Temperature Zonal Combustion Reactor for the 15 ... - ACS Publications

A temperature distribution was imposed on a solid−gas reactor to help accomplish the 15-nitrogen or 13-carbon isotopic determination of biosynthetic...
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Temperature Zonal Combustion Reactor for the 15-Nitrogen and 13-Carbon Isotopic Determination of Enriched Biosynthetic Materials Michael May,* John Kuo, Michael Gray, I. Knyazhansky, and C. T. Tan Quality Control Lab, Isotec Stable Isotopes, Sigma-Aldrich Corporation, Miamisburg, Ohio 45342

A temperature distribution was imposed on a solid-gas reactor to help accomplish the 15-nitrogen or 13carbon isotopic determination of biosynthetic materials such as algal protein-15N. The temperature zonal combustion reactor (TZCR) can operate offline from its mass spectrometer, and this reactor facilitates the stable isotopic determination of highly enriched biomaterials (>95 atom % isotope). The reactor structure is a glass pipe comprising a quartz segment, transition segments, and threaded sockets. Precise positioning of the reactor within a tubular furnace establishes a longitudinal temperature distribution that is used to drive solid-gas chemistry in distinct zones: sample, catalyst, and sorbent. The TZCR module typically includes a reactor pipe, gastight valve, sample, reactants, reactant containers, and furnace. An important advantage of TZCR-mass spectrometer is preliminary vacuum heat treatment of the crude sample to remove volatile impurities before sample combustion (enabling isotopic enrichment determination of the analyte). For the determination of 15N enrichment, sample combustion at 650 °C in oxygen was supplemented by Fe3O4 catalyst near 500 °C and Li2O at 25 °C that concertedly promoted the intrareactor depletion of CO, CO2, and H2O. As a result, the primary gaseous product was dinitrogen. After 15N sample conversion, the reactor was coupled to a quadrupole MS for product analysis (N2+ ions for 15N determination). Accordingly, uniformly enriched biomaterials were analyzed such as algal protein-15N that measured 99.0 ( 0.12 atom % 15N. By adapting the methodology for 13C determination, lyophilized algal cells-13C were determined to be 99.1 ( 0.06 atom % 13C. Stable isotope enrichments were computed by minimizing the numeric differences between theoretical and actual MS signals. Introduction Highly enriched isotopic biomaterials have become increasingly available in recent decades. Representative products isolated from algae biosynthesis include amino acids, fatty acids, algal protein, and algal cells. Even after multistep product isolation, determining the isotopic enrichment of complex biosynthetic materials is problematic. Classically, thermal oxidative combustion of organic samples was the major technology used to obtain the CHN elemental composition. Dennstedt constructed a thermal combustion apparatus that utilized CuO (for the conversion of NOx to N2) and chemical absorption traps to achieve quantitative elemental analyses.1 By 1960, Gustin had developed an automated combustion machine that accomplished nitrogen analysis.2 This multicompartment N2 analyzer contained a gas flowmeter, a combustion station (silica tube packed with CuO), a reduction station (silica tube packed with CuO/Cu), and a graduated syringe (to quantify the N2 volume). Employing a mass spectrograph, Aston definitively identified stable isotopes for neon, chlorine, and other elements.3 Technological progress led to specialized isotope mass spectrometers4,5 that provide precise isotope ratio values over a narrow abundance range (about 1 atom % isotope). In 1978, Matthews and Hayes reported a 15N determination system6 that mixed a gaschromatograph- (GC-) separated sample stream with dioxygen and directed it through a combustion zone, a MgClO4 moisture trap, a cryogenic CO2 trap, and a mass spectrometer. Contemporary versions of process-gas mass spectrometers provide very stable signal measurements across the entire mass range of interest for gas analysis, nominally from 2 to 200 Da. For the determination of stable isotope biomaterials that are highly * To whom correspondence should be addressed. E-mail: [email protected].

enriched, the process-gas mass spectrometry enables measurement over a wide dynamic range (0-100 atom % isotope, (0.1%) without preknowledge of the whole number enrichment value. Industrial quality-control (QC) laboratories tend to practice standard methods that achieve defined analytical objectives. After receiving QC service inquiries concerning biosynthetic algal samples that were enriched with stable isotopes (13C and/ or 15N) and spanned wide ranges (5-99.5 atom %), a combustion MS method was proposed for isotope determination. However, crude algal samples retain volatile (natural-abundance) impurities; furthermore, algal combustion generates distinct gases having equivalent nominal mass (such as 12C18O and 15 N2). Given such difficulties, the use of direct (sealed-tube) combustion quadrupole-MS instruments for algal material isotopic determination was found to be unsatisfactory. To address the aforementioned difficulties, a special (solid-gas) reactor was designed that would accomplish sample bake-out, oxidative sample combustion, and selective product gas removal. A unitary glass pipe provides the containment for desired chemical reactions, with each reaction expedited in one of the thermal zones established along the reactor length. For 15N sample determination, one multipurpose reactant is stationed to chemically convert the generated nitrogen oxides to N2 and the CO to CO2 (catalyst zone, ∼500 °C). Furthermore, one sorbent is separately stationed to absorb the generated CO2 and H2O. By appropriately positioning the sample, catalyst, and sorbent materials along the temperature-regulated reactor length, such a temperature zonal combustion reactor (TZCR) enables 15 N sample pretreatment, combustion, nitric oxide decomposition, carbon monoxide decomposition, carbon dioxide depletion, and water depletion within one common compartment.7 Alternatively, for 13C sample determination, the TZCR is adapted to

10.1021/ie100072z  2010 American Chemical Society Published on Web 03/31/2010

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Figure 1. Disassembled view of the temperature zonal combustion reactor showing (A) pressure transducer, (B) vacuum adapter, (C) seal, (D) reactor pipe, (E) reagent container, (F) vacuum adapter, (G) glass tee, and (H) valve stem with seals. During normal operation, the longitudinal z axis of the reactor pipe is horizontally oriented.

provide sample pretreatment, combustion, and water vapor absorption. Quantitative isotope determination utilizing TZCRMS is presented for significant biomaterials such as lyophilized algal cells-15N, algal protein-15N, and starch-13C. Experimental Section Temperature Zonal Combustion Reactor. The TZCR module operates offline from a quadrupole mass spectrometer, and it typically includes the reactor pipe, sample, supplemental reactants, reactant containers, tee, gas valve, pressure transducer (or plug), and flexible tube. An isometric section of a 0.15-L reactor is illustrated in Figure 1. The one-piece reactor pipe comprises a central quartz segment, two transition glass segments, and two internally threaded borosilicate sockets (Ace Glass, size 11). The overall pipe length along the (longitudinal) z axis is 1 m, the quartz segment is 540 mm long, and the inner diameter is 14 mm. Multiple quartz containers (10-mm o.d.) can be inserted within the reactor to hold sample and reagents.

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Vacuum or gas is supplied to the reactor through a glass tee that comprises a valve body (Chemglass CG-962; see Figure 1) and barb fitting. Thermal energy is obtained from a tubular electric furnace equipped with a digital controller (Carbolite MTF 12/38/400, furnace length ) 400 mm). The reactor pipe fits through the 38-mm furnace opening and extends beyond the furnace length (the quartz segment is positioned within the furnace). In this work, an electronic pressure transducer (Edwards EPS10) was attached to the reactor when pressure monitoring was needed. Also, for suitable mobility, the reactor module was assembled on a moveable cart. Gas Manifold. A reactor manifold provided vacuum service, gas transfer service, and pressure measurement (Teledyne Hastings VT-6 pressure transducer). Essentially, the reactor gas manifold is a 400-mm-long glass tube equipped with threaded access ports (size-7 glass sockets). The access ports were fitted with vacuum metering valves to enable adjustable gas flow. Typically, a sparse film of Krytox lubricant was applied to each valve stem O-ring. One access port was connected to a vacuum pump (Leybold Trivac D4B). Materials. In general, the analytes of interest were uniformly isotope-enriched. Algal cells-13C and -15N were biosynthetically grown and processed onsite. Algal cell biomass was generated from blue-green algae (spirulina or Agmenellum PR6). Crude protein extract-15N (denoted protein-15N) was isolated from the algae biomass through solvent extraction, hydrolysis, separation, and lyophilization. Materials such as algal amino acids-15N and algal starch-13C were also biosynthetically produced. Glycine15 N and 4-nitroaniline-15N2 were chemically synthesized. Potato starch-13C (Solanum tuberosum) was obtained from IsoLifeBV. High-purity solid reagents were used as received from Sigma-Aldrich. Black iron oxide (99.99% Fe3O4, particle size < 0.1 mm) functioned as the 15N determination catalyst. Lithium oxide powder (>97% Li2O, 60 mesh) functioned as a sorbent for CO2/H2O and was manipulated inside a fume hood. Anhydrous calcium sulfate powder was utilized for water sorption during 13C determination. Compressed oxygen gas (Linde, 99.999%) was used as received. Instrumentation. A process-gas mass spectrometer (Extrel QGP) was used to acquire the mass spectra of the combustion products. It contained an electron ionization source at 100 eV, a quadrupole mass filter (1-Da resolution), and an electron multiplier detector. Multiple background and product mass scans were acquired at 10 Da/s and then averaged for data storage. Routinely, ∼10-6 Torr of combustion product gas was admitted into the quadrupole region for measurement. An Agilent-6890 GC, specially modified for gas sampling, was employed for lowlevel chemical analysis. It was equipped with 0.53-mm capillary columns (molecular sieve PLOT and Pora-PLOT) and a pulsed discharge helium ionization detector. Alternatively, a GC equipped with a thermal conductivity detector was used (GowMac-580). To obtain the reactor temperature profile, a type-K thermocouple with a 1220-mm-long sheath was used (Omega Engineering, KQSS-18u-48). Procedure. Stepwise TZCR-MS operations depend on the sample type and the target isotope to be measured (15N or 13C). As a representative example, the method for algal protein-15N determination is outlined here. (Caution: Analysts should take appropriate safety measures including use of personal protective equipment.) For each experiment, a 0.15-L reactor pipe was cleaned, dried, inserted through the furnace opening, and horizontally oriented. Next, 0.06 g of protein-15N powder and 0.15 g of Fe3O4 powder were placed into quartz containers and each precisely positioned within the reactor (typically, the Fe3O4

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Figure 2. Measured longitudinal z-axis temperature profile (in air) along one-half of the reactor pipe with the furnace controller set to 650 °C. The axis location z ) 0 mm corresponds to the longitudinal center of the symmetric reactor pipe.

and sample were loaded at opposing ends of one 120-mm container, the sample being positioned near z ) 0). Anhydrous lithium oxide (2 g of Li2O) was loaded into a container and positioned at the periphery (z ) 400 mm). The reactor module was then assembled and connected to the gas manifold. Next, the protein sample and Fe3O4 were subjected to thorough vacuum heat treatment (VHT) with the furnace controller set to 250 °C. Following the VHT stage, oxygen gas was regulated through the manifold and into the reactor (a typical load provided 0.0025 mol of O2). Next, the reactor valve was closed, and the furnace controller was reset to 650 °C. Resultant heat flow established the nonuniform z-axial temperature profile along the reactor shown in Figure 2 (z ) 0 mm corresponds to the furnace center). This reactor temperature distribution gave rise to the denoted sample zone (650 °C), catalyst zone (500 °C), and sorbent zone (25 °C). For convenience, the sample chemical conversion, during which 15NOx was converted to 15N2, CO was converted to CO2, the CO2 was reactively absorbed, and water vapor was depleted, was executed overnight. The primary end product was N2. At this stage, the TZCR module was moved and attached to the mass spectrometer inlet. Product gas was metered into the spectrometer for analysis. Acquired spectral data were averaged (n ) 10 scans) and background-corrected. To perform 13C biomaterial enrichment determination utilizing the TZCR-MS, the 15N determination method was slightly modified. The crude sample was pulverized and subjected to preliminary VHT followed by oxidative combustion at 700 °C. Inclusion of a supplemental catalyst was not necessary, and the intrareactor water sorption was accomplished with calcium sulfate. The postcombustion gas contained CO2 as the major product. Results and Discussion Reactor Design. A primary objective for TZCR-MS was the quantitative isotope determination of highly enriched 15-nitrogen or 13-carbon biomaterials at nominal atom percent levels (precision of (0.2 atom %). The reactor and associated methods were adapted to analyze biosynthetic products exemplified by algal protein-15N, lyophilized algal cells-15N, mixed amino

acids-13C, and starch-13C. Such materials pose analytical difficulties that include compositional variability, production batch variability, limited solubility, and solvent retention. Established methods to measure the 15N and 13C enrichment of adequately soluble biomaterials by quantitative nuclear magnetic resonance (NMR) spectrometry, gas chromatography-mass spectrometry (GCMS), and liquid chromatography-mass spectrometry (LCMS) are widely applied in isotope laboratories. Fortunately, many isotopic biomaterials that cannot be solvated are amenable to combustive derivatization. Initial efforts to directly combust algal protein-13C samples in quartz vessels (without preliminary VHT) generated 13CO2 that was up to 10 atom % 13C lower than expected. A combustion reactor was designed that embodies a transparent glass pipe, facilitates biomaterial derivatization, and avoids wet sample digestion. The central segment comprises quartz so that a sample zone temperature of >600 °C can be repetitively applied, thus making the reactor reusable.8 One significant advantage of a zonal reactor is flexibility to position each solid reactant (sample/catalyst/sorbent) at a preferable temperature for chemical reaction. Insertable quartz containers (see Figure 1) function to enclose and localize each reactant. By allowing free airflow between the reactor pipe exterior and furnace interior, a smooth reactor temperature profile is established, as shown in Figure 2. The resultant z-axis temperature gradients (approximately the slope values between data points of Figure 2) are less steep than in a completely insulated furnace.9 The reactor temperature profile is advantageous for analyte conversion to N2: Protein-15N can undergo combustion in the 650 °C sample zone, while Fe3O4 operates in the 500 °C catalyst zone (z ) 120 mm) and Li2O operates in the 25 °C sorbent zone (z ) 400 mm). Moreover, the differential temperatures promote gas movement (intrareactor mass transfer). The reactor pipe comprises transparent glass segments that were fused into a unitary structure. When properly annealed, the reactor pipe provides reaction containment, heat-transfer service, sealable ports, and visual transparency. The primary port tee accommodates a high-vacuum valve (see Figure 1), thereby enabling adjustable gas flow between the reactor and coupled equipment. In addition, a secondary reactor port permits auxiliary operations such as pressure monitoring. At each port, the gas-tight connections are secured by compression of ring seals between a bushing and socket. To estimate seal integrity, an empty reactor pipe was evacuated to 10-2 Torr and valved off; atmospheric influx to the reactor was chromatographically measured to be 10 ppmv N2 per hour (about 8 × 10-7 mol of N2 after 15 h). Sample Vacuum Heat Treatment. To achieve quantitative isotopic determination for the biosynthetic analytes, it was crucial to first remove the sample residual solvents. Ordinarily, the crude sample was pulverized and subjected to intrareactor dynamic vacuum heat treatment (VHT). The VHT stage led to volatile chemical evaporation and sublimation, as evidenced by the appearance of deposits (at z > 200 mm) for some samples. To illustrate the VHT effect, Figure 3 displays a graph of several 13 C experiments for one particular algal protein batch that was preconditioned at various temperatures, combusted, and analyzed. Note that the 13C enrichment values progressively rise from 83.0 to 90.7 atom % 13C, with the latter value reported as the result. For algal biomaterials in general, the maximum obtained isotope enrichment value best agreed with the corresponding precursor (atom %) value. In the cases of 15N sample determination, the VHT stage further served to activate the Fe3O4 reagent.

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Figure 3. Plot of 13C test measurements for a 13C-enriched algal protein material that was preconditioned at various VHT temperatures prior to the protein combustion.

Oxygen Combustion with Supplemental Reagents. To supplement the 15N sample determinations, a transition metal oxide was incorporated that functioned to drive the reaction gas to N2 and CO2. Among others, candidate oxides included NiO, CuO,10 Co3O4,11 Ag2O, and iron oxides. Black iron oxide (Fe3O4) was chosen, in combination with oxygen gas, to promote the conversion of CO to CO2 because it contains both Fe3+ and Fe2+ cationic sites. During initial 15N experimentation, O2/ Fe3O4 was found to also promote the conversion of NOx to N2 under reaction conditions (activated Fe3O4 near 500 °C and a moist oxidative atmosphere). Kolesnik and St. Pierre previously investigated the effect of moisture concentration on CO decomposition using “reduced Fe2O3” pellets.12 Carbon monoxide can be efficiently dissociated in the presence of reduced Fe2O3 and water vapor (10-15 Torr of H2O at 513 °C). Recently, a potassium iron oxide, Fe19K1O30, was reported to catalyze the conversion of NOx to N2 and soot to CO2.13 The Fe19K1O30 catalyst (pellet) was active in synthetic exhaust gas for about 20 reaction cycles, with each cycle involving a thermal ramp from 150 to 500 °C. During 15N combustion, the application of a modest sample zone temperature (650 °C) helps to limit the total production of NO, a mass spectrometric interferent of N2. Empirical and theoretical O2-Fe phase diagrams14,15 imply that solid compositions more oxidized than Fe3O4 are generated during algal 15N determinations. To better understand the iron oxide phase during 15N sample combustion, a thermochemical calculation was performed for the iron oxide reaction 6Fe2O3(s) T O2(g) + 4Fe3O4(s) If O2 were instantly added to such an equilibrated system, the additional O2 pressure would shift the system leftward. For computational purposes, the number of components is three (O2, Fe3O4, and Fe2O3), the number of homogeneous phases is two (solid iron oxide and gaseous O2), and therefore the system variance is three (pressure, temperature, and solid-phase composition). Both the Gibbs (standard constant-volume) free energy of reaction and the equilibrium dioxygen pressure were calculated16 at 100 °C intervals, and the results are displayed in Figure 4. Over a temperature range from 25 to 1427 °C, the predicted O2 pressure would range from ∼10-65 Torr to 10+3 Torr. The thermochemical calculation confirms that, during an oxygenrich 15N sample determination, the Fe3O4 reagent will be oxidized to solid phases of progressively increasing O/Fe ratio. During actual 15N sample determinations, the nominal loading was 0.15 g of Fe3O4 for 0.06 g of sample (∼0.25 mol of Fe3O4

Figure 4. Theoretical results for the standard-state Gibbs free energy (left linear scale) and dioxygen pressure (right logarithmic scale) versus temperature for the thermochemical reaction 6Fe2O3(s) T O2(g) + 4Fe3O4(s).

per mole of sample carbon). The iron oxide color was observed to change from black to (postcombustion) red. To accomplish 15N sample determination using the TZCR with a process-gas mass spectrometer, CO and CO2 must be substantially removed from the final product gas. Anhydrous lithium oxide was found to effectively absorb both CO2 and H2O near room temperature (for 0.03 g of sample carbon, a loading of 2 g of Li2O corresponds to >20 mol of Li2O per mole of carbon). Referral to Figure 2 indicates that the Li2O reagent should be stationed near the reactor periphery (z ) 400 mm). Chemical sorption of H2O and CO2 from the combustion gas avoided certain procedural complexities associated with other gas separative options such as condensation or chromatography. Similarly, to accomplish the 13C sample determinations, anhydrous calcium sulfate was stationed to absorb water vapor. From an operational perspective, TZCR methodology can be viewed as a three-stage process (VHT/combustion/sorption) for the chemical conversion of analyte to simple gases. To help address process safety concerns, the reactor pressure response to an applied temperature increase was recorded. For a representative algal cell-15N combustion (using O2, Fe3O4, and Li2O reagents), the pressure-time response is shown in Figure 5. After the furnace controller had been reset from 250 °C (VHT) to 700 °C (combustion), the sample zone temperature increased at ∼50 °C/min, which coincided with a pressure rise from 255 to 363 Torr (over some 8 min). Subsequently, the pressure decreased to 285 Torr after 100 min. This latter pressure decline is attributable to ongoing gas depletion: O2 uptake by iron oxide and CO2/H2O sorption by lithium oxide. For several monitored 15N sample combustions, reactor peak pressure did not exceed 150% of the initial oxygen load. (Caution: Analysts should always be suitably shielded from operating reactors.) Biomaterial Isotopic Determination. Reactor coupling to a scanning quadrupole mass analyzer enables inspection of the product-gas mass spectrum, as the majority of pertinent combustion data fall in the range from 2 to 100 Da. For 15N sample determination, it was important to compare the magnitude of the m/z 30 signal (15N2+) relative to those at m/z 2 (1H2+) and m/z 32 (16O2+). The final product gas should contain only minor quantities of H2 so that spectral interferent formation is minimized. Problematic interferents during N2 analysis can

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Figure 5. Simultaneous time profiles of reactor pressure (right scale) and furnace readout temperature (left scale) during the combustion of lyophilized algal cells. Table 1. Summary of TZCR-MS Isotopic Determination Results with the Number of Replicate Analyses Noted in Parentheses glycine-15N (n ) 5) 4-nitroaniline-15N2 (n ) 5) algal protein-15N (batch A, n ) 5) algal protein-15N (batch B, n ) 10) lyophilized algal cells-15N (n ) 5) lyophilized algal cells-13C (n ) 5) lyophilized algal cells-13C (n ) 5) algal protein-13C (n ) 4) algal amino acids-13C (n ) 5) algal starch-13C (n ) 3) potato starch-13C (n ) 3) a

average

SDa (atom %)

99.2 atom % 15N 99.7 atom % 15N 99.0 atom % 15N 98.7 atom % 15N 98.8 atom % 15N 99.1 atom % 13C 5.8 atom % 13C 99.0 atom % 13C 99.1 atom % 13C 98.6 atom % 13C 98.3 atom % 13C

(0.08 (0.05 (0.12 (0.13 (0.17 (0.06 (0.04 (0.03 (0.05 (0.06 (0.03

Standard deviation abbreviated SD.

include ethane, ethylene, nitric oxide, carbon monoxide, and carbon dioxide; likewise, propane, propylene, nitrous oxide, and nitrogen dioxide can interfere during CO2 analysis. A minor amount of unreacted O2 was tolerable for the satisfactory 15N or 13C isotopic determination at atom percent levels. In other words, it is advantageous to ratio the starting materials so that the postcombustion signal magnitude of 15N2+ during 15N determination (or 13CO2+ during 13C determination) is very high relative to those of all other product ions (such as H2+, CO+, and O2+). Generally stated, isotope contamination must be minimized during the entire TZCR-MS process to obtain an accurate 15N sample enrichment. Because the reactor is normally operated at sub-barometric pressure during the pretreatment, reaction, and measurement stages, the primary contaminants are atmospheric N2 and CO2 (the latter generates CO+ fragments in the mass spectrometer). As a simple example, consider the combustion of 70 mg of algal protein (assumed to contain 15 mass % elemental nitrogen) to produce 0.00038 mol of N2. Based on the gas chromatographic analyses of an empty reactor maintained under static vacuum, the upper limit for overnight air leakage into the reactor is 8 × 10-7 mol of 14N2 (which equates to 0.15 mol % of the N2 combustion product). Thus, it is expected that 15N determinations of highly enriched biomaterials could be falsely low by 0.1 atom %. The nitrogen isotope TZCR-MS process was first evaluated using highly enriched glycine-15N and 4-nitroaniline-15N2 (see Table 1). Five replicate determinations for one batch of glycine15 N averaged 99.2 ( 0.08 atom % 15N. This same glycine-15N

was synthesized from ammonia-15N, where the ammonia was independently measured to contain 99.2 atom % 15N. Analogously, replicate analyses for 4-nitroaniline-15N2 averaged 99.7 ( 0.05 atom % 15N, and its synthetic precursor was analyzed as 99.8 atom % 15N. The 15N sample determinations for both evaluation materials, each known to be >99% chemically pure, thus strongly indicate that TZCR-MS is suitable for product quality control. Most stable isotopes of present consideration (protein/starch/ cells) exist as polymeric solids at ambient conditions. Within the isotope industry, biosynthetic algal materials have been especially troublesome to analyze. One major achievement of TZCR-MS has been reproducible quantitative isotope determination for many algal biomaterials. Referring to Table 1, batch A of algal protein-15N was subjected to overnight VHT at 250 °C and then combusted in 600 Torr of O2 to yield 99.0 ( 0.12 atom % 15N. The batch-A nitrogen precursor was reported to be 99.2 atom % 15N. Considering that more than 20 process steps were executed (including natural-abundance nitrogenous reagents) to grow the algal cells and ultimately isolate the protein, the 15N enrichment result is reasonable. By application of similar methodology, a production batch of lyophilized algal cells-15N was determined to be 98.8 atom % 15N. General quantitative reproducibility for 15N sample determinations was found to be better than 0.2 atom % 15N (see Table 1). Furthermore, the unitary reactor design, the specific reactor temperature profile, and the intrareactor chemistry clearly distinguish TZCR-MS from prior analytical methods.17 A simpler procedure was developed to perform the 13C determination of biosynthetic analytes for the following reasons: (1) The algal bioanalytes typically contain >50 mass % carbon. (2) After 13C sample derivatization, CO2 was the major gas product. (3) CO does not directly interfere with the 13C measurement of CO2+ ions. (4) The fraction of natural CO2 in air is ∼0.03 mol %. Reagents utilized for 13C sample combustion include dioxygen and (optionally) calcium sulfate. Each 13C-enriched biomaterial in Table 1 was analyzed three or more times, and as seen by inspection, the precision for 13C determination was better than (0.1 atom %. The appropriate VHT temperature for a given sample type was determined empirically. For instance, algal protein-13C should be pulverized and thoroughly pretreated at >200 °C in dynamic vacuum prior to combustion. To help characterize the postcombustion gas, algal protein-13C product gas was measured by quantitative gas chromatography. After five replicate protein-13C combustions (0.07 g of protein, 0.002 mol of O2, controller 700 °C), the product gas analyzed as 93 vol % CO2, 1.2 vol % CO, 0.08 vol % CH4, 95 atom % stable isotope (see Table 1). Within the reactor, preliminary sample bakeout was followed by sample combustion and selective gas sorption. A longitudinal temperature distribution was set up and maintained along the reactor (z-axis) length to facilitate the desired chemical conversions. During 15N sample determinations, O2, Fe3O4, and Li2O were cooperatively employed to direct the conversion of analyte nitrogen to N2 (the product gas was mostly N2). During 13C sample isotope determination, O2 was utilized to convert the analyte carbon to CO2, and calcium sulfate was stationed to absorb water. Significantly, the disparate operations of sample VHT, sample combustion, and gas sorption can all be executed within one special pipe. Compared to dynamic-flow combustion analyzers for isotope analysis (such as elemental analyzers with multiple reactors), the TZCR is simplified. Because each sample of Table 1 was received for analysis after considerable workup (isolation), no analytical chromatography was incorporated into the TZCR methodology. Postreaction gas injection into a quadrupole mass spectrometer enabled identification of the final product gases. In principle, a different MS analyzer might be substituted to obtain improved signal precision [isotope ratio mass spectrometry (IRMS)] or mass resolution [Fourier transform ion cyclotron resonance mass spectrometry (FT-ICRMS)]. For laboratories wherein a mass spectrometer is time-shared, the offline TZCR operations can be executed while the MS is analyzing other samples. With regard to process safety, no reactor has structurally failed over several years of ongoing activity. Rather, most reactor pipes were reused for about 50 analyses and then decommissioned. Primarily, the title combustion reactor functions to precondition the biosynthetic sample and selectively convert it to analyzable gas (N2 or CO2). Reactor application helped to solve a significant problem in biomaterial isotope analysis, exemplified by products such as lyophilized algal cells-15N and starch-13C. Furthermore, a transparent reactor design has enabled the visualization of certain (analyte-dependent) events such as sample vapor sublimation. Finally, the authors emphasize that offline TZCR-MS was developed for the quantitative stable isotope determination of highly enriched materials by contrast

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to more complex chromatographic combustion systems such as gas chromatography combustion isotope ratio mass spectrometry (GCC-IRMS, mainly used for analytes that contain modestly enriched isotopes). Therefore, TZCR-MS should prove beneficial to the technical communities that have need for biomaterial isotopic determination. Acknowledgment The authors thank the Aldrich Glass Shop group for reactor construction. It is a pleasure to acknowledge Scott Purdin, David Schory, David Kimmel, Lorrie Castro, Lane Dewees, Len Chandler, and John Shay for collaborative support. Furthermore, we thank each of our co-workers at the Miamisburg site of Sigma-Aldrich Corporation. Literature Cited (1) Dennstedt, M. Anleitung zur Vereinfachten Elementaranalyse fur wissenschaftliche und technische Zwecke; O. Meissner: Hamburg, Germany, 1903. (2) Gustin, G. M. A simple rapid automatic micro-Dumas apparatus for nitrogen determination. Microchem. J. 1960, 4, 43–54. (3) Aston, F. W. A positive ray spectrograph. Philos. Mag. 1919, 38, 707–715. (4) Nier, A. O. C. A mass spectrometer for isotope and gas analysis. ReV. Sci. Instrum. 1947, 18, 398–411. (5) McKinney, C. R.; McCrea, J. M.; Epstein, S.; Allen, H. A.; Urey, H. C. Improvements in mass spectrometers for the measurement of small differences in isotope abundance ratios. ReV. Sci. Instrum. 1950, 21, 724– 730. (6) Matthews, D. E.; Hayes, J. M. Isotope ratio monitoring gas chromatography mass spectrometry. Anal. Chem. 1978, 50, 1465–1473. (7) May, M.; Gray, M. Process and apparatus for isotope determination of condensed phase samples. U.S. Patent Application 2008/0035840a1. (8) Boutton, T. W.; Wong, W. W.; Hachey, D. L.; Lee, L. S.; Cabera, M. P.; Klein, P. D. Comparison of quartz and Pyrex tubes for combustion of organic samples for stable carbon isotope analysis. Anal. Chem. 1983, 55, 1832–1833.

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ReceiVed for reView January 15, 2010 ReVised manuscript receiVed March 13, 2010 Accepted March 14, 2010 IE100072Z