Quantitative determination of the average molecular weights of dextrans by diffusion ordered NMR spectroscopy

Diffusion ordered NMR (DOSY) was applied to the determination of the average molecular weights of polymers based on the dependence of the measured self-diffusion coefficient D on the corresponding weights of a number of polymers of the same type. As a rule, a calibration function is plotted by varying DOSY experimental parameters for each particular test sample; however, this approach is inapplicable to the development of a standard procedure for the quantitative assessment of the molecular weights of dextrans. In this article, an optimization method is considered to ensure high resolution in terms of the values of D for dextrans without varying experimental conditions; this method makes it possible to evaluate the average molecular weights of dextrans with a high accuracy over a wide range of their values.     

Pivotal steps towards quantification of molecular diffusion coefficients by NMR.

Using nuclear magnetic resonance (NMR) spectroscopy with a pair of pulsed field gradients (PFGs), Stajeskal and Tanner successfully measured molecular diffusion coefficients in solution in 1965. This method has since been used extensively in various applications, especially after the PFG was implemented in commercial NMR probes. Due to the nonuniformity of the PFG and radio frequency (RF) fields, molecules distributed throughout the sample experience different PFG and RF fields and contribute unevenly to the measured diffusion coefficients, resulting in considerable errors in conventional NMR diffusion experiments. By selective excitation of a central sample region with an offset-independent adiabatic inversion pulse and a PFG, a uniform RF field can be assumed, and the PFG can be represented as a linear approximation. Under these conditions, the molecules diffuse as if they were all experiencing the same effective gradient g(e), leading to a Gaussian signal decay as a function of the PFG strength. Quantitative measurement of molecular diffusion coefficients is therefore made possible. From the diffusion coefficient of a 90 % H(2)O/10 % D(2)O sample, it is convenient to calibrate g(e) with a Java program. In a similar way the nonlinearity of the PFG can be corrected.     

Determining accurate molecular sizes in solution through NMR diffusion spectroscopy

This tutorial review deals with the methodological procedures that can be used to obtain accurate molecular sizes in solution from diffusion NMR spectroscopy. The critical aspects associated with the estimation of the size of molecules from the measured translational self-diffusion coefficient, using the Stokes-Einstein equation, are highlighted and discussed. From a theoretical point of view, it is shown how to take into account the size of the solute with respect to that of the solvent and its non-spherical shape using the appropriate correction factors in the frictional coefficient. From a practical point of view, the advantages of introducing an internal standard in the sample are presented. Initially, non-aggregating systems are considered in an attempt to clarify what hydrodynamic dimensions mean. Successively, aggregating systems are addressed showing how it is possible to understand the aggregation level and derive the thermodynamic parameters for some illustrative aggregation processes.     

On measurement of molecular and thermal diffusion coefficients in multicomponent mixtures

We have developed the theory for using the deflection of laser beams of various wavelengths to determine molecular and thermal diffusion coefficients in multicomponent mixtures. In the past, simultaneous determination of molecular and thermal diffusion coefficients have only been achieved for binaries. Our procedure is faster and more accurate than the current techniques to determine molecular diffusion coefficients. The analysis for an N-component mixture requires deflections from (N - 1) beams of different wavelengths. We show in an example that the molecular and thermal diffusion coefficients can be determined reliably in a ternary mixture.     

Slow diffusion by singlet state NMR spectroscopy

Small diffusion coefficients can be measured by using populations of singlet states that have a relaxation time constant, T(s), which can be much longer than the longitudinal relaxation time, T1. Spatial information can be encoded with pulsed field gradients in the manner of stimulated echo sequences. Singlet states can be excited via double-quantum coherences to enhance the efficiency of phase encoding and decoding.     

NMR measurement of self-diffusion coefficients by slice selection

Most of the time, so-called inversion-recovery experiments concern longitudinal nuclear magnetization of the whole sample, the region of interest being limited by the transmitting-receiving coil. Here we address the question of what occurs if the region of interest is purposely limited to a thin slice selected by means of procedures employing magnetic field gradients. Gradients of both magnetic fields (B(0), the static magnetic field, and B(1), the radio-frequency magnetic field) can be used. In this study we resorted essentially to B(1) gradients and novel procedures, based on the natural inhomogeneity of the B(1) field delivered by a saddle coil, are described. It is obvious that molecules leaving and entering the slice during the evolution (recovery) period should influence the magnetization recovery. Molecular self-diffusion is responsible for such effects, experimentally visible and accounted for by an appropriate theory which has been approximated for by permitting an easy physical assessment. This approach should lead to alternative methods for measuring self-diffusion coefficients.     

Quantitative 15N NMR spectroscopy

Line intensities in ¹⁵N NMR spectra are strongly influenced by spin-lattice and spin–spin relaxation times, relaxation mechanisms and experimental conditions. Special care has to be taken in using ¹⁵N spectra for quantitative purposes. Quantitative aspects are discussed for the ¹⁵N NMR of molecules with different nitrogen functional groups and also mixtures of nitrogen-containing compounds. It is shown that, in general, quantitative data are obtainable from integration of ¹⁵N lines in proton decoupled ¹⁵N NMR spectra using NOE suppression. Addition of paramagnetic relaxation reagents (PARR) under controlled conditions is frequently needed to accomplish the experiment within reasonable time limits.     

The determination of molecular diffusion coefficients by the barometric method

The binary molecular diffusion coefficients D _AB of a vaporizing liquid in gases under atmospheric conditions were measured by the new simple barometric method. A change in the pressure of a vapor-gas mixture in a closed cell was determined as a function of time. Under experimental conditions, one can also find partial saturated vapor pressures of substances. The deviations of the coefficients D _AB obtained by the new procedure from the literature values were within the spread of the experimental data. The D _AB coefficients (absent in the literature) were measured for mixtures of acetone, ethyl acetate, carbon tetrachloride, difluoroethanol, and cis and trans isomers of perfluorodecalin with argon. The saturated vapor pressures were determined for difluoroethanol and cis-and trans-perfluorodecalins under argon.     

Kinetics of the grating formation in holographic polymer-dispersed liquid crystals: NMR measurement of diffusion coefficients

Polymer films with embedded liquid crystal inclusions (polymer-dispersed liquid crystals) are superb composites for addressable windows, flexible displays and optical storage. Their scattering behavior and electro-optic properties depend essentially on the shape and size of the liquid crystal inclusions, which are typically formed by phase separation from a multicomponent homogeneous mixture. Here, pulsed field gradient NMR is used to measure the self-diffusion coefficients of the liquid crystal and a photo-reactive monomer, which compose such a precursor mixture. The kinetics of holographic grating formation in this mixture can be predicted by inserting the NMR diffusion coefficient of the monomer and the polymerization rate in a reaction diffusion model. The ratio of diffusion rate over reaction rate is found to be in the limiting regime in which the kinetics of the grating formation is not sensitive to this parameter.     

Efficient determination of diffusion coefficients by monitoring transport during recovery delays in NMR

A novel NMR approach allows one to efficiently determine translational diffusion coefficients of macromolecules in solution. This method for Signal Optimization with Recovery in Diffusion Delays (SORDID) monitors transport occurring during the recovery times between consecutive scans so that the duration of the measurements can be reduced approximately by a factor two.     

Properties of small molecular drug loading and diffusion in a fluorinated PEG hydrogel studied by 1H molecular diffusion NMR and 19F spin diffusion NMR

R(f)-PEG (fluoroalkyl double-ended poly(ethylene glycol)) hydrogel is potentially useful as a drug delivery depot due to its advanced properties of sol-gel two-phase coexistence and low surface erosion. In this study, (1)H molecular diffusion nuclear magnetic resonance (NMR) and (19)F spin diffusion NMR were used to probe the drug loading and diffusion properties of the R(f)-PEG hydrogel for small anticancer drugs, 5-fluorouracil (FU) and its hydrophobic analog, 1,3-dimethyl-5-fluorouracil (DMFU). It was found that FU has a larger apparent diffusion coefficient than that of DMFU, and the diffusion of the latter was more hindered. The result of (19)F spin diffusion NMR for the corresponding freeze-dried samples indicates that a larger portion of DMFU resided in the R(f) core/IPDU intermediate-layer region (where IPDU refers to isophorone diurethane, as a linker to interconnect the R(f) group and the PEG chain) than that of FU while the opposite is true in the PEG-water phase. To understand the experimental data, a diffusion model was proposed to include: (1) hindered diffusion of the drug molecules in the R(f) core/IPDU-intermediate-layer region; (2) relatively free diffusion of the drug molecules in the PEG-water phase (or region); and (3) diffusive exchange of the probe molecules between the above two regions. This study also shows that molecular diffusion NMR combined with spin diffusion NMR is useful in studying the drug loading and diffusion properties in hydrogels for the purpose of drug delivery applications.     

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.     

On measurement of thermal diffusion coefficients in multicomponent mixtures

We investigate the steady-state separation of the individual components of an incompressible multicomponent liquid mixture in a narrow two-dimensional thermogravitational column. Analytic working equations for measuring thermal diffusion coefficients analogous to the existing equations for a binary mixture are derived. Similar to the binary results, we find that when compositional variation has negligible effect on fluid density and vertical diffusive flux can be ignored, molecular diffusion does not affect steady-state separation. However, when compositional effects on density are taken into account, molecular diffusion does affect the bulk convective flow and the steady-state separation of the components. There may be also two distinct trends in the velocity and separation profiles. With one or more negative thermal diffusion coefficients, there may be more than one convection cell resulting in oscillatory behavior of separation. The working equations presented can be used to measure thermal diffusion coefficients of multicomponent mixtures. Such measurements have not yet been reported in the literature.     

Effective diffusion coefficients for methanol in sulfuric acid solutions measured by Raman spectroscopy

The diffusion of methanol into 0-96.5 wt % sulfuric acid solutions was followed using Raman spectroscopy. Because methanol reacts to form protonated methanol (CH 3OH 2 (+)) and methyl hydrogen sulfate in H 2SO 4 solutions, the reported diffusion coefficients, D, are effective diffusion coefficients that include all of the methyl species diffusing into H 2SO 4. The method was first verified by measuring D for methanol into water. The value obtained here, D = (1.4 +/- 0.6) x 10 (-5) cm (2)/s, agrees well with values found in the literature. The values of D in 39.2-96.5 wt % H 2SO 4 range from (0.11-0.3) x 10 (-5) cm (2)/s, with the maximum value of D occurring for 61.6 wt % H 2SO 4. The effective diffusion coefficients do not vary systematically with the viscosity of the solutions, suggesting that the speciation of both methanol and sulfuric acid may be important in determining these transport coefficients.     

Weak molecular complexes as studied by NMR spectroscopy of oriented molecules

Weak molecular interactions such as those in pyridine—iodine, benzene—iodine and benzene—chloroform systems oriented in thermotropic liquid crystals have been studied from the changes of the order parameters as a result of complex formation. The results indicate the formation of at least two types of charge transfer complexes in pyridine—iodine solutions. The pi-complexes in benzene—chloroform and benzene—iodine mixtures have also been detected. No detectable changes in the inter-proton distances in these systems were observed.     

Quantitative analysis of a mixture of cresols by carbon-13 NMR spectroscopy

The suitability of ¹³C NMR as a technique for determining o-, m, and p-cresols in their mixtures was investigated. Spectra were determined in methanol, containing o-toluidine as an internal standard, without nuclear Overhauser suppression. Ratios of peak areas of each cresol isomer to o-toluidine were determined by means of the standard calibration method, and then mixtures of cresol isomers were prepared and quantified in varying concentrations from 8.30 to 66.84 mol% for each isomer, with an average absolute error of 0.78% for all isomers. The method described was convenient and the results were generally satisfactory.     

Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures

A simple and versatile methodology has been developed for the simultaneous measurement of multiple concentration profiles of colourants in transparent microfluidic systems, using a conventional transmitted light microscope, a digital colour (RGB) camera and numerical image processing combined with multicomponent analysis. Rigorous application of the Beer-Lambert law would require monochromatic probe conditions, but in spite of the broad spectral bandwidths of the three colour channels of the camera, a linear relation between the measured optical density and dye concentration is established under certain conditions. An optimised collection of dye solutions for the quantitative optical microscopic characterisation of microfluidic devices is proposed. Using the methodology for optical concentration measurement we then implement and validate a simplified and robust method for the microfluidic measurement of diffusion coefficients using an H-filter architecture. It consists of measuring the ratio of the concentrations of the two output channels of the H-filter. It enables facile determination of the diffusion coefficient, even for non-fluorescent molecules and nanoparticles, and is compatible with non-optical detection of the analyte.     

Quantitative determination of glyphosate in human serum by 1H NMR spectroscopy

The determination and quantification of glyphosate in serum using (1)H NMR spectroscopy is reported. This method permitted serum samples to be analysed without derivatization or any other sample pre-treatment, using 3-trimethylsilyl 2,2',3,3'-tetradeuteropropionic acid (TSP-d(4)) as a qualitative and quantitative standard. Characterization of the herbicide N-(phosphonomethyl)glycine was performed by analysing chemical shifts and coupling constant patterns. Quantification was performed by relative integration of CH(2)-P protons to the TSP-d(4) resonance peak. The method was tested for repeatability (n=5) and yielded coefficients of variation of 1% and 3%, respectively: detection and quantification limits were also determined and were 0.03 and 0.1mmol/L, respectively. The method was applied to the quantification of glyphosate in a case of acute poisoning.     

Feasibility study for the rapid screening of target molecules using translational diffusion coefficients: diffusion-ordered NMR spectroscopy of biological toxins

A panel of 15 biological toxins ranging between ~60–28,000 g/mol was used to evaluate the feasibility of screening aqueous samples for toxin analytes based on their translational diffusion coefficients, D _t. Toxin D _t values were measured by pulsed-field gradient ¹H NMR spectroscopy using a bipolar pulse pair, longitudinal eddy current delay pulse sequence incorporating water suppression to achieve the maximum dynamic range for toxin signals. To collect data for an effective screening protocol, reference D _t values were determined from five independent measurements at both 25 and 37 °C for all toxins in the panel. In the protocol, D _t values are measured at both temperatures for a suspected toxin target in a sample, and for assignment as a potential toxin analyte, the measurements are required to fall within ±0.25 × 10⁻⁶ cm²/s of both reference D _t values for at least one toxin in the panel. Only solution viscosity was found to influence sample D _t measurements appreciably; however, the measurements are easily corrected for viscosity effects by calculating the D _t value of the suspected toxin at infinite dilution. In conclusion, the protocol provides a rapid and effective means for screening aqueous samples for all toxins in the panel, narrowing toxin identification to ≤2 possibilities in virtually all cases.