Patent Application
20080035840
Corresponding reference characters indicate corresponding parts throughout the drawings.
In accordance with the present invention, an advantageous process and apparatus are provided by which a specimen of a condensed phase composition may be reacted to generate gases suitable for mass spectrometric determination of the proportion of the composition carbon content that is constituted by 13C, the proportion of the composition nitrogen content that is constituted by 15N, or the proportions of other elements that may be constituted of particular isotopes, such as, e.g., 33P or 34S. Preferably a specimen of the composition is reacted using dry combustion, i.e., without need for preliminary acid digestion of a composition such as a biosynthesized protein or peptide to break it down into its constituent amino acids. By directly combusting a specimen of an unknown composition, any isotopic contamination or adulteration that might otherwise arise in the digestion process may be avoided. Direct combustion also avoids the corrosive conditions typically incurred in wet digestion with hydrochloric acid. The process is relatively practical and straightforward, and the apparatus simple, economical and robust. Moreover, the system is amenable to the determination of 13C or 15N content by injecting isolated CO2 or N2 into a general-purpose mass spectrometer rather than a specialized isotope ratio mass spectrometer.
The process can be conducted in a batch mode to produce a convectively mixed carbon oxide or N2 gas fraction that can be injected into the mass spectrometer at the convenience of the operator. Unlike dynamic flow systems of the prior art, the operation of the process and apparatus of the invention does not require critical timing of the introduction of the gas stream into the mass spectrometer, or critical control of the interval between the combustion step and the gas analysis. This obviates the need for sophisticated, typically computer driven, control systems that are necessary to identify and divert an N2, CO2 or other isotopically representative fraction to the spectrometer. Moreover, the apparatus can be cleaned and reused a large number of times, e.g., ordinarily at least 10, more typically at least 50, and often at least 100 times before it must be discarded or overhauled.
The process and apparatus are particularly adapted for the determination of 15N/14N ratios in condensed phase nitrogenous compositions or 13C/12C ratios in condensed phase carbonaceous compositions, e.g., solid-phase organic compositions, and/or compositions of relatively low vapor pressure. A prominent application is for analysis of the 13C or 15N content of proteins, peptides, mixtures of proteins and peptides, lyophilized biological cells, algal cells, or a tissue specimen, but the process is also well adapted for the determination of 13C or 15N in chemically synthesized compositions such as solid asphalts, solid tars, polyaromatic materials, oxygenated or sulfur-bearing hydrocarbon polymers, nitroanilines, nitrophenols, phthalimides, phenylene diamines and/or other nitrogenous aromatic compounds. 15N-bearing and/or 13C-bearing amino acids are also subject to analysis according to the process of the invention. More generally, the process might be adapted for 13C or 15N-determination of solid-phase compositions that include amines, amides, nitro-group chemicals, nitrogenous heterocycles, enzymes, purines, pyrimidines, polyaromatic hydrocarbons, polyacrylics, polyethers, polyesters, polysulfones and select drugs. In various preferred applications, the composition subject to analysis is substantially in the solid phase at 100° C. and/or has a vapor pressure less than about 10 mm Hg, preferably less than about 1 mm Hg, at 100° C. and/or has an atmospheric boiling point greater than about 200° C.
Biosynthesized proteins, biosynthesized peptides, algal lyophilized cells, other cellular specimens, tissue culture or other tissue specimen and other carbonaceous or nitrogenous materials or specimens are often solvated with residual solvent used in their preparation. Although they are also commonly lyophilized after synthesis, lyophilization is generally effective only for removal of free solvent, not for removing bound solvent that may be bonded to the carbonaceous and/or nitrogenous product via hydrogen bonding or the like, and/or present as inclusions embedded within the solid mass. In various preferred embodiments of the process of the invention, a specimen of the composition is controllably desolvated within the process vessel prior to combustion thereof.
As illustrated in
In many if not most instances, the combustion product gas may also at least nascently contain CO and NOx. Especially where solid specimens are combusted, carbon contained in specimen compounds may not be fully oxidized, especially at the modest ratios of dioxygen to specimen described below. Moreover, under the prevailing combustion conditions, CO2 tends to partially dissociate. In various preferred embodiments, as described below, a supplemental reaction agent is provided to oxidize CO to CO2. The supplemental reaction agent may be provided within the combustion region where oxidation of CO to CO2 proceeds (step B). As described below, contamination with NOx has generally proven not to be a major problem in conducting the process using the apparatus of the invention. It is believed that during combustion NOx may be reduced (step C) by reaction with transient NHx, or by reaction with transient CHx, or by reaction with carbon char produced by incomplete combustion of the specimen. Optionally however, the gas phase may be contacted with a reductant for NOx within the generation and isolation zone, either inside or outside the combustion region. Where a reductant for NOx is used, a discrete reduction region may be preferably positioned within the generation and isolation zone at a location where a temperature effective, more preferably optimal, for NOx reduction may prevail during combustion, i.e., a location along the temperature profile of
Over the course of a combustion and sorption cycle, the composition of the combustion gas approaches a steady-state kinetic condition in which chemical reactions reach equilibrium, phase equilibrium is established with sorbent(s), and/or condensate at a cold trap, contained within the zone, and concentration gradients have been eliminated by axial (and radial) convective mixing. For the case of 15N-determination, both water vapor and CO2 are preferably depleted from the gas-phase.
Free-convective mixing of the gas phase within the space defining the generation and isolation zone yields a carbon oxide or nitrogen gas fraction of substantially steady-state and uniform composition within the generation and isolation zone, typically within the single chamber of the process vessel of
The combustion region 5 of generation and isolation zone 3 within tubular process vessel 1 is surrounded by a heat source, e.g., an electrical resistance furnace 23 such as a Carbolite MTF 12/38/9400 furnace, which is effective for heating the specimen in boat 11 to its reactive temperature. The furnace is adapted to supply heat substantially to the combustion region so that the sorption region 7 may be maintained at a lower temperature suitable for sorption of components such as CO2 and/or water vapor from the gas phase.
During combustion, there is dynamic movement of gas within the process vessel between the combustion region and other regions of the generation and isolation zone, as driven by turbulence within the zone and by axial gradients of concentration and temperature between the combustion region, typically located in a middle section of the generation and isolation zone, and other regions, such as the sorption and/or dehydration region, which are typically located in peripheral sections of the zone. The spacing between the combustion region 5 and the sorption/dehydration region 7 relative to the diameter and wall thickness of the tubular vessel is preferably selected so that environmental heat losses alone establish an axial thermal gradient along the longitudinal axis of the vessel sufficient that the sorption region is at a temperature low enough for effective sorption of CO2 and/or water vapor.
Further salient features of the apparatus are described hereinbelow, following detailed description of the process.
In carrying out the process of the invention, a specimen of a condensed phase composition to be analyzed, typically a solid specimen, is introduced into the combustion region within the generation and isolation zone, e.g., by placement in boat 11 within vessel 1. Often, the raw condensed phase composition comprises volatile components, for example a bound reagent that can cause complications in the isotopic analysis. In preferred embodiments of the process, such volatile components are removed by controllably heating the combustion region containing the specimen, exposing it to sub-atmospheric pressure, or both. Preferably gas flow port 17 is connected in gas flow communication to a vacuum pump or ejector via an adjustable orifice (such as a metering valve), and the pressure dynamically reduced to and maintained at less than about 1 torr, preferably less than about 0.1 torr, while heat is supplied by furnace 23 to promote removal of volatiles. Usage of an adjustable valve between process vessel 1 and an external vacuum source limits the movement of intravessel materials during vessel evacuation. The temperature of the specimen during removal of volatiles is typically in the range between about ambient and 250° C. Removal of volatiles may generally require exposure to dynamic vacuum for a period of about 1 to about 50 hours.
As needed for an isotopic determination, a sorbent may be loaded into boat 15 in sorption region 7 prior to evacuation of the generation and isolation zone 3 for removal of volatiles from the specimen. Where this procedure is followed, the sorbent is degassed during the vacuum heat treatment step. Degassing the sorbent functions primarily to remove air. Removal of air is desirable inter alia to avoid contamination of a product fraction with dinitrogen or CO2 from the air. Where a supplemental reaction agent is used, e.g., a metal oxide as described below, vacuum heating of the combustion region prior to combustion may serve the further purpose of increasing supplemental reaction agent activity by removing adsorbed impurities and converting metal carbonate to metal oxide.
To carry out the combustion step, a primary oxidant, preferably dioxygen (molecular oxygen), is introduced into the generation and isolation zone. Other oxidants, e.g., Cl2 or O3, may be used in some circumstances, but molecular oxygen is highly preferred. The dioxygen charge is preferably substantially dry, i.e., containing less than 1 volume % water vapor, preferably less than about 0.01 volume % water vapor, and chemically pure, containing at least 99 volume % O2, preferably greater than 99.9% by volume, more preferably greater than about 99.995 volume % O2. In order to minimize the presence of nitrogen from air, the generation and isolation zone is preferably evacuated prior to admission of oxygen, irrespective of whether evacuation is necessary for removal of undesired volatiles from the specimen. Oxygen is thereafter charged to any convenient pressure, for example, 10 to 1000 torr (absolute), more typically 100 to 1000 torr, still more typically about 125 to about 700 torr, and most preferably between about 150 to about 400 torr (absolute). After oxygen is admitted, heat is applied by furnace 23 to heat the specimen to its reactive temperature in contact with dioxygen, thus generating a combustion product gas typically comprising CO2 and water vapor, or CO2, water vapor and N2. Combustion may typically be conducted at a temperature between about 350° and about 1000° C., more typically between about 500° and about 800° C. A modest exotherm is typically expected during combustion. For example, if the furnace is controlled to bring the temperature of the specimen to a temperature in the range of 500° C. to 750° C. for reaction, the exotherm may typically involve an additional 0.5 to 5 kilojoules for organic combustions. Gradual low-temperature combustion, as preferably practiced in the process of the present invention, contrasts with GC-IRMS, which generally utilizes temperature greater than 900° C. for rapid organic combustion. Thus low-temperature combustion significantly simplifies process vessel design and the selection of construction materials. Optionally, a thermocouple (not shown) may be placed in contact with the outer wall of vessel region 5 or even more proximal to boat 11, and the heat generation by furnace 23 controlled in response to the combustion region temperature via a temperature controller (not shown) in order to initiate combustion at a target temperature. However, for routine repetitive processing of specimens of similar composition, furnace output can be controlled based on process operating experience without need for a temperature control loop in solid contact with vessel or specimen. It will be understood by those skilled in the art that if desired, other apparatus controllers may be additionally incorporated into the apparatus without departing from the scope of the invention (such as timer controller, mass flow controller, pressure controller, valve controller, programmable logic controller, or microprocessor).
Complete combustion of the specimen is not necessary. In fact, the process can proceed satisfactorily where the principal combustion step actually comprises a combination of oxidation and pyrolysis, yielding not only CO2, water and CO as products of combustion, but also hydrogen and carbon under particular conditions. Thus, e.g., the quantity of the specimen relative to the volume of the generation and isolation zone, i.e., the interior of tubular process vessel 1, may be such that a charge of 100% molecular oxygen occupying this zone at 25° C. and 500 torr (absolute) would be stoichiometrically equivalent to about 10% to about 150% of the carbon content of the specimen. The specimen is sized and molecular oxygen charged at such pressure that the measured signal magnitude for dioxygen in the process vessel after combustion/sorption is preferably less than about 50%, more preferably less than 5%, and still more preferably less than 1% of the signal magnitude for product CO2 or dinitrogen.
Preferably, there are no significant flow restrictions between the combustion and optional sorption and/or dehydration region, nor otherwise within the generation and isolation zone. More preferably, as shown, the combustion and sorption/dehydration regions are contained within a single chamber. As a consequence, no significant pressure drop is observed in flow of gas from the combustion region to the sorption/dehydration region, and sorption or other dehydration step is typically initiated during the combustion phase. As combustion and sorption/dehydration progress, the generation and isolation zone becomes mixed substantially via thermally-driven free convection between the combustion and sorption/dehydration regions, and typically throughout the entire zone. The total pressure within the generation and isolation zone is typically in the range of about 10 and about 1000 torr, more typically between about 25 and about 1000 torr, most typically between about 50 and about 800 torr, during the combustion/sorption cycle. During combustion, the peak pressure may typically be in the range of 100 to 1000 torr, the peak pressure being dependent upon the particular isotopic determination, the reactant quantities, the furnace temperature, the combustion time, etc. A modest positive gauge pressure can assist in preventing ingress of air during combustion and sorption. For example during 15N-determination, where dioxygen is introduced into the process vessel 1 at an absolute pressure in the range of 400 to 600 torr, peak pressure may be reached at 550 to 1000 torr, after which the pressure declines due to gas sorption and/or reactant depletion. Where combustion and sorption are conducted in a batch mode, the overall batch cycle is typically between about 1 and about 50 hours, more typically between about 2 and about 50 hours.
Preferably, a supplemental reaction agent is provided to promote the chemical conversion of CO and/or NOx. Conveniently, the supplemental reaction agent may be present in the combustion zone, e.g., mixed with the specimen in boat 11 at combustion station 9. In such embodiments, the supplemental reaction agent may be mixed with the specimen prior to introduction of the specimen into the combustion region, or mixed with the specimen at the combustion station, e.g., in boat 11, prior to combustion, and ordinarily prior to vacuum heating for removal of volatile components from the specimen. Alternatively, a separate station may be provided comprising a holder for the supplemental reaction agent.
Preferably, the supplemental reaction agent is a transition metal oxide, typically in particulate form with particle size preferably less than about 0.1 mm. Exemplary suitable supplemental reaction agents include Fe3O4, CuO, CO3O4, or NiO, although mixtures of a given metal oxide plus metal (such as Fe3O4—Fe) can also be exploited as supplemental reaction agent mixtures. Peroxides are generally not preferred as supplemental reactants. The supplemental reactant can directly function as an oxidizer of specimen material and/or CO, and may also catalyze the chemical conversion of CO and/or NOx. Preferably the specimen, or a mixture of specimen plus supplemental reaction agent, is mechanically pulverized prior to introduction into the process vessel. Preferably, the specimen and supplemental reaction agent may be mixed in a mass ratio between 10:1 and 1:10, more preferably between 5:1 and 1:5, and ordinarily between about 2:1 to 1:2.
The sorption region may comprise a plurality of sorption stages, but does not require a multi-stage separation system such as a chromatographic column. Ordinarily, not more than three sorption stages are needed and, regardless of the number of stages, the sorption region gas-phase is convectively mixed. During analysis for 15N-content, sorption is preferably conducted in a single stage using a sorbent that is effective for sorption of both water and CO2. During 13C-analysis, an optional sorbent may be utilized to selectively remove water vapor or carbon monoxide. Again sorption is preferably conducted in a single stage. Contact between the gas phase and the sorbent may be effected by allowing the gas to flow over the sorbent bed, e.g., by placing the sorbent in boat 15, rather than through the sorbent bed as in a conventional fixed bed adsorber or packed column. In this way, plugging of flow path is advantageously avoided, pressure drop across the sorbent region is minimized, and free convective gaseous mixing is promoted within the generation and isolation zone.
Any convenient sorbent may be used. It will be understood that the sorption station may comprise an adsorbent, an absorbent, or both. In the case of 15N determination, preferred sorbents for CO2 and water include alkali metal oxides such as lithium oxide, sodium oxide and potassium oxide, preferably in an anhydrous state as charged to the sorption station. Lithium oxide is a particularly preferred H2O and CO2 sorbent. While sorbent mixtures such as soda-lime can be used if desired, it is generally preferred that strong oxidants not be used. Typically, the solid sorbent may consist of particles sized less than 0.5 mm. Without being limited to any single theory, it is believed that dry alkali metal oxide sorbents may function as either adsorbents or absorbents. Under initial gas/solid contact, the dry alkali metal oxide may function primarily as an adsorbent. However, once an alkali metal oxide sorbent has sorbed a modest quantity of water, a caustic mixture may be formed at the sorption station which can function as an absorbent for both CO2 and water. In the case of 13C-determination, particular sorbents that may be useful for water sorption include anhydrous calcium sulfate, phosphorus pentoxide, activated alumina, and activated silica gel; similarly, an exemplary material that may be useful for carbon monoxide removal at the sorption station is hopcalite.
If desired for purposes of 15N determination, separate sorbents may be provided at separate sorption stations for CO2 and water vapor, respectively, within the sorption region. For example, water vapor can be sorbed by anhydrous calcium sulfate at one sorption station, whilst carbon dioxide is sorbed by soda-lime at a separate sorption station. Where a specimen containing sulfur is reacted for purposes of either 15N or 13C determination, a sorbent effective for removal of SOx may be provided. Certain metal oxide sorbents are effective for SOx removal, for example CaO (quicklime). Where reactive absorption is involved, e.g., between CO2 and/or SOx and alkaline metal hydroxide solution, the quantity of sorbent(s) is preferably at least stoichiometrically equivalent to the total carbon, hydrogen, and sulfur contained in the specimen. Typically, an alkali metal oxide is provided in an excess ratio of at least ten, more typically at least fifteen, with respect to the sum of carbon, hydrogen, and sulfur. If a separate sorbent is provided for water, such as CaSO4 or P2O5, it is preferably provided in stoichiometric excess vs. the hydrogen content of the specimen.
The apparatus of the invention, as typically used in the process for determination of 15N content, enables recovery of a nitrogen gas fraction that is substantially devoid of CO2 without the necessity of using a cold trap. Avoidance of cold traps significantly simplifies the process and apparatus for isotopic determination. However, it will be understood by those skilled in the art that, if desired, a containment module can be provided, either ported to the tubular process vessel (for example such a module may be sealably connected via port 19), or as an appendage to the vessel or connected between process vessel and spectrometer during discharge of the nitrogen gas fraction to a mass spectrometer, or in a combination of two or more such arrangements. The use of a sorbent material positioned at a favorable location along the axial temperature gradient, i.e., a temperature environment in which sorption equilibria and mass transfer rates are conducive to sorption, is desirable for most applications, and the removal of H2O, CO2, and/or SOx by sorption rather than by cold trap represents a significant advantage of various preferred embodiments of the present invention.
The generation and isolation zone within tubular process vessel 1 is preferably configured so that environmental heat losses during combustion cause sorption to take place at a temperature less than 100° C., preferably less than about 50° C., typically at ambient or slightly above. Optionally, the sorption region may be cooled by forced circulation of ambient air around the portion of the tubular process vessel surrounding the sorption region, or by circulation of another cooling fluid, e.g., cooling water or coolant gas through an annular jacket surrounding the sorption region. The pressure in the sorption region is essentially the same as in the combustion region, i.e., during the combustion step. During 15N-determination it may typically rise early in the process to a peak level in the range 550 to 1000 torr, then drop back, e.g., to a level in the range of 10 to 500 torr as gaseous material is sorbed. Pressures in these ranges are generally suitable for effecting sorption of water vapor and/or CO2.
Because combustion is practically conducted with a slight deficiency or slight excess of oxidant, combustion of the specimen is not necessarily complete. Often the specimen may partly char during combustion instead of being entirely consumed. Under such conditions, most NOx generated during the combustion may typically be reduced by reaction with carbon, transient CHx species, or transient NHx species produced by combustion. The presence of H2 (dihydrogen) has been conditionally observed in the product carbon oxide or dinitrogen gas fraction, indicating that hydrogen species could also have a role in reducing NOx or inhibiting its formation. Moreover, within a preferred combustion temperature range, e.g., 500° C. to 750° C., even nascent NOx formation may not be favored. In any event, the carbon oxide or nitrogen gas fraction appropriately produced in accordance with the invention does not typically contain any substantially interfering fraction of NOx. In addition to the potential function of carbon char, CHx species, NHx species, and/or hydrogen as reductants, a metal oxide used as a supplemental reactant may conceivably function as a catalyst for the decomposition of NOx to N2 and O2. As a still further possibility, some NOx might be captured in the sorption region by the sorbent for water vapor and/or CO2.
Alternatively, as illustrated schematically in
In preferred embodiments of the invention, as combustion and sorption proceed, the gas phase equilibrates by free convective axial (longitudinal) and radial back mixing so that the composition of the gas phase becomes substantially steady-state throughout the generation and isolation zone. After combustion and any sorption or other dehydration step(s) have been completed in the course of a 13C determination, a product carbon oxide gas fraction is obtained wherein the sum of the H2 and CO content is preferably not greater than about 5 volume %, more preferably not greater than about 1 volume %, basis CO2. Though the 13C-process can proceed stoichiometrically deficient of dioxygen, it is preferred that the combination of combustion and sorption/dehydration conditions be such that the sum of the water vapor content, CO content and H2 content is not greater than about 10 volume %.
After the combustion and sorption steps are complete in the course of 15N determination, a product dinitrogen gas fraction is obtained wherein the sum of the water vapor content and CO2 content is not more than about 5 volume %, preferably not more than 1 volume %, and more preferably not more than 0.5 volume %. Preferably, the carbon monoxide content is not more than about 0.5 volume %, more preferably not more than about 0.2 volume %. It is desirable to establish both a low CO content and a low CO2 content, especially for higher precision determinations because at typical ionization conditions CO2 tends to fragment into CO and oxygen in a mass spectrometer. While 12C16O2 as such, which has a nominal mass of 44, does not directly interfere with the determination of 15N, any 12C16O (nominal mass=28) or 13C16O (nominal mass=29), whether present in the sample or formed within the spectrometer, has a deleterious effect. A sorbent or combination of sorbents effective for removal of both CO2 and water is preferred because the presence of water vapor can apparently influence the process conversion of CO to CO2. Preferably, the product nitrogen gas fraction is essentially free of NOx, a result that is achievable on the basis described hereinabove.
On the basis of actual process results, it is preferred that a product nitrogen gas fraction contains minimal quantities of both dioxygen and dihydrogen (that is, minimal amounts of gaseous oxidant and reductant).
Because molecular oxygen has nominal molecular masses of 32 through 36, the presence of a minor proportion of dioxygen and/or dihydrogen in the product nitrogen gas fraction does not directly affect mass spectral analysis. For purposes of 15N-determination, it is preferred that the dioxygen content be not greater than about 50 volume % in the product gas, more preferred that the dioxygen content be not greater than 5 volume %, even more preferred that the dioxygen content be not greater than 1 volume %. With regard to residual hydrogen gas after combustion, it is preferred that the dihydrogen content be not greater than 5 volume % or more preferably not greater than 1 volume %.
For purposes of 13C determination, it is preferred that the dioxygen content of the carbon oxide gas fraction comprise not more than about 50 volume %, more preferably no more than about 10 volume %, still more preferably not more than about 5 volume %, most preferably no more than about 1 volume %.
In the system described above, an appropriate thermal gradient, such as that illustrated in
It will be understood that
After the combustion and sorption cycle is complete, the process vessel containing the product nitrogen gas fraction may be wheeled to the location of a mass spectrometer, and a sample comprising at least a portion of the carbon oxide or nitrogen gas fraction introduced into the injection port of the spectrometer. The 15N-isotopic content of a sample, and thus of the nitrogen contained in the specimen, is ordinarily determined from mass spectrometric signals observed at nominal m/z ratios 30, 29, and 28 (N2), though it is conditionally conceivable that spectrometric signals at nominal m/z ratios 15, 14.5, and 14 (N22+) might instead be utilized for 15N-determination. It has been found that, when the apparatus of
In combination, the process and apparatus are useful for performing 13C or 15N-determinations of various uniformly isotope-enriched specimens. A major advantage of the invention is capability to controllably vacuum heat treat a carbonaceous and/or nitrogenous specimen within the process vessel itself, some controllable parameters being intravessel pressure, treatment temperature, and treatment time. The vacuum heat treatment step, performed prior to and within the same chamber as combustion, primarily functions to remove natural abundance volatile materials from the condensed phase specimen. Thereby the measured 13C or 15N-content of a treated specimen is representative of the actual analyte. Advantageously, any supplemental reaction agent (such as Fe3O4) that may be mixed together with specimen in a first station within the combustion region can be activated simultaneously during the vacuum heat treatment step. After the combustion cycle is complete, the process vessel containing the combustion product gas may be wheeled near a mass spectrometer, and some portion of the product gas introduced into the spectrometer injection port. The 13C-isotopic content of a sample, and thus of the corresponding specimen, is preferably determined from mass spectrometric signals observed at nominal m/z ratios 44, 45, 46, 47, 48, and 49 (CO2 ions). Alternatively however, it may be desirable in particular cases to instead determine sample 13C-content derived from mass spectrometric signals at nominal m/z ratios 28, 29, 30, and 31 (CO ions), or derived from signals at nominal m/z ratios 12 and 13 (C1 ions).
Referring again to the apparatus as depicted in
One or more O-rings 37 may assist in hermetically sealing the connection between socket 36 and bushing 35. The primary gas port 17 comprising socket 36 is adapted for gas flow connection to a source of molecular oxygen, a gas analyzer, a vacuum source or a gas manifold such as that illustrated in
Combustion region segment 31 preferably extends beyond combustion station 9 in both longitudinal directions, i.e. in the direction of both port 19 and port 17. This dual extension typically extends beyond the region to which heat is actively supplied by furnace 23, and thus provides intermediate regions within which the temperature may progressively decrease from peak combustion temperature to a temperature favorable for selective sorption and/or condensation during the combustion cycle, as illustrated in
Secondary access port 19 comprising threaded socket 38 at the outer end of sorption region segment 39 is adapted for direct connection to a threaded and flanged bushing 41. The latter connection may be established with the assistance of one or multiple O-rings 43. By appropriate combination of tube, bushing, O-ring, and socket subassemblies, the apparatus of the invention is effectively sealed against influx of contaminating air, without the necessity of vacuum grease.
Tee 45 (
Illustrated in
Referring to
It has been found convenient for process vessel 1 to have an internal volume in the range of 0.02 to 2 liters, more conveniently 0.05 to 0.5 liters, advantageously between about 0.1 and about 0.25 liters. In such instance, the amount of specimen charged to combustion station 9 may typically be in the range between about 15 mg and about 1.5 g, more typically between about 25 mg and about 500 mg, most typically between about 50 mg and about 250 mg, where O2 is initially charged to a pressure of about 100 to about 800 torr. The supplemental reaction agent charge is preferably within the range between about 5 mg and about 1.5 g, between about 50 mg and about 500 mg, or between about 100 and about 250 mg. At the scale defined by the aforesaid quantities of specimen and a preferred process vessel L/D aspect between about 10 and about 250, the distance from combustion station 9 to sorption station 13 is preferably between about 5 cm and about one meter, more preferably between about 10 cm and about 75 cm, more typically between about 15 cm and about 50 cm.
Illustrated in
The apparatus and process of the invention are adapted for conducting a relatively high volume of repetitive isotopic determinations. After each combustion cycle, combustion and sorption cycle, or combustion and water vapor condensation cycle, the apparatus may be cleaned and re-used to generate and isolate another carbon oxide or N2 fraction. The interior of the vessel and the quartz boats may be cleaned with a brush and abrasive, after which fresh sorbent may be supplied to the sorption station and a new specimen, mixed with fresh supplemental reaction agent as needed, may be supplied to the combustion station. By use of boats for specimen and sorbent, the apparatus may be readily re-used without laborious repacking of reactors, sorption columns, and the like. Moreover, the process vessel/furnace module can be mobile thus easily transported and attached to available gas analyzers and manifolds. The unified reactor/sorption process pipe is refractory, transparent and grease-free. Greaseless connections eliminate any possible isotopic contamination from grease usage and they facilitate apparatus cleanup and re-use.
The use of boats for specimen, supplemental reaction agent, sorbent and reductant minimizes movement of any of these materials outside its proper region during combustion and sorption operations.
Gastight process vessel 1 also affords convenient storage of the carbon oxide or nitrogen fraction pending analysis by mass spectrometry or alternative techniques. For example, in higher volume operations, a plurality of process vessels may be used to generate a plurality of carbon oxide or N2 fractions which can be accumulated to be run successively through a single mass spectrometer. This allows the mass spectrometer to be usefully employed in making other analyses, if desired, without compromising analyses of specimens for 13C or 15N content. The sample or accumulated samples of carbon oxide or nitrogen gas fraction may be analyzed at any time as determined by instrumentation availability.
The apparatus and process of the invention are capable of making 13C and 15N determinations with accuracy and analytical precision. In various preferred embodiments, however, the process and apparatus of the invention are not designed for ultra-precise isotopic analysis equivalent to that provided by a GC-IRMS system. Generally, the system is adapted to determine overall 13C or 15N condensed-phase specimen content, being reproducible to about 0.1 atom % 13C or 15N. At this level of precision, which is entirely sufficient for most 13C or 15N-enriched commercial materials and many research applications, the apparatus can be provided at modest cost for processing of a high volume of either routine samples or samples for which such precision is otherwise satisfactory. Because of its batch operation and lack of multi-stage adsorption, the apparatus of the invention is not typically used for determination of both carbon and nitrogen isotopic ratios on the same sample. However, if desired, the apparatus and process might be adapted to firstly measure 15N-isotopic content of a sample as described, next remove residual gases from the process vessel, and afterward desorb or by some other mechanism liberate carbonaceous gas from a sorbent at sorbent station 13 and in this manner secondly measure the 13C-isotopic content for the same sample.
The natural abundance of 15N is about 0.4 atom %. The process of the invention is particularly effective for determination of 15N isotope content in a specimen wherein 15N constitutes between about 5 atom % and about 99.5 atom % of the total nitrogen present. For determinations at the lower and especially at the upper end of this range, or anywhere in the range where maximum precision is desired, the water, CO and CO2 content are preferably as low as practicable. However, over a wide spectrum within the range, and depending on the purpose and use of the analytical data, analyses of practical value may be achieved even though the nitrogen sample contains minor concentrations of CO2, water, or even CO.
The process and apparatus of the invention are effective for measurement of the 13C-proportion where 13C constitutes between about 5 atom % and about 99.5 atom % of the total sample carbon present. The natural abundance of 13C is about 1.1 atom %. For samples in which the 13C-content approaches either extreme, it is desirable to utilize relatively pure O2 for combustion and to further utilize a supplemental reactant that converts CO to CO2, thereby increasing the proportion of CO2 in the combustion product gas.
Additional logistical advantages can be afforded by the preferred use of a general-purpose mass spectrometer, such as (but not limited to) quadrupole-based spectrometers, as made feasible by the process and apparatus of the invention. Although special isotope ratio mass spectrometers can provide exceptionally high precision, they are not generally versatile, being adapted for simultaneous determination of only 3 or 4 different masses. Thus, when there is less than a constant demand for isotope ratio analyses, these highly specialized and expensive instruments may be idle. By contrast, a process gas mass spectrometer is a versatile instrument having a typical scanning range of m/z 2 to 250 or higher; and may, thus, be used for a wide variety of other applications in addition to the isotopic ratio determinations that are made according to the process of the invention.
The invention having been described in detail, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims.
The following non-limiting examples are provided to further illustrate the present invention.
Glycine-15N specimen (0.15-gram) and Fe3O4 (0.15-gram) were loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Biosynthesized lyophilized protein-15N specimen (0.15-gram) and Fe3O4 (0.15-gram) were loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Para-nitrophenol specimen (0.13-gram) and Fe3O4 (0.15-gram) were loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Lyophilized algal cell-15N specimen (0.08-gram) and Fe3O4 (0.15-gram) were loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Biosynthesized lyophilized protein-13C specimen (0.105-gram) was loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Algal cells-13C specimen (0.100-gram) was loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
Starch-13C specimen (0.105-gram) from Solanum tuberosum was loaded into quartz boat 11 within the combustion region of a tubular process vessel (internal volume 0.15-liter with L/D 60) typical of that depicted in
When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.
In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.
As various changes could be made in the above constructions and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11423317 | Jun 2006 | US |
| Child | 11756865 | US |
