Adiabatic pulses in 1H-15N direct and long-range heteronuclear correlations

The application of adiabatic inversion pulses to the detection of (1)H-(15)N heteronuclear correlations is described. The pulse sequences studied were gHSQC, CRISIS-gHSQC, gHMBC and CRISIS-gHMBC. The poor inversion quality of rectangular 180 degrees X pulses can lead to a loss of signal at the peripheries of the spectrum. Replacing these pulses with adiabatic sweeps significantly improves sensitivity across the potentially large (15)N spectral window. Satellite spectrum profiles are shown to demonstrate the increase in sensitivity when employing adiabatic pulses on wide spectral widths. Additionally, the active pharmaceutical nizatidine was used as a model compound to demonstrate the improvements in the long-range correlation data.     

J-modulated ADEQUATE experiments using different kinds of refocusing pulses

Owing to the recent developments concerning residual dipolar couplings (RDCs), the interest in methods for the accurate determination of coupling constants is renascenting. We intended to use the J-modulated ADEQUATE experiment by K?vér et al. for the measurement of (13)C - (13)C coupling constants at natural abundance. The use of adiabatic composite chirp pulses instead of the conventional 180 degrees pulses, which compensate for the offset dependence of (13)C 180 degrees pulses, led to irregularities of the line shapes in the indirect dimension causing deviations of the extracted coupling constants. This behaviour was attributed to coupling evolution, during the time of the adiabatic pulse (2 ms), in the J-modulation spin echo. The replacement of this pulse by different kinds of refocusing pulses indicated that a pair of BIPs (broadband inversion pulses), which behave only partially adiabatic, leads to correct line shapes and coupling constants conserving the good sensitivity obtained with adiabatic pulses.     

Doubly compensated multiplicity-edited HSQC experiments utilizing broadband inversion pulses

We propose a family of doubly compensated multiplicity-edited heteronuclear single quantum coherence (HSQC) pulse sequences. The key difference between our proposed sequences and the compensation of refocusing inefficiency with synchronized inversion sweeps (CRISIS)-HSQC experiments they are based on is that the conventional rectangular 180 degrees pulses on the proton channel in the latter have been replaced by the computer-optimized broadband inversion pulses (BIPs) with superior inversion performance as well as much improved tolerance to B(1) field inhomogeneity. Moreover, all adiabatic carbon 180 degrees pulses during the INEPT and reverse-INEPT periods in the CRISIS-HSQC sequences have also been replaced with the much shorter BIPs, while the adiabatic sweeps during the heteronuclear spin echo for multiplicity editing are kept in place in order to maintain the advantage of the CRISIS feature of the original sequences, namely J-independent refocusing of the one-bond (1)H--(13)C coupling constants. These modifications have also been implemented to the preservation of equivalent pathways (PEP)-HSQC experiments. We demonstrate through a detailed comparison that replacing the proton 180 degrees pulses with the BIPs provide additional sensitivity gain that can be mainly attributed to the improved tolerance to B(1) field inhomogeneity of the BIPs. The proposed sequences can be easily adapted for (19)F--(13)C correlations.     

A modified CRISIS-HSQC for band-selective IMPRESS

CRISIS (Compensation of Refocusing Inefficiency with Synchronized Inversion Sweep) is a powerful technique for obtaining multiplicity-edited HSQC spectra without compromising sensitivity. However, the stringent requirement for the duration of the CRISIS waveforms makes them unsuitable for other functions, such as band selection or IMPRESS (IMProved REsolution using Symmetrically Shifted pulses). We report here a modified CRISIS-gHSQC pulse sequence employing time-reversed 13C pi/2 EBURP-2 pulses. This IC-bs-gHSQC (IMPRESS-CRISIS-bs-gHSQC) sequence was found to be equally useful for acquiring multiplicity-edited, band-selective spectra individually or in tandem with IMPRESS. Remarkably, the latter provides multiple spectra in significantly less time and is the preferred approach when several crowded regions need to be assigned unambiguously. The use of adiabatic sweeps and the CRISIS pulses enable IC-bs-gHSQC to give better sensitivity than the original IMPRESS sequence for band-selective spectra.     

Next‐Generation Heteronuclear Decoupling for High‐Field Biomolecular NMR Spectroscopy

Ultra‐high‐field NMR spectroscopy requires an increased bandwidth for heteronuclear decoupling, especially in biomolecular NMR applications. Composite pulse decoupling cannot provide sufficient bandwidth at practical power levels, and adiabatic pulse decoupling with sufficient bandwidth is compromised by sideband artifacts. A novel low‐power, broadband heteronuclear decoupling pulse is presented that generates minimal, ultra‐low sidebands. The pulse was derived using optimal control theory and represents a new generation of decoupling pulses free from the constraints of periodic and cyclic sequences. In comparison to currently available state‐of‐the‐art methods this novel pulse provides greatly improved decoupling performance that satisfies the demands of high‐field biomolecular NMR spectroscopy.     

New DEFT sequences for the acquisition of one-dimensional carbon NMR spectra of small unlabelled molecules

The acquisition time and quality of 1D 13C{1H} spectra can be improved substantially by using a modified driven equilibrium Fourier transform (DEFT) sequence, which is specifically designed to compensate for the effects of B1 inhomogeneity, pulse miscalibration and frequency offsets. The new sequence, called uniform driven equilibrium Fourier transform (UDEFT), returns the carbon magnetization with a high accuracy along its equilibrium position after each transient is complete. Thus, the sequence allows the use of relaxation delays (RD), which are much shorter than the carbon T1 of the molecule, thereby speeding up the acquisition process of 1D 13C{1H} spectra. To achieve this level of performance, UDEFT employs a refocusing element constituted by a composite adiabatic carbon pulse surrounded by two 90 degrees carbon pulses whose phases are designed to compensate for 90 degrees pulse miscalibrations in an MLEV manner (90 degrees+x-tau(FID)-180+y(Adia)-tau-90 degrees+x-180 degrees+x(Adia)). A version of the UDEFT sequence allows recording 1D 13C{1H} spectra devoid of heteronuclear NOE by using a matched adiabatic 1H decoupling scheme where an even number of 180 degrees adiabatic pulses is applied during the UDEFT module. Spectra of a solution of 300 mM camphor that contains some carbon nuclei with very long T1 relaxation times (90 s and 78 s) were acquired with 128 scans in 10 min using a 5 s relaxation delay.     

Chemical imaging by single pulse interferometric coherent anti-stokes Raman scattering microscopy

A single pulse interferometric coherent anti-Stokes Raman (CARS) spectroscopy method is used to obtain broadband CARS spectra and microscopy images of liquid and polymer samples. The pump, Stokes, and probe pulses are all selected inside a single broadband ultrafast pulse by a phase- and polarization-controlled pulse shaping technique and used to generate two spectral interference CARS signals simultaneously. The normalized difference of these two signals provides an amplified background-free broadband resonant CARS spectrum over the 400-1500 cm(-1) range with 35 cm(-1) spectral resolution. Chemically selective microscopy images of multicomponent polymer and liquid samples are investigated with this new CARS method. Multiplex CARS spectra at 10,000 spatial points are measured within a few minutes, and used to construct chemically selective microscopy images with a spatial resolution of 400 nm. The spectral bandwidth limits, sensitivity, homodyne amplification advantages, spatial resolution, depolarization, chromatic aberration, and chemical imaging aspects of this new technique are discussed in detail.     

Improved resolution in two-dimensional 1H NMR spectra of peptides by band-selective, homonuclear decoupling during both the evolution and acquisition periods: application to characterization of the binding of peptides by heparin

Two-dimensional 1H NMR experiments that achieve band-selective, homonuclear decoupling in both the indirectly detected F1 and directly detected F2 dimensions were used to assign the highly overlapped 1H NMR spectrum of the peptide Ac-SRGKARVRAKVKDQTK-NH2, both free in solution and bound to heparin. Band-selective, homonuclear decoupling during the evolution period was achieved using a double pulsed field gradient spin-echo (DPFGSE) with semi-selective shaped pulses; band-selective, homonuclear decoupling during the acquisition period was achieved by time-shared semi-selective shaped pulse decoupling. Regular TOCSY, ROESY and NOESY spectra and TOCSY, ROESY and NOESY spectra measured with band-selective, homonuclear decoupling in the evolution (F1) dimension (BASHD-TOCSY, ROESY and NOESY spectra) and with band-selective, homonuclear decoupling in both the F1 and F2 dimensions (D-(or Double)-BASHD-TOCSY, ROESY and NOESY spectra) are reported and compared for the peptide and its heparin complex. Complete assignment of the 1H-NMR spectra of the free and heparin-complexed peptide was achieved with the high resolution of the D-BASHD-TOCSY, ROESY and NOESY spectra. Characterization of the heparin-complexed peptide is of interest because of the ability of the peptide to neutralize the anticoagulant activity of heparin.     

Low-power composite CPMG HSQMBC experiment for accurate measurement of long-range heteronuclear coupling constants

A modified version of CPMG-HSQMBC pulse scheme is presented for the measurement of long-range heteronuclear coupling constants. The method implements adiabatic inversion and refocusing pulses on the heteronucleus. Low-power composite 180° XY-16 CPMG pulse train is applied on both proton and X nuclei during the evolution of long-range couplings to eliminate phase distortions due to co-evolution of homonuclear proton-proton couplings. The pulse sequence yields pure absorption antiphase multiplets allowing precise and direct measurement of the (n)J(XH) coupling constants regardless from the size of the proton-proton couplings. The applicability of the method is demonstrated using strychnine as a model compound. The selective 1D version of the method is also presented.     

Constant amplitude broadband refocusing pulses from numerical optimization

Broadband refocusing pulses for high-field NMR can be constructed with broadband 90× pulses from numerical optimization of Bloch simulations concatenated with their time and phase reversed transformations. This work describes the search for minimal duration 18-kHz modulation frequency constant amplitude refocusing pulses made in this manner for bandwidths of 40, 60 and 80 kHz. Variants optimized at multiple frequencies and with sine squared amplitude truncation also are described. The resulting pulses are expected to have immediate application especially for (13)C refocusing in multidimensional experiments.     

Profiles of paint layer samples obtained in the fringe field of a high field magnet by means of very short broadband frequency-modulated pulses

In this article, we describe the acquisition of depth profiles, in particular of paint layers, in the static gradient of a high field magnet, providing a superior sensitivity. The main objective are reference profiles that help to understand scans made with noninvasive unilateral nuclear magnetic resonance (NMR), which often suffers from poor signal-to-noise ratio when working with real samples. Various technical aspects like the coil geometry and the limit of resolution are investigated. A major advancement is the use of frequency-modulated pulses that are very broadband and at the same time very short (25 μs). The latter is necessary to allow the acquisition of a CPMG echo train of old, rigid paint material. Despite being far from adiabatic, they provide uniform excitation and refocusing over 1 MHz, which corresponds to about 400 μm with the used gradient. We show that the uniformity is even sufficient to obtain biexponential relaxation profiles. With these tools, a paint sample from a restoration campaign is analyzed with different contrast criteria: The original and two layers from former restoration attempts can be visualized, and furthermore, the relaxation profiles allow to study the migration of plasticizing molecules.     

CRAPT: an improved version of APT with compensation for variations in JCH

A modified version of the attached proton test (APT) sequence for ¹³C spectral editing, which we call CRisis‐APT (CRAPT), is developed and tested on representative organic compounds. CRAPT incorporates ¹³C compensation for refocusing inefficiency with synchronized inversion sweeps (CRISIS) pulses in combination with ¹H broadband inversion pulses to give improved compensation for variations in ¹J_CH along with improved refocusing efficiency. It is shown that CRAPT gives edited ¹³C spectra with only small losses in sensitivity (between 8% and 15% for strychnine, 1 , menthol, 2 , cholecalciferol, 3 , and isotachysterol, 4 ), compared with basic ¹³C spectra obtained on the same compounds. CRAPT also gives significantly better signal/noise than DEPTQ for nonprotonated carbons. Therefore, we conclude that CRAPT is an improvement over APT or DEPTQ or a combination of DEPT135 with a full ¹³C spectrum for routine ¹³C spectral editing of organic compounds. Copyright © 2014 John Wiley & Sons, Ltd.     

Nuclear Magnetic Resonance Metabolomics with Double Pulsed-Field-Gradient Echo and Automatized Solvent Suppression Spectroscopy for Multivariate Data Matrix Applied in Novel Wine and Juice Discriminant Analysis

The quality of foods has led researchers to use various analytical methods to determine the amounts of principal food constituents; some of them are the NMR techniques with a multivariate statistical analysis (NMR-MSA). The present work introduces a set of NMR-MSA novelties. First, the use of a double pulsed-field-gradient echo (DPFGE) experiment with a refocusing band-selective uniform response pure-phase selective pulse for the selective excitation of a 5–10-ppm range of wine samples reveals novel broad ¹H resonances. Second, an NMR-MSA foodomics approach to discriminate between wine samples produced from the same Cabernet Sauvignon variety fermented with different yeast strains proposed for large-scale alcohol reductions. Third a comparative study between a nonsupervised Principal Component Analysis (PCA), supervised standard partial (PLS-DA), and sparse (sPLS-DA) least squares discriminant analysis, as well as orthogonal projections to a latent structures discriminant analysis (OPLS-DA), for obtaining holistic fingerprints. The MSA discriminated between different Cabernet Sauvignon fermentation schemes and juice varieties (apple, apricot, and orange) or juice authentications (puree, nectar, concentrated, and commercial juice fruit drinks). The new pulse sequence DPFGE demonstrated an enhanced sensitivity in the aromatic zone of wine samples, allowing a better application of different unsupervised and supervised multivariate statistical analysis approaches.     

Improved multiplicity-editing of HMBC NMR spectra

A new improved multiplicity-edited HMBC experiment is introduced that leads to better J cross-talk suppression in the even (i.e. C + CH2 groups) and odd (i.e. CH + CH3 groups) subspectra. By combining data recorded with three different pulse sequences J cross-talk becomes a second-order effect in Delta1J, i.e. the deviation of an actual 1J coupling constant from the value 1J0 used in setting delays tau = (1J0)(-1/2), which is adequate for most applications. As for the original multiplicity-edited HMBC experiment, the improved experiment can be performed with a single excitation delay or implemented in a broadband version similar to broadband HMBC.     

Complete characterization of molecular vibration using frequency resolved gating

The authors propose a new approach to vibration spectroscopy based on the coherent anti-Stokes Raman scattering of broadband ultrashort laser pulses. The proposed method reveals both the amplitude and the phase of molecular vibrations by utilizing the cross-correlation frequency resolved optical gating (XFROG) technique. The spectrum of the anti-Stokes pulse is measured as a function of the time delay between the laser-induced molecular vibrations and a well characterized broadband femtosecond probe pulse. The iterative XFROG algorithm provides a simultaneous complete characterization of molecular vibrations both in frequency and time domains with high resolution. They demonstrate experimentally the feasibility of the proposed method and show one of its potential applications in disentangling the time behavior of a mixture of vibrationally excited molecules. The technique of femtosecond pulse shaping is used for further improvement of accuracy and stability against noise.     

Multi-frequency improved constant amplitude pulses for broadband inversion

Optimization of constant amplitude broadband inversion pulses for maximum inversion over a range of multiple fields yields more regular pulses with better tolerance to B(1) errors than those optimized for a single field. These multi-frequency improved constant amplitude (MICA) pulses as constructed for (13)C broadband inversion give best results for HMQC and especially HSQC type experiments. Most of the advantages of MICA pulses versus other inversion pulses in these experiments can be attributed to their relatively short durations. Linearly truncated versions of these pulses do not offer any advantage over MICA pulses for these applications. MICA inversion pulses can also be used for decoupling and a (13)C decoupling example gives twice the decoupling bandwidth as the GARP1 sequence at the same power level with no appreciable increase in decoupling sidebands.     

Broadband RF‐amplitude‐dependent flip angle pulses with linear phase slope

Pulse sequences in NMR spectroscopy sometimes require the application of pulses with effective flip angles different from 90° and 180°. Previously (Magn. Reson. Chem. 2015, 53, 886‐893), offset‐compensated broadband excitation pulses with RF‐amplitude‐dependent effective flip angles (RADFA) were introduced that are applicable in such cases. However, especially RF‐amplitude‐restricted RADFA pulses turned out to perform not as good as desired in terms of achievable bandwidths. Here, a class of RF‐amplitude‐restricted RADFA pulses with linear phase slope is introduced that allows excitation over much larger bandwidths with better performance. In this theoretical work, the basic principle of the pulse class is explained, their physical limits explored, and their properties, also compared with other pulse classes, discussed in detail. Copyright © 2017 John Wiley & Sons, Ltd.     

Extending the Scope of Singlet‐State Spectroscopy

Different decoupling sequences are tested—using various shaped radio‐frequency (RF) pulses—to achieve the longest possible lifetimes of singlet‐state populations over the widest possible bandwidths, that is, ranges of offsets and relative chemical shifts of the nuclei involved in the singlet states. The use of sinc or refocusing broadband universal rotation pulses (RE‐BURP) for decoupling during the intervals where singlet‐state populations are preserved allows one to extend the useful bandwidth with respect to prior state‐of‐the‐art methods based on composite‐pulse WALTZ decoupling. The improved sinc decoupling sequences afford a more reliable and sensitive measure of the lifetimes of singlet states in pairs of spins that have widely different chemical shifts, such as the two aromatic protons H⁵ and H⁶ in uracil. Similar advantages are expected for nucleotides in RNA and DNA. Alternative approaches, in particular frequency‐modulated decoupling sequences, also appear to be effective in preserving singlet‐state populations, even though the profiles of the apparent relaxation rate constants as a function of the offset are somewhat perturbed. The best decoupling sequences prove their utility in sustaining longer lifetimes of singlet states than previously achieved for the side‐chain tyrosine protons in bovine pancreatic trypsin inhibitor (BPTI) at 600 MHz (14.1 T), where the differences of chemical shifts between coupled protons are a challenge.     

Technological innovations in high count-rate instrumentation

Measurements at counting rates above 50,000 counts per second require specialized pulse processing electronics. New preamplifier technology incorporating an injection junction field effect transistor with integral reset gate provides superior throughput with better low energy resolution. Data acquisition at short peaking times permits maximum throughput with minimum pile-up. Over the past ten years, amplifier throughput of unpiled-up pulses improved. Now it is possible to correct the pulse amplitudes as they are processed, improving performance over older methods. Trapping and ballistic deficit are both greater in high rate measurements, where the shorter amplifier peaking times employed result in larger amplitude variations of the amplifier output pulse. Charge trapping may occur in any sized detector. The Goulding-Landis method corrects best for charge carrier trapping while the Hinshaw method works best for ballistic deficit effects.     

An improved 15N relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins

A new (15)N constant-time relaxation dispersion pulse scheme for the quantification of millisecond time-scale exchange dynamics in proteins is presented. The experiment differs from previously developed sequences in that it includes (1)H continuous-wave decoupling during the (15)N Carr-Purcell-Meiboom-Gill (CPMG) pulse train that significantly improves the relaxation properties of (15)N magnetization, leading to sensitivity gains in experiments. Moreover, it is shown that inclusion of an additional (15)N 180 degrees refocusing pulse (phase cycled +/- x) in the center of the CPMG pulse train, consisting of 1(5)N 180 degrees (y) pulses, provides compensation for pulse imperfections beyond the normal CPMG scheme. Relative to existing relaxation-compensated constant-time relaxation dispersion pulse schemes, nu(CPMG) values that are only half as large can be employed, offering increased sensitivity to slow time-scale exchange processes. The robustness of the methodology is illustrated with applications involving a pair of proteins: an SH3 domain that does not show millisecond time-scale exchange and an FF domain with significant chemical exchange contributions.