Conventional Cross-polarization Research Articles

Quantifying Micromolar Crystallinity in Pharmaceutical Materials Utilizing 19F Solid-State NMR.

Quantifying low-level components in solid-state analysis presents a significant challenge for most thermal, diffractometric, vibrational, and spectroscopic techniques. In pharmaceutical analysis, identifying and quantifying the physical form of the drug substance in solid dosages is a critical task to ensure the quality of drug products. For example, recrystallization of active pharmaceutical ingredients in amorphous solid dispersions can compromise the stability and bioavailability of drug products. Herein, we have developed and demonstrated fluorine-19 solid-state nuclear magnetic resonance (19F ssNMR) methods and pushed the boundary to quantify minor crystalline contents in amorphous pharmaceuticals. Calibration curves suggest that 19F direct polarization and 1H-19F cross-polarization ssNMR can readily quantify 0.1% w/w crystalline compound I, a commercial fluorinated drug molecule developed by Merck & Co., Inc., Rahway, NJ, U.S.A., in its amorphous formulation. 1H-19F multiple cross-polarization (MultiCP) has been implemented, for the first time, and compared with conventional cross-polarization methods. Most importantly, a relaxation-filtered 19F ssNMR method was utilized to unambiguously identify and quantify as low as 0.04% w/w crystalline components, that is, 6 μmol in a 100 mg tablet at 25% drug loading, by suppressing the signal from the amorphous counterpart. Such a low level of detection offers high confidence and sensitivity to quantify trace amounts of phase change in pharmaceutical amorphous materials in the solid state, which can facilitate formulation development as well as quality control.

Maximizing efficiency of dipolar recoupling in solid-state NMR using optimal control sequences.

Dipolar recoupling is a central concept in the nuclear magnetic resonance spectroscopy of powdered solids and is used to establish correlations between different nuclei by magnetization transfer. The efficiency of conventional cross-polarization methods is low because of the inherent radio frequency (rf) field inhomogeneity present in the magic angle spinning (MAS) experiments and the large chemical shift anisotropies at high magnetic fields. Very high transfer efficiencies can be obtained using optimal control–derived experiments. These sequences had to be optimized individually for a particular MAS frequency. We show that by adjusting the length and the rf field amplitude of the shaped pulse synchronously with sample rotation, optimal control sequences can be successfully applied over a range of MAS frequencies without the need of reoptimization. This feature greatly enhances their applicability on spectrometers operating at differing external fields where the MAS frequency needs to be adjusted to avoid detrimental resonance effects.

Quantification of magic angle spinning dynamic nuclear polarization NMR spectra

Dynamic nuclear polarization (DNP) allows to dramatically enhance the sensitivity of magic angle spinning nuclear magnetic resonance (MAS NMR). DNP experiments usually rely on the detection of low-γ nuclei hyperpolarized from 1H with the use of cross polarization (CP), which assures more efficient signal enhancement. However, CP is usually not quantitative. Here we determine the quantification performance of three different approaches used in MAS NMR, (conventional CP, variable contact time CP, and multiple-contact CP) under DNP conditions, and we show that absolute quantification in MAS DNP NMR is possible, with errors below 10%.

Probing membrane protein ground and conformationally excited states using dipolar- and J-coupling mediated MAS solid state NMR experiments.

The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1H or 13C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca2+-ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments.

Hybridizing cross-polarization with NOE or refocused-INEPT enhances the sensitivity of MAS NMR spectroscopy

Heteronuclear cross polarization (CP) has been commonly used to enhance the sensitivity of dilute low-γ nuclei in almost all solid-state NMR experiments. However, CP relies on heteronuclear dipolar couplings, and therefore the magnetization transfer efficiency becomes inefficient when the dipolar couplings are weak, as is often the case for mobile components in solids. Here, we demonstrate methods that combine CP with heteronuclear Overhauser effect (referred to as CP-NOE) or with refocused-INEPT (referred to as CP-RINEPT) to overcome the efficiency limitation of CP and enhance the signal-to-noise ratio (S/N) for mobile components. Our experimental results reveal that, compared to the conventional CP, significant S/N ratio enhancement can be achieved for resonances originating from mobile components, whereas the resonance signals associated with rigid groups are not significantly affected due to their long spin–lattice relaxation times. In fact, the S/N enhancement factor is also dependent on the temperature, CP contact time as well as on the system under investigation. Furthermore, we also demonstrate that CP-RINEPT experiment can be successfully employed to independently detect mobile and rigid signals in a single experiment without affecting the data collection time. However, the resolution of CP spectrum obtained from the CP-RINEPT experiment could be slightly compromised by the mandatory use of continuous wave (CW) decoupling during the acquisition of signals from rigid components. In addition, CP-RINEPT experiment can be used for spectral editing utilizing the difference in dynamics of different regions of a molecule and/or different components present in the sample, and could also be useful for the assignment of resonances from mobile components in poorly resolved spectra. Therefore, we believe that the proposed approaches are beneficial for the structural characterization of multiphase and heterogeneous systems, and could be used as a building block in multidimensional solid-state NMR experiments.

Optimization of cross-polarization at low radiofrequency fields for sensitivity enhancement in solid-state NMR of membrane proteins reconstituted in magnetically aligned bicelles

Solid-state NMR (ssNMR) of oriented membrane proteins (MPs) is capable of providing structural and dynamic information at nearly physiological conditions. However, NMR experiments performed on oriented membrane proteins generally suffer from low sensitivity. Moreover, utilization of high-power radiofrequency (RF) irradiations for magnetization transfer may give rise to sample heating, thereby decreasing the efficiency of conventional cross-polarization schemes.Here we have optimized the recently developed repetitive cross-polarization (REP-CP) sequence (Tang et al., 2011) to further increase the magnetization transfer efficiency for membrane proteins reconstituted in magnetically aligned bicelles and compared its performance to single-contact Hartmann–Hahn cross-polarization (CP), CP-MOIST and the adiabatic transfer. It has been found that employing the REP-CP sequence at RF amplitudes of 19kHz instead of the commonly used higher RF fields (>45kHz) enhances the efficiency of REP-CP. An additional 30% signal can be obtained as compared to the previously published REP-CP, and 20% when compared to the re-optimized REP-CP at 50kHz RF fields. Moreover, the 15N signal gain of low-power REP-CP was found to be 40% over the adiabatic CP and up to 80% over CP-MOIST. Thus, the low-power REP-CP sequence surpasses all of the previous CP schemes in addition of having the tremendous advantage of reducing the RF powers by a factor of seven, thereby preserving the liquid-like bicelle sample. By contrast, in purely static (NAL crystal) and semi-rigid systems (Pf1 phage), the adiabatic CP was found to be more effective. Periodic oscillations of the intensity profile (distinct from the transient oscillations) as a function of the CP contact time and B1 RF field strengths were observed during the REP-CP optimization with the oscillations becoming more pronounced with lower RF fields. Many-spin simulations were performed to explain the oscillations and their periodicity.

Broadband adiabatic inversion pulses for cross polarization in wideline solid-state NMR spectroscopy

Efficient acquisition of ultra-wideline solid-state NMR powder patterns is a continuing challenge. In particular, when the breadth of the powder pattern is much larger than the cross-polarization (CP) excitation bandwidth, transfer efficiencies suffer and experimental times are greatly increased. Presented herein is a CP pulse sequence with an excitation bandwidth that is up to ten times greater than that available from a conventional spin-locked CP pulse sequence. The pulse sequence, broadband adiabatic inversion CP (BRAIN-CP), makes use of the broad, uniformly large frequency profiles of chirped inversion pulses, to provide these same characteristics to the polarization transfer process. A detailed theoretical analysis is given, providing insight into the polarization transfer process involved in BRAIN-CP. Experiments on spin-1/2 nuclei including 119Sn, 199Hg and 195Pt nuclei are presented, and the large bandwidth improvements possible with BRAIN-CP are demonstrated. Furthermore, it is shown that BRAIN-CP can be combined with broadband frequency-swept versions of the Carr–Purcell–Meiboom–Gill experiment (for instance with WURST–CPMG, or WCPMG for brevity); the combined BRAIN-CP/WCPMG experiment then provides multiplicative signal enhancements of both CP and multiple-echo acquisition over a broad frequency region.

Sensitizing solid state nuclear magnetic resonance of dilute nuclei by spin-diffusion assisted polarization transfers

Recent years have witnessed efforts geared at increasing the sensitivity of NMR experiments, by relying on the suitable tailoring and exploitation of relaxation phenomena. These efforts have included the use of paramagnetic agents, enhanced (1)H-(1)H incoherent and coherent transfers processes in 2D liquid state spectroscopy, and homonuclear (13)C-(13)C spin diffusion effects in labeled solids. The present study examines some of the opportunities that could open when exploiting spontaneous (1)H-(1)H spin-diffusion processes, to enhance relaxation and to improve the sensitivity of dilute nuclei in solid state NMR measurements. It is shown that polarization transfer experiments executed under sufficiently fast magic-angle-spinning conditions, enable a selective polarization of the dilute low-γ spins by their immediate neighboring protons. Repolarization of the latter can then occur during the time involved in monitoring the signal emitted by the low-γ nuclei. The basic features involved in the resulting approach, and its potential to improve the effective sensitivity of solid state NMR measurements on dilute nuclei, are analyzed. Experimental tests witness the advantages that could reside from utilizing this kind of approach over conventional cross-polarization processes. These measurements also highlight a number of limitations that will have to be overcome for transforming selective polarization transfers of this kind into analytical methods of choice.

A Modified XPC Characterization for Polarimetric Channels

Models describing the polarimetric response of a multipath channel are considered. Conventional cross-polarization coupling (XPC) models are found to provide an ambiguous polarization representation and are sensitive to the receive antenna polarization configuration. These limitations are contrasted with polarization mode dispersion (PMD) models, which provide unambiguous frequency-dependent polarization characterizations. The XPC model is augmented using received signal power ratios associated with three orthogonally polarized antenna pairs. The revised model is shown to provide higher fidelity polarization characterizations. A comparison of the polarization behavioral models is illustrated using measured data from channel sounding experiments conducted in the 2.4-GHz industrial, scientific, and medical band in an urban propagation environment.

Two-dimensional 43Ca– 1H correlation solid-state NMR spectroscopy

Calcium-43 (nuclear spin, S=7/2) is an NMR insensitive low- γ quadrupolar nucleus and up until recently only one-dimensional solid-state 43Ca NMR spectra have been reported. Through-space correlation experiments are challenging between spin- 1 2 and low- γ quadrupolar nuclei because of the intrinsically weak dipolar interaction and the often-low natural abundance of the quadrupolar nucleus. Rotary-resonance recoupling ( R 3) has recently been used to re-introduce hetero-nuclear dipolar interactions for sensitive high- γ quadrupolar nuclei, but has not yet been applied in the case of low- γ half-integer quadrupolar nuclei. Here an effective and robust 2D 1H- 43Ca NMR correlation experiment combining the R 3 dipole-recoupling scheme with 2D HMQC is presented. It is demonstrated that the weak 43Ca– 1H dipolar coupling in hydroxyapatite and oxy-hydroxyapatite can be readily re-introduced and that this recoupling scheme is more efficient than conventional cross-polarization transfer. Moreover, three 43Ca– 1H dipolar coupled calcium environments are clearly resolved in the structurally unknown oxy-hydroxyapatite. This local information is not readily available from other techniques such as powder XRD and high resolution electron microscopy. R 3-HMQC is also a desirable experiment because the set-up is simple and it can be applied using conventional multi-resonance probes.

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance 13C and 14N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of 13C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using 14N experiments on bicelles is also discussed.

A new technique for cross polarization in solid-state NMR compatible with high spinning frequencies and high magnetic fields

The idea of integrated cross polarization which has been utilized for electron-to-nucleus polarization transfer is applied to nucleus-to-nucleus polarization transfer. Instead of using a π 2 pulse followed by a π 2 phase-shifted locking pulse like in the conventional cross polarization, irradiation with a single phase is applied together with adiabatic frequency sweep from far off-resonance toward on-resonance. This is capable of locking individual spin packets even in the presence of considerable spectral distribution and/or line broadening. Thus, this technique can provide efficient polarization transfer for spin species having large chemical shifts and experiments in high static fields.

Adiabatic cross-polarization via intermediate dipolar-ordered state

It is experimentally demonstrated that an adiabatic demagnetization–remagnetization in the laboratory frame, where the Zeeman order of abundant nuclei is first adiabatically converted into the dipolar order, and then, into the Zeeman order of rare nuclei, can significantly increase polarization of rare nuclei compared to the conventional cross-polarization technique.

Enhancing the frequency response of cross-polarization wavelength conversion

This work presents a novel wavelength conversion scheme, differential cross-polarization modulation (DXPoM), using an extra birefringence delay to enhance the performance of the conventional cross-polarization modulation. Simulation and experimental results confirm that the predicted performance is enhanced. Using the proposed scheme improves rise time by over 300%, reduces timing jitter by 50%, and increases extinction ratio by 9%.

H 1 to H2 uniform cross-polarization nuclear magnetic resonance using H2 Lee–Goldburg irradiation in static powders

Application of conventional cross polarization (CP) to (2)H results in only a narrowband enhancement of the powder line shape due to the quadrupole interaction. We propose a CP scheme to uniformly enhance (2)H spectra in static powders. In this method, a Hartmann-Hahn matched (2)H rf field is applied on the Lee-Goldburg (LG) condition to remove the zeroth-order quadrupole interaction. In order to achieve a uniformly enhanced (2)H powder line shape with a limited (2)H rf intensity, the (1)H rf amplitude in CP is stepwise altered during the contact time. We develop a spin-thermodynamic theory to describe polarization transfer due to CP with LG irradiation (LG-CP) under the influence of the quadrupole interaction, which can successfully reproduce the LG-CP line shapes observed under various experimental conditions. Experimental and simulated (2)H powder spectra are reported for some compounds.

Quasi-isotropic single-transition cross-polarization in nuclear magnetic resonance

The theory of single-transition cross-polarization in nuclear magnetic resonance is presented and verified by experimental evidence. In comparison to conventional cross-polarization a qualitative change in the mechanism is observed. Under the influence of matched radio-frequency fields with amplitudes that are smaller than the scalar coupling constant JIS for a two-spin system with I=12 and S=12 in isotropic solution, two simultaneous coherence transfer processes are observed between single-transition coherences which have phases that are parallel to those of the radio-frequency fields, an on-resonance transfer from SxIα to SαIx and an off-resonance transfer from SxIβ to SβIx, without mixing between the two pathways. Coherence transfer is also observed between single-transition coherences with phases that are perpendicular to the radio-frequency fields, from SyIα to SαIy and from SyIβ to SβIy, as well as between longitudinal components, from SzIα to SαIz and from SzIβ to SβIz. The transfer may therefore be considered quasi-isotropic. We consider the conditions under which such transfer processes can be observed. Coherence transfer is affected by differential relaxation due to cross-correlation effects.

Structural characterization of monomeric and oligomeric arylamides by solution and solid‐state NMR spectroscopy

AbstractAn extensive study of both liquid‐ and solid‐state NMR spectroscopy was undertaken in order to elucidate the structural features of a phenyleneterephthalamide oligomer (OPTA) and of some related diarylamides. 1D‐ and 2D‐COSY measurements allowed us to assign completely the proton signals of the title compounds in solution, while 1D‐, 2D‐HETCOR and 2D‐COLOC measurements were used to assign 13C resonances. Solid‐state 13C NMR experiments, by conventional cross‐polarization (CP) at different contact times and with the dipolar dephased CP technique, were used to characterize these molecules in the solid state. Such techniques allowed us to differentiate among different carbon atoms; in the resulting spectra it was then possible to observe the selective appearance of signals from protonated and quaternary carbon atoms. It was also ascertained that the limited structural mobility of the insoluble OPTA, existing as a single monophasic species, can be explained in terms of hydrogen‐type bonds present in the solid state. Copyright © 2002 John Wiley & Sons, Ltd.

Suitability of Different 13C Solid-state NMR Techniques in the Characterization of Humic Acids

Qualitative and quantitative analyses of humic acids (HAs) with five different 13C solid-state NMR techniques were assessed using HAs of various origins and locations. The NMR techniques compared are: (1) direct polarization/magic angle spinning (DP/MAS) at 13 kHz, (2) conventional cross polarization (CP)/MAS at 5 kHz, (3) ramp-CP/MAS at 8 kHz, (4) CP/total sideband suppression (TOSS) at 4.5 kHz, and (5) DP/MAS corrected by CP/spin-lattice relaxation with TOSS. The spectra from the five techniques were first compared qualitatively. Then, each spectrum was divided into eight regions for quantitative evaluation. DP/MAS spectra were used as quantitative references. Ramp-CP/MAS and CP/TOSS spectra gave consistently better results than those of the conventional CP/MAS spectra at a 13C frequency of 75 MHz, which were incorrect due to spinning sidebands. CP/MAS at low magnetic fields (22.6 and 50.6 MHz 13C frequency) indicated improved integration results but lower resolution. Correction factors calculated by comparison with DP/MAS will be useful to convert the non-quantitative peak areas in the CP/TOSS and ramp-CP/MAS spectra into more quantitative results.

Cross polarization with long delayed contact in rotating solids

The cross polarization NMR spectra and cross relaxation rates of polycrystalline hexamethylbenzene and adamantane were obtained by employing a long delay before contact for samples spinning at, and off, the magic angle with respect to the static magnetic field. The results are compared with those obtained by using the conventional cross polarization pulse sequence. The results support the memory effect explanation of origin of the SPEDA spectra in contrast with the assumptions that the high resolution SPEDA spectra arising either, from the nearly isotropic motion of a very small fraction, or a pool of highly thermo-activated molecules, or from special spin pairs within the crystallites which are orientated at the magic angle with respect to the static magnetic field.

Rotor-synchronized amplitude-modulated nuclear magnetic resonance spin-lock sequences for improved cross polarization under fast magic angle sample spinning

We demonstrate that in solid-state nuclear magnetic resonance the Hartmann–Hahn cross-polarization efficiency in the presence of fast magic angle sample spinning can be improved by the application of rotor-synchronized amplitude-modulated radio-frequency irradiation during the mixing time (AMCP pulse sequences). The modulation reintroduces time-independent coupling elements between dressed eigenstates of the Floquet Hamiltonian which lead to flip–flop transitions. By numerical optimization, an optimal AMCP pulse sequence is designed which provides a significant improvement over conventional cross polarization. The matching condition can be broadened by rotor-synchronized 180° phase shifts to render the transfer less sensitive to exact matching of the applied radio-frequency field strengths.