Local covariance order diffusion-ordered spectroscopy: a powerful tool for mixture analysis

Diffusion-ordered spectroscopy (DOSY) is an important tool in NMR mixture analysis that has found use in most areas of chemistry, including organic synthesis, drug discovery, and supramolecular chemistry. Typically the aim is to disentangle the overlaid, and often overlapped, NMR spectra of individual mixture components and/or to obtain size and interaction information from their respective diffusion coefficients. The most common processing method, high-resolution DOSY, breaks down where component spectra overlap; here multivariate methods can be very effective, but only for small numbers (2-5) of components. In this study, we present a hybrid method, local covariance order DOSY (LOCODOSY), that breaks a spectral data set into suitable windows and analyzes each individually before combining the results. This approach uses a multivariate algorithm (e.g., SCORE or DECRA) to resolve only a small number of components in any given window. Because a small spectral region should contain signals from only a few components, even when the spectrum as a whole contains many more, the total number of resolvable chemical components rises dramatically. It is demonstrated here that complete resolution of component spectra can be achieved for mixtures that are much more complex than could previously be analyzed with DOSY. Thus, LOCODOSY is a powerful, flexible tool for processing NMR diffusion data of complex mixtures.     

Ultrahigh‐Resolution Diffusion‐Ordered Spectroscopy

Diffusion‐ordered spectroscopy (DOSY) is an effective method for the analysis of intact mixtures, but the quality of results is critically limited by resolution in the NMR dimension. A new experiment integrating diffusion weighting into the PSYCHE method for pure shift NMR spectroscopy allows DOSY spectra to be measured with ultrahigh NMR resolution at improved sensitivity.     

Real-life applications of the MULVADO software package for processing DOSY NMR data

MULVADO is a newly developed software package for DOSY NMR data processing, based on multivariate curve resolution (MCR), one of the principal multivariate methods for processing DOSY data. This paper will evaluate this software package by using real-life data of materials used in the printing industry: two data sets from the same ink sample but of different quality. Also a sample of an organic photoconductor and a toner sample are analysed. Compared with the routine DOSY output from monoexponential fitting, one of the single channel algorithms in the commercial Bruker software, MULVADO provides several advantages. The key advantage of MCR is that it overcomes the fluctuation problem (non-consistent diffusion coefficient of the same component). The combination of non-linear regression (NLR) and MCR can yield more accurate resolution of a complex mixture. In addition, the data pre-processing techniques in MULVADO minimise the negative effects of experimental artefacts on the results of the data. In this paper, the challenges for analysing polymer samples and other more complex samples will also be discussed.     

Using perdeuterated surfactant micelles to resolve mixture components in diffusion‐ordered NMR spectroscopy

Diffusion‐ordered NMR spectroscopy resolves mixture components on the basis of differences in their respective diffusion coefficients or molecular sizes. However, when components have near‐identical diffusion coefficients, they are not resolved in the diffusion dimension of a diffusion‐ordered spectroscopy (DOSY) spectrum. Adding surfactant micelles to these mixtures has been shown to enhance resolution when the component molecules interact differentially with the micelles. This approach is similar to that used in electrokinetic chromatography (EKC) where modifiers like micelles or polymers are used to enhance the separation of mixture components. In this study, perdeuterated surfactants are added to analyte mixtures studied with the DOSY technique. Since no micelle resonances appear in the mixture spectra, the difficulty associated with performing biexponential analyses in spectral regions where analyte and surfactant resonances overlap is avoided. The approach is demonstrated using mixtures of peptides with near‐identical diffusion coefficients. Copyright © 2008 John Wiley & Sons, Ltd.     

Matrix-Assisted DOSY for Analysis of Indole Alkaloid Mixtures

Diffusion-ordered spectroscopy (DOSY) is a powerful tool for investigating mixtures and identifying peaks of chemical components. However, similar diffusion coefficients of the components, particularly for complex mixtures that contain crowded resonances, limit resolution and restrict application of the DOSY technique. In this paper, matrix-assisted DOSY were used to explore whether the diffusion resolution of a complex model involving indole alkaloid mixtures can be realized. Furthermore, we investigated the influence of different factors on the separation effect. The results showed that the changes in diffusion coefficient differences were achieved more obviously when using sodium dodecyl sulfate (SDS) micelles as the matrix. In addition, we also found that increasing the concentration of SDS can improve the resolution of the DOSY spectrum. Finally, after investigating the influence factors and NMR conditions, we demonstrated the applications of the SDS-assisted DOSY on analyzing the total alkaloid extract of Alstonia Mairei, and the virtual separation of mixtures was achieved.     

Measurement of aroma compound self-diffusion in food models by DOSY

Self-diffusion measurement of solutes in polymer gels has been investigated using pulsed gradient spin echo NMR spectroscopy. However, few data are available on the self-diffusion of small solutes in natural polysaccharide polymers used as thickeners in the food industry. Since aroma diffusion in food matrices could have an impact on flavor release, this is an interesting and economic challenge. Diffusion ordered spectroscopy (DOSY) resolves diffusion data for each component in complex mixtures. We used DOSY with the inverse Laplace transform approach with the maximum entropy algorithm to investigate diffusion of two aroma compounds, ethyl butanoate and linalool, in an iota-carrageenan matrix as the food model. We showed that the self-diffusion coefficient values of small molecules in a polysaccharide matrix could be easily extracted using this method. We then investigated the impact of the gelling state of iota-carrageenan matrices on the self-diffusion of ethyl butanoate.     

Single-Scan Diffusion-Ordered NMR Spectroscopy of SABRE-Hyperpolarized Mixtures

The analysis of complex mixtures of dissolved molecules is a major challenge, especially for systems that gradually evolve, e. g., in the course of a chemical reaction or in the case of chemical instability. 1D NMR is a fast and non-invasive method suitable for detailed molecular analysis, though of low sensitivity. Moreover, the spectral resolution of proton, the most commonly used and most sensitive stable isotope in NMR, is also quite limited. Spatially encoded (SPEN) experiments aim at creating in one acquisition a 2D data set by simultaneously performing different 1D sub-experiments on different slices of the NMR tube, at the price of an extra loss of sensitivity. Choosing translational diffusion coefficients as the additional dimension (the so-called DOSY approach) helps to recover proton spectra of each molecule in a mixture. The sensitivity limitation of SPEN NMR can, on the other hand, be addressed with hyperpolarization methods. Within hyperpolarization methods, signal amplification by reversible exchange (SABRE), based on parahydrogen, is the cheapest and the easiest one to set up, and allows multi-shot experiments. Here we show that the spectra of a mixture's components at millimolar concentration are resolved in few seconds by combining the SABRE, SPEN and DOSY concepts.     

Online Reaction Monitoring with Fast and Flow-Compatible Diffusion NMR Spectroscopy

Online monitoring by flow NMR spectroscopy is a powerful approach to study chemical reactions and processes, which can provide mechanistic understanding, and drive optimisations. However, some of the most useful methods for mixture analysis and reaction monitoring are not directly applicable in flow conditions. This is the case of classic diffusion-ordered NMR spectroscopy (DOSY) methods, which can be used to separate the spectral information for mixture's components. We describe a fast and flow-compatible diffusion NMR experiment that makes it possible to collect accurate diffusion data for samples flowing at up to 3 mL/min. We use it to monitor the synthesis of a Schiff base with a flow-tube with a time resolution of approximately 2 minutes. The one-shot flow-compatible diffusion NMR described here open many avenues for reaction monitoring applications.     

Improved DECRA processing of DOSY data: correcting for non-uniform field gradients

Mixture analysis using PFG-NMR (DOSY) data is, for many chemists, a valuable and increasingly popular technique where the NMR signals of different species are separated according to their diffusion coefficients. Where NMR signals overlap, however, it is often difficult to extract the spectra of pure components from experimental data. In such situations, it can often be helpful to use multivariate methods, which exploit all the available signal covariance, to resolve the spectra of the components of a mixture. The best-established and by some way the quickest such method, DECRA (Direct Exponential Curve Resolution Algorithm), unfortunately requires that data conform to a pure exponential decay as a function of gradient strength squared, while experimental data typically deviate significantly from this. If this deviation is known, the performance of DECRA can be greatly improved for components with similar diffusion coefficients by adjusting the choice of gradient strengths used.     

Structure elucidation of the intermediate in triethylborane-mediated radical addition of oxime ethers with 2D- and 3D-DOSY NMR

The structures of the components in the triethylborane-mediated radical addition reaction of oxime ether were investigated by 1H- and 3D-DOSY NMR methods. It has been impossible to physically separate the unstable intermediates; therefore, the structures were thus far unidentified. It has been possible to elucidate the structures of these unstable intermediates using Diffusion-Ordered Spectroscopy (DOSY) methods for the reaction in an NMR tube. The DOSY methods resolved the spectra of each starting compound, intermediate and product having different diffusion coefficients. The structure of the intermediate was shown to be due to the bonding of diethylborane to the nitrogen atom of the alkoxyamino group.     

Detection of Potential TNA and RNA Nucleoside Precursors in a Prebiotic Mixture by Pure Shift Diffusion‐Ordered NMR Spectroscopy

In the context of prebiotic chemistry, one of the characteristics of mixed nitrogenous‐oxygenous chemistry is its propensity to give rise to highly complex reaction mixtures. There is therefore an urgent need to develop improved spectroscopic techniques if onerous chromatographic separations are to be avoided. One potential avenue is the combination of pure shift methodology, in which NMR spectra are measured with greatly improved resolution by suppressing multiplet structure, with diffusion‐ordered spectroscopy, in which NMR signals from different species are distinguished through their different rates of diffusion. Such a combination has the added advantage of working with intact mixtures, allowing analyses to be carried out without perturbing mixtures in which chemical entities are part of a network of reactions in equilibrium. As part of a systems chemistry approach towards investigating the self‐assembly of potentially prebiotic small molecules, we have analysed the complex mixture arising from mixing glycolaldehyde and cyanamide, in a first application of pure shift DOSY NMR to the characterisation of a partially unknown reaction composition. The work presented illustrates the potential of pure shift DOSY to be applied to chemistries that give rise to mixtures of compounds in which the NMR signal resolution is poor. The direct formation of potential RNA and TNA nucleoside precursors, amongst other adducts, was observed. These preliminary observations may have implications for the potentially prebiotic assembly chemistry of pyrimidine threonucleotides, and therefore of TNA, by using recently reported chemistries that yield the activated pyridimidine ribonucleotides.     

Studies on supramolecular gel formation using DOSY NMR

Herein, we present the results obtained from our studies on supramolecular self‐assembly and molecular mobility of low‐molecular‐weight gelators (LMWGs) in organic solvents using pulsed field gradient (PFG) diffusion ordered spectroscopy (DOSY) NMR. A series of concentration‐dependent DOSY NMR experiments were performed on selected LMWGs to determine the critical gelation concentration (CGC) as well as to understand the behaviour of the gelator molecules in the gel state. In addition, variable‐temperature DOSY NMR experiments were performed to determine the gel‐to‐sol transition. The PFG NMR experiments performed as a function of gradient strength were further analyzed using monoexponential DOSY processing, and the results were compared with the automated Bayesian DOSY transformation to obtain 2D plots. Our results provide useful information on the stepwise self‐assembly of small molecules leading to gelation. We believe that the results obtained from these experiments are applicable in determining the CGC and gel melting temperatures of supramolecular gels. Copyright © 2015 John Wiley & Sons, Ltd.     

Slice-selected LED and BPPLED: application of slice selection to DOSY

High-resolution DOSY (Diffusion-ordered spectroscopy) is a series of 2-dimensional and 3-dimensional NMR techniques based on the differing diffusivity of constituent molecules in the solution state, with which the individual NMR spectrum of each component in a chemical mixture can be observed. All of the DOSY pulse sequences are derived from the spin-echo or stimulated-echo techniques under the effect of PFG (pulsed field gradient). One of the requirements for successful DOSY experiments and data fitting is that PFG must be uniform across the active sample volume. However, PFG, in general, is not uniform across the active sample volume in commercial high-resolution NMR probes and this nonuniformity of PFG is known to produce systematic errors in DOSY experiments. In fact, a strong and uniform gradient field can be realized only in the central region of the gradient coil and the slice-selection technique, widely used in Magnetic Resonance (MR) imaging, can be employed in resolving problems associated with the nonuniformity of PFG. We have developed a slice-selection pulse block, which can be generally applied to any DOSY pulse sequence with proper care of the phase cycling and experimental parameters. We applied the slice-selection technique to LED and BPPLED pulse sequences, which are among the most popular DOSY pulse sequences, and obtained good experimental results for a chemical mixture.     

DOSY Analysis of Micromolar Analytes: Resolving Dilute Mixtures by SABRE Hyperpolarization

DOSY is an NMR spectroscopy technique that resolves resonances according to the analytes’ diffusion coefficients. It has found use in correlating NMR signals and estimating the number of components in mixtures. Applications of DOSY in dilute mixtures are, however, held back by excessively long measurement times. We demonstrate herein, how the enhanced NMR sensitivity provided by SABRE hyperpolarization allows DOSY analysis of low‐micromolar mixtures, thus reducing the concentration requirements by at least 100‐fold.     

Fine-tuning of the diffusion dimension of –OH groups for high resolution DOSY NMR applications in crude enzymatic transformations and mixtures of organic compounds

A convenient DOSY methodology was developed that can be applied directly in crude reaction products or mixtures containing polyphenol organic compounds, for the rapid identification of their various components without any prior separation or isolation. The method is based on the resolution enhancement of the resonances of the –OH protons and the fine-tuning of their diffusion coefficients to the molecular diffusion coefficient; this can be achieved in DMSO-d₆ in combination with the addition of picric acid and the use of temperatures near the freezing point of the solution. This method, which does not modify the apparent molecular diffusion, allowed the recording of high resolution DOSY spectra, both in crude enzymatic reactions and mixtures of organic compounds based on the phenolic OH NMR spectral region which is much less crowded and, thus, much more informative compared to the aromatic region.     

Characterization of titanium dioxide nanoparticles dispersed in organic ligand solutions by using a diffusion-ordered spectroscopy-based strategy

Diffusion-ordered NMR spectroscopy (DOSY NMR) is presented as a tool for the determination of the diffusion coefficients of organic ligands in suspensions of titanium dioxide nanoparticles. The nanoparticles were prepared by a sol-gel process by hydrolysis and condensation reactions of titanium tetra-n-butoxide in the presence of pentane-2,4-dione (acacH: acetylacetone), as well as para-toluenesulfonic acid (pTsA) and n-butanol (nBuOH). NMR spectroscopic studies were performed in various deuterated solvents, on both dispersed xerosols and diluted sols. The bipolar-pulsed field gradient longitudinal eddy-current delay (LED) pulse sequence was used for data acquisition. The data were processed by inverse Laplace transformation (ILT), by using a maximum entropy algorithm, to afford 2D DOSY spectra. Different diffusion regimes for organic ligands in the bound and unbound states were successfully discriminated, more particularly in [D3]acetonitrile, thus allowing assessment of their interactions with the nanoparticles.     

Enhanced chromatographic NMR with polyethyleneglycol. A novel resolving agent for diffusion ordered spectroscopy

NMR analysis of complex mixtures can be significantly simplified using polyethyleneglycol (PEG) as resolving additive in DOSY NMR technique, which allows the extraction of individual spectra of mixture components with differing polarity. Resolving power of PEG‐assisted DOSY was demonstrated with natural product mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Polydimethylsiloxane: A General Matrix for High‐Performance Chromatographic NMR Spectroscopy

The detection and structural characterization of the components of a mixture is a challenging task. Therefore, the development of a facile and general method that enables both the separation and the structural characterization of the components is desired. Diffusion‐ordered NMR spectroscopy (DOSY) with the aid of a matrix is a promising tool for this purpose. However, because the currently existing matrices only separate limited components, the application of the DOSY technique is restricted. Herein we introduce a new versatile matrix, poly(dimethylsiloxane), which can fully separate many mixtures of different structural types by liquid‐state NMR spectroscopy. With poly(dimethylsiloxane), liquid‐state chromatographic NMR spectroscopy could become a general approach for the structural elucidation of mixtures of compounds.     

NMR experiments for the analysis of mixtures: beyond 1D 1H spectra

State-of-the-art technologies and methodologies in NMR spectroscopy make it possible to obtain very informative and high-quality spectra in much less experimental time than classical methods by making better choices of NMR pulse sequences and acquisition parameters. This review presents some recent NMR methods allowing rapid identification, assignment and structural characterization of the components in mixtures. The relative merits of the different NMR pulse sequences are briefly discussed and recommendations are made for the preferred choice of sequences to obtain rapidly artifact-free data. This review covers diffusion experiments (DOSY), HSQC and HMBC experiments, ultra-resolved 2D spectra exploiting the property of aliasing and NOESY/ROESY experiments. It will be in particular shown that selective 1D NOESY/ROESY sequences can be more informative and reach higher resolution in less experimental time than the corresponding 2D sequences.     

New validation of molecular mass measurements by means of 2D DOSY 1H NMR experiments: Application to surfactants

This study proposes a new correlation equation between surfactant diffusion coefficients and molecular mass using 2D DOSY ¹H NMR experiment. Indeed, DOSY is a suitable NMR technique to discriminate the different components in a mixture. Using Inverse Laplace Transform (ILT), all chemical shifts in the direct dimension are dispersed along the second dimension in relation to the diffusion coefficient. This approach does not make any assumption about the number of exponential components. The empiric equation was determined for anionic and non-ionic surfactants dissolved in dimethylsulfoxide (DMSO). The parameters, K = C_r = 5.18 ± 0.70 and a shape factor α = 0.45 ± 0.02 that is related to the molecular family fractal dimension constant δ_F = 2.21 ± 0.11, have been extracted from the data. The equation validity was checked for simple commercial raw material and a simple mixture composed of three surfactants classically used in detergent formulations. This equation, which describes quite satisfactorily the diffusion phenomenon of surfactants, could be used for deformulation purpose to check commercial products in case of quality control or adulteration assessment.