Anal. Chem. 1995,67, 3086-3088
Rapid On=LineCombustion System for I3C Analysis of Nonvolatile Compounds Amy H. Luke and Dale A. SchoelleP
Department of Medicine, University of Chicago, Chicago, Illinois 60637
A novel on-line combustion system was designed and tested for dynamic 13C analysis of minute quantities of previously isolated, nonvolatile organic compounds. The analyte was loaded onto a tungsten iilament, sealed in a helium/oxygen carrier gas stream, and heated to combustion by passing an electrical current through the iilament. The accuracy of the filament system was 1-2%0for natural abundance and highly enriched (-25 to 500%0vs PDB) nonvolatile bioorganic compounds. The precision was 1-2%0at natural abundance and 4%0at high 13C enrichments. These precisions were obtained with -500 ng of sample. Memory limited the accuracy of the filament inlet when the envelope was not changed between samples. This on-line combustion system represents a new approach to isotope ratio analysis of nonvolatile organics. It has the potential for total carbon analysis of complex organic mixtures and has sample requirements 100-fold lower than those of other systems designed for nonvolatile organics. As recently reviewed,' gas chromatography/combustion/ isotope ratio mass spectrometry (GC/C/IRMS) as introduced by Matthews and HayesZ is being utilized for compound-specific carbon isotope ratio analysis in the field of geochemistry and in the biological sciences. The technique is advantageous because it combines the capabilities of GC for the analysis of small samples with the precision of isotope ratio mass spectrometry. At the same time, however, the requirements for sample presentation typically associated with GC, namely volatility and thermal stability, have limited the possible applications of this technique. While derivatization is an option for extending the applicability of GC/C/IRMS to many compounds of interest to biomedical investigators,there are many compounds for which appropriate derivatives are not available. Furthermore, in the cases where derivatization is possible, the process itself alters the natural 13C abundance by adding carbon with the derivative moiety and by the potential isotope effects created at the reaction site.3 Recently, Caimi and Brenna4presented a moving wire interface to couple a high-performance liquid chromatograph to a combustion interface of a dynamic IRMS for the analysis of nonvolatile bioorganic compounds. This system expands the type and number of compounds that can be analyzed, but it suffers the sample transfer limitations inherent in a moving wire interface, with only 0.1%of the compound being transferred to the wire and 2%of that reaching the IRMS. In addition, HPLC cannot be used for all biorganic compounds, nor can it be used for total carbon (1) Brenna, T. J. Act. Chem. Res. 1994,27, 340-346.
(2) Matthews, D. E.; Hayes, J. M. Anal. Chem. 1978,50, 1465-1473. (3) Hayes, J. M. Spectra 1982,8, 3-8. (4) Caimi, R J.; Brenna, J. T. Anal. Chem. 1993,65, 3497-3500. 3086 Analytical Chemistry, Vol. 67, No. 17, September 1 , 1995
II A
D
TO
E
Figure 1. Diagram of the filament inlet system. (A) Source of carrier gas (helium). (B) Four-port valve. (C) Reservoir with helium/On mixture. (D) Threaded inlet: (1) brass reducing union (10 mm to 1.6 mm); (2)Teflon ferrule; (3) disposable Pyrex envelope; (4) tungsten filament; (5) electrical leads to autotransformer. (E) Autotransformer. (F) Thermal reactor. (G) Nafion tubing for water removal. (H) Open split.
analysis of complex biological materials. The complex biological materials can be isotopically analyzed using an elemental analyzer interfaced to an IRMS, but sample requirements are on the order of 30-100 p g 5 To fill this gap, we have designed and tested an interface for the carbon isotope analysis of minute quantities of previously isolated compounds that avoids the sample losses of the directly coupled HPLC. With this device, analyte is loaded on a filament, sealed in a helium/oxygen carrier gas stream, and heated to combustion by passing an electrical current through the filament, with the resulting COZbeing carried into the ion source of the IRMS. EXPERIMENTAL SECTION
Instrumentation. (i) Filament Inlet. The filament inlet system was constructed as an adaptation of the GC/C interface of a Finnigan MAT Delta S IRMS (Finnigan MAT, San Jose, CA). The inlet was positioned upstream from the vendor's thermal reactor and the open-split interface (Figure 1). The inlet consisted of a 6cm length of 10-mm4.d. Pyrex tubing for simple disposal after each sample. An &m x 0.20." tungsten filament was wrapped into a 3-mm-0.d. coil and inserted into the Pyrex tube, with 1cm extending out of both ends. The Pyrex tube was fitted with two brass reducing unions (10 mm x 1.6 mm; Swagelok, Bedford, MA) using Teflon ferrules such that the tungsten filament made electrical contact with the unions at both ends. (ii) Carrier Gas Configuration. Because the filament inlet was placed upstream from the thermal reactor, we were able to use the existing helium carrier gas lines from the GC to provide a 3-4 mWmm regulated flow of helium (ultrahigh purity; Linde (5) Wong, W. W.; Clark, L. L; Johnson, C. A: Llaurador. M.; Klein, P. D. Anal. Chem. 1992,64, 354-358. 0003-2700/95/0367-3086$9.00/0 0 1995 American Chemical Society
Gas, East Chicago, IN). Additional flow rates were used as indicated during initial testing. The gas was directed to a Cport valve (Valco Instruments, Houston,TX) so that it could be directed to the filament inlet or diverted through a 125mL reservoir filled with helium and oxygen. The oxygen (extra dry, Linde) was loaded into a syringe and injected into the reservoir via a septum and thus could be easily varied from 1 to 20%by volume. The thermal reactor positioned downstream from the filament inlet was a 3 k m x l.&mm-0.d.ceramic tube, into which two 2 k m x 0.001-mm-0.d. copper wires had been inserted. The reactor was operated at 600 "C to remove excess oxygen from the postcombustion carrier gas stream. Oxygen removal was confirmed by monitoring the m/z 32 ion current, which was
