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.     

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.     

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.     

Assessment of techniques for DOSY NMR data processing

Diffusion-ordered spectroscopy (DOSY) NMR is based on a pulse-field gradient spin-echo NMR experiment, in which components experience diffusion. Consequently, the signal of each component decays with different diffusion rates as the gradient strength increases, constructing a bilinear NMR data set of a mixture. By calculating the diffusion coefficient for each component, it is possible to obtain a two-dimensional NMR spectrum: one dimension is for the conventional chemical shift and the other for the diffusion coefficient. The most interesting point is that this two-dimensional NMR allows non-invasive “chromatography” to obtain the pure spectrum for each component, providing a possible alternative for LC-NMR that is more expensive and time-consuming. Potential applications of DOSY NMR include identification of the components and impurities in complex mixtures, such as body fluids, or reaction mixtures, and technical or commercial products, e.g. comprising polymers or surfactants.

Data processing is the most important step to interpret DOSY NMR. Single channel methods and multivariate methods have been proposed for the data processing but all of them have difficulties when applied to real-world cases. The big challenge appears when dealing with more complex samples, e.g. components with small differences in diffusion coefficients, or severely overlapping in the chemical shift dimension. Two single channel methods, including SPLMOD and continuous diffusion coefficient (CONTIN), and two multivariate methods, called direct exponential curve resolution algorithm (DECRA) and multivariate curve resolution (MCR), are critically evaluated by simulated and real DOSY data sets. The assessments in this paper indicate the possible improvement of the DOSY data processing by applying iterative principal component analysis (IPCA) followed by MCR-alternating least square (MCR-ALS).     

Matrix‐assisted diffusion‐ordered spectroscopy: choosing a matrix

Diffusion‐ordered spectroscopy (DOSY) is an important technique for separating the NMR signals of the components in a mixture, and relies on differences in diffusion coefficient. Standard DOSY experiments therefore struggle when the components of a mixture are of similar size, and hence diffuse at similar rates. Fortunately, the diffusion coefficients of solutes can be manipulated by changing the matrix in which they diffuse, using matrix components that interact differentially with them, a technique known as matrix‐assisted DOSY. In the present investigation, we evaluate the performance of a number of new, previously used, and mixed matrices with an informative test mixture: the three positional isomers of dihydroxybenzene. The aim of this work is to present the matrix‐assisted DOSY user with information about the potential utility of a set of matrices (and combinations of matrices), including ionic and non‐ionic surfactants, complexing agents, polymers, and mixed solvents. A variety of matrices improved the diffusion resolution of the signals of the test system, with the best separation achieved by mixed micelles of sodium dodecyl sulfate and cetyl trimethylammonium bromide. The use of mixed matrices offers great potential for the analyst to tailor the matrix to a particular sample under study. © 2016 The Authors Magnetic Resonance in Chemistry Published by John Wiley & Sons, Ltd.     

Matrix‐assisted diffusion‐ordered spectroscopy: application of surfactant solutions to the resolution of isomer spectra

The component spectra of a mixture of isomers with nearly identical diffusion coefficients cannot normally be distinguished in a standard diffusion‐ordered spectroscopy (DOSY) experiment but can often be easily resolved using matrix‐assisted DOSY, in which diffusion behaviour is manipulated by the addition of a co‐solute such as a surfactant. Relatively little is currently known about the conditions required for such a separation, for example, how the choice between normal and reverse micelles affects separation or how the isomer structures themselves affect the resolution. The aim of this study was to explore the application of sodium dodecyl sulfate (SDS) normal micelles in aqueous solution and sodium 1,4‐bis(2‐ethylhexyl)sulfosuccinate (AOT) aggregates in chloroform, at a range of concentrations, to the diffusion resolution of some simple model sets of isomers such as monomethoxyphenols and short chain alcohols. It is shown that SDS micelles offer better resolution where these isomers differ in the position of a hydroxyl group, whereas AOT aggregates are more effective for isomers differing in the position of a methyl group. For both the normal SDS micelles and the less well‐defined AOT aggregates, differences in the resolution of the isomers can in part be rationalised in terms of differing degrees of hydrophobicity, amphiphilicity and steric effects. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Resolving natural product epimer spectra by matrix-assisted DOSY

High resolution diffusion-ordered NMR spectroscopy allows the separation of signals from different species based on their diffusion coefficients. In general this requires that the NMR spectra of the components do not have overlapping signals, and that the diffusion coefficients are significantly different. Modifying the solvent matrix in which a sample is dissolved can change the diffusion coefficients observed, allowing resolution ("matrix-assisted DOSY"). We show here that dissolving the two naturally-occurring epimers of naringin in an aqueous solution of β-cyclodextrin causes both shift and diffusion changes, allowing the signals of the epimers to be distinguished. Chiral matrix-assisted DOSY has the potential to allow simple resolution and assignment of the spectra of epimers and enantiomers, without the need for derivatisation or for titration with a shift reagent.     

NMR chromatography using microemulsion systems

NMR spectroscopy is an excellent tool for structural analysis of pure compounds. However, for mixtures, it performs poorly because of overlapping signals. Diffusion ordered NMR spectroscopy (DOSY) can be used to separate the spectra of compounds with widely differing molecular weights, but the separation is usually insufficient. NMR "chromatographic" methods have been developed to increase the diffusion separation but these usually introduced solids into the NMR sample that reduce resolution. Using nanostructured dispersed media, such as microemulsions, eliminates the need for suspensions of solids and brings NMR chromatography into the mainstream of NMR analytical techniques. DOSY was used in this study to resolve spectra of mixtures with no increase in line-width as compared to regular solutions. Components of a mixture are differentially dissolved into the separate phases of the microemulsions. Several examples of previously reported microemulsions and those specifically developed for this purpose were used here. These include a fully dilutable microemulsion, a fluorinated microemulsion, and a fully deuterated microemulsion. Log(diffusion) difference enhancements of up to 1.7 orders of magnitude were observed for compounds that have similar diffusion rates in conventional solvents. Examples of commercial pharmaceutical drugs were also analyzed via this new technique, and the spectra of up to six components were resolved from one sample.     

Matrix‐assisted diffusion‐ordered spectroscopy: mixture resolution by NMR using SDS micelles

Diffusion‐ordered spectroscopy (DOSY) is a powerful technique for mixture analysis, but in its basic form it cannot separate the component spectra for species with very similar diffusion coefficients. It has been recently demonstrated that the component spectra of a mixture of isomers with nearly identical diffusion coefficients (the three dihydroxybenzenes) can be resolved using matrix‐assisted DOSY (MAD), in which diffusion is perturbed by the addition of a co‐solute such as a surfactant [R. Evans, S. Haiber, M. Nilsson, G. A. Morris, Anal. Chem. 2009, 81, 4548–4550]. However, little is known about the conditions required for such a separation, for example, the concentrations and concentration ratios of surfactant and solutes. The aim of this study was to explore the concentration range over which matrix‐assisted DOSY using the surfactant SDS can achieve diffusion resolution of a simple model set of isomers, the monomethoxyphenols. The results show that the separation is remarkably robust with respect to both the concentrations and the concentration ratios of surfactant and solutes, supporting the idea that MAD may become a valuable tool for mixture analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Natural products dereplication by diffusion ordered NMR spectroscopy (DOSY)

Diffusion-ordered NMR spectroscopy (DOSY) can be used to analyze mixtures of compounds since resonances deriving from different compounds are distinguished by their diffusion coefficients (D). Previously, DOSY has mostly been used for organometallic and polymer analysis, we have now applied DOSY to investigate diffusion coefficients of structurally diverse organic compounds such as natural products (NP). The experimental Ds derived from 55 diverse NPs has allowed us to establish a power law relationship between D and molecular weight (MW) and therefore predict MW from experimental D. We have shown that D is also affected by factors such as hydrogen bonding, molar density and molecular shape of the compound and we have generated new models that incorporate experimentally derived variables for these factors so that more accurate predictions of MW can be calculated from experimental D. The recognition that multiple physicochemical properties affect D has allowed us to generate a polynomial equation based on multiple linear regression analysis of eight calculated physicochemical properties from 63 compounds to accurately correlate predicted D with experimental D for any known organic compound. This equation has been used to calculate predicted D for 217 043 compounds present in a publicly available natural product database (DEREP-NP) and to dereplicate known NPs in a mixture based on matching of experimental D and structural features derived from NMR analysis with predicted D and calculated structural features in the database. These models have been validated by the dereplication of a mixture of two known sesquiterpenes obtained from Tasmannia xerophila and the identification of new alkaloids from the bryozoan Amathia lamourouxi. These new methodologies allow the MW of compounds in mixtures to be predicted without the need for MS analysis, the dereplication of known compounds and identification of new compounds based solely on parameters derived by DOSY NMR.

We report accurate DOSY NMR based molecular weight and diffusion coefficient prediction tools. These tools can be used to dereplicate known natural products from databases using structurally rich NMR data as a surrogate for mass spectrometric data.     

Independent component analysis applied to diffusion‐ordered spectroscopy: separating nuclear magnetic resonance spectra of analytes in mixtures

Nuclear magnetic resonance spectrum of a mixture contains the overall peaks of all the analytes. It is impossible to perform structural assignment on the mixture without the knowledge of individual spectra of the components. Spectral separation is thus an important means of teasing out pure components of a mixture before spectral assignment. We propose a strategy called diffusion‐ordered independent component analysis (DIFFICA) to achieve this task. This strategy applies independent component analysis algorithms to diffusion‐ordered spectroscopy (DOSY) to extract spectra of pure components in a mixture. DIFFICA was tested in a simulation and experimentally in two three‐component systems with and without water suppression, in 1D and 2D DOSY data. Pure spectra were achieved in both cases. The selection of diffusion parameters to guarantee pure spectra is guided by the distance correlation between separated spectra. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Diffusion-Ordered Spectroscopy (DOSY) as a Powerful NMR Method to Investigate Molecular Mobilities and Intermolecular Interactions in Complex Oil Mixtures

Studying the characteristics and molecular mechanisms of liquid self-diffusion coefficient and viscosity changes is of great significance for, e. g., chemical and petroleum processing. As examples of highly complex liquid,an asphaltene-free high-acid and high-viscosity crude oil and its extracted fractions were studied by comparing their ¹H DOSY diffusion maps. The crude oil exhibited a polydisperse diffusion distribution, including multiple diffusion portions with diffusion coefficients much smaller than that of any single fraction in independent diffusion. The main mechanism that leads to the decreases in the diffusion coefficients of crude oil is attributed to diffusion resistance enhanced by Dynamical Molecular-Interaction Networks (DMINs), rather than by enlargement of the diffusion species caused by molecular aggregation. Constructed through the synergistic interactions of various polar molecules in crude oil, DMINs dynamically bind polar molecules, trap polarizable molecules, and spatially hinder the free motion of non-polar molecules. Overall, this reduces the mobility of all molecular species, as illustrated by the decreased diffusion coefficients. This study demonstrates that DOSY is a powerful NMR method to investigate molecular motion abilities also in complex mixtures. In addition, the insights in the influence of the interaction matrix on the molecular mobility also help to understand the contribution of “structural viscosity” to the viscosity of heavy oil.     

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.     

Orthogonal extraction ion mobility spectrometry

Ion Mobility Spectrometry is a powerful tool for the study of molecular conformations, separation of mass isomers, and analysis of complex mixtures and suppression of chemical background. The factors that limit the capabilities of the technique include its relatively low resolving power and duty cycle. New principle of gas-phase ion separation, based on ion focusing under the influence of electrostatic field and stationary in time gas flow, is proposed. Both analytical calculations and a numerical simulation show that a diffusion-limited resolution of several hundred can be achieved. The new type of ion mobility analyzer is called orthogonal extraction IMS. The proposed ortho-IMS can be interfaced with commercial mass spectrometers and offers the theoretical resolution of several hundred and ion transmission close to 100%.     

Comparison of oil-in-water and water-in-oil microemulsion electrokinetic chromatography as methods for the analysis of eight phenolic acids and five diterpenoids

Oil-in-water (O/W) and water-in-oil (W/O) MEEKC were compared for their abilities to separate and detect eight phenolic acids and five diterpenoids in Radix et Rhizoma Salviae Miltiorrhizae (RRSM). The effects of oil type and concentration, organic modifier, SDS, and buffer concentration on separation were examined in order to optimize the two methods. Oil contents and organic modifier were found to markedly influence the separation selectivity for both O/W and W/O systems. SDS concentration rarely affected separation resolution for O/W MEEKC, and separation of eight phenolic acids and five diterpenoids could be improved by changing the buffer concentration for W/O MEEKC. A highly efficient O/W MEEKC separation method, where the 13 compounds were separated with baseline resolution, was achieved by using a microemulsion solution of pH 8.0 containing 0.6% cyclohexane, 3.0% SDS, 6.0% 1-butanol, and 3.0% ACN. The W/O MEEKC was unable to resolve all the components. In addition, the analytic time in O/W MEEKC was shorter than that in W/O MEEKC. Finally, the developed O/W MEEKC method was successfully applied to determine analytic compounds in RRSM samples.