumvent the difficulties associated with measurement of the quadrupolar, spin-1 ¹⁴N nuclide, ¹⁵N NMR is employed in samples for detection since it has a ground-state spin of ½. Since¹⁴N is 99.64% abundant, incorporation of ¹⁵N into samples often requires novel synthetic techniques.
Nitrogen-15 is frequently used in nuclear magnetic resonance spectroscopy, because unlike the more abundant nitrogen-14, that has an integer nuclear spin and thus a quadrupole moment, ¹⁵N has a fractional nuclear spin of one-half, which offers advantages for NMR like narrower line width. Proteins can be isotopically labeled by cultivating them in a medium containing nitrogen-15 as the only source of nitrogen. In addition, nitrogen-15 is used to label proteins in quantitative proteomics.

Implementation

¹⁵N NMR has complications not encountered in ¹H and ¹³C NMR spectroscopy. The 0.36% natural abundance of ¹⁵N results in a major sensitivity penalty. Sensitivity is made worse by its low gyromagnetic ratio, which is 10.14% that of ¹H. The signal-to-noise ratio for ¹H is about 300-fold greater than ¹⁵N at the same magnetic field strength.

Physical properties

The physical properties of ¹⁵N are quite different from other nuclei. Its properties along with several common nuclei are summarized in the below table.

Isotope	Magnetic dipole moment	Nuclear spin number	Natural abundance	Gyromagnetic ratio	NMR frequency at 11.7T
¹H	2.79284734	1/2	~100	267.522	-500
²H	0.857438228	1	0.015	41.066	-76.753
³H	2.97896244	1/2	0	285.349	-533.32
¹⁰B	1.80064478	3	19.9	28.747	-53.718
¹¹B	2.6886489	3/2	80.1	85.847	-160.42
¹³C	0.7024118	1/2	1.1	67.238	-125.725
¹⁴N	0.40376100	1	99.6	19.338	-36.132
¹⁵N	-0.28318884	1/2	0.37	-27.126	50.782
¹⁷O	-1.89379	5/2	0.04	-36.281	67.782
¹⁹F	2.628868	1/2	~100	251.815	-470.47
³¹P	1.13160	1/2	~100	108.394	-202.606

From these data, one can see that at full enrichment, ¹⁵N is about one tenth as sensitive as ¹H.

Chemical shift trends

The International Union of Pure and Applied Chemistry recommends using CH₃NO₂ as the experimental standard; however in practice many spectroscopists utilize pressurized NH₃ instead. For ¹⁵N, chemical shifts referenced with NH₃ are 380.5 ppm upfield from CH₃NO₂. Chemical shifts for ¹⁵N are somewhat erratic but typically they span a range of -400 ppm to 1100 ppm with respect to CH₃NO₂. Below is a summary of ¹⁵N chemical shifts for common organic groups referenced with respect to NH₃, whose chemical shift is assigned 0 ppm.

Gyromagnetic ratio

Unlike most nuclei, the gyromagnetic ratio for ¹⁵N is negative. With the spin precession phenomenon, the sign of γ determines the sense of precession. Most common nuclei have positive gyromagnetic ratios such as ¹H and ¹³C.

Applications

Tautomerization

¹⁵N NMR is used in a wide array of areas from biological to inorganic techniques. A famous application in organic synthesis is to utilize ¹⁵N to monitor tautomerization equilibria in heteroaromatics because of the dramatic change in ¹⁵N shifts between tautomers.

Protein NMR

¹⁵N NMR is also extremely valuable in protein NMR investigations. Most notably, the introduction of three-dimensional experiments with ¹⁵N lifts the ambiguity in ¹³C–¹³C two-dimensional experiments. In solid-state nuclear magnetic resonance, for example, ¹⁵N is most commonly utilized in NCACX, NCOCX, and CANcoCX pulse sequences.

Investigation of nitrogen-containing heterocycles

¹⁵N NMR is the most effective method for investigation of structure of heterocycles with a high content of nitrogen atoms. ¹⁵N labeling followed by analysis of ¹³C–¹⁵N and ¹H–¹⁵N couplings may be used for establishing structures and chemical transformations of nitrogen heterocycles.

INEPT

Insensitive nuclei enhanced by polarization transfer is a signal resolution enhancement method. Because ¹⁵N has a gyromagnetic ratio that is small in magnitude, the resolution is quite poor. A common pulse sequence which dramatically improves the resolution for ¹⁵N is INEPT. The INEPT is an elegant solution in most cases because it increases the Boltzmann polarization and lowers T₁ values. Additionally, INEPT can accommodate negative gyromagnetic ratios, whereas the common nuclear Overhauser effect cannot.