WO2007115423A1

WO2007115423A1 - Equilibration chamber, apparatus and method for online determination of the isotopic composition of non-exchangeable stable hydrogen in a substance sample

Info

Publication number: WO2007115423A1
Application number: PCT/CH2007/000175
Authority: WO
Inventors: Markus Leuenberger; Marc Filot
Original assignee: Universität Bern
Priority date: 2006-04-07
Filing date: 2007-04-05
Publication date: 2007-10-18

Abstract

An apparatus for online determination of the isotopic composition of nonex- changeable stable hydrogen in a substance sample, comprises: a) an equilibration chamber (1) comprising a sample compartment (26); b) a detection device comprising a pyrolysis chamber (13) and a continuous flow isotope mass spectrometer (19) connected thereto; c) means for loading said sample compartment with a substance sample; d) means for controlled supply of a purging fluid (3) and of a hydrogen exchange fluid (6) to said sample compartment when it contains a substance sample; and e) means (11) for transferring a substance sample from said sample compartment to said detection device under medium tight-conditions.

Description

Equilibration chamber, apparatus and method for online determination of the iso- topic composition of non-exchangeable stable hydrogen in a substance sample

Technical Field of the Invention The present invention relates to an equilibration chamber and to an apparatus for online determination of the isotopic composition of non-exchangeable stable hydrogen in a substance sample. Moreover the invention relates to a method for online determination of the isotopic composition of non-exchangeable stable hydrogen in a substance sample.

Background of the Invention

The stable hydrogen isotope composition of organic substrates is of interest in environmental studies. Although most of the hydrogen in organic substrates is bound to carbon and is not exchangeable with ambient moisture, a considerable part of the total organic hydrogen, mainly in form of hydroxyl, acid and thiol functional groups readily exchanges hydrogen atoms with ambient water or vapor and should therefore not be considered in these studies. However, stable hydrogen isotope measurements determine the isotope ratio of the total hydrogen content of the sample.

One approach to determine the isotope composition of the nonexchangeable hydrogen only is to replace all the exchangeable hydrogen by a hydrogen free group. This can be achieved by nitration of substrates which only have exchangeable hydrogen in form of hydroxyl groups. Many nitration methods de- scribed in literature have several deficiencies like (1) long preparation times, (2) hazardous processing (explosive products), (3) significant sample loss and (4) rather high minimal sample amount requirements.

Another approach to determine the isotopic composition of nonexchangeable hydrogen is to control the isotopic composition of the exchangeable hydrogen by

ϊθsϊ&ϊigiϊrϊg .*' -s=. rk,s<opje equilibration with water of known isotopic composition. In the following some of the more prominent of the equilibration techniques are briefly summarized.

A static equilibration technique for determination of D/H ratios of nonexchange- able hydrogen in cellulose is discussed by Feng et al (Feng, X. (1993). "Determination of D/H ratios of nonexchangeable hydrogen in cellulose: A method based on the cellulose-water exchange reaction". Geochimica et Cosmochimica Acta 57: 4249-4256). Cellulose is equilibrated close to the freezing point with an excess of water of known isotopic composition in a vycor tube sealed off at one end. The sample boat is placed in a larger vycor tube containing activated cupric oxide and is attached to a vacuum line. The sample is rapidly cooled below the freezing point to minimize reequilibration of cellulose during the transition of water to ice followed by evacuation. The vycor tube is sealed off, when the sample ^■ is dry. Subsequently the vycor tube is pyrolyzed and the reaction products, car- bon dioxide and water are separated cryogenically and the water converted to hydrogen over hot uranium, which is then analyzed using a dual inlet isotopic ratio mass spectrometer.

The measured deuterium to hydrogen D/H ratio is then the weighted average of the D/H ratio of the nonexchangeable hydrogen and that of the exchanged hydrogen. If the hydrogen exchange has reached equilibrium with the water, the D/H ratio of exchanged hydrogen is related to the D/H ratio of water through an equilibrium fractionation factor which depends on temperature. If the fraction of exchangeable hydrogen is constant among aliquots of the sample, then a plot of total measured D/H ratio vs. D/H ratio of water is linearly correlated. The slope is the product of the fraction of exchangeable hydrogen and fractionation factor. The intercept is a function of the fraction of exchangeable hydrogen, equilibrium fractionation factor and nonexchanged hydrogen. Thus it is possible to determine fraction of exchangeable hydrogen and fractionation factor for specific cellulose samples. Some deficiencies of this method are (1) the slow equilibration process due to the low temperature, (2) removal of excess of water is difficult and time- consuming and (3) the higher fractionation between water and exchangeable hydrogen at lower temperatures (temperature dependence).

Another method for the equilibration of organic samples with water vapor is described by Schimmelmann (Schimmelmann, A. (1991). "Determination of the concentration and stable isotopic composition of nonexchangeable hydrogen in organic mater." Analytical Chemistry 63: 2456-2459). The sample to be equili- brated is loaded in a vycor ampule together with all reagents necessary for combustion. Several vycor ampules are loaded to a temperature controlled (150- 200⁰C) oven system which can be linked either to a supply line of dry nitrogen gas or to a reservoir of deionized water with known isotopic composition for equilibration. During heating the oven, samples are purged with dry nitrogen gas. As soon as the desired equilibration temperature is reached the manifold is supplied with water which evaporates in the hot stainless steel capillary. After equilibration the manifold is again connected to the dry gas supply and the tubes are sealed off at one end and subsequently connected to a vacuum line to seal off the other end. For isotopic measurement the samples are combusted and the cryogenically separated water reduced over hot chromium to hydrogen gas. Subsequently the D/H ratio of the hydrogen gas is measured using a dual inlet isotopic ratio mass spectrometer and the D/H ratio of the nonexchangeable hydrogen is calculated according to the same procedure as in Feng et al.

Some deficiencies of this method are (1) that the analysis method is slow, (2) the rather large amount of required sample and (3) the potential of fractionation effects during the different steps of this method.

The development of continuous flow stable isotope mass spectrometry (see, e.g. Matthews, D. E., Hayes, J. M., (1978). Isotope-ratio-monitoring gaschromatogra- phy-massspectrometry. Analytical Chemistry 50, 1465-1473) has dramatically reduced the sample amount and time required for isotopic analysis. Systems comprising a pyrolysis chamber and a continuous flow isotope mass spectrometer are commercially available for stable hydrogen isotope analysis of solid and liquid samples (e.g. Finnigan TC/EA from Thermo Electron Corporation). Dry solid samples in tin foil capsules are pyrolyzed in a stream of helium at 1000⁰C (or above). The pyrolysis products are resolved using gas chromatography prior to mass spectrometry.

However, many systems currently used for the determination of the stable hy- drogen isotope composition of nonexchangeable hydrogen require large sample sizes and have relatively long process times due to the complicated and time- consuming preparation of the samples. Accordingly there is a need to determine the stable hydrogen isotope composition of nonexchangeable hydrogen with a system that simplifies sample preparation and reduces process time.

Summary of the Invention

It is the principal object of the present invention to overcome the limitations and disadvantages of currently known devices and methods for determination of the isotopic composition of non-exchangeable stable hydrogen in a substance sam- pie.

The foregoing and further objects are achieved by the equilibration chamber of claim 1 , the apparatus of claim 4 and the method of claim 6.

According to a first aspect of this invention, an equilibration chamber for determining the isotopic composition of nonexchangeable stable hydrogen in a substance sample, comprises: a chamber body with a piston slidingly disposed within a main channel of said chamber body; said piston having a piston borehole extending through said piston substantially perpendicularly to a longitudinal piston axis and forming a sample compartment terminated by inner wall portions of said channel; said chamber body having an entrance borehole disposed proximate to a first end of said main channel and an exit borehole substantially parallel to said entrance borehole and disposed proximate to a second end of said main channel, said entrance borehole and said exit borehole each extending from said main channel in mutually opposite directions to a respective outer face portion of said chamber body so as to define thereon an en- trance aperture and an exit aperture, respectively; said chamber body further having a flushing borehole disposed between said entrance borehole and said exit borehole and extending through said chamber body substantially parallel to said entrance borehole; said piston being slidingly movable from a first position wherein said piston borehole is in line with said entrance borehole to a second position wherein said piston borehole is in line with said flushing borehole and further to a third position wherein said piston borehole is in line with said exit borehole; the equilibration chamber further comprising temperature control means for maintain said chamber body and said piston at a predetermined equilibra- tion temperature.

According to a further aspect of this invention, an apparatus for online determination of the isotopic composition of nonexchangeable stable hydrogen in a substance sample, comprises: a) an equilibration chamber comprising a sample compartment and means for maintaining the chamber at a predetermined equilibration temperature; b) a detection device comprising a pyrolysis chamber and a continuous flow isotope mass spectrometer connected thereto; c) means for loading said sample compartment with a substance sample; d) means for controlled supply of a purging fluid and of a hydrogen exchange fluid to said sample compartment when it contains a substance sample; and e) means for transferring a substance sample from said sample compartment to said detection device under medium tight-conditions.

It will be understood that the apparatus will generally comprise additional fea- tures known in the art, e.g. control and processor units interacting with the functional components of the apparatus. In particular, it may be contemplated to adopt or appropriately modify available laboratory equipment to control and interact with the apparatus of this invention.

According to yet a further aspect of this invention, a method of online determination of the isotopic composition of nonexchangeable stable hydrogen in a substance sample using the apparatus defined in claim 4, comprising at least one of the following sequence of steps: a) loading a substance sample into said sample compartment; b) supplying a flow of hydrogen exchange fluid to said sample compartment during a predefined equilibration period thereby forming an equilibrated sample; c) while supplying a flow of purging fluid to said sample compartment, transferring said equilibrated sample to said detection device; d) measuring the isotopic composition of nonexchangeable stable hydrogen of said equilibrated sample; the above steps being carried out with said equilibration chamber maintained at a predefined equilibration temperature.

It will be understood that the supply of purging fluid does not need to be limited to step c) and could even be kept on during a whole series of measurements. In general, the purging fluid will be used to avoid any contact of ambient air and humidity with an equilibrated sample.

With the above features it becomes possible to carry out determinations of the isotopic composition of nonexchangeable stable hydrogen online, i.e. a given substance sample is used for equilibration and subsequent isotope determination without the need for offline pre-processing of a larger amount of substance. This allows one to use small amounts of sample and to carry out the entire measurement rapidly.

Although the above defined sequence of steps could be performed just once so as to measure one sample, it will generally be desirable to repeat the sequence of steps several times so as to measure a whole series of samples.

Advantageous embodiments are defined in the dependent claims.

In principle, the piston could have one of several cross sectional shapes. According to claim 2, the piston is substantially cylindrical in order to have good sliding properties and being easily machined.

While various embodiments of temperature control means for the equilibration chamber may be envisaged, the embodiment of claim 3 comprises heating elements that are arranged within respective boreholes in the chamber body and in the piston.

According to an advantageous embodiment of the apparatus, the equilibration chamber is configured according to any one of claims 1 to 3 and is arranged with said exit borehole aligned in a substantially downward vertical direction with said exit aperture being positioned at a bottom face portion of said chamber body and wherein said exit aperture is connected in medium-tight fashion by means of a substantially vertical duct to a detection inlet of said pyrolysis chamber arranged thereunder.

According to the preferred embodiment of claim 7, the method of this invention uses an apparatus according to the preferred embodiment defined in claim 5, and said loading step a) comprises: a1) moving said piston to said position 1; a2) dropping said substance sample into said entrance aperture, thereby causing said sample to fall into said sample compartment within said piston; and a3) moving said piston to said position 2; said supplying steq b) comprises supplying a flow of hydrogen exchange fluid to said flushing borehole; and said transferring step c) comprises: c1) while supplying a flow of purging fluid to said flushing borehole, moving said piston to said position 3, thereby causing the equilibrated sample to fall into the detection inlet; c2) moving said piston away from said position 3, thereby isolating said sample compartment from said detection inlet.

According to the preferred embodiment of claim 8, the method of this invention further comprises the step of carrying out at least one calibration measurement before and/or after step d).

According to the preferred embodiment of claim 9, the sequence of steps a) to d) is completed within 500 seconds. This means that a considerable number of samples may be analyzed in rapid succession, as only a few minutes are required for each sample.

In principle, the purging fluid could be any suitable inert liquid or gas. According to the preferred embodiment of claim 10, the purging fluid is an inert gas, for ex- ample helium.

In principle, the hydrogen exchange fluid could be any suitable liquid or gas that contains readily exchangeable hydrogen. According to the preferred embodiment of claim 11 , the hydrogen exchange fluid is water vapor. It will be appreciated that such water vapor may be supplied by streaming a flow of carrier gas through liquid water with appropriate heating. For other applications, e.g. if the sample substance is a sugar, it is preferable to use liquid water as the hydrogen exchange fluid as defined in claim 12.

The method of this invention can be applied to a large variety of sample sub- stances. In particular, it may be applied to organic molecules. According to the embodiment of claim 13, the sample substance is cellulose. In another embodiment, the sample substance is a sugar. It has been found that for sugars it is preferable to use a hydrogen exchange fluid in liquid form in order to achieve sufficiently rapid hydrogen equilibration.

This invention relates to a method and a device to determine the deuterium to hydrogen ratio of nonexchangeable hydrogen in organic samples by coupling an equilibration unit/device with a device comprising a pyrolysis chamber and a continuous flow isotope mass spectrometer. Some embodiments of the invention are directed to an apparatus that comprises an equilibration unit, a pyrolysis chamber and a continuous flow isotope mass spectrometer. In certain embodiments of the invention the equilibration unit could be directly coupled to the pyrolysis chamber or an autosampler unit. In another embodiment of the invention, the equilibration unit is integrated into an autosampler unit. Another embodiment of the invention comprises an adaptable equilibration unit including an adapter that can be linked to a pyrolysis chamber or an autosampler unit. More particularly this invention relates to a method and a device to equilibrate organic samples in a continuous water vapor flow prior to pyrolysis. In one embodiment of the invention, equilibration may be carried out in an equilibration unit such as a heated chamber supplied with water of known isotopic composition and a gas such as helium or argon. The chamber may be heated or cooled. The chamber may be a metal chamber, such a chamber made from stainless steel or copper. In another embodiment of this invention equilibration on organic samples packed in elemental analysis capsules may be carried out in an equilibration unit such as a cham- ber supplied with a continuous flow of a liquid or gas of known isotopic composition and dry purge gas such as helium or argon. In another embodiment of the invention, the equilibration unit may have provisions to load the sample, equilibrate it and subsequently transfer it to a pyrolysis chamber or an autosampler unit. In still another embodiment of the equilibration unit the sample may be directly transferred to a valve, such as an autosampler valve. In one embodiment of the invention the sample is packed in capsules such as often used for elemental analysis, for example capsules obtained from Brechbϋhler AG, Schlieren, Switzerland, Art. Nr. 176.9807.26. In one embodiment of the invention, sample amounts with a hydrogen content ranging from 5-100 micrograms can be equilibrated within a few minutes, for example 500 micrograms of cellulose corre- sponding to a hydrogen content of approx. 25 micrograms is equilibrated within 8 minutes prior to analysis. Lower sample amounts are possible in certain embodiments. In another embodiment of the invention, the equilibration is at high enough temperatures and fast enough to substantially prevent water to condensate. Temperature and equilibration time are dependent on certain parameters. In a further embodiment of the invention, while a sample is pyrolyzed and the resulting gases monitored with the IRMS, the next sample is being equilibrated.

Brief description of the drawings

The above mentioned and other features and objects of this invention and the manner of achieving them will become more apparent and this invention itself will be better understood by reference to the following description of various embodiments of this invention taken in conjunction with the accompanying drawings, wherein:

Fig. 1 is a schematic representation of an embodiment of the apparatus and process of this invention;

Fig. 2 shows different views of an embodiment of the equilibration chamber block;

Fig. 3 shows different views of an embodiment of the piston of the equilibration chamber; Fig. 4 shows an embodiment of a time diagram of the process of this invention.

Detailed description of the invention The present invention is based on coupling the equilibration chamber with a continuous flow stable isotope mass spectrometer. A continuous gas flow such as a helium or a nitrogen (could potentially form CN, a highly toxic compound) or an argon gas flow purges the organic sample in a temperature controlled chamber. This substantially prevents the sample from equilibration with moist ambient air. Water of known isotopic composition can be added to the gas prior to entering to the chamber. This results in a mixture of the gas and water vapor within the chamber. The water vapor equilibrates with the exchangeable hydrogen atoms of the sample. The equilibrated sample may be directly transferred to a valve such as a autosampler valve of a continuous flow stable isotope mass spectrometry and subsequently pyrolyzed in the oven. The obtained products are transported by a carrier gas, generally helium, through a gas chromatographic (GC) column for gas separation. The gas mixture is then introduced through an open split interface into an isotopic ratio mass spectrometer (IRMS) for analysis.

As shown in FIG. 1 , in one embodiment of the invention the sample is placed into a borehole of a piston 2 of the chamber body 1. The piston can be moved between three positions, loading position, equilibration position and the sample dropping position. A detailed description of one embodiment of the chamber is given below. The chamber is steadily flushed with a helium flow 3 of 40 ml/min supplied from a helium high pressure tank 4. This allows a sealing of the chamber against ambient air. A tubing pump 5 controls and mixes selectively a water flow 6 of 7.8 μl/min and known isotopic composition to the helium through a t- piece 7 prior to the chamber. The water rapidly vaporizes when entering the heated chamber (1+2) controlled at 110⁰C by a microprocessor 8. The ex- changeable hydrogen of the sample equilibrates with the water vapor hydrogen. The helium water vapor mixture leaves the chamber through a borehole (vent) 9. The chamber body 1 is connected through a glass tube 10 with an autosampler 11 , which is purged by a helium flow 12 of 40 ml/min. The autosampler 11 drops the sample into the pyrolysis tube 13 held at approximately 1400⁰C. A second helium flow 14 of 120 ml/min carries the sample gas products through a 1/16" tubing 15 (300 mm in length) to a GC column 16 held at 70⁰C where the gas products are separated. The sample products are flushed through another 1/16" tubing 17 (1000 mm in length) to an open split 18 (Conflow Il system of Ther- moFinnigan). The open split 18 allows diluting the sample by adding helium and acts also as a reference gas supply. A sniffle capillary is placed to the open split which directs the sample gases into the isotope ratio mass spectrometer 19.

In one embodiment of the invention, the equilibration chamber comprises of a metal chamber body 1 and a piston 2, which are depicted in more details in FIG. 2 and FIG. 3, respectively. Both parts are heated with electrical high perform- ance heating cartridges (Electrolux type HHP, 40mm in length, 6.5mm outer diameter, 100 Watt power at 230 volt) controlled by a microprocessor 8 (Jumo Ir- ton 16).

As shown in FIG. 2 the metal chamber body 1 has four boreholes 20 for the heating cartridges around the main bore hole 21 which contains the piston 2. Through the aperture 22 in the chamber body 1 samples are introduced into a borehole 26 of the piston 2. On the orifice 23 a 1/16" t-piece 7 connects to the helium 3 and the water supply 6. In the t-piece 7 these flows are mixed to yield moist helium flowing over the sample and leaving the chamber body 1 through the vent 9. The equilibrated sample is dropped through the aperture 24 into the autosampler 11. The piston 2 which can be moved in the main borehole 21 of the metal chamber body 1 is depicted in FIG. 3. The cylindrical piston 2 has two longitudinal boreholes 25 to contain two heating cartridges and a borehole 26 to carry the sample. There are three distinct positions for the piston 2. In the loading position the borehole 26 in the piston 2 and the aperture 22 in the metal chamber body 1 are in line to introduce the sample. In the equilibration position the borehole 26 in the piston, the orifice 23 and the vent 9 of the metal chamber body 1 are in line to equilibrate the sample. In the third position the borehole 26 in the piston 2 is above the aperture 24 in the metal chamber body 1 to drop the equilibrated sample into the autosampler 11.

In the following a detailed description of an embodiment of a single analysis of cellulose is given. To determine the deuterium to hydrogen ratio of nonex- changeable hydrogen a sample of 0.5 mg cellulose is weighed either into a silver or a into tin capsule such as often used for elemental analysis and which should be wrapped loose enough to allow water vapor to enter the capsule, but tight enough to prevent sample dropping out of the capsule. The sample is dropped inside the borehole 26 of the piston 2 inside the metal chamber body 1. At that time both helium flows 3 and 14 are active. The piston 2 is then brought into the equilibration position and the tubing pump 5 is switched on for 500s supplying the water of known isotopic composition for isotopic equilibration. After 400s the IRMS 19 acquisition is started. Then the piston 2 is moved to the dropping posi- tion. The equilibrated sample is dropped into the autosampler 11 and further into the pyrolysis oven within 5 seconds. The resulting hydrogen from the pyrolysis is monitored using an IRMS 19.

The evaluation of the deuterium to hydrogen ratio of the nonexchangeable hy- drogen accords to the proceeding of Schimmelmann et. al. with a unique calibration of the equilibrium fractionation factor for a given temperature and a provisional exchangeability of the theoretically exchangeable hydrogen for cellulose.

For one embodiment, the simultaneous overlapping cycles of sample equilibra- tion and isotopic measurement are illustrated in FIG. 4. The signal amplitude obtained for mass to charge 2 is plotted as a function of time. The isotopic ratio is 00175

- 14 -

obtained by dividing the signal for mass to charge 3 by the signal for mass to charge 2. Each isotopic measurement may comprise several working standard peaks (ST) (in this embodiment three pulses were applied) before and after a sample peak (SA) lasting for 500s which corresponds to the required time to equilibrate a cellulose sample. 400s before the data acquisition of sample n starts, the equilibration is launched (Os). At 500s the equilibrated sample is transferred from the equilibration chamber to the pyrolysis oven and the next sample n+1 is loaded and equilibrated. Within 50s the hydrogen from the pyrolyzed sample is detected in the IRMS. After three more working standard peaks acqui- sition of measurement n is stopped (900s), to launch the acquisition of the sample n+1 which is already in the equilibration chamber for 400s.

While a sample is pyrolyzed and the resulting gases monitored with the IRMS the next sample can already be equilibrated.

It will be appreciated that modifications to the embodiments described above are of course possible. Accordingly the present invention is not limited to the embodiments described above.

Claims

1. An equilibration chamber for determining the isotopic composition of non- exchangeable stable hydrogen in a substance sample, comprising: - a chamber body (1) with a piston (2) slidingly disposed within a main channel (21) of said chamber body; said piston having a piston borehole (26) extending through said piston substantially perpendicularly to a longitudinal piston axis and forming a sample compartment terminated by inner wall portions of said channel; said chamber body having an entrance borehole (22) disposed proximate to a first end of said main channel and an exit borehole (24) substantially parallel to said entrance borehole and disposed proximate to a second end of said main channel, said entrance borehole and said exit borehole each extending from said main channel in mutually opposite directions to a respective outer face portion of said chamber body so as to define thereon an entrance aperture and an exit aperture, respectively; said chamber body further having a flushing borehole (23) disposed between said entrance borehole and said exit borehole and extending through said chamber body substantially parallel to said entrance borehole; said piston being slidingly movable from a first position wherein said piston borehole is in line with said entrance borehole to a second posi- tion wherein said piston borehole is in line with said flushing borehole and further to a third position wherein said piston borehole is in line with said exit borehole; the equilibration chamber further comprising temperature control means for maintain said chamber body and said piston at a predeter- mined equilibration temperature.

2. The equilibration chamber as defined in claim 1 , wherein said piston is substantially cylindrical.

3. The equilibration chamber as defined in claim 1 or 2, wherein said tempera- ture control means comprise heating elements that are arranged within respective boreholes (20, 25) in said chamber body and in said piston.

4. An apparatus for online determination of the isotopic composition of nonex- changeable stable hydrogen in a substance sample, comprising: a) an equilibration chamber (1) comprising a sample compartment (26) and means for maintaining the chamber at a predetermined equilibration temperature; b) a detection device comprising a pyrolysis chamber (13) and a continuous flow isotope mass spectrometer (19) connected thereto; c) means for loading said sample compartment with a substance sample; d) means for controlled supply of a purging fluid (3) and of a hydrogen exchange fluid (6) to said sample compartment when it contains a substance sample; and e) means (11) for transferring a substance sample from said sample compartment to said detection device under medium tight-conditions.

5. The apparatus as defined in claim 4, wherein said heatable equilibration chamber is configured according to any one of claims 1 to 3 and is ar- ranged with said exit borehole (24) aligned in a substantially downward vertical direction with said exit aperture being positioned at a bottom face portion of said chamber body (1) and wherein said exit aperture is connected in medium-tight fashion by means of a substantially vertical duct (10) to a detection inlet of said pyrolysis chamber (13) arranged thereunder.

6. A method of online determination of the isotopic composition of nonex- changeable stable hydrogen in a substance sample using the apparatus defined in claim 4, comprising at least one of the following sequence of steps: a) loading a substance sample into said sample compartment; b) supplying a flow of hydrogen exchange fluid to said sample compartment during a predefined equilibration period thereby forming an equilibrated sample; c) while supplying a flow of purging fluid to said sample compartment, transferring said equilibrated sample to said detection device; d) measuring the isotopic composition of nonexchangeable stable hydrogen of said equilibrated sample; the above steps being carried out with said equilibration chamber maintained at a predefined equilibration temperature.

7. The method as defined in claim 6 using the apparatus as defined in claim 5, wherein said loading step a) comprises: a1) moving said piston to said position 1 ; a2) dropping said substance sample into said entrance aperture, thereby causing said sample to fall into said sample compartment within said piston; and a3) moving said piston to said position 2; and wherein said supplying step b) comprises supplying a flow of hydrogen exchange fluid to said flushing borehole; and wherein said transferring step c) comprises: c1) while supplying a flow of purging fluid to said flushing borehole, moving said piston to said position 3, thereby causing the equilibrated sample to fall into the detection inlet; c2) moving said piston away from said position 3, thereby isolating said sample compartment from said detection inlet.

8. The method as defined in claim 6 or 7, further comprising the step of carrying out at least one calibration measurement before and/or after step d).

9. The method as defined in any one of claims 6 to 8, wherein said sequence of steps a) to d) is completed within 500 seconds.

10. The method as defined in any one of claims 6 to 9, wherein said purging fluid is an inert gas.

11. The method as defined in any one of claims 6 to 10, wherein said hydrogen exchange fluid is water vapor.

12. The method as defined in any one of claims 6 to 10, wherein said hydrogen exchange fluid is liquid water.

13. The method as defined in any one of claims 6 to 12, wherein said sample substance is cellulose.