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.     

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.     

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.     

Computer simulation studies of surfactant monolayer mixtures at the water/oil interface: charge distribution effects

Charge distribution effects on polar head groups for a mixture of amphiphilic molecules at the water/oil interface were studied. For this purpose a model which allowed us to investigate the charge effects exclusively was created. As a molecular model we used the structure of sodium dodecyl sulfate. Then we prepared molecules with the same molecular structure but with different charge distributions in order to have one cationic and one nonionic molecule. So, in this way, we were able to focus only in the charge effects. The monolayer mixtures were composed of anionic/nonionic and cationic/nonionic surfactants. Simulations of these systems show that the location of the different surfactants at the interface is determined by the interaction and the charge distribution of the molecules. Due to the difference in the charge distribution of the surfactant monolayers, the water molecules present distinct orientations in the mixture. Finally, it was found that the electrostatic potential difference across the interface depended on the interactions (charge distribution) of the anionic, cationic, and nonionic molecules in the mixture.     

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.     

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.     

19F DOSY NMR analysis for spin systems with nJFF couplings

NMR is a powerful method for identification and quantification of drug components and contaminations. These problems present themselves as mixtures, and here, one of the most powerful tools is DOSY. DOSY works best when there is no spectral overlap between components, so drugs containing fluorine substituents are well‐suited for DOSY analysis as ¹⁹F spectra are typically very sparse. Here, we demonstrate the use of a modified ¹⁹F DOSY experiment (on the basis of the Oneshot sequences) for various fluorinated benzenes. For compounds with significant ⁿJ_FF coupling constants, as is common, the undesirable J‐modulation can be efficiently suppressed using the Oneshot45 pulse sequence. This investigation highlights ¹⁹F DOSY as a valuable and robust method for analysis of molecular systems containing fluorine atoms even where there are large fluorine–fluorine couplings. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

Correction of systematic errors in CORE processing of DOSY data

DOSY data for mixtures are commonly processed either by single channel methods (e.g. HR-DOSY) or multichannel methods (e.g. CORE). Both aim to separate the signals from species of different molecular sizes by their diffusion coefficients; the result is displayed either as a 2D plot (as in HR-DOSY) or as individual spectra (as in CORE). Both types of methods are sensitive to any systematic errors in the experimental data. The effects of, and remedies for, two such sources of error, spatially non-uniform pulsed field gradients (PFGs) and instrument instability, are demonstrated for CORE processing, using a corrected form of the Stejskal-Tanner equation and reference deconvolution, respectively.     

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.     

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.     

Isotope beating effects in the analysis of polymer distributions by Fourier transform mass spectrometry

For Fourier transform mass spectrometry analysis of high mass ions, the signals from closely spaced isotope peaks undergo periodic destructive interference, producing a beat pattern in the time-domain signal. The mass spectra that are obtained by sampling transient signals for less than two beat periods exhibit an error in the relative abundances that are measured. This effect is shown to cause significant errors in the measurement of the relative abundances of the components of polymer distributions, leading to errors in the derived average molecular weights for such samples. Computer simulations show that isotope beating causes this error to increase as the duration of an acquired transient becomes short compared to the beating period. This error becomes insignificant when the transient is acquired for longer than twice the beat period. Experimental data are presented for polymers in which an oligomeric distribution of monoisotopic peaks is produced by stored waveform inverse Fourier transform ejection of all ¹³C-containing isotope peaks. The data show that the isotope beating-induced abundance errors are eliminated when there are no isotope peaks present.     

Synthesis of Nonionic Oligomeric Manganese(Ⅱ) Complexes and Investigation of Their Toxicity and Τ1-Relaxation Enhancement

Six nonionic oligomeric manganese(Ⅱ) complexes with oligomeric phosphate-polyglycol-EDTA ester ligands were synthesized and characterized. The longitudinal relaxivities of these complexes were measured. One of these complexes, which showed the highest relaxivity, was chosen to be used in the acute toxicity test and the Τ1-weighted imaging experiment. It has been found that compared to Gd-DTPA, this nonionic oligomeric Mn(Ⅱ) complex exhibits no acute toxicity, generates highly enhanced MRI signals and increases the intention time in the rat liver tissue.     

Synthesis of Nonionic Oligomeric Manganese（ Ⅱ ） Complexes and Investigation of Their Toxicity and T1 -Relaxation Enhancement

Six nonionic oligomeric manganese （ Ⅱ ） complexes with oligomeric phosphate-polyglycol-EDTA ester ligands were synthesized and characterized. The longitudinal relaxivities of these complexes were measured. One of these complexes, which showed the highest relaxivity, was chosen to be used in the acute toxicity test and the T1-weighted imaging experiment. It has been found that compared to Gd-DTPA, this nonionic oligomeric Mn（ Ⅱ ） complex exhibits no acute toxicity, generates highly enhanced MRI signals and increases the intention time in the rat liver tissue.     

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.     

On-Line HPLC-UV-mass spectrometry and tandem mass spectrometry for the rapid delineation and characterization of differences in complex mixtures: a case study using toxic oil variants

An integrated differential approach to the characterization of complex mixtures is presented which includes the targeting of liquid chromatography (LC) peaks for identification using characteristic UV adsorption of the LC peak, subsequent molecular weight and formula determination using accurate mass LC mass spectrometry (MS), and structure characterization using accurate mass LC-tandem mass spectrometry. The use of differential UV adsorption aids in narrowing the scope of the study to only specific peaks of interest. Accurate mass measurement of the molecular ion species provides molecular weight information as well as atomic composition information. The tandem MS (MS/MS) spectra provide fragmentation information which allows for structural characterization of each component. Accurate mass assignment of each of the fragment ions in the MS/MS spectrum provides atomic composition for each of the fragment ions and thus further aids in the structural characterization. These experiments are facilitated through the use of on-line LC-MS and LC-MS/MS with in-line UV detection. A synthetic toxic oil (STO) related to Toxic Oil Syndrome is studied with a focus on possible contaminants resulting from the interaction of aniline, used as a denaturant, with the normal components of the oil. A differential analysis between the STO and a control oil is performed. LC peaks were targeted using UV absorbance to indicate the possible presence of the aniline moiety. Further differential analysis was performed through the determination of the MS signals associated with each component separated on the LC. Finally, the MS/MS data was also used to determine if the fragmentation of the targeted components indicated the presence of aniline. The MS/MS and accurate mass data were used to assign the structures for the targeted components.     

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).     

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.     

Protic ionic liquids based on the dimeric and oligomeric anions: [(AcO)xH(x-1)]-

We describe a fluidity and conductivity study as a function of composition in N-methylpyrrolidine-acetic acid mixtures. The simple 1 : 1 acid-base mixture appears to form an ionic liquid, but its degree of ionicity is quite low and such liquids are better thought of as poorly dissociated mixtures of acid and base. The composition consisting of 3 moles acetic acid and 1 mole N-methylpyrrolidine is shown to form the highest ionicity mixture in this binary due to the presence of oligomeric anionic species [(AcO)(x)H(x-1)](-) stabilised by hydrogen bonds. These oligomeric species, being weaker bases than the acetate anion, shift the proton transfer equilibrium towards formation of ionic species, thus generating a higher degree of ionicity than is present at the 1 : 1 composition. A Walden plot analysis, thermogravimetric behaviour and proton NMR data, as well as ab initio calculations of the oligomeric species, all support this conclusion.