Elsevier

Journal of Magnetic Resonance

Volume 258, September 2015, Pages 86-95
Journal of Magnetic Resonance

Comparison of various NMR methods for the indirect detection of nitrogen-14 nuclei via protons in solids

https://doi.org/10.1016/j.jmr.2015.06.008Get rights and content

Highlights

  • 1H–{14N} D-HMQC HETCOR.

  • Frequency selective single-quantum excitation.

  • HMQC detection of 14N overtone.

  • HMQC detection of 14N single- and double-quantum.

  • Hard-pulse and WURST overtone excitation.

Abstract

We present an experimental comparison of several through-space Hetero-nuclear Multiple-Quantum Correlation experiments, which allow the indirect observation of homo-nuclear single- (SQ) or double-quantum (DQ) 14N coherences via spy 1H nuclei. These 1H–{14N} D-HMQC sequences differ not only by the order of 14N coherences evolving during the indirect evolution, t1, but also by the radio-frequency (rf) scheme used to excite and reconvert these coherences under Magic-Angle Spinning (MAS). Here, the SQ coherences are created by the application of center-band frequency-selective pulses, i.e. long and low-power rectangular pulses at the 14N Larmor frequency, ν0(14N), whereas the DQ coherences are excited and reconverted using rf irradiation either at ν0(14N) or at the 14N overtone frequency, 2ν0(14N). The overtone excitation is achieved either by constant frequency rectangular pulses or by frequency-swept pulses, specifically Wide-band, Uniform-Rate, and Smooth-Truncation (WURST) pulse shapes. The present article compares the performances of four different 1H–{14N} D-HMQC sequences, including those with 14N rectangular pulses at ν0(14N) for the indirect detection of homo-nuclear (i) 14N SQ or (ii) DQ coherences, as well as their overtone variants using (iii) rectangular or (iv) WURST pulses. The compared properties include: (i) the sensitivity, (ii) the spectral resolution in the 14N dimension, (iii) the rf requirements (power and pulse length), as well as the robustness to (iv) rf offset and (v) MAS frequency instabilities. Such experimental comparisons are carried out for γ-glycine and l-histidine.HCl monohydrate, which contain 14N sites subject to moderate quadrupole interactions. We demonstrate that the optimum choice of the 1H–{14N} D-HMQC method depends on the experimental goal. When the sensitivity and/or the robustness to offset are the major concerns, the D-HMQC sequence allowing the indirect detection of 14N SQ coherences should be employed. Conversely, when the highest resolution and/or adjusted indirect spectral width are needed, overtone experiments are the method of choice. The overtone scheme using WURST pulses results in broader excitation bandwidths than that using rectangular pulses, at the expense of reduced sensitivity. Numerically exact simulations also show that the sensitivity of the overtone 1H–{14N} D-HMQC experiment increases for larger quadrupole interactions.

Introduction

Nitrogen atoms are ubiquitous in nature and are present in organic molecules, such as pharmaceuticals or biological molecules, e.g. proteins or DNA, as well as organic, hybrid and inorganic materials, such as polyamides, functionalized silica, metal–organic frameworks or nitrides. Nevertheless, nuclear magnetic resonance (NMR) observation of nitrogen nuclei still remains a difficult task owing to the unfavorable properties of the two magnetic isotopes, 14N and 15N. The latter is a spin-1/2 nucleus, and hence it can easily lead to high resolution NMR spectra, even for solids. However, its low gyromagnetic ratio, γ(15N)/γ(1H) = 0.1, and its very low natural abundance of 0.35% often prevent its NMR detection in isotopically unmodified samples. Furthermore, 15N enrichment can be expensive and difficult.

Conversely, the 14N isotope has a very high natural abundance of 99.65%. However, and as explained in recent review articles [1], [2], this nucleus presents three main difficulties from the NMR point of view. First, it has a low gyromagnetic ratio, γ(14N)/γ(1H) = 0.072, that leads to a low Larmor frequency, even at high magnetic fields: e.g. ν0(14N) = 57.8 MHz at B0 = 18.8 T. This low gyromagnetic ratio decreases the sensitivity, which is proportional to γ3/2 in the case of 14N detection. It also results in additional experimental difficulties, including weak maximum radio-frequency (rf) fields and long electronic dead-times, since the rf field and dead-time are roughly proportional to γ and 1/γ, respectively. These two limitations often further decrease the sensitivity. Second, 14N has a relatively large electric quadrupole moment of Q = 2.04 fm2 and thus it is subject to large quadrupole interactions with CQ = e2qQ/h of up to 6 MHz [3]. Third, it is a spin-1 nucleus, hence has no central transition, and the signal from the two single quantum transitions, 14NSQ: ΔmN = ±1, for a powder sample is broadened by the very large first-order quadrupole interaction (HQ1) over a width of 3CQ/2, which reaches 9 MHz for CQ = 6 MHz. The combination of these three unfavorable properties leads to a very weak and broad 14NSQ NMR signal.

Generally, the direct detection of one-dimensional (1D) 14NSQ signal of solids is a challenging task, which has notably been achieved in static samples using piecewise acquisition [4], or frequency-swept excitation [5] It must be mentioned that 14N nuclei have also been directly detected under MAS, which improves the resolution [6]. However, this type of experiment is quite experimentally demanding and requires optimization of the spectrometer bandwidth and impedance as well as a highly stable and accurate MAS angle and frequency. Furthermore, given the low rf field delivered at ν0(14N) by standard solid-state NMR probes, small flip-angles must then be used to broaden the excitation bandwidth, bringing a corresponding reduction in sensitivity. Trains of rotor-synchronized short rectangular pulses in the manner of Delays Alternating with Nutation for Tailored Excitation (DANTE) can achieve efficient broadband excitation at lower rf fields than short pulses by matching the rf excitation profile to the spinning sidebands [7](a), [7](b). However, globally these 1D methods are only applicable to samples containing a small number of different nitrogen species.

Recently, in order to increase the sensitivity and resolution, two-dimensional (2D) methods have been proposed, in which the 14NSQ signal is observed under MAS indirectly via a more sensitive ‘spy’ nucleus, such as 1H [8]. These hetero-nuclear correlations are denoted 1H–{14NSQ} in the following. For solid samples, most of these indirect detection methods are based on the hetero-nuclear multiple-quantum coherence (HMQC) scheme [8]. It should be mentioned that the hetero-nuclear single-quantum coherence (HSQC) scheme, has also been used, but this sequence is less sensitive than HMQC [9]. For these HMQC experiments, the coherence transfer has been achieved (i) through a combination of J-couplings and second-order quadrupolar–dipolar cross-terms, also known as residual dipolar splittings (RDS), using the (J + RDS)-HMQC sequence [8], and (ii) more recently, through dipolar couplings, using the D-HMQC one [10]. In the 1H–{14NSQ} D-HMQC sequence, the 1H–14N dipolar couplings are reintroduced during the defocusing and refocusing delays by applying hetero-nuclear dipolar recoupling sequences, such as SR412, on the 1H channel [10]. As for the 1D NMR observation of 14NSQ signals, the challenge for all 1H–{14N} HMQC experiments is the excitation of the very wide 14NSQ spectra. A first approach consists of the broadband excitation of 14NSQ. Optimal broadband excitation using single rectangular pulses is often not compatible with the rf specifications of the common probes. Conversely, DANTE schemes made of short pulses can achieve efficient broadband excitation using a low rf field [7](a), [7](b). A second approach relies on sideband selective excitation of the 14NSQ signal using a single long and low-power rectangular pulse or a train of those pulses in the manner of DANTE [11]. In HMQC experiments used for the indirect detection of 14NSQ, the indirect evolution period, t1, defined as the interval between the centers of the two 14N pulses or DANTE schemes, must be rotor-synchronized, i.e. equal to an integer number of rotor periods, and the rotor axis must be precisely set to the magic-angle in order to refocus the first-order quadrupole interaction.

An alternative approach for the acquisition of high-resolution 14N spectra from a powder sample is to observe homo-nuclear double-quantum (14NDQ) transitions, ΔmN = ±2, since these are not broadened by the first-order (HQ1) but only by the second-order (HQ2) quadrupole interaction.

The 14NDQ coherences can be excited from quadrupolar order, Qz, i.e. the population distribution determined by the quadrupole interaction, by applying a selective [11] or broadband excitation pulse [12]. Such coherences can also be excited from the z-magnetization by applying: either a ‘classical’ fundamental DQ excitation, 14NFDQ [13] or a ‘forbidden’ overtone DQ excitation, 14NOTDQ. [14], [15], [16] The fundamental excitation employs rf irradiation at ν0(14N) and relies on a second-order perturbation of HQ1 by the rf field. This was first demonstrated for static single-crystals and powders [13], and more recently for powders under MAS conditions [11]. The overtone excitation is performed at 2ν0(14N) and is based on the admixture of the Zeeman states, e.g. some mN = 0 character in the mN = ±1 states when the magnitudes of Zeeman and quadrupole interactions are comparable. The admixture of Zeeman states also allows the direct observation of the overtone signal, i.e. the 14NOTDQ transition, at 2ν0(14N) in 1D experiment.

Overtone excitation and observation was first reported for single-crystals and powders under static conditions [14], [15]. Direct observation of overtone signals under MAS remained elusive for a long time. Nevertheless, the first 1D 14NOTDQ powder patterns were recently reported under MAS [16a], and their widths are significantly narrowed owing to the elimination of CSA and the partial averaging of HQ2 terms. MAS causes the preferential selection of the 2nd spinning sideband which exhibits a frequency shift of twice the spinning speed, 2νR, with respect to the center-band. Hence, and contrary to the ‘classical’ 14NSQ and 14NFDQ signals, the apparent position of the overtone signal depends on the spinning speed value and also on the sense of rotation [16c]. Drawbacks of overtone NMR include (i) bandwidth limitations owing to the long length of the overtone pulse and (ii) its low sensitivity. The excitation bandwidth of overtone pulses can be increased by using amplitude and frequency modulated Wide-band, Uniform-Rate, and Smooth-Truncation (WURST) pulses, at the expense of the sensitivity [16a]. Therefore, polarization transfers, including 1H  14N cross-polarization and Dynamic Nuclear Polarization, have been employed to enhance the sensitivity of this technique under static [15] and MAS conditions [17], [18]. We have chosen to use WURST pulses herein because they achieve more uniform broadband excitation than other frequency-swept pulse shapes, such as hyperbolic secant.

The 14NDQ transition can also be observed indirectly using 2D hetero-nuclear correlation experiments, such as HMQC and HSQC [8], [9]. The indirect detection via protons improves sensitivity and HMQC is more sensitive than HSQC [9c]. In these experiments, the 14NDQ coherences evolve during the indirect evolution period, t1. At the end of the defocusing and refocusing periods, the 14N population distribution for the terms contributing to the detected HMQC signals are a combination of Qz and Iz terms. Therefore, the 14NDQ coherences can be excited and reconverted by either broadband, or selective irradiation. The indirect detection of 14NDQ coherences via protons using fundamental irradiation at ν0(14N) is denoted 1H–{14NFDQ} hereafter. It has been shown that its sensitivity is lower than that of 1H–{14NSQ} [8], [11]. Overtone irradiation can also be used for the excitation and the reconversion of 14NDQ coherences in HMQC experiments [16](c), [19]. Such experiments with 1H detection are denoted 1H–{14NOTDQ} in the following. The sensitivity of 1H–{14NOTDQ} HMQC is comparable to that of 1H–{14NSQ} [16](c), [19], and its 14N excitation bandwidth can be increased with WURST pulses, again at the expense of the sensitivity [16c].

The present article presents a comprehensive comparison of 1H–{14NSQ}, 1H–{14NFDQ} and 1H–{14NOTDQ} D-HMQC experiments. Using a sample of γ-glycine, we compare their sensitivity, their resolution along the 14N dimension, and their rf field requirements as well as their robustness to rf offset and MAS instabilities. The performances of these methods are also assessed by numerical simulations of the spin dynamics as well as D-HMQC experiments on l-histidine.HCl monohydrate.

Section snippets

Pulse sequences

The 1H–{14N} D-HMQC sequence is a variant of the (J + RDS)-HMQC experiment, but hetero-nuclear dipolar recoupling schemes are applied on 1H channel during the defocusing and refocusing periods to reintroduce the 1H–14N dipolar couplings. These interactions are usually larger than the sum of J-couplings and residual dipolar splittings and hence shorter recoupling delays are employed in the D-HMQC experiment compared to the (J + RDS)-HMQC one, which minimizes signal loss. Consequently, D-HMQC

Experiments

The 1H–{14N} D-HMQC spectra were recorded at 18.8 T on an Avance-III Bruker spectrometer. Experiments were carried out at a high magnetic field because it decreases the second-order quadrupolar broadening and increases the chemical shift range, thus enhancing the resolution along the F1 (14N) and F2 (1H) dimensions. It also increases the Larmor frequency and hence contributes to an improved sensitivity by increasing the Boltzmann factor and by decreasing the second-order quadrupolar broadening

Optimum ‘on-resonance’ rf parameters

We first optimized the amplitude and the length of the 14N rf pulses in order to maximize the signal in the case of ‘on-resonance’ excitation. Fig. 1 shows the 1H signal intensity observed in a 1D 1H–{X} D-HMQC spectrum of γ-glycine, with X = 14NSQ (a), 14NFDQ (b), and 14NOTDQ (c, d) versus the rf field amplitude, ν1N (or ν1Nmax), and the pulse length, τp. In the case of Fig. 1d, the 14NOT,WTDQ excitation was performed using two WURST80 pulses with same direction for the frequency modulation and

Simulations of 14N overtone experiments under MAS

For a better understanding of the 1H–{14NOT,SPDQ} D-HMQC experiments, exact numerical simulations of a single pulse experiment with overtone excitation were performed at B0 = 18.8 T and νR = 62.5 kHz, using methods previously described [16b]. Fig. 5a maps the intensity of +2νR overtone sideband of the nitrogen site in γ-glycine (CQ = 1.18 MHz and ηQ = 0.53) excited by an ‘on-resonance’ single rectangular overtone pulse as a function of rf field strength and pulse length. The simulations of Fig. 5a are in

Conclusion

We have compared three different 1H–{X} D-HMQC experiments with X = 14NSQ, 14NFDQ and 14NOTDQ for the indirect detection of 14N isotopes in solids. The 1H–{14NSQ} single-quantum D-HMQC experiment using center-band selective excitation is very efficient and easy to optimize. Its pulses on the 14N channel require a moderate rf field amplitude of ca. 2νR/3 with durations of ca. one to two rotor periods. Its broadband version with single 14N rectangular pulses is typically difficult to use

Acknowledgments

This work was supported by: French contract ANR-2010-jcjc-0811-01. FP, JT, OL, and JPA are grateful for funding provided by Région Nord/Pas de Calais, European Union (FEDER), CNRS, French Ministry of Scientific Research, Université de Lille, ENSCL, and CortecNet. MS is grateful for the financial support from China Scholarship Council. Financial support from the TGIR-RMN-THC Fr3050 CNRS for conducting the research is gratefully acknowledged. Authors would like to thank Z. Gan for fruitful

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