Use our NMR service that provides ¹⁴N, ¹⁵N-NMR and many other NMR techniques. We recommend ¹H-¹⁵N heteronuclear correlation as a more sensitive alternative to ¹⁵N-NMR in most cases.

Nitrogen has two NMR active nuclei (fig. 1). ¹⁵N yields sharp lines but is very insensitive. ¹⁴N is a medium sensitivity nucleus but its signals are usually significantly broadened by quadrupolar interactions sometimes to the extent that they are unobservable on a high-presolution NMR spectrometer.

Fig. 1. Comparison of ¹⁴N and ¹⁵N spectra of urea. The ¹⁴N signal is extremely broad because it is quadrupolar.

A typical analysis of an N NMR spectrum consists of matching expected chemical shifts (fig. 2) to the expected moieties. Our NMR service provides ¹⁴N and ¹⁵N NMR along with many other NMR techniques. However, we recommend ¹H-¹⁵N heteronuclear correlation as a more sensitive alternative to ¹⁵N-NMR in most cases. Each type of signal has a characteristic chemical shift range that can be used for assignment. The chemical shift ranges are the same for both nitrogen isotopes. IUPAC recommends CH₃NO₂ (90% in CDCl₃) as the chemical shift standard for both nucleides (¹⁴N and ¹⁵N). However, most spectroscopists reference nitrogen spectra to liquid NH₃ and IUPAC recommends this as an alternative for ¹⁵N. Here, the chemical shifts are referenced to liquid NH₃ (under pressure) at 25°C even though this is against IUPAC recommendations for ¹⁴N. To convert ¹⁵N chemical shifts to the IUPAC CH₃NO₂ standard, subtract 380.5 ppm and for ¹⁴N, subtract 381.6 ppm.

Fig. 2. Chemical shift ranges of nitrogens according to their chemical environment

Choose the structure that most closely represents the nitrogen in question.

¹⁴N NMR

The 1D ¹⁴Nitrogen NMR experiment is much less sensitive than Proton (¹H) but has a much larger chemical shift range. Its signals are broadened by quadrupolar interactions. The larger the molecule and the more asymmetric the nitrogen's environment, the broader the signal. Hence, the small and highly symmetric aqueous ammonium ion gives a very sharp line (fig. 3), less than one Hertz wide. Liquid ammonia, being less symmetric, is 16 Hz wide (on a 400 MHz spectrometer at room temperature). Urea is larger and asymmetric so the line-width is approximately 1 KHz. Molecules that are significantly larger than urea yield signals too broad to be observed with a high-resolution NMR spectrometer. However, the nitrogen chemical shift range is wide and so may be readily used for distinguishing nitrogen species for very small molecules.

Fig. 3. Comparison of line-widths of ¹⁴N signals. The larger and less symmetric the molecule, the wider the signal.

Heteronuclear coupling is rarely observed with ¹⁴N because of its quadrupolar broadening. It can be observed with protons of the ammonium ion (figs. 4, 5) at room temperature and liquid ammonia at low temperatures. Carbon signals of nitriles and NH protons are often broadened by residual coupling to ¹⁴N.

Fig. 4. ¹H spectrum of natural abundance NH₄Cl (1.5 M) in 1M HCl/H₂O showing coupling to ¹⁴N as a (spin 1) triplet and coupling to ¹⁵N as a weak doublet. Note that the ¹⁴N coupling constant is smaller than that of ¹⁵N because of ¹⁴N's lower resonant frequency.

Fig. 5. ¹⁴N spectrum of NH₄Cl (1.5 M) in 1M HCl/H₂O showing coupling to ¹H as a quintet due to the presence of four protons in an AX₄ coupling pattern.

Properties of ¹⁴N

(Click here for explanation)

Property	Value
Spin	1
Natural abundance	99.63%
Chemical shift range	900 ppm, from 0 to 900
Frequency ratio (Ξ)	7.226317% (NH3 7.223561%)
Reference compound	90% CH3NO2 in CDCl3
Linewidth of reference	19 Hz
T1 of reference	19 ms
Alternative reference (not IUPAC)	NH3 liquid, Ξ = 7.223561
Linewidth of NH3	16 Hz
T1 of NH3	0.2 s
Receptivity rel. to 1H at natural abundance	1.01×10-3
Receptivity rel. to 1H when enriched	1.01×10-3
Receptivity rel. to 13C at natural abundance	5.74
Receptivity rel. to 13C when enriched	5.74
Linewidth parameter	21 fm4

¹⁵N NMR

The 1D ¹⁵Nitrogen NMR experiment is much less sensitive than Proton (¹H) and ¹³Carbon. Consequently, the ¹⁵Nitrogen is rarely sensitive enough and it is necessary to isotopically enrich (fig. 6) or to use the projection of a heteronuclear correlation (short range and long range) to improve sensitivity (Fig. 7). In order to obtain a short-range heteronuclear correlation, the NH protons must be in slow exchange and not deuterated. This precludes D₂O and deuterated alcohol solutions. A common practice in biochemistry is either to use a non-exchanging solvent such as DMSO-d₆ or 1:9 D₂O/H₂O combined with water suppression techniques. Long range heteronuclear correlation requires very sharp proton signals that undergo two or three bond coupling with a practical line-width limit of approximately 3 Hz but has the advantage of not requiring the presence of sharp NH signals.

Fig. 6. ¹⁵N NMR spectrum of ¹⁵N enriched Cyclosporin A (25 mM) in CDCl₃. This spectrum was acquired in one hour. To obtain similar sensitivity without enriched would take about 10 years.

Fig. 7. ¹⁵N NMR spectrum of Cyclosporin A (25 mM) in DMSO-d₆ obtained from the projection of a heteronuclear correlation. The upper red spectrum is of nitrogens directly attached to hydrogen. The lower blue spectrum includes weak signals from long-range correlations and suppresses nitrogens without long-range correlations.

The 1D ¹⁵Nitrogen NMR experiment yields narrow lines and has a large chemical shift range. Its low natural abundance (0.37%) makes its low sensitivity even worse so it is often enriched for NMR.

A typical analysis of a ¹⁵N NMR spectrum consists of matching expected chemical shifts to the expected moieties. A simple example is that of sodium azide (fig. 8) where the center nitrogen has a different chemical shift from the terminal nitrogens.

Fig. 8. ¹⁵N NMR spectrum of sodium azide (1M) in D₂O

An alternative to using high concentrations and a necessity for large molecules is to enrich with ¹⁵N. However, this is very expensive and the cost of producing the example in fig. 9 is several thousand dollars.

Fig. 9. ¹⁵N NMR spectrum of enriched HIV-1 protease

Decoupling leads to negative NOE, reducing or negating the signal (fig. 10). In order to prevent this, A DEPT experiment or very long relaxation delays of minutes or more are required.

Fig. 10. Decoupled ¹⁵N NMR spectrum of enriched HIV-1 protease showing signal inversion due to negative NOE. The peak on the right is not attached to protons so does not invert.

Heteronuclear coupling is observed with protons. One-bond couplings are of the order of 90 Hz and smaller 2, 3 and 4-bond coupling are observed. In some cases, very weak satellite signals can be observed in the ¹H spectrum of NH signals (fig. 11) and can be used to confirm the assignment of NH signals.

Fig. 11. ¹H spectrum of natural abundance urea (5 mM) in DMSO-d₆ showing signal broadening arising from residual coupling to ¹⁴N and coupled ¹⁵N satellites. The satellites combined with main-signal broadening are a good indicator of NH signals in the ¹H spectrum.

¹⁵N DEPT

Sensitivity of ¹⁵N can be greatly improved using the DEPT experiment (fig. 12), however, unprotonated nitrogens are suppressed.

Fig. 12. ¹⁵N DEPT spectrum of enriched HIV-1 protease. This experiment is much more sensitive and can be acquired faster but only protonated nitrogens appear.

Properties of ¹⁵N

(Click here for explanation)

Property	Value
Spin	½
Natural abundance	0.37%
Chemical shift range	900 ppm, from 0 to 900
Frequency ratio (Ξ)	10.136767% or 10.1329111%
Reference compound	90% CH3NO2 in CDCl3 or NH3 liquid
Linewidth of CH3NO2	0.14 Hz
T1 of CH3NO2	43 s
Linewidth of NH3	0.2 Hz
T1 of NH3	5 s
Receptivity rel. to 1H at natural abundance	3.85×10-6
Receptivity rel. to 1H when enriched	1.04×10-3
Receptivity rel. to 13C at natural abundance	0.0219
Receptivity rel. to 13C when enriched	5.91

Safety note:

Safety note: Some of the materials mentioned here are very dangerous. Ask a qualified chemist for advice before handling them. Qualified chemists should check the relevant safety literature before handling or giving advice about unfamiliar substances. NMR solvents are toxic and most are flammable. Specifically, ammonia is toxic and the liquid is pressurized at room temperature: work in a hood. Ammonium chloride may be toxic: use protective gloves.