Patent Description:
Isotope Ratio Mass Spectrometry (IRMS) is a technique that finds application across many fields including geosciences, archaeology, medicine, geology, biology, food authenticity and forensic science. Accurate and precise measurement of variations in the abundances of isotopic ratios of light elements in a sample such as <NUM>C/<NUM>C (δ<NUM>C), <NUM>N/<NUM>N (δ<NUM>N), <NUM>O/<NUM>O (δ<NUM>O), D/H, and <NUM>S/<NUM>S (δ<NUM>S), relative to an isotopic standard, can provide information on the geographical, chemical and biological origins of substances, allowing differentiation between samples that are otherwise chemically identical. The δ values are defined in a specific way. For example, δ<NUM>C is defined as: <MAT>.

A typical EA-IRMS instrument is formed of six main sections: a sample introduction system, a sample preparation system, an electron ionisation source, a magnetic sector analyser, a Faraday collector detector array, and a computer controlled data acquisition system. The sample is split into atoms/ molecules and/or compounds by the sample preparation system. The electron ionisation source ionizes the prepared sample and the resulting sample ions are spatially separated in the magnetic sector analyser. The Faraday collector comprises a detector array which detects the spatially separated ions, and the computer controlled data acquisition system generates mass spectra from the Faraday collector outputs.

Sample preparation may be achieved in a number of different ways, with advantages and disadvantages to each. The two best-known groups of techniques for sample preparation are those which carry out elemental analysis for the whole sample (EA-IRMS), and those which first separate the chemical substances of the sample by gas chromatography before splitting the separated substances into atoms/ molecules and/or compounds (GC-IRMS). Liquid chromatography (LC-IRMS) has also been explored for sample preparation but is less commonly used.

EA-IRMS is a measurement technique which analyses the whole sample at the same time, to investigate the variations in the abundances of isotope ratios in the whole sample. <FIG> shows a highly schematic arrangement of the sample introduction and preparation part (see above) of an EA-IRMS system. The system is under the control of a system controller <NUM> as may be seen.

A sample (not shown in <FIG>) is weighed and placed in a combustible capsule (also not shown in <FIG>). The combustible capsule is sealed with the sample inside and is usually made of tin, although aluminium or silver may be used instead.

An autosampler carousel <NUM> is positioned above a combustion furnace <NUM>. Helium purge gas is supplied to the autosampler <NUM>, typically at a rate of <NUM> - <NUM>/min, by a first gas supply control <NUM> from a first Helium bottle <NUM> to reduce air intake. The He purge gas flows out of the sampler via the outlet pipe <NUM>. The autosampler carousel <NUM> injects the sealed sample capsule into the combustion furnace <NUM> in a carrier gas flow of helium supplied by a second gas supply control <NUM> from a second Helium bottle <NUM>. The sample is combusted in the combustion furnace <NUM>, under the control of the system controller <NUM>. Pulsed oxygen may optionally be employed to aid combustion. The oxygen is supplied from an oxygen bottle <NUM>, also under the control of the second gas supply control <NUM>.

The sample matrix breaks down into its constituent elemental components (mostly atoms) and is conveyed by the carrier gas flow of Helium from the second Helium bottle <NUM>, across an oxygen donor compound such as Cr<NUM>O<NUM>, WO<NUM>, or CuO. The oxygen donor is present to ensure complete oxidation of the elemental components, particularly of carbon, nitrogen and sulfur evolved from the sample matrix. Typically the reactor zone (containing the oxygen donor) in the combustion furnace <NUM> is held at a temperature of between <NUM> and <NUM> degrees Celsius, with an ideal range of between <NUM> and <NUM> degrees Celsius. The Helium carrier gas employs a maximum flow rate of up to <NUM>/min, but typically in the range of <NUM> to <NUM>/min.

The resulting products may be one or more of NOx, CO<NUM>, SO<NUM> and/or H<NUM>O. After the oxidation a reduction takes place. For example, to measure δ<NUM> N, NOx has to be reduced to N<NUM>. This may be carried out either using separate, serially arranged combustion and reduction furnaces (as shown in <FIG>), or alternatively by combining both into a single reactor heated by the same furnace.

In particular, the arrangement shown in <FIG>, employs a separate reduction oven <NUM>, arranged downstream of the combustion furnace <NUM> and heated separately to the combustion furnace <NUM>. In the arrangement of <FIG>, the sample is generally swept across the oxygen donor material in the reactor zone of the combustion furnace <NUM> using the Helium carrier gas, and then transferred to the reduction oven <NUM>, via a stainless steel/sulfinert capillary or heated bridge, which contains metallic copper (not shown in <FIG>). The reduction oven <NUM> is generally held at a temperature between <NUM> - <NUM> and is designed to reduce NOx and NO gas species (for example) to N<NUM>, reduce SO<NUM> to SO<NUM> and absorb excess O<NUM> not used in the combustion reaction.

In the alternative arrangement, where the combustion and reduction processes may instead be combined in the same reactor, heated by the same furnace, the analyte gases first pass across the oxygen donor compound. The gases are then conveyed onward to metallic copper within the same reactor. Here, they undergo the same chemical reaction as described above in respect of the serially arranged furnaces illustrated in <FIG>.

In either case (separate or combined combustion and reduction furnaces/ovens), the resultant gases are then directed through a moisture trap <NUM> (<FIG>). Optionally, a chemical trap <NUM> can also be provided, which may contain soda lime, NaOH on a silica substrate, Carbosorb® or the like. The chemical trap <NUM> may allow removal of carbon dioxide from the analyte gases when it is only desired to look at nitrogen isotope ratios. The moisture trap <NUM> usually contains Magnesium perchlorate to trap any water generated during the combustion process. Depending upon the nature of the reagents, the chemical trap <NUM> and moisture trap <NUM> may be placed in the reverse order to that shown in <FIG>.

The dried gaseous output is introduced into a separation column <NUM> that serves to separate the output into its constituent atoms, molecules or compounds, e.g. carbon dioxide and nitrogen or carbon dioxide, nitrogen and sulphur dioxide. The separation column <NUM> may be a packed column for gas chromatography (GC) having a constant temperature when the dried gaseous output flows through the GC column, the GC column being heated by a resistance heater <NUM> surrounding the GC column <NUM>. The resistance heater <NUM> is controlled by a heater controller <NUM> to keep the temperature of the GC column constant. This heater controller <NUM> is triggered to start the heating by the system controller <NUM>. The arrangement of <FIG> shows a separation column <NUM> in the form of a GC column, with the moisture trap <NUM> arranged before the separation column <NUM> as described above.

Once the analyte gas has been separated into its combustion components based on their interaction with the separation column <NUM>, they are conveyed through a thermal conductivity detector (TCD) <NUM>, which forms the basis of weight % determinations. Detection by the TCD <NUM> is non-destructive. Therefore, after detection, the gas can be conveyed to an isotope ratio mass spectrometer, via an interface capable of diluting the gas if required (not shown in <FIG>), for simultaneous measurement, in particular of δ<NUM>C, δ<NUM>N and/or δ<NUM>S values.

Before or after the measurement of an isotope ratio by IRMS, or in parallel with the measurement of an isotope ratio by IRMS, a reference gas of the investigated isotope ratio can be supplied to the IRMS in order to allow a reference measurement to be carried out. The reference gas may be supplied via a gas supply pipe <NUM> and is under the control of a reference gas supply controller <NUM> The reference gas supply controller <NUM> is connected with a bottle <NUM> of N<NUM>, a bottle <NUM> of CO<NUM> and a bottle <NUM> of SO<NUM>. The measured isotopic ratio is an average for the whole sample. EA-IRMS is particularly suited to non-volatile substances such as soils, sediments, plants, foods, drugs, amino and fatty acids, and many more. Although an average isotope ratio value for the whole sample is obtained, nevertheless analysis of very small samples is possible.

The separation column <NUM> could also be a thermal desorption unit for gas separation. In such a desorption unit, the thermal desorption temperature is varied as described in <CIT>. If the separation column is instead a thermal desorption unit, the moisture trap <NUM> may be also arranged after the separation column <NUM>.

The thermal desorption unit uses the principle of thermal desorption. Gases emerging from the reduction oven are supplied to the desorption unit. The entire mixture of components of the gas is adsorbed by the adsorbing material of the thermal desorption unit. This adsorption takes place at temperatures between room temperature and <NUM> degrees Celsius, in systems having a single thermal desorption unit (systems having multiple thermal desorption units are also known, and in these, the lower end of the temperature range may be above room temperature).

The whole gas is stored and can be concentrated by the adsorbing material. Separation of the components of the gas takes place based on different desorption temperatures. Thus, the thermal desorption unit has to be heated to various temperatures to supply specific components of the gas to the EA-IRMS. Due to the control of the desorption of specific elements by the heating temperature it is possible to control the time of the supply of specific component of the gas to the EA-IRMS and the time between the supply of two specific component of the gas to EA-IRMS to be analysed.

GC-IRMS, by contrast, permits separation of the sample prior to isotope ratio analysis. This in turn permits isotopic analysis of complex mixtures by a specific isotope analysis of each chemical substance contained in the mixture, which can reveal additional information not normally available using EA-IRMS, as well as better discrimination. <FIG> shows a typical arrangement of a GC-IRMS system, again in highly schematic form. Components common to <FIG> and <FIG> are labelled with like reference numbers.

Liquid samples (not shown) are provided in small vials (not shown) and loaded into an autosampler <NUM>. The samples are injected by the autosampler <NUM> into a gas chromatograph (GC) column <NUM> e.g. by a syringe system (not shown). The gas chromatograph (GC) <NUM> can be heated in a GC oven <NUM> under the control of a system controller <NUM> to improve the separation of the chemical substances contained in the mixture of the investigated sample. The GC oven <NUM> includes a vent <NUM>. The sample elutes from the column of the GC <NUM> into an oxidation chamber <NUM>, such as a non-porous alumina tube, usually mounted on the side of the GC oven <NUM>. The eluents from the GC <NUM> are combusted at elevated temperatures e.g. into NOx, CO<NUM>, and/or H<NUM>O. As with the EA-IRMS of <FIG>, to measure e.g. δ<NUM>C, the resulting products are carried in a stream of dry Helium through a reduction oven <NUM> that converts the nitrous oxides into N<NUM> and removes any excess O<NUM>. Water (which is a byproduct of the combustion) is removed using a counterflow of dry helium in a dryer <NUM>, and the dried gaseous output may be introduced into a Thermal Conductivity Detector (TCD) <NUM>.

The gases exiting the TCD <NUM> are carried into an IRMS (again not shown in <FIG>) using CO<NUM> from a reference CO<NUM> supply <NUM> that is introduced at an open split.

As with the arrangement of <FIG>, various components in <FIG> are under the control of the system controller <NUM>. The system controller <NUM> controls the autosampler <NUM> as it supplies a sample to the combustion oven <NUM>, triggers the supply of the purge gas to the autosampler <NUM> via the first gas supply control <NUM>, and triggers the supply of the carrier gas flow and the (optional) combustion-assisting oxygen pulse via the second gas supply control <NUM>. The system controller also sets the set-points of the temperature of the combustion oven <NUM> and the temperature of the reduction oven <NUM>. Finally the system controller <NUM> controls the temperature of the GC oven <NUM> which heats the GC column <NUM>. As noted above, EA-IRMS and GC-IRMS are complementary techniques. GC-IRMS allows a specific analysis of each chemical substance contained in a sample, e.g. an organic matter sample (for example, individual amino acids in a protein), but requires that any compound constituting the sample mixture can be made sufficiently volatile and thermally stable to permit initial elution in a GC. It also allows analysis of very small sample quantities (nanogram to picogram range; the typical sample weight in an EA-IRMS experiment is in the milligram to microgram range). The main drawbacks of GC-IRMS are the considerably longer analysis time (typically hours rather than minutes as with EA-IRMS), loss of sample integrity during sample preparation, cost and user complexity. Due to the separation of the chemical substances of the sample by the GC column, the different atoms, molecules and/or compounds of each separated chemical substance are supplied to the mass analyser simultaneously during the GC-IRMS measurement. The different atoms, molecules and/or compounds such as N<NUM>, CO<NUM> and SO<NUM> of each chemical substance are very difficult to resolve in such systems. Therefore the measurement results of GC-IRMS are much more complex, or on the other hand different isotope ratios have to be measured one after the other which is very time consuming.

The present invention relates to EA-IRMS, which allows isotopic analysis of the whole samples. One of the key benefits of EA-IRMS is the relatively short time needed for sample analysis. In recent years, simultaneous δ<NUM>C, δ<NUM>N and δ<NUM>S measurements have become a more common approach in EA-IRMS across all application fields. This is because of the ability to produce accurate and precise data from one sample drop, thus increasing system productivity and reducing sample analysis costs. However, such simultaneous measurements in EA-IRMS present a number of challenges. <FIG> illustrates a chromatogram for simultaneous δ<NUM>C, δ<NUM>N and δ<NUM>S analysis of sulfanilamide using GC separation at a constant temperature (isothermal) in an EA-IRMS experiment such as that described in connection with <FIG> above.

Carbon dioxide, nitrogen and sulfur dioxide molecules generate peaks in the chromatogram of <FIG>. These molecules are contained in the dried gaseous output of the moisture trap <NUM> after a sample has been introduced into the sample introduction system shown in <FIG>. To the left of <FIG>, mass peaks of N<NUM> molecules (having isotopic masses <NUM> u (peak <NUM>) and <NUM> u (peak <NUM>)) may be observed. The mass peaks of CO<NUM> molecules having isotopic masses <NUM> u (peak <NUM>), <NUM> u (peak <NUM>) and <NUM> u (peak <NUM>) are also visible in <FIG>. Mass peaks of SO<NUM> molecules having isotopic masses <NUM> u (peak <NUM>) and <NUM> u (peak <NUM>) may be seen towards the right of <FIG>. To determine the isotope ratios δ<NUM>C, δ<NUM>N and δ<NUM>S, reference gases are supplied to the IRMS via the gas supply <NUM> in parallel with the measurement of the molecules originating from the investigated sample. Those peaks in the chromatogram arising from reference gases are labelled with the same reference number as the corresponding sample gas peak, save for the addition of a prefixed "R". So, for example the peak labelled "<NUM>" in <FIG> represents the mass peak of N<NUM> molecules having the isotopic mass <NUM> u, and which originate from the investigated sample. The peak of N<NUM> molecules having the isotopic mass <NUM> and which are derived from the N<NUM> reference gas is labelled with the reference number "R128".

The chromatogram of <FIG> exhibits relatively poor N<NUM> and CO<NUM> separation (less than <NUM> seconds, with some loss of N<NUM> peak tail), a high peak width for SO<NUM> (greater than <NUM> seconds) and long retention time of SO<NUM>, which is the time the SO<NUM> need to pass the GC column, resulting in a total analysis time of in excess of <NUM> minutes, although this time can often be even longer. The GC column is held at a temperature of around <NUM>-<NUM> degrees Celsius, in the experiment in which the <FIG> data are derived and is based on a sample of sulfanilamide (C/S ratio of around <NUM>). Sufficient baseline separation between N<NUM> and CO<NUM> on the one hand, and CO<NUM> and SO<NUM> is particularly challenging in samples with large CO<NUM> amounts relative to N<NUM> and SO<NUM>. For example, analysis of high C/S ratio samples, such as wood (><NUM>:<NUM>), would result in chromatographic separation compromises that would make the analysis using an isothermal technique impossible for δ<NUM>C, δ<NUM>N and δ <NUM>S from a single sample drop, because separation of N<NUM> and CO<NUM> peaks would not be achieved, and the SO<NUM> peak shape for small S concentrations would result in poor reproducibility.

So the CO<NUM> peaks, the N<NUM>peaks and the SO<NUM> peaks show a peak tailing, that is, exhibit peaks that are not very sharp on their tail side. Sharp peaks permit better peak separation, particularly for the N<NUM> peaks and the CO<NUM> peaks, because the tail side of the N<NUM> peaks do not then extend so close to the front side of the CO<NUM> peaks. Also, for peaks that do not exhibit peak tailing, data integration of the peak is better, and determination of the ratio of the various isotopes is improved. This improvement arises particularly from the fact that, for sharp peaks, it is much easier to distinguish the noise measured in an measurement signal of an EA-IRMS, from the signal of a peak. This results in a more accurate data integration of the peak and consequently a more accurate determination of the ratio of the various isotopes measured by the peak. By contrast, peak tailing results in an extension of the measuring time.

It is possible to reduce the analysis time slightly by operating the GC column at a higher constant temperature, in some prior art systems. However, raising the temperature of the GC column results in poorer N<NUM> and CO<NUM> separation. Thus there is a compromise between achieving analytically acceptable data and the time taken to obtain that. To date, an optimal compromise of around <NUM> minutes per simultaneous NCS analysis, per sample, has been employed.

The alternative, which is to analyse each of δ<NUM>C, δ<NUM>N and δ<NUM>S separately, has its own drawbacks, in terms of an increase in initial sample weighing and preparation time, along with a requirement for at least three times the amount of the sample. In fact, some prior art EA-IRMS systems require repetition of an experiment once or twice before a statistically acceptable accuracy of the data can be achieved. In such cases, attempting to analyse δ<NUM>C, δ<NUM>N and δ<NUM>S separately can in fact result in up to <NUM> times more analyses than a simultaneous δ<NUM>C, δ<NUM>N and δ<NUM>S analysis. This results in additional costs per analysis, a longer overall sample preparation time, and lower system productivity (that is, a lower throughput of a specific sample).

Various solutions to these problems have been proposed. One solution employs two GC columns, an S column for the SO<NUM>, and an NC column for the N<NUM> and CO<NUM> molecules. The dried gaseous output of a moisture trap <NUM>, containing of N<NUM>, CO<NUM> and SO<NUM>, flows initially into the S column. The gas flow downstream of the S column can be switched by way of a valve. The valve is initially in a first position which directs the gas flow out of the S column directly to the IRMS, in order that it may be analysed thereby. Once the SO<NUM> has passed through the S column, the valve is switched into a second position so that the gas flowing out of the S column is instead directed next to the NC column. The gas flow out of the NC column is then directed to the IRMS to be analysed. Using this arrangement, the sequence of the molecules to be analysed is changed: initially the SO<NUM> peak is measured by the IRMS, and subsequently the N<NUM> and CO<NUM> peaks are measured by the IRMS. Measurement time can be reduced by the use of a shorter column length of the S column, and larger quantities of CO<NUM> can be measured. Overall, however, the measurement time for the method may be increased, because (at least for a part of the analysis period), the gas is required to flow through two columns (the S and the NC columns) before being measured. Moreover, the costs for this arrangement are higher because of the use of two GC columns as well as an additional controlling system for controlling the additional switching valve.

Also the use of a thermal desorption unit as separation column <NUM> has its disadvantages. A process of continuously flowing gas into the separation column <NUM> is not employed. Instead, it is necessary initially to adsorb the whole mixture of gases to be analysed, with the separation column <NUM> at a low temperature. Only then, by controlled elevation of the temperature, are the specific components to be analysed set free (by a process of desorption) and supplied to the EA-IRMS. This process is time consuming and more difficult to control. Also, the accuracy of the measurement suffers, because it is possible that the specific elements to be analysed are not completely adsorbed during the initial phase of analysis, so that they cannot subsequently be desorbed.

The present invention seeks to address these challenges with existing EA-IRMS devices and methods. It is one of the objects of the invention to reduce the measurement time for the elemental analysis system. It is another one of the objects of the invention to improve the distance between the peaks of different atoms, molecules and/or compounds in the measurement results of the elemental analysis device and to achieve a better peak separation. It is still another one of the objects of the invention to improve the peak shape of the detected atoms, molecules and/or compounds by minimising peak tailing and reducing the peak width. It is still another one of the objects of the invention to expand the range of sample types that may be analysed; for example, it is an object to permit analysis of samples having a high C/S value such as wood. It is still another one of the objects of the invention to reduce the experimental costs associated with the elemental analysis system, for example by reducing the amount of the investigated sample that is needed for successful analysis, and/or by reducing the amount of the flow gases that are needed.

<CIT>, <CIT>, <CIT> and <CIT> each disclose a method for analysing samples containing Carbon, Nitrogen, Sulphur and the like. A sample is combusted and the effluent elutes through a heated gas chromatograph to separate the constituents of the sample. <CIT> discloses a fast gas chromatography apparatus and method. <CIT> discloses a hand-held sampling and gas chromatographic separation detection system and method. <CIT> discloses a fast gas chromatograph (GC) method and device for obtaining fast gas chromatography analysis.

According to a first aspect of the present invention, there is provided a sample preparation apparatus for an EA-IRMS, in accordance with claim <NUM>.

The invention also extends to an EA-IRMS apparatus including such a sample preparation apparatus, in accordance with claim <NUM>.

In another aspect of the invention, there is provided an EA-IRMS method in accordance with claim <NUM>.

By increasing the GC temperature according to a temperature gradient during analysis, many of the problems of the prior art EA-IRMS techniques are avoided or at least ameliorated. For example, when the GC temperature is held static during simultaneous δ<NUM>C, δ<NUM>N and δ<NUM>S analysis, the chromatographic peaks are not as sharp as desired and do not exhibit the lowest possible retention times. Peak tailing can also be exaggerated, especially for sulphur dioxide.

A GC temperature profile with a temperature gradient, by contrast, can optimize data integration, improve the determination of isotope ratios , and lower sample analysis times. In particular, increasing the temperature of the GC during analysis can reduce the
data acquisition time and achieve complete separation of N<NUM> CO<NUM> and SO<NUM>, with sharp peak shapes and lower retention times, resulting in accurate and precise data. The temperature gradient GC technique makes it possible to investigate samples with a high content of carbon atoms.

The invention pertains to an apparatus according to claim <NUM>, an EA-IRMS according to claim <NUM> and a method according to claim <NUM>. The method and apparatus of the present invention employ a continuous flow of gas into the GC column to which the temperature gradient is applied. This is in contrast to a thermal desorption unit, when it is used as separation column <NUM>. If the temperature of at least a part of the GC column is increased whilst the sample gas flow in the GC column elutes, it has been found that the elution time of the atoms, molecules and/or compounds contained in the sample gas flow can be changed. In consequence, it is possible to change the chronological distance between two peaks of different atoms, molecules and/or compounds contained in the sample gas flow as they leave the GC column. The result is both a change in the chronological distance between the centre of the peaks, and a change in the chronological distance between the peaks, where no peak is detected. As a further benefit, some of the atoms, molecules and/or compounds contained in the sample gas flow leave the GC column after a relatively shorter time period. This decreases the measurement time for some experiments considerably.

For example, to date, when the dried gaseous output of a moisture trap <NUM> flows, as a sample gas flow, through a GC column held at a constant temperature, there are markedly different speeds of elution of N<NUM> and CO<NUM> on the one hand, and SO<NUM> on the other. It has been found that, by increasing the temperature of the GC column whilst the sample gas flow in the GC column elutes, the SO<NUM> peak can be expected much sooner. It has also been found that, by increasing the temperature of the GC column whilst the sample gas flow in the GC column elutes, the chronological distance between the N<NUM> peaks and CO<NUM> peaks where no peak is detected, is increased.

It has still further been found that, by increasing the temperature of at least a part of the GC column whilst the sample gas flow in the GC column elutes, the peak shape of atoms, molecules and/or compounds contained in the sample gas flow detected by the IRMS can be changed in a manner such that the shape of the peaks is sharpened. Peak tailing can in particular be reduced or avoided. This improves the data integration of the peak and the determination of the ratio of the isotopes to be detected, whether by the use of an elemental analysis system such as an IRMS, by the use of a thermal conductivity detector, or otherwise. The reduced or removed peak tailing allows peaks that have been eluted in rapid succession to be better distinguished by the elemental analysis system (IRMS, thermal conductivity detector or otherwise).

For example, it has been found that increasing the temperature of the GC column whilst the sample gas flow in the GC column elutes results in an improvement in the peak shapes of N<NUM>, CO<NUM> and SO<NUM> when they are in the sample gas flow. Due to the applied temperature gradient, the peaks appear sharper and the peak tailing of the peaks can be reduced significantly and sometimes totally. So the N<NUM> peaks and CO<NUM> peaks can be better distinguished. This results in an increase of the chronological distance between the N<NUM> peaks and CO<NUM> peaks where no peak is detected.

In an embodiment, the GC temperature profile may be such that for a first time period there may be a first fixed temperature, Tstart, whilst during a second time period there may be a second fixed temperature Tend. Between these times the temperature is increased. The rate of ramping of the temperatures in the GC may be linear or non-linear, ie, ∂T/∂t may be constant or variable. The result is a system and method offering higher system productivity through greater sample throughput, and accurate and precise analysis isotope ratios like δ<NUM>C, δ<NUM>N and δ<NUM>S. A single sample drop can be employed, whereas, with prior art isothermal GC analysis, often the experiment must be carried out twice or three times using additional material from the same sample, in order to achieve an acceptable accuracy level. Thus embodiments of the present invention permit a significant workflow enhancement in the form of a reduction in the cost per sample analysis.

Preferred embodiments of the invention also allow a data acquisition time reduction of at least <NUM>-<NUM>% relative to the time taken in the traditional isothermal GC approach. For example, the typical <NUM>-minute data acquisition time (a result of the trade-off between acquisition time and peak shape/baseline separation explained in the Background section above) with an isothermal GC, may be reduced to as low as <NUM> minutes. A desirable consequence of the reduction in acquisition time is a reduction in the amount of helium gas required for sample purge and drying and as flow gas during analysis.

Although the technique is useful in respect of samples having a wide variety of ratios of N:C:S, it is particularly attractive when seeking to analyse samples having high (eg, <NUM> or greater) ratios of carbon to sulphur.

The invention may be put into practice in a number of ways and some specific embodiments will now be described by way of example only and with reference to the accompanying drawings in which:.

Referring first to <FIG>, a highly schematic arrangement of a sample preparation section of an EA-IRMS in accordance with an embodiment of the present invention is shown. Those components common to <FIG> and <FIG> are labelled with like reference numerals.

The sample preparation and combustion/reduction proceeds, in the embodiment of <FIG>, in the same manner as was described in the Background section above, in respect of <FIG>. To avoid unnecessary repetition, this part of the process will only be summarised here.

A sample (not shown in <FIG>) is weighed and placed in a combustible capsule that is sealed and placed into an autosampler carousel <NUM> positioned above a combustion furnace <NUM>. The autosampler carousel <NUM> injects the sealed sample capsule into the combustion furnace <NUM> under the control of a system controller <NUM>. As before, Helium may be supplied to the autosampler <NUM> as a purge gas, and combustion in the combustion furnace <NUM> may be carried out in the presence of pulsed oxygen.

Helium carrier gas is employed to carry the sample across an oxygen donor compound. The flow rate of the helium carrier gas is again optimally between <NUM> and <NUM>/min, but can be up to <NUM>/min. The reaction zone in the combustion furnace <NUM> is typically held at a temperature between <NUM> and <NUM> degrees Celsius, with an ideal range of between <NUM> and <NUM> degrees Celsius.

The resulting NOx, CO<NUM>, SO<NUM> and/or H<NUM>O products are reduced in a reduction oven <NUM>, which may be a separate component as shown schematically in <FIG>, or may form a part of a single, combined combustion/reaction unit.

The reduction oven <NUM> is generally held at a temperature between <NUM> - <NUM> and the gases exiting that reduction oven are then directed through optionally a chemical trap <NUM> and a moisture trap <NUM>, again as previously described; the order of the chemical and moisture traps <NUM>, <NUM> may be reversed depending upon the reagents employed in each.

The dried gaseous output of the moisture trap <NUM> is introduced into a GC column <NUM>, for separation of the gases. The GC column <NUM> of preferred embodiments of the present invention will be described in further detail below, but in general terms, the GC column <NUM> may preferably incorporate a carbon molecular sieve.

The GC column <NUM> of <FIG> is mounted within a GC chamber <NUM> whose interior is heated by halogen lamps <NUM>. The halogen lamps <NUM> are controlled by a heater controller <NUM> which is connected to the system controller <NUM>. A fan <NUM> draws ambient (cool) air into the interior of the chamber <NUM>.

<FIG> shows an embodiment of the GC chamber <NUM> of <FIG>, in schematic sectional view. The GC chamber <NUM> contains the GC column <NUM> which is positioned generally centrally of the GC chamber <NUM>. The GC chamber <NUM> has outer side walls 64a, a base 64b and a closure 64c, each of which are formed of an insulating material. The inner surfaces of the outer side walls 64a and the closure 64c have a reflective coating. The outer side walls 64a are separated from the closure 64c by openings <NUM>, <NUM>'.

Extending in an axial direction of the GC chamber <NUM> are inner walls <NUM>. The inner walls are also coated or formed from a reflective material. The inner walls <NUM> are spaced inwardly of the outer side walls 64a of the GC chamber <NUM> so as to define fluid channels <NUM> which communicate with a central region of the GC chamber at a first end proximal the GC column <NUM> and the base 64b, and which communicate with the openings <NUM>, <NUM>' at a second end. The halogen lamps <NUM> are mounted outwardly of the GC column <NUM>, upon the inner walls <NUM>, so that, in use, heat is radiated from the halogen lamps <NUM> towards the GC column <NUM>. Electrical power is supplied from the exterior of the GC chamber <NUM> to the halogen lamps <NUM> via electrical standoffs <NUM> extending outwardly across the fluid channels <NUM>. A gas supply inlet <NUM> and a gas outlet <NUM>' are also provided which extends outwardly through the outer side walls 64a to the GC column <NUM> so that the sample and/or reference gases generated upstream of the GC column <NUM> (<FIG> again) can be introduced into the GC column <NUM> and leave the GC column <NUM> as gaseous output.

The fan <NUM> is, as noted above in connection with <FIG>, mounted externally of the GC chamber <NUM> and, in use, draws ambient (relatively cool) air from outside of the GC chamber <NUM> and blows it into the central part of the GC chamber <NUM>. The relatively cool air forces any relatively warm or hot air present in the vicinity of the GC column <NUM> to be expelled from the GC chamber <NUM> along the fluid channels <NUM> and out via the openings <NUM>, <NUM>'.

Rapid ramping up (heating) and down (cooling) of the temperature of the GC column <NUM> can thus be achieved. To achieve rapid heating, the system controller <NUM> sends a trigger signal to the heater controller <NUM> which applies electrical power to the halogen lamps in order to cause the temperature in the GC chamber <NUM> to be increased. The heater controller <NUM> may be programmed with one or many temperature profiles (some examples of which will be described in respect of later Figures) that cause the temperature of the GC column <NUM> to be ramped up to one or more temperature set points. The skilled person will recognise that proportional-integral-differential (PID) or other known feedback control techniques may be employed in order that the set point temperatures are reached without excessive overshoot or oscillations.

The temperature may be ramped between first and second set points at a constant (or substantially constant) rate. The heater controller <NUM> may be configured to ramp between different set point temperatures at different constant rates, depending for example upon the experiment being carried out and the constituent compounds, molecules etc. Additionally or alternatively, the rate of temperature change between two set points may be non-linear, or may be linear over a part of the time and non linear at other times. It is moreover to be understood that the temperature gradient does not even need to be constantly positive between the two set points, provided only that, during elution of gases through the GC column, there is a net positive increase in temperature.

For example, it appears that providing a small temperature change even at the start of the experiment, when the GC column <NUM> is eluting the N<NUM> and CO<NUM>, can improve further the baseline separation. So the temperature ramp could start slowly and then increase in rate as the temperature of the GC column <NUM> rises.

The arrangement described above in connection with <FIG> also allows rapid cooling of the GC column <NUM> between experiments. In particular the fan <NUM> and the arrangement of the outer side walls 64a and the inner walls <NUM>, resulting in the fluid channels <NUM>, allows the cool air blown by the fan <NUM> to rapidly purge the GC chamber <NUM> of warm or hot air in order to allow a lower starting set point temperature to be rapidly attained.

Separated gases eluting from the GC column <NUM> are then conveyed through a thermal conductivity detector (TCD) <NUM> for weight percent measurements. After (non destructive) analysis by the TCD <NUM>, the analyte gases are directed into an isotope ratio mass spectrometer for simultaneous measurement of δ<NUM>C, δ<NUM>N and δ<NUM>S values.

In the IRMS (not shown in <FIG>), the combusted, reduced gases are ionized and passed through a magnetic sector analyser where they separate in space according to their mass to charge ratios. The resulting spatially separated ion species are detected at a plurality of Faraday detectors in a detector array.

Techniques for ionization, separation and detection in the IRMS will be familiar to the skilled reader. The details of the IRMS do not in any event form a part of the present invention and will not be discussed further.

Turning now to <FIG>, a flow chart illustrating the steps carried out during Gas Chromatography is shown. At step <NUM>, the system controller <NUM> sends a set/reset signal to the heater controller <NUM> of the halogen lamps <NUM> to hold or move the temperature measured at the GC column <NUM> to a start temperature Tstart. The start temperature Tstart of the GC column <NUM> is held between <NUM> and <NUM> degrees Celsius, but is ideally in the range of <NUM> to <NUM> degrees Celsius. In a most preferred embodiment, the system controller <NUM> sends a set/reset signal to the heater controller <NUM> so that the GC column <NUM> is held at <NUM> degrees Celsius for N<NUM> and CO<NUM> separation.

Once the temperature of the GC column <NUM> is stabilized at the desired start temperature Tstart, a ramp up trigger signal is generated. This ramp up trigger signal may be generated based upon a predetermined time - for example, the ramp up trigger signal may be generated at a time t<NUM> after the system controller has instructed the autosampler <NUM> to inject the sample billet into the combustion oven <NUM>. The time t<NUM> may itself be predetermined through factory or user calibration or may be user settable. Alternatively, the ramp up trigger signal may be generated based upon detection of a threshold gas flow rate of N<NUM>/CO<NUM> at the GC column <NUM> and/or the GC chamber entrance, for example.

At step <NUM> of <FIG>, the heater controller <NUM> receives the ramp up trigger signal from the system controller <NUM> and commences a temperature ramp (step <NUM>). In the preferred embodiment, this results in a rapid rise in the temperature of the GC column <NUM> from the start temperature Tstart (preferably <NUM> degrees Celsius) to an end temperature Tend, which is (again in the preferred case) <NUM> degrees Celsius. By "rapid rise" is meant a change from Tstart to Tend over several tens of seconds, and most preferably over <NUM> to <NUM> minutes.

As noted above, the heater controller <NUM> controls the temperature of the GC column <NUM> so as to ramp up at a linear rate, a non linear rate, or a combination of the two.

At step <NUM>, once one or more temperature sensors in the GC chamber <NUM>/GC column <NUM> (not shown in <FIG> or <FIG>) determine that the end temperature Tend has been reached, the heater controller <NUM> of the halogen lamps <NUM> then controls the temperature of the GC column <NUM>, so that the temperature of the GC column <NUM> is held constant at the temperature Tend. The time over which the GC column <NUM> is held at temperature Tend may, as with Tstart, either be pre-programmed within the heater controller <NUM> based upon factory or user calibration, or may be user selected, or may be based upon detection of a threshold of gas flow. As was explained in the Background section, SO<NUM> elutes more slowly than N<NUM> and CO<NUM> so the system controller <NUM> may look for a threshold of SO<NUM> gas flow into the GC column <NUM> for example.

Once system controller <NUM> determines, based on a time, a user input or a threshold gas flow rate, that the GC column temperature is to be reset, a ramp down trigger signal is generated by the system controller <NUM> and sent to the controller <NUM> of the halogen lamps <NUM>. This results in a rapid cooling of the GC column <NUM>: see step <NUM> of <FIG>. The temperature drop is (as with the temperature rise) typically tens of seconds and optimally <NUM> minutes. As explained in connection with <FIG> and <FIG> above, rapid cooling is preferably facilitated by the use of the fan <NUM> which blows cool air into the GC chamber <NUM> in order to displace warm or hot air adjacent the GC column <NUM> The final temperature following ramp down is Tstart again.

Once the temperature has reached Tstart, the control loop reverts to step <NUM> again, ready for a next sample to be loaded into the EA-IRMS by the autosampler <NUM>.

<FIG>, <FIG>, <FIG> and <FIG> show chromatograms measured with EA-IRMS. For the sake of clarity, reference peaks are not shown in those chromatograms, and only the peaks of the isotope of the molecules having the highest abundance ( N<NUM>: isotope mass <NUM> u, CO<NUM>: isotope mass <NUM> u and isotope mass SO<NUM>: <NUM> u) are shown.

<FIG> shows a chromatogram of N<NUM> and CO<NUM> peaks obtained from a prior art EA-IRMS with an isothermal GC column, using caffeine as a sample. The left hand peak <NUM> in <FIG> arises from N<NUM>, whilst the right hand peak <NUM> is derived from CO<NUM>. Peak tailing is apparent in <FIG>.

<FIG> shows a first exemplary temperature profile that may be applied to the temperature variable GC column <NUM> of <FIG> and <FIG>. The temperature profile of <FIG> is, in particular, applied to the GC column <NUM> by the heater controller <NUM> based upon a trigger signal from the system controller <NUM>. It will be seen that the start temperature Tstart is <NUM> degrees Celsius and the heater controller <NUM> holds the GC column <NUM> at that temperature for <NUM> seconds. At that point, the heater controller <NUM> causes the power supplied to the halogen lamps <NUM> to be increased so that the GC column temperature rises in a linear manner from <NUM> up to <NUM> degrees Celsius over a period of <NUM> seconds. The heater controller <NUM> then maintains the GC column <NUM> at the upper set temperature Tend of <NUM> degrees Celsius until the experiment is concluded. The temperature is then ramped back down again but this is not shown in <FIG>. <FIG> shows a chromatogram of N<NUM> and CO<NUM> peaks obtained from the EA-IRMS embodying the present invention, such as is shown in <FIG> and <FIG>, to which the temperature profile of <FIG> is applied during sample elution, again using caffeine as a sample. It will be seen that the N<NUM> peak <NUM> and the CO<NUM> peak <NUM> are each much narrower than in <FIG>, with the peak tailing much reduced. The separation between the two peaks <NUM>, <NUM> is thus greatly increased.

The GC column employed to generate the chromatograms of <FIG> and <FIG> contains a porous material. The pore mean diameter of the porous material is preferably larger than <NUM> Angstrom, particularly preferably larger than <NUM> Angstrom, and in the specific embodiment employed to obtain the chromatograms of <FIG> and <FIG>, is <NUM> Angstrom (<NUM> Angstrom = <NUM>*<NUM>-<NUM> m).

The material in the GC column has a a large surface area (preferably larger than <NUM><NUM>/ g, particularly preferably larger than <NUM><NUM>/g. ) Again in the embodiment employed to obtain the chromatograms of <FIG> and <FIG>, the material in the GC column has a surface area of larger than <NUM><NUM>/ g.

The GC column can be filled with spherical carbon. The GC column employed to obtain the chromatograms of <FIG> and <FIG> is filled with a spherical carbon molecular sieve.

The GC column is preferably filled with a spherical material having a diameter between <NUM> and <NUM>, preferably between <NUM> and <NUM> and particularly preferably between <NUM>,<NUM> and <NUM>. The GC column employed to generate the chromatograms of <FIG> and <FIG> is filled with a spherical material having a diameter between <NUM> and <NUM>.

<FIG> shows a chromatogram of N<NUM>, CO<NUM> and SO<NUM> peaks obtained from a prior art EA-IRMS with an isothermal GC, using sulfanilamide as a sample. The N<NUM>, and CO<NUM> peaks <NUM>, <NUM> in <FIG> are close together and again exhibit peak tailing; the tail of the N<NUM> peak <NUM> runs into the leading edge of the CO<NUM> peak <NUM>. The SO<NUM> peak <NUM> is broad with a FWHM (full width of half maximum) of around <NUM> seconds.

<FIG> shows a second exemplary temperature profile that may be applied to the temperature variable GC column <NUM> of <FIG> and <FIG>. The temperature profile of <FIG> is, in particular, applied to the GC column <NUM> by the heater controller <NUM> based upon a trigger signal from the system controller <NUM>. It will be seen that the start temperature Tstart in the profile of <FIG> is <NUM> degrees Celsius and the heater controller <NUM> holds the GC column <NUM> at that temperature for <NUM> seconds. At that point, the heater controller <NUM> causes the power supplied to the halogen lamps <NUM> to be increased so that the GC column temperature rises in a linear manner from <NUM> up to <NUM> degrees Celsius over a period of <NUM> seconds. The heater controller <NUM> then maintains the GC column <NUM> at the upper set temperature Tend of <NUM> degrees Celsius until the experiment is concluded. The temperature is then ramped back down again but this is not shown in <FIG>. The benefit of this heat and cool strategy is based upon the strongly differing elution speeds of N<NUM> and CO<NUM> on the one hand, and SO<NUM> on the other. As the three gases arrive at the GC column <NUM> with the latter held at Tstart (<NUM> degrees Celsius for example), the SO<NUM> is relatively slowly eluting over the column. Once the temperature is ramped up to Tstop, the SO<NUM> experiences a higher temperature and this reduces the SO<NUM> elution time.

Reduction in the SO<NUM> elution time causes the peak in the resulting mass spectrum to be sharper and with minimal tailing. This beneficial effect is clearly seen in <FIG>, which shows EA-IRMS analysis of the same sample (sulfanilamide) as was employed to generate the prior art isothermal mass spectrum of <FIG>. Comparing <FIG> and <FIG>, the SO<NUM> peak <NUM> at the right hand side of the chromatogram is seen to be much sharper. The temperature ramping scheme of <FIG> results in an SO<NUM> peak width (full width at half maximum) of around <NUM>-<NUM> seconds (time is shown on the horizontal axis). This is nearly half of the peak width shown in <FIG> that employs isothermal GC, where the broad flat peak (full width at half maximum) there is around <NUM> wide.

The GC column used to generate the chromatogram of <FIG> and <FIG> contains a porous material, again preferably with large pores (eg pore mean diameter greater than <NUM> Angstroms ). The column is filled with a material having a large surface area, eg at least <NUM><NUM>/ g. The filler is a polymer having a spherical shape and a silanised surface. The column is filled with a spherical material preferably having a diameter between <NUM> and <NUM> and in the specific arrangement employed to generate the chromatogram of <FIG> and <FIG>, it is between <NUM> and <NUM>. Overall, the total analysis time employing the scheme described above is less than <NUM> minutes, and all peak integration is concluded in around <NUM>-<NUM> minutes. Thus there is at least a <NUM>% improvement in analysis time when changing the temperature of the GC column <NUM> during an analysis, relative to the prior art isothermal GC analysis (where, as discussed in the Background section, compromise times of <NUM> minutes are employed). A reduction in sample analysis time improves sample throughput and system productivity.

A further benefit of the reduced analysis time is that the volume of Helium purge/carrier gas needed to complete each experiment can be reduced. A flow of helium gas only needs to be present during the sample analysis phase. At other times, the flow can be throttled. If the time taken to carry out each experiment can be reduced by a third, this offers the opportunity to save very significant amounts of helium over an extended period of use of the improved EA-IRMS device of the present invention. Reactor lifetime and chemical trap lifetime may also be extended when using a non-isothermal temperature profile, since the improved analytical and workflow procedures outlined above reduce the time per experiment, and provide an increased maintenance interval.

One further surprising consequence of the use of a non-isothermal temperature profile during EA-IRMS is that simultaneous δ<NUM>C, δ<NUM>N and δ<NUM>S measurements, along with %C, %N and %S measurements, are achievable even for those bulk organic samples such as wood or bone collagen, where the ratio of Carbon to Sulphur can exceed <NUM>:<NUM>, preferably <NUM>:<NUM> and particularly preferably <NUM>,<NUM>:<NUM>. As a result, it is often not necessary to repeat an experiment multiple times (in order to obtain a statistically acceptable result), as can often be the case with isothermal GC analyses.

Turning now to <FIG>, various different exemplary temperature ramping schemes are shown. In <FIG>, the temperature gradient is constant (ie the slope is linear). In <FIG>, the temperature gradient is non linear between the start and finish temperature, and in particular the rate of change of temperature is relatively low at the start and finish of the temperature ramping, reaching a maximum around half way between Tstart and Tend.

<FIG> illustrates the use of two plateaus with a linear gradient between the two. <FIG> by contrast employs a non-linear gradient between two plateaus, again with the rate of change of temperature being slowest towards the start and end temperatures Tstart and Tend, and with the most rapid change being between those two temperatures.

<FIG> employs two plateaus again, but this time has zero gradient at the start temperature Tstart up to t<NUM> (to form the first plateau), a constant gradient between t<NUM> and t<NUM>, then a non constant gradient between t<NUM> and t<NUM> and finally a zero gradient after t<NUM> at the end temperature Tend (to form the second plateau).

<FIG> employs three plateaus rather than two, with a constant gradient between the first and second, and another constant gradient between the second and third plateaus (which may be the same as or different to the gradient between the first and second plateaus).

Finally <FIG> employs three plateaus, but this time has zero gradient at the start temperature Tstart up to t<NUM> (to form the first plateau), a constant gradient between t<NUM> and t<NUM>, then a zero gradient (to form the second plateau) between t<NUM> and t<NUM> a non constant gradient between t<NUM> and t<NUM> and finally a zero gradient after t<NUM> at the end temperature Tend (to form the third plateau).

Although some specific embodiments have been described, it will be understood that these are merely for the purposes of illustration and that various modifications or alternatives may be contemplated by the skilled person. For example, although a single GC column has been described, it will be understood that the invention is equally applicable to a system involving multiple (eg, <NUM>) GC columns. In particular, it is possible to use a second (additional) GC or LC column before any combustion or reduction etc takes place. This allows the constituents of the sample to be chromatographically separated before they are each (potentially separately) combusted, reduced or otherwise. Each set of combustion or reaction products (eg N, C or S) can then be separately analysed using the temperature variable GC column <NUM> described above.

It will of course be understood that the temperatures and ramping rates employed to generate the chromatograms of <FIG> and <FIG> are exemplary in nature. In general terms, the parameters chosen (temperature(s); ramping rate(s); ramping rate profiles, ie linear, non linear or combined ramping rates; no, one, or multiple intermediate plateaus during ramping from start to finish temperatures, etc) will depend upon multiple factors such as (but not limited to) the sample to be analysed, the configuration (size, shape, phases etc) of the GC column <NUM>, and so forth. The skilled person will have no difficulty in identifying and optimising the parameters. So, for example, although a starting temperature of <NUM> degrees Celsius was employed to generate the chromatogram of <FIG>, a range of temperatures from around <NUM> degrees Celsius up to around <NUM> degrees Celsius, preferably a range of temperatures from around <NUM> degrees Celsius up to around <NUM> degrees Celsius may in fact be employed. Likewise, a range of end temperatures in <FIG> between around <NUM> and <NUM> degrees Celsius, preferably between around <NUM> degrees and <NUM> degrees Celsius may be used. The rate of temperature increase (indicated as <NUM> degree per second in <FIG> may be anywhere between around <NUM> degrees per second up to around <NUM> degrees per second. It will be understood that the rate of temperature increase needs to be correlated with the peak positions, and these are dependent upon both the sample and the GC column. Likewise in respect of <FIG>, a range of temperature gradients between <NUM> degrees per second and <NUM> degrees per second is possible, the start temperature may be anywhere from around <NUM> degrees Celsius up to around <NUM> degrees Celsius, preferably anywhere from around <NUM> degrees Celsius up to around <NUM> degrees Celsius, and a range of end temperatures in <FIG> between around <NUM> and <NUM> degrees Celsius, preferably between around <NUM> and <NUM> degrees Celsius may be used.

Claim 1:
A sample preparation apparatus for an elemental analysis system, particularly an elemental analysis isotope ratio mass spectrometer, i.e. EA-IRMS, comprising:
a sample combustion and/or reduction arrangement (<NUM>) and/or pyrolysis arrangement (<NUM>) for receiving a sample of material to be analysed, and producing therefrom a sample gas flow containing atoms, molecules and/or compounds;
a gas chromatography, i.e. GC, column (<NUM>) into which the sample gas flow is directed;
a heater (<NUM>) for heating at least a part of the GC column (<NUM>); and
a controller (<NUM>) for controlling the heater (<NUM>), wherein
the gas chromatography column (<NUM>) is contained centrally in a GC chamber (<NUM>), characterized in that the GC chamber (<NUM>) has outer side walls (64a), a base (64b), a closure (64c), each of which are formed from an insulating material, wherein the outer side walls (64a) are separated from the closure (64c) by openings (<NUM>, <NUM>'),
wherein inner surfaces of the outer side walls (64a) and the closure (64c) have a reflective coating, and
wherein the GC chamber (<NUM>) has inner walls (<NUM>) coated or formed from a reflective material and extending in an axial direction of the GC chamber (<NUM>), wherein the inner walls (<NUM>) are spaced inwardly of the outer side walls (64a) of the GC chamber (<NUM>) so as to define fluid channels (<NUM>) which communicate with a central region of the GC chamber (<NUM>) at a first end proximal the GC column (<NUM>) and the base (64b), and with the openings (<NUM>, <NUM>') at a second end,
wherein
the controller (<NUM>) is configured to control the heater (<NUM>) so as to increase the temperature of at least the part of the GC column (<NUM>) whilst the sample gas flow in the GC column (<NUM>) elutes.