Modification of a commercial gas chromatography isotope ratio mass spectrometer for on-line carbon isotope dilution: Evaluation of its analytical characteristics for the quantification of organic compounds
Introduction
GC–MS and GC–MS/MS with electron ionization (EI) have become one of the most widely used techniques in laboratories all over the world for the quantification of volatile or semivolatile compound (directly or after chemical derivatization) in a wide variety of samples. Of course, as EI is a molecular-ion source, its response is molecule-specific for each single molecule to be analysed [1]. Therefore, traceable analytical standards for every analyte are needed to carry out external calibrations or standard additions when quantification is sought [2]. Additionally, and for more than sixty years now, analytical procedures for the determination of organic compounds by mass spectrometry have made also use of isotopically labelled compounds (deuterated or 13C-labelled internal standards) for accurate quantification, based on the isotope dilution (ID) concept [3], [4]. This method relies on the fact that the instrumental response is identical for both, the natural abundance and the isotopically labelled compound and that the recoveries for non-chromatographic separation procedures are also identical. Therefore it can provide excellent sensitivity, accuracy and precision. Unfortunately, a limited number of isotopically labelled internal standards are commercially available.
In the search for quantitative estimation of various compounds lacking standards or surrogates using GC–MS, predictive equations have been derived for many Volatile Organic Compounds (VOCs) from the linear regression analysis between their actual response factors and their physicochemical parameters, mostly the number of carbons [5]. The use of such predictive statistical approaches has been further refined using the effective carbon number, which takes into account the functional groups present [6], [7], providing adequate levels of reliability for some limited mixtures of compounds (10–17% percentage difference between predicted and actual values). Of course, the nature and number of these functional groups and the number of carbons present determine such deviation, which can increase up to 20–30% in more complex mixtures.
The on-line carbon isotope dilution concept, introduced in 2009 [8], opened the door to the generic quantification of any organic compound present in a sample without resorting to specific standards. A constant flow of a non-specific isotopic tracer (i.e. 13CO2) was added continuously to the sample after the chromatographic separation. Isotopic equilibration was obtained using a combustion furnace located after the GC column which converted all compounds into CO2. The continuous measurement of the isotope ratio 12C/13C (obtained by measuring the signals at m/z 44 and 45 corresponding to 12CO2 and 13CO2, respectively) in a quadrupole mass spectrometer after electron ionization was the basis of the on-line carbon IDMS procedure developed [8]. Later, the same authors applied this concept to the evaluation of Solid Phase Micro Extraction (SPME) procedures as the absolute amount of an organic compound fixed to the SPME fibre could be evaluated accurately by this method [9]. However, there is one important limitation to this on-line carbon IDMS procedure: any loss of substance occurring before isotopic equilibration could lead to biased results. Isotopic equilibration occurs here after the combustion furnace where all organic compounds are converted into carbon dioxide. So, losses of substance before or at the combustion oven will be problematic. When working with GC, recoveries from the column are typically quantitative [2], however fractionation effects during injection are likely to occur in standard GC injectors if mixtures of compounds with very different boiling points are analysed [10]. Moreover, the presence of heteroatoms in the structure of the organic compounds could negatively affect their combustion efficiency and therefore the trueness of the results [11]. Obviously, losses occurring before the injection in the gas chromatograph will not be corrected for unless a suitable internal standard is employed.
On the other hand, natural variations in carbon isotope compositions of volatile compounds are measured routinely with GC–combustion–IRMS instruments [12] in which a combustion oven is employed to transform all organic compounds into carbon dioxide. Modern GC–IRMS instruments, such as the Delta V Advantage from Thermo Scientific, employ a CuO/NiO/Pt tube at ca. 1000 °C for combustion. Additionally, a Nafion membrane tube is employed to eliminate water vapour from the combustion gases reducing potential isobaric interferences at m/z 45 from proton transfer reactions. These instruments are ideal to evaluate the ultimate potential of the on-line carbon isotope dilution procedure as, in contrast to previous home-made instruments [8], [9], they provide highly precise isotope ratios and have been optimized for the elimination of potential interferences.
Therefore, in the present study a commercial GC–IRMS instrument has been modified for the first time to evaluate its applicability for on-line carbon isotope dilution. In addition, the use of a Programmed Temperature Vaporizer (PTV) has demonstrated to be ideal to prevent fractionation in the GC injector and thus any loss of compounds before isotopic equilibration with the non-specific isotopic tracer (13CO2) had taken place. In this way, the concept of on-line carbon isotope dilution could be extended to mixtures of compounds with very different boiling points (n-alkanes and PAHs) and heteroatom-containing organic compounds (PCBs and benzothiophenes). The analytical characteristics of the proposed compound-independent quantification procedure are described.
Section snippets
Reagents and standards
Solid enriched NaHCO3 (99% 13C enrichment) was purchased as a high purity chemical reagent (purity > 98%) from Cambridge Isotopes Laboratories (Andover, Massachusetts, USA). Phosphoric acid (99% puriss.), n-hexane and dichloromethane for organic trace analysis grade, benzo(b)fluorene (BbF, 99.4% puriss.) and 3-methylbenzothiophene (3-Me-BT, certified purity 96%) were obtained from Sigma–Aldrich (St. Louis, USA). QTM PAHs Mix (2000 μg mL−1 each in dichloromethane) and CEN PCBs Mix (10 μg mL−1 each in n
Modification of the GC–combustion–IRMS instrument
The GC–combustion–IRMS instrument at the University of Oviedo was factory configured only for the measurement of carbon isotope ratios. After the combustion oven no reduction oven was installed (required for nitrogen isotope ratios) and the output of the oven was directly connected to the Nafion membrane tube for water removal. Also, no liquid nitrogen trap was installed in our system. In our previous quadrupole home-made system for on-line IDMS [8], [9] we added the spike flow just after the
Conclusions
We have successfully modified a commercial GC–IRMS instrument and evaluated its performance characteristics for on-line carbon isotope dilution using a series of different organic compounds. Additionally, the use of a PTV injector was investigated for the elimination of injection-derived fractionation allowing the use of a single internal standard to quantify different compounds with a wide range of sizes and boiling points in a single chromatographic run. In comparison to standard GC–QMS
Acknowledgements
This research was supported by the Spanish Ministry of Science and Innovation (MICINN; project CTQ2009-12814, cofunded by FEDER) and FICYT (PC10-58). The provision of FEDER funds for the purchase of the IRMS instrument is also acknowledged.
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