Improving pulse sequences for 3D DOSY: convection compensation

Signal overlap in the NMR dimension significantly complicates the construction and analysis of 2D diffusion-ordered (DOSY) spectra. Such problems can often be reduced or even eliminated by extending the NMR domain of a DOSY experiment into two dimensions, giving a 3D-DOSY spectrum. To date such experiments have generally sacrificed some signal-to-noise ratio and have required extensive and time-consuming phase cycling. A new family of pulse sequences with internal diffusion encoding (IDOSY) has been introduced which avoids both of these problems. It is often straightforward to incorporate convection compensation in such sequences at no cost in signal-to-noise ratio. Here, some of the problems caused by convection in DOSY are described and illustrated, and the efficacy of convection compensation in the 2DJ-IDOSY and COSY-IDOSY experiments is demonstrated.     

The 2D-J-DOSY experiment: resolving diffusion coefficients in mixtures

Many diffusion-ordered spectroscopy (DOSY) NMR techniques have recently been developed to aid in the deconvolution of complex mixtures. Spectroscopic separation based on chemical and physical properties facilitates the identification of mixture components while eliminating time-consuming separation steps and preserving the chemical environment. One way to improve resolution in such experiments is to spread the spectroscopic information into two dimensions. The 2D-J-DOSY experiment has been designed to resolve mixture components in terms of a chemical shift and proton coupling constant as well as distinguishing them on the basis of translational diffusion. Acquiring a series of spectra as a function of gradient amplitude permits the determination of diffusion coefficients for components that cannot be resolved in the one-dimensional (1D) (1)H NMR spectrum. Comparison of the resulting values with those obtained through the traditional 1D diffusion experiment for a mixture of sugars validates The 2D-J-DOSY technique.     

High-resolution NMR "chromatography" using a liquids spectrometer

NMR spectroscopy is an excellent tool for the structural analysis of pure compounds. However, for mixtures it performs poorly because of overlapping signals. Diffusion can be used to separate compounds of widely differing molecular weight but the amount of separation is usually insufficient.Addition of a solid medium, analogous to the stationary phase in chromatography, can preferentially slow the diffusion of some components of a mixture permitting separation in the diffusion dimension. However, this would usually require a solid-state NMR spectrometer otherwise the signals would be too broad.Susceptibility matching the solvent to the solid medium yields a spectrum with narrow signals allowing the measurement of a DOSY spectrum with enhanced separation in the diffusion dimension.     

High resolution 13C DOSY: the DEPTSE experiment

High Resolution Diffusion-ordered Spectroscopy (HR-DOSY) is a valuable tool for mixture analysis by NMR. It separates the signals from different components according to their diffusion behavior, and can provide exquisite diffusion resolution when there is no signal overlap. In HR-DOSY experiments on (1)H (by far the most common nucleus used for DOSY) there is frequent signal overlap that confuses interpretation. In contrast, a (13)C spectrum usually has little overlap, and is in this respect a much better option for a DOSY experiment. The low signal-to-noise ratio is a critical limiting factor, but with recent technical advances such as cryogenic probes this problem is now less acute. The most widely-used pulse sequences for (13)C DOSY perform diffusion encoding with (1)H, using a stimulated echo in which half of the signal is lost. This signal loss can be avoided by encoding diffusion with (13)C in a spin echo experiment such as the DEPTSE pulse sequence described here.     

扩散序谱(DOSY)实验中扩散系数维数字分辨率的影响

核磁共振二维扩散序谱（DOSY）实验测定溶液中分子自扩散系数时,扩散系数维的数据点及其数字分辨率直接影响了测定值的精度.在较系统地确定了DOSY实验本身偏差范围的基础上,本文研究了扩散系数维不同的数字分辨率对测定值的影响,包括其引入偏差的来源以及形成偏差的大小.由于不同的溶液条件下分子扩散系数的改变可直接用于表征分子结构或状态的变化,本文提出的相对数字分辨率与扩散系数相对变化值的直接比较,可直观地表明数字分辨率对扩散系数测定值的影响.     

High-resolution DOSY NMR with spins in different chemical surroundings: influence of particle exchange

The effect of chemical exchange in the diffusion-ordered (DOSY) spectra of a two-site system in the slow-exchange limit with respect to the chemical shift is studied. The problem is addressed both theoretically and experimentally. The relationship between diffusion time (t) and mean lifetimes (tau) is studied by the simulation of the magnetization attenuations as a function of the gradient strength, under PFG conditions. The influence of the difference in populations and diffusion coefficients of the two sites is also considered. In analogy to the limiting cases of fast- and slow-exchange with respect to the chemical shift, limiting cases with respect to the diffusion dimension are defined. The slow-exchange limit in diffusion corresponds to the relation of t and tau that allows us to observe the two spins in exchange associated with the individual diffusion coefficients of the two sites when no exchange is present. The fast-exchange limit in diffusion is reached when the relation of t and tau is such that the two spins present the same apparent diffusion coefficient. By using a model system consisting of water/t-butanol it is shown that by recording several DOSY experiments with increasing diffusion times it is possible to estimate the value of the exchange rate.     

Polydispersity index of polymers revealed by DOSY NMR

The polydispersity of a polymer chain is usually measured by its polydispersity index (PDI). In this study we present a method which allows to estimate the PDI of linear polymers from a simple diffusion experiment. The approach is based on the differential diffusion profile observed for the main polymer chain signal versus the extremity signal. From this difference, a statistical analysis of the DOSY spectrum allows the PDI to be estimated accurately, to the condition that the Flory coefficient of the polymer chain is known. Alternatively, the mass average molar mass Mw and the number average molar mass Mn can be extracted separately from the same spectrum. Results on PEO mixes reveal that, using this new method, PDI can be estimated with a very good accuracy. This method can easily be applied to almost any kind of linear polymers.     

Applications of DOSY NMR in the Chemical IndustryApplications of DOSY NMR in the Chemical Industry

DOSY(diffusion ordered spectroscopy)NMR is a technique that separates NMR signals of different species according to their diffusion coefficients.It is a powerful technique to provide solid characterization evidence for various applications in the chemical industry.Encapsulation and functional polymer grafting are two important capabilities in the chemical industry for new product development with challenging characterization requirements.Model systems of encapsulation and grafted polymer were studied and the characterization methods were set up by DOSY NMR.     

Recoupling of chemical-shift anisotropy powder patterns in MAS NMR

A comparison of three different implementations of the chemical-shift recoupling experiment of Tycko et al. [R. Tycko, G. Dabbagh, P.A. Mirau, Determination of chemical-shift-anisotropy lineshapes in a two-dimensional magic-angle-spinning NMR experiment, J. Magn. Reson. 85 (1989) 265-274] is presented. The methods seek to reduce the effects of artefacts resulting from pulse imperfections and residual C-H dipolar coupling in organic solids. An optimised and constant time implementation are shown to give well-defined and artefact free powder pattern lineshapes in the indirectly observed dimension for both sp2 and sp3 carbon sites. Experimental setup is no more demanding than for the original experiment, and can be implemented using standard commercial hardware.     

HMQC-TOCSY技术在植物环肽和配糖体中的应用

核磁共振HMQC-TOCSY二维技术可在氢谱方向和碳谱方向分别得到独立的自旋系统内氢核和碳核的全相关信息.该技术对于分子内具有多个自旋系统的植物环肽和配糖体的结构解析尤为适用.本文以环肽annosquamosin A和三萜皂甙prostratoside A为例说明该技术在这两类化合物中的应用.     

Simplifying DOSY spectra with selective TOCSY edited preparation

Diffusion-ordered NMR spectroscopy, while quite powerful, is limited by its inability to resolve signals that are severely overlapped in the proton spectrum. We present here a DOSY experiment that uses selective TOCSY as an editing/preparation period. With this method, well-resolved signals of the analytes are selectively excited and the magnetization subsequently transferred by isotropic mixing to resonances buried in the matrix background, which are then resolved by the ensuing DOSY sequence. Key to the success of our proposed method is the incorporation of a highly effective zero-quantum filter into the selective TOCSY preparation period, which prevents zero-quantum coherence from being carried into the DOSY part of the pulse sequence. Further improvement in spectral resolution can be obtained by expanding the proposed experiment into a 3D sequence and utilizing the homonuclear decoupling feature of the BASHD-TOCSY technique. Both pulse sequences were found to greatly simplify the DOSY spectrum of a 'dirty' sucrose/raffinose mixture, as the complex matrix background is no longer present to obscure or overlap with the signals of interests. Furthermore, complete resolution of the relevant signals was achieved with the 3D sequence.     

THE APPLICATION OF HMQC-TOCSY TO THE PLANT GLYCOSIDES AND PLANT CYCLOPEPTIDES

核磁共振HMQC-TOCSY二维技术可在氢谱方向和碳谱方向分别得到独立的自旋系统内氢核和碳核的全相关信息.该技术对于分子内具有多个自旋系统的植物环肽和配糖体的结构解析尤为适用.本文以环肽annosquamosin A和三萜皂甙prostratoside A为例说明该技术在这两类化合物中的应用.     

J-modulation effects in DOSY experiments and their suppression: the Oneshot45 experiment

Diffusion-ordered spectroscopy (DOSY) is a powerful NMR method for identifying compounds in mixtures. DOSY experiments are very demanding of spectral quality; even small deviations from expected behaviour in NMR signals can cause significant distortions in the diffusion domain. This is a particular problem when signals overlap, so it is very important to be able to acquire clean data with as little overlap as possible. DOSY experiments all suffer to a greater or lesser extent from multiplet phase distortions caused by J-modulation, requiring a trade-off between such distortions and gradient pulse width. Multiplet distortions increase spectral overlap and may cause unexpected and misleading apparent diffusion coefficients in DOSY spectra. These effects are described here and a simple and effective remedy, the addition of a 45° purging pulse immediately before the onset of acquisition to remove the unwanted anti-phase terms, is demonstrated. As well as affording significantly cleaner results, the new method allows much longer diffusion-encoding pulses to be used without problems from J-modulation, and hence greatly increases the range of molecular sizes that can be studied for coupled spin systems. The sensitivity loss is negligible and the added phase cycling is modest. The new method is illustrated for a widely-used general purpose DOSY pulse sequence, Oneshot.     

On the role of experimental imperfections in constructing 1H spin diffusion NMR plots for domain size measurements

We discuss the precision of 1D chemical-shift-based ¹H spin diffusion NMR experiments as well as straightforward experimental protocols for reducing errors. The ¹H spin diffusion NMR experiments described herein are useful for samples that contain components with significant spectral overlap in the ¹H NMR spectrum and also for samples of small mass (<1 mg). We show that even in samples that display little spectral contrast, domain sizes can be determined to a relatively high degree of certainty if common experimental variability is accounted for and known. In particular, one should (1) measure flip angles to high precision (≈±1° flip angle), (2) establish a metric for phase transients to ensure their repeatability, (3) establish a reliable spectral deconvolution procedure to ascertain the deconvolved spectra of the neat components in the composite or blend spin diffusion spectrum, and (4) when possible, perform 1D chemical-shift-based ¹H spin diffusion experiments with zero total integral to partially correct for errors and uncertainties if these requirements cannot fully be implemented. We show that minimizing the degree of phase transients is not a requirement for reliable domain size measurement, but their repeatability is essential, as is knowing their contribution to the spectral offset (i.e. the J₁ coefficient). When performing experiments with zero total integral in the spin diffusion NMR spectrum with carefully measured flip angles and known phase transient effects, the largest contribution to error arises from an uncertainty in the component lineshapes which can be as high as 7%. This uncertainty can be reduced considerably if the component lineshapes deconvolved from the composite or blend spin diffusion spectra adequately match previously acquired pure component spectra.     

Single-scan 2D DOSY NMR spectroscopy

Spatial encoding is a particular kind of spin manipulation that enables the acquisition of multidimensional NMR spectra within a single scan. This encoding has been shown to possess a general applicability and to enable the completion of arbitrary nD NMR acquisitions within a single transient. The present study explores its potential towards the acquisition of 2D DOSY spectra, where the indirect dimension is meant to encode molecular displacements rather than a coherent spin evolution. We find that in its simplest form this extension shows similarities with methods that have been recently discussed for the single-scan acquisition of this kind of traces; still, a number of advantageous features are also evidenced by the “ultrafast” modality hereby introduced. The principles underlying the operation of this new single-scan 2D DOSY approach are discussed, its use is illustrated with a variety of sequences and of samples, the limitations of this new experiment are noted, and potential extensions of the methodology are mentioned.     

Processing DOSY spectra using the regularized resolvent transform

A new method for processing diffusion ordered spectroscopy (DOSY) data is presented. This method, the regularized resolvent transform (iRRT-the i denoting the adaptation of the method to evaluate the inverse Laplace transform), is better than conventional processing techniques for generating 2D DOSY spectra using data that has poor chemical shift resolution. From the same data, it is possible to use the iRRT to generate 1D subspectra corresponding to different components of the sample mixture; these subspectra compare favorably to 1D spectra of the pure substances. Both the 2D spectra and the 1D subspectra offer a vast improvement over results generated using a conventional processing technique (non-linear least-squares fitting). Consequently, we present the iRRT as a stable and reliable tool for solving the inverse Laplace transform problem present in experiments such as DOSY.     

ANAFOR: application of a restricted linear least squares procedure to NMR data processing

Experimental data collection time in multidimensional nuclear magnetic resonance experiments can be significantly decreased if the lineshapes of all the components of one of the ID summations of the spectrum are known. When this condition is fulfilled, a simple linear least squares fit of the time-domain signal taking the lineshapes into account not only allows saving time in data collection, but also improves sensitivity and resolution. The reliability of the proposed procedure is carefully addressed in the particular case of Lorentzian lines. This strategy applied to a 3Q-REDOR experiment reduced experimental time by a factor of 6.     

STD-DOSY: A new NMR method to analyze multi-component enzyme/substrate systems

A new approach to analyze multi-component Saturation Transfer Difference (STD) NMR spectra by combining the STD and the DOSY experiment is proposed. The resulting pulse sequence was successfully used to simplify an exemplary multi-component protein/substrate system by means of standard DOSY processing methods. Furthermore, the same experiment could be applied to calculate the ratio of saturated substrate molecules and its saturation rate in the case of competitive interactions. This ratio depends on the strength of this interaction between the substrates and the protein, so that this kind of information could be extracted from the results of our experiment.     

WIM/WISE NMR studies of chain dynamics in solid polymers and blends

Wideline separation (WISE) NMR with window-less isotropic mixing (WIM) is developed as a method to study the dynamics of polymers and blends. This experiment is designed to measure the dynamics of polymers through the proton lineshapes that are correlated with the carbon chemical shifts in two-dimensional NMR experiments. If the atoms experience large amplitude fluctuations that are fast compared to the dipolar broadening, then the proton lines will be narrowed relative to rigid solids. We have modified the WISE experiment by replacing the cross polarization step with WIM to quench spin diffusion during the cross polarization so that the proton linewidths can be directly related to the chain dynamics. Two-dimensional WIM/WISE has been used to measure the main-chain and side-chain dynamics in poly(n-butyl methacrylate) and blends of polystyrene and poly(vinyl methyl ether).     

Program for the analysis of pulse height spectra and the background from a proportional detector

A PC-Fortran program is presented for fitting of lineshapes and the analysis of pulse height spectra obtainable with proportional detectors. The common fitting and analysis of pulse height spectra by means of mixed Gaussian lineshapes is readily improved by using Voigt lineshapes. In addition, the background can be evaluated during the fitting process without the need of extra measurements. As an application of the program, a pulse height transmission spectrum accumulated with a static⁵⁷Co source and detected with an argon-metane proportional detector, was least squares fitted to an elaborated complex trial lineshape function containing two Voigt lines plus a straight line. The fitting straight line parametersa andb characterize quantitatively the background. The very good PC-fitting obtained shows that the fitting of experimental spectra with the more realistic Voigt lineshapes is no longer a formidable task and that it is possible to evaluate and subtract the background inherent to the experiment during the fitting process.