Conditions to obtain precise and true measurements of the intramolecular C distribution in organic molecules by isotopic C nuclear magnetic resonance spectrometry

Intramolecular ¹³C composition gives access to new information on the (bio) synthetic history of a given molecule. Isotopic ¹³C NMR spectrometry provides a general tool for measuring the position-specific ¹³C content. As an emerging technique, some aspects of its performance are not yet fully delineated. This paper reports on (i) the conditions required to obtain satisfactory trueness and precision for the determination of the internal ¹³C distribution, and (ii) an approach to determining the “absolute” position-specific ¹³C content. In relation to (i), a precision of <1% can be obtained whatever the molecule on any spectrometer, once quantitative conditions are met, in particular appropriate proton decoupling efficiency. This performance is a prerequisite to the measurement of isotope fractionation either on the transformed or residual compound when a chemical reaction or process is being studied. The study of the trueness has revealed that the response of the spectrometer depends on the ¹³C frequency range of the studied molecule, i.e. the chemical shift range. The “absolute value” and, therefore, the trueness of the ¹³C NMR measurements has been assessed on acetic acid and by comparison to the results obtained on the fragments from COOH and CH₃ by isotopic mass spectrometry coupled to a pyrolysis device (GC-Py–irm-MS), this technique being the reference method for acetic acid. Of the two NMR spectrometers used in this work, one gave values that corresponded to those obtained by GC-Py–irm-MS (thus, the “true” value) while the other showed a bias, which was dependent to the range covered by the resonance frequencies of the molecule. Therefore, the former can be used directly for studying isotope affiliations, while the latter can only be used directly for comparative data, for example in authenticity studies, but can also be used to obtain the true values by applying appropriate correction factors. The present study assesses several key protocol steps required to enable the determination of position-specific ¹³C content by isotopic ¹³C NMR, irrespective of the NMR spectrometer: parameters to be adjusted, performance test using [1,2-¹³C₂]acetic acid, generation of correction factors.     

Species specific isotope dilution for the accurate and SI traceable determination of arsenobetaine and methylmercury in cuttlefish and prawn

Methods based on species specific isotope dilution were developed for the accurate and SI traceable determination of arsenobetaine (AsBet) and methylmercury (MeHg) in prawn and cuttlefish tissues by LC-MS/MS and SPME GC-ICPMS. Quantitation of AsBet and MeHg were achieved by using a ¹³C-enriched AsBet spike (NRC CRM CBET-1) and an enriched spike of Me¹⁹⁸Hg (NRC CRM EMMS-1), respectively, wherein analyte mass fractions in enriched spikes were determined by reverse isotope dilution using natural abundance AsBet and MeHg primary standards. Purity of these primary standards were characterized by quantitative ¹H-NMR with the use of NIST SRM 350b benzoic acid as a primary calibrator, ensuring the final measurement results traceable to SI. Validation of employed methods of ID LC-MS/MS and ID SPME GC-ICPMS was demonstrated by analysis of several biological CRMs (DORM-4, TORT-3, DOLT-5, BCR-627 and BCR-463) with satisfying results.     

Unique challenges with recent gamma spectroscopy measurements at Los Alamos National Laboratory

A variety of unique radioactive samples have been measured recently at Los Alamos National Laboratory (LANL) using an electrically-cooled high-purity germanium detector. In each case the purpose of the measurements included one or more of the following objectives: (1) an accurate determination of the isotopic weight fractions of different plutonium or uranium materials; (2) an accurate determination of the isotopic quantity in the absence of relevant calibration standards; and (3) a qualitative determination of various sample impurities for additional forensic information. This paper discusses how simple modifications to the PC-FRAM parameter sets enabled a better determination of the isotopic content of the following samples: (1) high-purity plutonium metal, (2) plutonium-beryllium (PuBe) neutron sources, (3) neutron-irradiated natural uranium, and (4) re-processed HEU fuel with elevated ²³⁶U content. The isotopic quantity in a variety of samples was determined using a combination of the Spectral Nondestructive Assay Platform (SNAP™) routine from Eberline Services and the Monte Carlo Neutral Particle (MCNP) code developed at LANL. The non-traditional sources that were quantified with these gamma ray modeling codes included dozens of neutron-irradiated targets of natural uranium, several plutonium-beryllium neutron sources, and three high-purity samples of plutonium metal.     

Trace analysis of high-purity graphite by LA–ICP–MS

Laser-ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) has been established as a very efficient and sensitive technique for the direct analysis of solids. In this work the capability of LA–ICP–MS was investigated for determination of trace elements in high-purity graphite. Synthetic laboratory standards with a graphite matrix were prepared for the purpose of quantifying the analytical results. Doped trace elements, concentration 0.5 μg g^–1, in a laboratory standard were determined with an accuracy of 1% to ± 7% and a relative standard deviation (RSD) of 2–13%. Solution-based calibration was also used for quantitative analysis of high-purity graphite. It was found that such calibration led to analytical results for trace-element determination in graphite with accuracy similar to that obtained by use of synthetic laboratory standards for quantification of analytical results. Results from quantitative determination of trace impurities in a real reactor-graphite sample, using both quantification approaches, were in good agreement. Detection limits for all elements of interest were determined in the low ng g^–1 concentration range. Improvement of detection limits by a factor of 10 was achieved for analyses of high-purity graphite with LA–ICP–MS under wet plasma conditions, because the lower background signal and increased element sensitivity. Received: 4 January 2001 / Revised: 27 March 2001 / Accepted: 28 March 2001     

Qualitative and quantitative characterization of a complex mixture of hexylbenzenes by 13C nuclear magnetic resonance and gas chromatography-mass spectrometry

Summary A considerable amount of byproducts is formed in the production of isopropylbenzene (cumene). Recently, a distillation fraction of these byproducts became commercially attractive which justified its compositional analysis. All the signals in the complex ¹³C nuclear magnetic resonance (NMR) spectrum of this material have been assigned. The mass spectra of the individual constituents were recorded by the application of capillary gas chromatography-mass spectrometry. The data obtained with both analytical techniques revealed that the product is composed of a complex mixture of 3 isomeric diisopropylbenzenes and 10 different hexyl-substituted benzenes. The mass spectra and carbon-13 NMR chemical shift data of most of the compounds presented here have not been previously reported. NMR data of all possible butyl- and a number of pentyl-substituted benzenes are included in this report for assignment purposes. Quantitative studies on this material and some of its distillation fractions by ¹³C NMR and gas chromatography enabled the assignment of individual peaks in the gas chromatogram.

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Qualitative und quantitative Charakterisierung eines komplexen Gemisches von Hexylbenzolen durch ¹³C-NMR und GC/MS

     

Group IV organometal derivatives of pyridine. I—A 1H and 13C NMR investigation of trialkylmetal derivatives of pyridine

The proton and carbon-13 NMR spectra of thirteen trialkylmetal derivatives of pyridine, several of which were previously unknown, have been recorded and analysed. The proton NMR spectra show variations in proton chemical shifts but not in proton-proton coupling constants when the metal substituent is changed; the ring proton-metal coupling constants ⁿJ(M? H) in the tin and lead derivatives correspond closely with the corresponding proton-proton couplings ⁿJ(H? H) in pyridine. The carbon-13 chemical shifts of the carbons bound to the metal can apparently be correlated with the electron-donating ability of the trialkylmetal group. In the trimethylstannylpyridines the value of ¹J(Sn? C_ring) varies greatly with the position of the Me₃Sn group.     

A NMR method for the analysis of mixtures of alkanes with different deuterium substitutions

¹³C NMR at 125.76 MHz with ¹H and ²H decoupling, ²H NMR at 76.77 MHz with ¹H decoupling, and ¹H NMR at 500.14 MHz with ²H decoupling were employed as analytical tools to study the complex mixtures of deuterated ethanes resulting from the catalytic H–D exchange of normal ethane with gas-phase deuterium in the presence of a platinum foil. Reference samples consisting of 1:1 binary mixtures of pure normal ethane and ethane-d_n (n=1–6) were used to identify the peak positions in the ¹³C, ²H, and ¹H NMR spectra due to each individual isotopomer, and the effect of isotopic substitution on the chemical shifts was determined in each case. While the NMR of all three nuclei worked well for the identification of the individual components of the 1:1 standard mixtures, both ¹H and ²H NMR suffered from inadequate resolution when studying complex reaction mixtures because of the broadening of the lines due to ¹H–¹H (¹H NMR) and ²H–²H (²H NMR) couplings. ¹³C NMR was therefore determined to be the method of choice for the quantitative analysis of the reaction mixtures. Using the ¹³C NMR results, a correlation that takes into account the primary and secondary isotope substitution effects on chemical shifts was deduced. This equation was used for the identification of the individual components of the mixtures, and integration of the individual observed resonances was then employed for quantification of their composition. This study shows that ¹³C NMR with ¹H and ²H decoupling is a viable procedure for studying mixtures of deuterated ethanes. Furthermore, the additivity of the isotopic effects on chemical shifts and the transferability of the values obtained with ethane to other molecules makes this approach general for the analysis of other isotopomer mixtures.     

Preparation and certification of arsenobetaine reference material NMIJ CRM 7901-a

An arsenobetaine [(CH₃)₃As⁺CH₂COO⁻] solution reference material, NMIJ CRM 7901-a, intended for use in the speciation of arsenic compounds, was developed and certified by the National Metrology Institute of Japan (NMIJ), part of the National Institute of Advanced Industrial Science and Technology (AIST). The high-purity arsenobetaine powder was synthesized from trimethylarsine [(CH₃)₃As], and it was dissolved in water in order to prepare 20 mg kg⁻¹ of arsenobetaine standard solution. The solution was bottled in 500 bottles (each containing 10 ml). Certification of the CRM for arsenobetaine was conducted by NMIJ. The concentration of As was determined by four independent analytical techniques (ICP–MS, ICP–OES, GFAAS and LC–ICP–MS), and each result was converted to the arsenobetaine concentration by applying an appropriate factor. The arsenobetaine concentration in the CRM was thus certified.     

Carbon-13 isotope enrichment in the photolysis of CO with the 123.58 nm Kr resonance radiation

The 123.58 nm Kr resonance radiation was shown to excite selectively the ¹³CO(A ¹Π-X¹Σ⁺) 13,0 transition in natural isotopic composition CO. The photolysis products, CO₂ andC₃O₂, were enriched in carbon-13. Carbon dioxide was enriched ten-fold and the central carbon of C₃O₂ was enriched sixty-fold.     

High yield synthesis and characterization of isotopically enriched monoethylmercury chloride (C2H5201HgCl)

An important but commercially unavailable compound isotopically enriched monoethylmercury chloride (C₂H₅²⁰¹HgCl), has been synthesized from commercially available ²⁰¹HgO (98.11% enriched isotopic purity) and tetraethyltin. The required synthesis time is 1 h at 90 °C, and the product is the single product of monoethylmercury chloride, yielding more than 95% as ²⁰¹Hg in C₂H₅²⁰¹Hg⁺ (98.19 ± 0.22% enriched isotopic purity). The synthesized product was analyzed with high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC‐ICP‐MS) to determine its concentration, isotopic composition and purity. The synthetic isotopically enriched monoethylmercury synthesized can be used in speciated isotope dilution mass spectrometry (SIDMS) and isotope dilution mass spectrometry (IDMS) analyses as a standard. Copyright © 2005 John Wiley & Sons, Ltd.     

Pulsed fourier transform NMR of substituted aryltrimethyltin derivatives : IV. Proton and carbon-13 NMR data for ortho-, 2,6-, and poly-substituted aryltrimethyltins

Proton and carbon-13 NMR data recorded in the Fourier transform mode are reported for ten ortho-substituted, six 2,6-disubstituted, and six miscellaneous polysubstituted aryltrimethyltin compounds. Although ¦¹J(¹³C¹H)¦ and ¦²J(¹¹⁹SnC¹H)¦ coupling constants are rather insensitive to substituent variation, tin methyl proton chemical shifts reflect the increasing inductive effects as methyl-, chloro-, fluoro-, and trifluoromethyl-groups are brought into juxtaposition with the trimethyltin moiety. Resonances in the natural-abundance carbon-13 NMR spectra for the tin derivatives are assigned on the basis of additivity relationships, proton undecoupled spectra, and relative magnitudes of ¦J(¹¹⁹Sn¹³C)¦ and ¦J(¹³C¹⁹F)¦ coupling constants. Mutually deshielding γ-, δ-, and ?-effects in the carbon-13 chemical shifts of substituent carbons are rationalized in terms of steric crowding between the trimethyltin group and neighboring substituents. Deshieldings in ring carbons formally para- to conjugating substituents are discussed in terms of the steric inhibition of resonance model. Previous conclusions concerning lack of significant higher coordination at tin in aryltin derivatives bearing substituents with lone pair electrons are corroborated in this work.     

Determination of the orientational ordering of 4′-cyanophenyl-4-alkylbenzoates by 13C NMR

Abstract

The orientational ordering of three 4′-cyanophenyl-4-alkylbenzoates (with number of carbons in the alkyl chain, n = 6,7 and 8; hereafter abbreviated as n-CPBs) has been investigated by ¹³C NMR. The order parameters of different molecular segments in the nematic phase of the n-CPBs were determined by the two-dimensional technique of separated local field (SLF) spectroscopy combined with off-magic-axis, variable-angle spinning (VAS) of the sample. The carbon-13 chemical shifts for each carbon nucleus in these compounds were determined by slowly spinning the sample parallel to the applied magnetic field. The order parameters obtained from SLF/VAS studies are linearly related to the corresponding anisotropic carbon-13 chemical shifts. These results provide a convenient way to obtain the order parameters for other homologous members of this liquid crystal series by direct measurement of only their carbon-13 chemical shifts in conjunction with the observed linear relationship between order parameters and chemical shifts.     

NMR analysis of antitumor drugs: Doxorubicin,daunorubicin and their functionalized derivatives

¹H, ¹H–¹H COSY and ¹³C NMR techniques were proven as effective methods for the analysis of functionalized doxorubicin (DOX) and daunorubicin (DAU) derivatives. These compounds represent important drug conjugate intermediates that can be coupled to a variety of nanocarriers. NMR spectroscopy was shown to be an efficient method for following the progress of drug modification and can also be useful for evaluation of the functionalized structures purity. Additionally, a correction of the chemical shifts reported in the literature for the relevant drugs e.g. DOX·HCl and DAU·HCl is presented.     

Silicon-29 and carbon-13 NMR studies of organosilicon chemistry. II—The Methylethoxysilanes MenSi(OEt)4−n

Silicon-29, carbon-13 and oxygen-17 NMR data are reported for the methylethoxysilanes, Me_nSi(OEt)_4?n with n = C to 3. The values of ¹J(SiC) vary greatly, in a manner which appears to be inconsistent with an effect of s-character variation alone. The chemical shifts are discussed in terms of possible π-bonding. Silicon-29 and carbon-13 spin-lattice relaxation times and nuclear Overhauser effects are also reported and discussed.     

Force field and multinuclear nmr study of the conformational properties of thiolane-1-oxide and its mono and dimethyl derivatives

Force field calculations and ¹H, ¹³C and ¹⁷O NMR data are reported for thiolane S-oxide and several of its mono and dimethyl derivatives. For comparison carbon-13 and oxygen-17 chemical shifts of the corresponding S-dioxides are also reported. According to force field calculations and NMR data the conformation of S-oxides depends on the number and on the position of ring substituents. Oxygen-17 chemical shifts of thiolane S-oxides are apparently not very sensitive to ring substitution and to the configuration of the sulfinyl group. This is however the result of conformational changes which cannot be easily predicted. For 2-methyl derivatives δ₁₇₀ allows an easy identification of the cis and trans isomers.     

Carbon-14 kinetic isotope effect in the decarbonylation of lactic acid[1-14C]

The carbon-14 kinetic isotope effect for the decarbonylation of lactic acid[1-¹⁴C] in sulfuric acid has been measured in the temperature interval of 20–90°C. The experimental values of (k_12C/k_14C) are compared with the theoretical¹⁴C kinetic isotope effect calculated assuming that one carbon-oxygen stretching vibration is lost in the rate-determining step. The discrepancy between experimentally observed temperature dependence of the¹⁴C kinetic isotope effect and the theoretical one is explained by the possible side reactions which change the apparent degrees of decarbonylation and isotopic composition of CH₃CHOHCOOH[1-¹⁴C] used in experiments aiming at the determination of carbon-14 kinetic isotope effect in the decarbonylation process itself.     

Synthesis and structure of allylated derivatives of fullerenes C<Subscript>60</Subscript> and C<Subscript>70</Subscript>

New chromatographically pure monoand hexamethanofullerenes C₆₀ and C₇₀ containing active allylic groups were synthesized by Bingel—Hirsch reaction. These compounds are promising for the studies of biological activity, as well as for obtaining on their basis new fullerenecontaining materials. The purity and composition of the synthesized compounds were confirmed by MALDI-TOF mass spectrometry and HPLC, their structure was established by ¹H and ¹³C NMR spectroscopy and X-ray diffraction.     

The chemical form and acute toxicity of arsenic compounds in marine organisms

A method for the separation and identification of inorganic and methylated arsenic compounds in marine organisms was constructed by using a hydride generation/cold trap/gas chromatography mass spectrometry (HG/CT/GC MS) measurement system. The chemical form of arsenic compounds in marine organisms was examined by the HG/CT/GC MS system after alkaline digestion. It was observed that trimethylarsenic compounds were distributed mainly in the water-soluble fraction of muscle of carnivorous gastropods, crustaceans and fish. Also, dimethylated arsenic compounds were distributed in the water-soluble fraction of Phaeophyceae. It is thought that most of the trimethylated arsenic is likely to be arsenobetaine since this compound released trimethylarsine by alkaline digestion and subsequent reduction with sodium borohydride. The major arsenic compound isolated from the water-soluble fraction in the muscle and liver of sharks was identified as arsenobetaine from IR, FAB Ms data, NMR spectra and TLC behaviour. The acute toxicity of arsenobetaine was studied in male mice. The LD₅₀ value was higher than 10 g kg⁻¹. This compound was found in urine in the non-metabolized form. No particular toxic symptoms were observed following administration. These results suggest that arsenobetaine has low toxicity and is not metabolized in mice. The LD₅₀ values of other minor arsenicals in marine organisms, trimethylarsine oxide, arsenocholine and tetramethylarsonium salt, were also examined in mice.     

Differentiation and quantification of synthetic phosphatidylethanol (PEth) homologues by 1H- and 13C-NMR in polar organic solvents

Various phosphatidylethanol (PEth) derivatives, the corresponding reversed positional isomers (RPI-PEths), lyso-PEth-16:0, and penta-deuterium-labeled PEth analogs (d5-PEths), were synthesized by enzyme-independent synthetic routes. A general solvent system consisting of a mixture of acetone-d6 and methanol-d4 (97:3; v/v) was found to provide a good solubilizing capacity and excellent hydrogen-1 NMR (¹H-NMR) peak resolution of various PEth homologues. Analytical differentiation of PEth from the corresponding RPI-PEth by carbon-13 NMR (¹³C-NMR) was demonstrated by comparison of the ¹³C-NMR signals of the carbonyl groups, the allylic positions, and of the β-carbons. An exemplary stable long-term room temperature, DMSO-d6-based, and proton-sensitive quantitative nuclear magnetic resonance (¹H-qNMR) independently quantified calibrator comprising PEth-16:0/18:1 for liquid chromatography (tandem) mass spectrometry (LC-MS/MS) analytical applications were prepared by employment of sodium dodecyl sulfate (SDS) as a solubilizing additive. In summary, novel hypothetically occurring PEth derivatives, e.g., RPI-PEths, have been independently synthesized with regio- and stereochemical control. Use of polar organic solvents, e.g., mixtures of acetone-d6 and methanol-d4 or DMSO-d6, improves spectral line shapes as compared to traditional hydrophobic solvents and allow for analytical differentiation between closely related PEth derivatives, as well as LC-MS/MS-independent concentration determination of dissolved single species by employment of ¹H-qNMR.