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An improved technique for the determination of oxygen stable isotope ratios in wood cellulose
0969-8043/94 $6.00 + 0.00 Copyright 0 1994 Pergamon Press Ltd
Appl. Radial. Isot. Vol. 45, No. 2, pp. 177-181, 1994 Printed in Great Britain. All rights reserved
An Improved Technique for the Determination of Oxygen Stable Isotope Ratios in Wood Cellulose E. M. FIELD’, V. R. SWITSUR’ and J. S. WATERHOUSE2 ‘Godwin
Laboratory, ‘Anglia
Cambridge Polytechnic
University, Free School Lane, Cambridge, University, East Road, Cambridge, U.K.
(Received 28 March
1993; in revised form
U.K. and
22 June 1993)
A technique is described for the accurate and reproducible (0.2 per mille) determination of stable oxygen isotope ratios from wood alpha-cellulose using an improved version of the mercuric chloride pyrolysis method. In this a macroreticular resin, Amberlyst A-21, is used as absorbent for hydrogen chloride. The case for use of quartz rather than borosilicate reaction vessels is presented.
nickel powder. Whilst all of these techniques, with care, are capable of yielding accurate and consistent results, each technique has its own drawbacks in terms of cost, complexity of equipment or inconvenience. It is in an attempt to reduce the inconveof the mercuric chloride technique niences (Rittenberg and Ponticorvo, 1956; Dunbar and Wilson, 1983) that the process described here was perfected. The mercuric chloride technique is probably one of the simplest and involves heating the purified alphacellulose with an excess of mercuric chloride for several hours so that the reaction proceeds to completion. The chloride is partially decomposed and the organic material is oxidized with the mercury acting as catalyst. The reaction products include carbon, carbon monoxide, carbon dioxide and hydrogen chloride. The oxygen from the cellulose thus appears in a mixture of carbon monoxide and dioxide, the proportion of each depending on the conditions such as the temperature reached and the rate of heating. Early workers found that the isotopic composition of the oxides differed slightly and that in order to measure the stable oxygen isotope ratio accurately in a mass spectrometer, the oxygen is required to be in the form of carbon dioxide. The monoxide is converted quantitatively to dioxide by disproportionation in a high voltage electrical discharge. Unfortunately the presence of hydrogen chloride in the sample of carbon dioxide adversely affects the stable isotope measurement and so it is essential that it is removed. The gases are difficult to separate physically since their boiling points are similar. The separation is normally accomplished by the reaction chloride with weakly-basic of the hydrogen compounds of low volatility, such as quinoline or
Background Of the several techniques described during the past few decades for determining the stable isotope ratios of oxygen in organic materials, only a few have proved suitable, in terms of accuracy and reproducibility, for measuring the natural abundances of ‘80/‘60, such that they may be used in investigations of palaeoclimate based on alpha-cellulose from deciduous tree species. An isotope ratio mass spectrometer of sufficient sensitivity is used for measuring the ‘sO/‘6O ratio. In practice the ratio variations are reported as differences from an arbitrary standard. These are calculated from the expression: a=[*-l]xlOoOpermille where R = ‘*O/‘6O. The standard used here is V-SMOW (Vienna Standard Mean Ocean Water) issued by the I.A.E.A. which is a version of the SMOW defined by Craig (1961). Of the published techniques only one (Mullane et al., 1988) utilizes a stepwise chemical degradation procedure that may be modified as required for the study of oxygen atom ratios at specific positions in the cellulose molecule. The other methods rely on non-specific thermal degradation to generate carbon dioxide having the same isotopic composition as the parent material (Rittenberg and Ponticorvo, 1956; Hardcastle and Friedman, 1974; Epstein et al., 1977; Thompson and Gray, 1977; Brenninkmeijer and Mook, 1981; Brenninkmeijer, 1983; Dunbar and Wilson, 1983; Ramesh, 1984). These workers use equipment of widely different complexity and various catalysts including mercuric chloride, diamond and 177
E. M. FIELD et al.
178
5,6benzoquinohne. These substances have to be highly purified, typically by multiple distillation shortly before use. Experiments during our work demonstrated that quinoline retained substantial quantities of gases and that it was necessary to degas the sample in uacuo before use. Similarly, after the reaction and the removal of hydrogen chloride, the quinoline retained some of the carbon dioxide. It was found that the quinohne had to be subjected to at least three freeze-thaw cycles in uacuo to obtain a quantitative yield of the carbon dioxide. Quinoline is toxic and unpleasant to work with and since a large number of isotope determinations are required in any palaeoclimatic investigation, an alternative substance was sought that was readily available and easy to use. Its properties needed to be sufficiently specific so as to absorb the hydrogen chloride, but without absorbing the carbon dioxide, and hence perform the necessary separation. (i) Absorbents for hydrogen chloride We first investigated weakly basic anionic resins as reagents, potentially having suitable properties for this requirement. Most resins of this type, however, are supplied in a wet form and the water thus contained would render the sensitive oxygen isotopic results meaningless unless thorough desiccation were possible before use. If the resins remained active after this thorough drying they could be suitable for the reaction. Generally, two types of resin are available,
Fig.
known as gel and macroreticular. With the former resins we found that, on desiccation, the pores collapsed and no hydrogen chloride was absorbed. The latter type, however, retain their pore structure on desiccation and we found that hydrogen chloride could be absorbed efficiently. The initial experiments were performed using the macroreticular resin Amberlyst IRA -93 (Sigma Chemicals). As supplied this contains 61% water. It was dried in a vacuum oven at 60°C for ca 40 h, after which successive weighings became constant, thus indicating that the water had been removed. This was used in place of quinoline in the reaction chamber (Fig. 1). It was pumped to high vacuum for 30min before use to ensure complete removal of the water. After the reaction the measured yield of carbon dioxide was only 80% of theoretical but a mass spectrometer scan between mass numbers 25560 indicated, as also in the case of quinoline, that all hydrogen chloride had been removed. Gentle warming of the resin had the effect of raising the yield to 95-100%. Further investigation, however, revealed that this resin had significant shortcoming in that some of the carbon dioxide that was collected originated, not from the sample, but from the resin itself. The amine groups present in Amberlyst IRA-93 are mostly tertiary but some primary and secondary amines are also present and these could absorb carbon dioxide from the atmosphere with the production of carbamates. These carbamates would not
Apparatus for determination of stable oxygen isotope ratios.
Oxygen isotopes in wood cellulose
release carbon dioxide during the drying procedure but would do so on acidification with hydrochloric acid. In order to check this, a sample of hydrogen chloride was admitted to the reaction chamber containing only dried IRA-93. Carbon dioxide was indeed released, ca 3% of that expected from a cellulose sample. It may be assumed that a resin containing solely tertiary amines would be without these shortcomings. Accordingly Amberlyst A-21 (BDH), which is such a resin, was tested in a similar manner. It was found that no carbon dioxide was generated from this material, which behaved otherwise in a similar manner to the Amberlyst IRA-93. It was decided to use Amberlyst A-21 in all further work. (ii) Borosilicate
glass us quartz
One of the original aims was to determine whether the pyrolysis could be carried out in vessels of Pyrex glass at 450°C for 4 h instead of quartz at a higher temperature, owing to the greater ease of use of the former. Experiments demonstrated that values of the oxygen isotope ratio obtained at this temperature gave a reproducibility of only 0.94.5 per mille. Replacement of the Pyrex vessels by quartz vessels and raising the pyrolysis temperature to 550°C for 6 h immediately improved the reproducibility to 0.2 per mille. It was found that a similar reproducibility of 0.2 per mille could also be obtained using Pyrex vessels by increasing the reaction time to 18 h at 450°C however, the average of the values from these was consistently 1.6 per mille lower than that from the reactions in quartz. In order to investigate which of these values gave the correct result, quartz and Pyrex vessels were filled with standard grade carbon dioxide of known delta IgO value. The quartz tubes were heated in the furnace to 550°C for 6 h, whilst the borosilicate tubes were heated at 450°C for 18 h. Our findings were that for the quartz vessels there was no change in the isotopic ratios, whereas for the Pyrex vessels, the value was lowered consistently by 1.6 per mille. Obviously, some interaction occurs between Pyrex and carbon dioxide even at this lower temperature which indicates that Pyrex is not suitable for use in this research. Observations of a reaction of carbon dioxide with borosilicate glass were made by Epstein et al. (1977). (iii) Comparison of delta I80 values of standard cellulose with another technique A sample of a standard commercial cellulose, cellulose CF-11 (Whatman) as used in his tests was kindly supplied by C.A.M. Brenninkmeijer for comparison of the two techniques. The measurements were made with respect to the international standard, V-SMOW supplied by the I.A.E.A. The mean of 7 determinations of delta IgO by the Cambridge technique, as described in this paper, was 26.67 f 0.20 per mille with respect to V-SMOW and this may be
179
compared with a mean for 17 determinations of 26.4 f 0.1 per mille reported by Brenninkmeijer using the sealed nickel tube technique (Brenninkmeijer, 1983). The agreement between the techniques is satisfactory. The consequence of this work is the development of an improved and very effective technique for the analysis of oxygen isotopes in wood cellulose having excellent reproducibility for the complete process through alpha-cellulose extraction, pyrolysis, disproportionation, purification and mass spectrometric analysis. The techniques used from the pyrolysis stage are given in detail below; the alpha-cellulose preparation is described in Mullane et al. (1988).
Apparatus and Methods It is important that all materials and samples used in oxygen isotope analyses must be thoroughly dried to a constant weight in a vacuum oven before attempting the determinations. Dried reagents and samples may be maintained in that state by storing in tightly stoppered vials or jars in a vacuum desiccator. Desiccators using sulphuric acid, phosphorus pentoxide and similar materials alone as drying agents require a considerable time to reach equilibrium and are generally unsuitable for this work. The pyrolysis tubes (9 mm o.d. by 125 mm length) are fabricated from high quality quartz tubing. Before use each tube is suitably inscribed with a diamond pen, dried at 550°C for 1 h and stored in a vacuum desiccator. Approximately 34mg of dried alpha-cellulose is accurately weighed into the pyrolysis tube and mixed with 130 mg mercuric chloride. A breakseal is formed ca 1.5 cm from the end of the tube which is attached via a Cajon ultratorr “0”-ring vacuum fitting to a high vacuum line and pumped overnight to ensure that all moisture is removed. The tube is then flame sealed under vacuum. The pyrolysis is performed by heating in an electrically heated tube furnace for 6 h at 550°C. It is convenient to process 8-10 samples in one batch. “Amberlyst A-21” resin (BDH) and mercuric chloride (BDH) are prepared by heating in a vacuum oven at 50°C for 48 h and stored in sealed bottles in a vacuum desiccator. The procedure, as detailed in the following, is carried out using the apparatus shown in Fig. 1. In this figure the vacuum valves (Tl-T8) are all 9 mm. “O”-ring type (Lowers Hapert). Vessel A is the disproportionation discharge tube (22 cm x 5 cm dia) fitted with platinum mesh electrodes, Pt (Johnson Matthey) (2.5 x 2.5 cm). These are cleaned from deposited carbon after the reaction by heating to incandescence in air. The high voltage applied to these electrodes is produced by a transformer capable of attaining 10,000 V. The output voltage is varied using a manually variable transformer (Variac) connected to the EHT input. The vessel is demountable via an “0”-ring joint “0”. The reaction vessel B
180
E. M.
FIELD et al.
(22 cm x 2 cm dia) having a demountable base (B-25 “0”-ring joint) contains the breaking device, K, consisting of a vacuum “0”-ring valve (Youngs) sealed into the side of the tube. Rotation of the valve head drives the glass barrel against the pyrolysis sample tube, S, causing it to shatter. During evacuation of vessel B, the pyrolysis tube is held gently against the side of vessel by the barrel of the cracker at a point above the removable base of the vessel in which the Amberlyst A-21 resin is weighed. This vessel is connected to the vacuum line via a 9 mm dia “0”-ring vacuum valve through a 3/8” dia ultratorr connector. Reaction vessel C (20 cm x 2.5 cm dia) is also demountable through an “0”-ring joint for cleaning. E is a small volume cylindrical finger connected to a mercury manometer. The removable receiver, D (10 cm x 0.9 cm dia) is fabricated from an “0”-ring vacuum valve (Lowers Hapert) modified so as to connect to the vacuum system via a stainless steel l/4” dia ultratorr connector. The system is evacuated via rotary and oil diffusion pumps and the vacuum measured with a high sensitivity (to 10-4Torr) Pirani gauge (Edwards High Vacuum).
Techniques (A) “Amberlyst A-21” resin (0.25 g), previously dried to constant weight, is placed in the breaker tube B together with a scored pyrolysis sample tube, S. All the taps, 1 to 9, are opened fully and the complete apparatus is pumped for 45 min to high vacuum with a Dewar vessel containing freshly boiled water placed around B. After this time the Dewar vessel is removed. (B) Vacuum valves T4, T5 and T6 are closed and a Dewar containing liquid nitrogen placed around vessel B. The pyrolysis tube is broken by rotating the valve head K, and forcing the barrel against it. The pyrolysis products, carbon dioxide and hydrogen chloride, are allowed to freeze on to the resin for about 1 min, whilst the carbon monoxide remains largely in the gaseous phase. (C) Valves T3 and Tl are closed to isolate the system and the finger on the discharge tube, A, is cooled by a Dewar of liquid nitrogen. Valve T4 is opened to enable the pressure to be recorded on the Pirani gauge. Valve T3 is opened and the pressure allowed to stabilize as carbon monoxide enters A. (D) High voltage is applied to the platinum electrodes, Pt, of the discharge tube, A, until the light eggshell-blue discharge strikes. The value of the high voltage required depends on the gas pressure and the separation of the electrodes, but is typically 2000 V. The carbon monoxide is disproportionated to carbon dioxide. The discharge soon quenches but is renewed by gradually increasing the high voltage via the variable transformer. Eventually when all the carbon monoxide has been converted to dioxide and the
discharge can not be maintained, the high voltage is reduced to zero. The carbon dioxide produced by the reaction condenses in the cooled finger of A. (E) Valve T4 is closed and the liquid nitrogen trap removed from vessel B. The hot water Dewar is returned to B for 1 min after which the resin is left to absorb the hydrochloric acid gas for IO min. (F) Any residual carbon monoxide remaining adsorbed in the frozen carbon dioxide in the discharge tube is released by removing the liquid nitrogen Dewar from the discharge tube finger A, thawing the solid carbon dioxide with hot water to release carbon monoxide and refreezing with liquid nitrogen. The high voltage is once more gradually applied to the electrodes, Pt, by increasing the t:ariac voltage. until the maximum is reached and the residual carbon monoxide is disproportionated. This stage is repeated to extract the final traces of carbon monoxide and convert to the dioxide. (G) Valve T3 is closed and when sufficient time has elapsed enabling the resin to absorb the hydrochloric acid valve T4 is opened and the pressure recorded. Valve T3 is again opened and the carbon dioxide frozen into the discharge tube A. When the pressure becomes stable once more the glow discharge is carefully restruck in vessel A. The procedure of section (F) is repeated. (H) The residual pressure in absorption vessel B is recorded after closing valve T3 and opening valve T4. The remaining gas is then transferred to the discharge tube, A, by opening valve T3 and freezing the carbon dioxide in the cold finger with liquid nitrogen. The high voltage is once more carefully applied to the electrodes, Pt, until the discharge ceases. when it is removed. (I) After 10 min valve T3 is closed for 2 min and re-opened. The pressure is noted; there should be no significant change in pressure indicating that all the gas has been processed. (J) Valve T4 is closed next and, with a liquid nitrogen trap surrounding A, valve Tl is opened and the line pumped to high vacuum. This processing is tedious; hoaerw. our ,findings are that these steps are essential (f the reaction is to proceed to completion and a quantitative yield obtained. The total yield of carbon dioxide, held frozen in the finger of vessel A is next transferred to the receiver, D for measurement by mass spectrometry. This is accomplished by the following sequence. (K) Valve T2 is closed and it is ensured that valves T5, T6, T7, T8 and T9 are open and this system pumped to high vacuum after which valves T6 and Tl are closed and valve T2 is opened. (L) The liquid nitrogen trap is removed from around the carbon dioxide in discharge vessel A and replaced with the hot water Dewar. The liquid nitrogen trap is replaced around vessel C and the carbon dioxide allowed to sublime into it. The return of the Pirani gauge reading to high vacuum is the signal that the transfer is complete. The line is taken to high
Oxygen
isotopes
vacuum by opening valve Tl and valve T5 is then closed to isolate the carbon dioxide. Valve T6 is opened and the line is once more pumped to high vacuum. (M) The liquid nitrogen trap is removed from around vessel C and the carbon dioxide vaporized with the hot water trap. A slurry of ca 50% each of liquid nitrogen and methylated spirit is prepared in a Dewar vessel and used to re-freeze vessel C. This mixture is sufficiently cold to freeze out and retain hydrocarbon impurities but allows the carbon dioxide to remain in the gas phase. Valves T7, T3 and T6 are closed, valve T5 opened and the pressure in vessel C is recorded. (N) A Dewar of liquid nitrogen is placed around finger E attached to the manometer. When the Pirani gauge reading is steady valve T6 is opened and the carbon dioxide sublimed into finger E. (0) Any residual carbon dioxide in trap C is scavenged by the process of thawing and re-freezing, as follows. Valve T6 is closed and trap C thawed with hot water to release carbon dioxide. It is then recooled with the liquid nitrogen/methylated spirit slurry. Valve T6 is reopened to allow transfer of this carbon dioxide to E. Valve T5 is closed to isolate the trap and valve T3 opened. The system is once more pumped to high vacuum. (P) The yield of carbon dioxide is next obtained using a manometer previously calibrated with known quantities of carbon dioxide. Valve T8 is closed and valve T7 opened. The carbon dioxide is vaporized by warming trap E with warm water and after leaving sufficient time to gain room temperature the manometer pressure is recorded. From the temperature, pressure and volume of carbon dioxide, the yield is calculated. (Q) The carbon dioxide is next collected for the mass spectrometric assay. A Dewar of liquid nitrogen is placed around the receiver, D. With valves T8, T7 and T9 open the gas transferred to the receiver. The process is monitored by the pirani gauge readings. When complete the valve T3 is opened and the system pumped to high vacuum. The valve, T9, of the receiver D, is closed to isolate the sample. (R) Valve T7 is closed and the receiver removed from the ultratorr connection, U, and transferred to the mass spectrometer for stable isotope measurement.
in wood cellulose
181
We now use this technique regularly in a project concerning measurements of the stable isotope ratios of hydrogen, carbon and oxygen in alpha-cellulose obtained from individual oak growth rings. The efficiency of the procedure could be greatly improved by automating the valve and pumping systems and controlling the operations from a microcomputer. This would greatly simplify the numerous sequences of valve manipulations which depend on parameters of time, temperature and pressure which could be determined by appropriate sensors. The sample throughput would be increased significantly and the possibility of operator error would be eliminated. Acknowledgemeno-We acknowledge gratefully the help and advice of our colleagues, Mike Hall, Nick Shackleton, Henry Schwartz and others in this work. We thank Tony Carter for his kindness in drawing the diagram. We thank the NERC for a Special Project grant to VRS and JSW for the funding of this work and for the larger project of isotopes in the environment.
References Brenninkmeijer C. A. M. (1983) Deuterium, oxygen-18 and carbon-13 in tree rings and peat deposits in relation to climate. Thesis, University of Groningen. Brenninkmeijer C. A. M. and Mook W. G. (1981) A batch process for direct conversion of organic oxygen and water to carbon dioxide for ‘8O/‘6O analysis. Int. J. Appl. Radiat. Isot. 32, 137. Craig H. (1961) Standards for reporting concentrations of deuterium and oxygen-18 in natural water. Science 133, 1833. Dunbar J. and Wilson A. T. (1983) Re-evaluation of the HgCl, pyrolysis technique for oxygen isotope analysis. Int. J. Appl. Radial. Isor. 34, 932. Epstein S., Thompson P. and Yapp C. J. (1977) Oxygen and hydrogen ratios in plant cellulose. Science 198, 1209. Hardcastle K. G. and Friedman I. (1974) A method for oxygen isotope analysis for organic material. Geophys. Res. Lett. 1, 6. Mullane M. V., Waterhouse J. S. and Switsur V. R. (1988) On the development of a novel method for the determination of stable oxygen isotope ratios in cellulose. Appl. Radiat. lsot. 39, 1029. Ramesh R. (1984) Stable isotope systematics in plant cellulose: implications for past climate. Thesis. Gujarat University. Rittenberg D. and Ponticorvo L. (1956) A method for the determination of the “0 concentration of oxygen of organic compounds. Int. J. Appl. Radial. Isor. 1, 208. Thompson P. and Gray J. (1977) Determination of the ‘“O/‘6O ratios in compounds containing C, H and 0. Int. J. Appl. Radiat. Isot. 28, 411.
Appl. Radial. Isot. Vol. 45, No. 2, pp. 177-181, 1994 Printed in Great Britain. All rights reserved
An Improved Technique for the Determination of Oxygen Stable Isotope Ratios in Wood Cellulose E. M. FIELD’, V. R. SWITSUR’ and J. S. WATERHOUSE2 ‘Godwin
Laboratory, ‘Anglia
Cambridge Polytechnic
University, Free School Lane, Cambridge, University, East Road, Cambridge, U.K.
(Received 28 March
1993; in revised form
U.K. and
22 June 1993)
A technique is described for the accurate and reproducible (0.2 per mille) determination of stable oxygen isotope ratios from wood alpha-cellulose using an improved version of the mercuric chloride pyrolysis method. In this a macroreticular resin, Amberlyst A-21, is used as absorbent for hydrogen chloride. The case for use of quartz rather than borosilicate reaction vessels is presented.
nickel powder. Whilst all of these techniques, with care, are capable of yielding accurate and consistent results, each technique has its own drawbacks in terms of cost, complexity of equipment or inconvenience. It is in an attempt to reduce the inconveof the mercuric chloride technique niences (Rittenberg and Ponticorvo, 1956; Dunbar and Wilson, 1983) that the process described here was perfected. The mercuric chloride technique is probably one of the simplest and involves heating the purified alphacellulose with an excess of mercuric chloride for several hours so that the reaction proceeds to completion. The chloride is partially decomposed and the organic material is oxidized with the mercury acting as catalyst. The reaction products include carbon, carbon monoxide, carbon dioxide and hydrogen chloride. The oxygen from the cellulose thus appears in a mixture of carbon monoxide and dioxide, the proportion of each depending on the conditions such as the temperature reached and the rate of heating. Early workers found that the isotopic composition of the oxides differed slightly and that in order to measure the stable oxygen isotope ratio accurately in a mass spectrometer, the oxygen is required to be in the form of carbon dioxide. The monoxide is converted quantitatively to dioxide by disproportionation in a high voltage electrical discharge. Unfortunately the presence of hydrogen chloride in the sample of carbon dioxide adversely affects the stable isotope measurement and so it is essential that it is removed. The gases are difficult to separate physically since their boiling points are similar. The separation is normally accomplished by the reaction chloride with weakly-basic of the hydrogen compounds of low volatility, such as quinoline or
Background Of the several techniques described during the past few decades for determining the stable isotope ratios of oxygen in organic materials, only a few have proved suitable, in terms of accuracy and reproducibility, for measuring the natural abundances of ‘80/‘60, such that they may be used in investigations of palaeoclimate based on alpha-cellulose from deciduous tree species. An isotope ratio mass spectrometer of sufficient sensitivity is used for measuring the ‘sO/‘6O ratio. In practice the ratio variations are reported as differences from an arbitrary standard. These are calculated from the expression: a=[*-l]xlOoOpermille where R = ‘*O/‘6O. The standard used here is V-SMOW (Vienna Standard Mean Ocean Water) issued by the I.A.E.A. which is a version of the SMOW defined by Craig (1961). Of the published techniques only one (Mullane et al., 1988) utilizes a stepwise chemical degradation procedure that may be modified as required for the study of oxygen atom ratios at specific positions in the cellulose molecule. The other methods rely on non-specific thermal degradation to generate carbon dioxide having the same isotopic composition as the parent material (Rittenberg and Ponticorvo, 1956; Hardcastle and Friedman, 1974; Epstein et al., 1977; Thompson and Gray, 1977; Brenninkmeijer and Mook, 1981; Brenninkmeijer, 1983; Dunbar and Wilson, 1983; Ramesh, 1984). These workers use equipment of widely different complexity and various catalysts including mercuric chloride, diamond and 177
E. M. FIELD et al.
178
5,6benzoquinohne. These substances have to be highly purified, typically by multiple distillation shortly before use. Experiments during our work demonstrated that quinoline retained substantial quantities of gases and that it was necessary to degas the sample in uacuo before use. Similarly, after the reaction and the removal of hydrogen chloride, the quinoline retained some of the carbon dioxide. It was found that the quinohne had to be subjected to at least three freeze-thaw cycles in uacuo to obtain a quantitative yield of the carbon dioxide. Quinoline is toxic and unpleasant to work with and since a large number of isotope determinations are required in any palaeoclimatic investigation, an alternative substance was sought that was readily available and easy to use. Its properties needed to be sufficiently specific so as to absorb the hydrogen chloride, but without absorbing the carbon dioxide, and hence perform the necessary separation. (i) Absorbents for hydrogen chloride We first investigated weakly basic anionic resins as reagents, potentially having suitable properties for this requirement. Most resins of this type, however, are supplied in a wet form and the water thus contained would render the sensitive oxygen isotopic results meaningless unless thorough desiccation were possible before use. If the resins remained active after this thorough drying they could be suitable for the reaction. Generally, two types of resin are available,
Fig.
known as gel and macroreticular. With the former resins we found that, on desiccation, the pores collapsed and no hydrogen chloride was absorbed. The latter type, however, retain their pore structure on desiccation and we found that hydrogen chloride could be absorbed efficiently. The initial experiments were performed using the macroreticular resin Amberlyst IRA -93 (Sigma Chemicals). As supplied this contains 61% water. It was dried in a vacuum oven at 60°C for ca 40 h, after which successive weighings became constant, thus indicating that the water had been removed. This was used in place of quinoline in the reaction chamber (Fig. 1). It was pumped to high vacuum for 30min before use to ensure complete removal of the water. After the reaction the measured yield of carbon dioxide was only 80% of theoretical but a mass spectrometer scan between mass numbers 25560 indicated, as also in the case of quinoline, that all hydrogen chloride had been removed. Gentle warming of the resin had the effect of raising the yield to 95-100%. Further investigation, however, revealed that this resin had significant shortcoming in that some of the carbon dioxide that was collected originated, not from the sample, but from the resin itself. The amine groups present in Amberlyst IRA-93 are mostly tertiary but some primary and secondary amines are also present and these could absorb carbon dioxide from the atmosphere with the production of carbamates. These carbamates would not
Apparatus for determination of stable oxygen isotope ratios.
Oxygen isotopes in wood cellulose
release carbon dioxide during the drying procedure but would do so on acidification with hydrochloric acid. In order to check this, a sample of hydrogen chloride was admitted to the reaction chamber containing only dried IRA-93. Carbon dioxide was indeed released, ca 3% of that expected from a cellulose sample. It may be assumed that a resin containing solely tertiary amines would be without these shortcomings. Accordingly Amberlyst A-21 (BDH), which is such a resin, was tested in a similar manner. It was found that no carbon dioxide was generated from this material, which behaved otherwise in a similar manner to the Amberlyst IRA-93. It was decided to use Amberlyst A-21 in all further work. (ii) Borosilicate
glass us quartz
One of the original aims was to determine whether the pyrolysis could be carried out in vessels of Pyrex glass at 450°C for 4 h instead of quartz at a higher temperature, owing to the greater ease of use of the former. Experiments demonstrated that values of the oxygen isotope ratio obtained at this temperature gave a reproducibility of only 0.94.5 per mille. Replacement of the Pyrex vessels by quartz vessels and raising the pyrolysis temperature to 550°C for 6 h immediately improved the reproducibility to 0.2 per mille. It was found that a similar reproducibility of 0.2 per mille could also be obtained using Pyrex vessels by increasing the reaction time to 18 h at 450°C however, the average of the values from these was consistently 1.6 per mille lower than that from the reactions in quartz. In order to investigate which of these values gave the correct result, quartz and Pyrex vessels were filled with standard grade carbon dioxide of known delta IgO value. The quartz tubes were heated in the furnace to 550°C for 6 h, whilst the borosilicate tubes were heated at 450°C for 18 h. Our findings were that for the quartz vessels there was no change in the isotopic ratios, whereas for the Pyrex vessels, the value was lowered consistently by 1.6 per mille. Obviously, some interaction occurs between Pyrex and carbon dioxide even at this lower temperature which indicates that Pyrex is not suitable for use in this research. Observations of a reaction of carbon dioxide with borosilicate glass were made by Epstein et al. (1977). (iii) Comparison of delta I80 values of standard cellulose with another technique A sample of a standard commercial cellulose, cellulose CF-11 (Whatman) as used in his tests was kindly supplied by C.A.M. Brenninkmeijer for comparison of the two techniques. The measurements were made with respect to the international standard, V-SMOW supplied by the I.A.E.A. The mean of 7 determinations of delta IgO by the Cambridge technique, as described in this paper, was 26.67 f 0.20 per mille with respect to V-SMOW and this may be
179
compared with a mean for 17 determinations of 26.4 f 0.1 per mille reported by Brenninkmeijer using the sealed nickel tube technique (Brenninkmeijer, 1983). The agreement between the techniques is satisfactory. The consequence of this work is the development of an improved and very effective technique for the analysis of oxygen isotopes in wood cellulose having excellent reproducibility for the complete process through alpha-cellulose extraction, pyrolysis, disproportionation, purification and mass spectrometric analysis. The techniques used from the pyrolysis stage are given in detail below; the alpha-cellulose preparation is described in Mullane et al. (1988).
Apparatus and Methods It is important that all materials and samples used in oxygen isotope analyses must be thoroughly dried to a constant weight in a vacuum oven before attempting the determinations. Dried reagents and samples may be maintained in that state by storing in tightly stoppered vials or jars in a vacuum desiccator. Desiccators using sulphuric acid, phosphorus pentoxide and similar materials alone as drying agents require a considerable time to reach equilibrium and are generally unsuitable for this work. The pyrolysis tubes (9 mm o.d. by 125 mm length) are fabricated from high quality quartz tubing. Before use each tube is suitably inscribed with a diamond pen, dried at 550°C for 1 h and stored in a vacuum desiccator. Approximately 34mg of dried alpha-cellulose is accurately weighed into the pyrolysis tube and mixed with 130 mg mercuric chloride. A breakseal is formed ca 1.5 cm from the end of the tube which is attached via a Cajon ultratorr “0”-ring vacuum fitting to a high vacuum line and pumped overnight to ensure that all moisture is removed. The tube is then flame sealed under vacuum. The pyrolysis is performed by heating in an electrically heated tube furnace for 6 h at 550°C. It is convenient to process 8-10 samples in one batch. “Amberlyst A-21” resin (BDH) and mercuric chloride (BDH) are prepared by heating in a vacuum oven at 50°C for 48 h and stored in sealed bottles in a vacuum desiccator. The procedure, as detailed in the following, is carried out using the apparatus shown in Fig. 1. In this figure the vacuum valves (Tl-T8) are all 9 mm. “O”-ring type (Lowers Hapert). Vessel A is the disproportionation discharge tube (22 cm x 5 cm dia) fitted with platinum mesh electrodes, Pt (Johnson Matthey) (2.5 x 2.5 cm). These are cleaned from deposited carbon after the reaction by heating to incandescence in air. The high voltage applied to these electrodes is produced by a transformer capable of attaining 10,000 V. The output voltage is varied using a manually variable transformer (Variac) connected to the EHT input. The vessel is demountable via an “0”-ring joint “0”. The reaction vessel B
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FIELD et al.
(22 cm x 2 cm dia) having a demountable base (B-25 “0”-ring joint) contains the breaking device, K, consisting of a vacuum “0”-ring valve (Youngs) sealed into the side of the tube. Rotation of the valve head drives the glass barrel against the pyrolysis sample tube, S, causing it to shatter. During evacuation of vessel B, the pyrolysis tube is held gently against the side of vessel by the barrel of the cracker at a point above the removable base of the vessel in which the Amberlyst A-21 resin is weighed. This vessel is connected to the vacuum line via a 9 mm dia “0”-ring vacuum valve through a 3/8” dia ultratorr connector. Reaction vessel C (20 cm x 2.5 cm dia) is also demountable through an “0”-ring joint for cleaning. E is a small volume cylindrical finger connected to a mercury manometer. The removable receiver, D (10 cm x 0.9 cm dia) is fabricated from an “0”-ring vacuum valve (Lowers Hapert) modified so as to connect to the vacuum system via a stainless steel l/4” dia ultratorr connector. The system is evacuated via rotary and oil diffusion pumps and the vacuum measured with a high sensitivity (to 10-4Torr) Pirani gauge (Edwards High Vacuum).
Techniques (A) “Amberlyst A-21” resin (0.25 g), previously dried to constant weight, is placed in the breaker tube B together with a scored pyrolysis sample tube, S. All the taps, 1 to 9, are opened fully and the complete apparatus is pumped for 45 min to high vacuum with a Dewar vessel containing freshly boiled water placed around B. After this time the Dewar vessel is removed. (B) Vacuum valves T4, T5 and T6 are closed and a Dewar containing liquid nitrogen placed around vessel B. The pyrolysis tube is broken by rotating the valve head K, and forcing the barrel against it. The pyrolysis products, carbon dioxide and hydrogen chloride, are allowed to freeze on to the resin for about 1 min, whilst the carbon monoxide remains largely in the gaseous phase. (C) Valves T3 and Tl are closed to isolate the system and the finger on the discharge tube, A, is cooled by a Dewar of liquid nitrogen. Valve T4 is opened to enable the pressure to be recorded on the Pirani gauge. Valve T3 is opened and the pressure allowed to stabilize as carbon monoxide enters A. (D) High voltage is applied to the platinum electrodes, Pt, of the discharge tube, A, until the light eggshell-blue discharge strikes. The value of the high voltage required depends on the gas pressure and the separation of the electrodes, but is typically 2000 V. The carbon monoxide is disproportionated to carbon dioxide. The discharge soon quenches but is renewed by gradually increasing the high voltage via the variable transformer. Eventually when all the carbon monoxide has been converted to dioxide and the
discharge can not be maintained, the high voltage is reduced to zero. The carbon dioxide produced by the reaction condenses in the cooled finger of A. (E) Valve T4 is closed and the liquid nitrogen trap removed from vessel B. The hot water Dewar is returned to B for 1 min after which the resin is left to absorb the hydrochloric acid gas for IO min. (F) Any residual carbon monoxide remaining adsorbed in the frozen carbon dioxide in the discharge tube is released by removing the liquid nitrogen Dewar from the discharge tube finger A, thawing the solid carbon dioxide with hot water to release carbon monoxide and refreezing with liquid nitrogen. The high voltage is once more gradually applied to the electrodes, Pt, by increasing the t:ariac voltage. until the maximum is reached and the residual carbon monoxide is disproportionated. This stage is repeated to extract the final traces of carbon monoxide and convert to the dioxide. (G) Valve T3 is closed and when sufficient time has elapsed enabling the resin to absorb the hydrochloric acid valve T4 is opened and the pressure recorded. Valve T3 is again opened and the carbon dioxide frozen into the discharge tube A. When the pressure becomes stable once more the glow discharge is carefully restruck in vessel A. The procedure of section (F) is repeated. (H) The residual pressure in absorption vessel B is recorded after closing valve T3 and opening valve T4. The remaining gas is then transferred to the discharge tube, A, by opening valve T3 and freezing the carbon dioxide in the cold finger with liquid nitrogen. The high voltage is once more carefully applied to the electrodes, Pt, until the discharge ceases. when it is removed. (I) After 10 min valve T3 is closed for 2 min and re-opened. The pressure is noted; there should be no significant change in pressure indicating that all the gas has been processed. (J) Valve T4 is closed next and, with a liquid nitrogen trap surrounding A, valve Tl is opened and the line pumped to high vacuum. This processing is tedious; hoaerw. our ,findings are that these steps are essential (f the reaction is to proceed to completion and a quantitative yield obtained. The total yield of carbon dioxide, held frozen in the finger of vessel A is next transferred to the receiver, D for measurement by mass spectrometry. This is accomplished by the following sequence. (K) Valve T2 is closed and it is ensured that valves T5, T6, T7, T8 and T9 are open and this system pumped to high vacuum after which valves T6 and Tl are closed and valve T2 is opened. (L) The liquid nitrogen trap is removed from around the carbon dioxide in discharge vessel A and replaced with the hot water Dewar. The liquid nitrogen trap is replaced around vessel C and the carbon dioxide allowed to sublime into it. The return of the Pirani gauge reading to high vacuum is the signal that the transfer is complete. The line is taken to high
Oxygen
isotopes
vacuum by opening valve Tl and valve T5 is then closed to isolate the carbon dioxide. Valve T6 is opened and the line is once more pumped to high vacuum. (M) The liquid nitrogen trap is removed from around vessel C and the carbon dioxide vaporized with the hot water trap. A slurry of ca 50% each of liquid nitrogen and methylated spirit is prepared in a Dewar vessel and used to re-freeze vessel C. This mixture is sufficiently cold to freeze out and retain hydrocarbon impurities but allows the carbon dioxide to remain in the gas phase. Valves T7, T3 and T6 are closed, valve T5 opened and the pressure in vessel C is recorded. (N) A Dewar of liquid nitrogen is placed around finger E attached to the manometer. When the Pirani gauge reading is steady valve T6 is opened and the carbon dioxide sublimed into finger E. (0) Any residual carbon dioxide in trap C is scavenged by the process of thawing and re-freezing, as follows. Valve T6 is closed and trap C thawed with hot water to release carbon dioxide. It is then recooled with the liquid nitrogen/methylated spirit slurry. Valve T6 is reopened to allow transfer of this carbon dioxide to E. Valve T5 is closed to isolate the trap and valve T3 opened. The system is once more pumped to high vacuum. (P) The yield of carbon dioxide is next obtained using a manometer previously calibrated with known quantities of carbon dioxide. Valve T8 is closed and valve T7 opened. The carbon dioxide is vaporized by warming trap E with warm water and after leaving sufficient time to gain room temperature the manometer pressure is recorded. From the temperature, pressure and volume of carbon dioxide, the yield is calculated. (Q) The carbon dioxide is next collected for the mass spectrometric assay. A Dewar of liquid nitrogen is placed around the receiver, D. With valves T8, T7 and T9 open the gas transferred to the receiver. The process is monitored by the pirani gauge readings. When complete the valve T3 is opened and the system pumped to high vacuum. The valve, T9, of the receiver D, is closed to isolate the sample. (R) Valve T7 is closed and the receiver removed from the ultratorr connection, U, and transferred to the mass spectrometer for stable isotope measurement.
in wood cellulose
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We now use this technique regularly in a project concerning measurements of the stable isotope ratios of hydrogen, carbon and oxygen in alpha-cellulose obtained from individual oak growth rings. The efficiency of the procedure could be greatly improved by automating the valve and pumping systems and controlling the operations from a microcomputer. This would greatly simplify the numerous sequences of valve manipulations which depend on parameters of time, temperature and pressure which could be determined by appropriate sensors. The sample throughput would be increased significantly and the possibility of operator error would be eliminated. Acknowledgemeno-We acknowledge gratefully the help and advice of our colleagues, Mike Hall, Nick Shackleton, Henry Schwartz and others in this work. We thank Tony Carter for his kindness in drawing the diagram. We thank the NERC for a Special Project grant to VRS and JSW for the funding of this work and for the larger project of isotopes in the environment.
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