Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements

ISBN-13: 978-1-4665-9407-4

9 781466 594074

9 0 0 0 0

K20665

C H E M I S T R Y

Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements explores different methods of isotope analysis, including spark, secondary ion, laser, glow discharge, and isotope ratio mass spectrometry. It explains how to evaluate the isotopic composition of light elements (H, C, N, O) in solid, liquid, and gaseous samples of organic and inorganic substances, as well as:

• Presents a universal, economical, simple, and rapid technique for sample preparation of organic substances to measure the isotopic composition of carbon• Describes how to determine microbial mineralization of organic matter in soil and the effect of exogenous substrates on environmental sustainability• Examines use of the isotopic composition of n-alkanes from continental vegetation to study the paleoclimate and plant physiology• Proposes a systematic approach to identifying tobacco areas of origin and tobacco products based on data from the isotopic composition of light elements• Discusses ways to detect doping drugs and suggests results assessment criteria based on determining reference intervals for endogenous markers• Reviews methods of release of gases from inclusions of rocks and minerals for further implementation of isotope mass spectrometric analysis • Considers use of optical isotope analyzers for determining the isotopic composition of carbon in CO2 and of hydrogen and oxygen in water

Providing a complete picture of the latest advancements in the field, Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements aids readers from a variety of disciplines in identifying the fundamental processes in biological, ecological, and geological systems and in revealing the subtle features of many physicochemical processes and chemical transformations.

Isotope Ratio Mass Spectrometry of Light Gas-Forming ElementsEdited by V. S. Sevastyanov

Isotope Ratio Mass Spectrom

etry of Light Gas-Forming Elem

entsSevastyanov

K20665_Cover.indd All Pages 6/11/14 3:11 PM

Edited by V. S. Sevastyanov

Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements

CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742

© 2015 by CISPCRC Press is an imprint of Taylor & Francis Group, an Informa business

No claim to original U.S. Government worksVersion Date: 20140611

International Standard Book Number-13: 978-1-4665-9408-1 (eBook - PDF)

This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint.

Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information stor-age or retrieval system, without written permission from the publishers.

For permission to photocopy or use material electronically from this work, please access www.copy-right.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that pro-vides licenses and registration for a variety of users. For organizations that have been granted a pho-tocopy license by the CCC, a separate system of payment has been arranged.

Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.com

and the CRC Press Web site athttp://www.crcpress.com

Contents i

Contents

Preface v1. Isotope ratio mass spectrometry: Devices, methods,

applications 1V.S. Sevastyanov

General characteristics of mass spectrometric methods for the determination of isotopic composition of light elements 1

Improvement of equipment for the determination isotope ratios of light elements 14

Mass spectrometric methods for determining the isotopic composition of light elements 24Metrological characteristics of mass spectrometry

of isotope ratios 36The effect of the strength of the analytical signal on the results of measuring the isotopic composition of light elements 36Study of the completeness of combustion of difficult to oxidise compounds 53Developing a new method of sample preparation based on solid electrolytes for isotope mass spectrometric analysis The electrochemical decomposition of water using a solid electrolyte based on zirconium dioxide to determine the isotopic composition of hydrogen 73The distribution of the isotopes of light elements in various objects 77Finding the source of drugs and explosives 77The effects of isotope fractionation, accompanying organic

synthesis 91Isotope effects in carbonaceous chondrites 96Determination of the isotopic composition of hydrogen and oxygen of water, isotope effects during evaporation 99The distribution of carbon isotopes in complex organic compounds of biological origin (oil, hydrocarbon gases) 103Determination of the isotopic composition of carbon in the collagen of bones of ancient tombs 108

ii

Conclusions 113References 113

2. Universal method for preparation of liquid, solid and gaseous samples for determining the isotopic composition of carbon 119T.A. Velivetskaya, A. Ignat'ev, S. Kiyashko

Introduction 119The experimental part 122Equipment and materials 122Combustion of solid and low-volatility liquid samples to determine the isotopic composition of carbon 124Combustion of volatile liquids to determine carbon isotopic composition 126Combustion of methane for the determination of the isotopic composition of carbon 127Results and discussion 128Conclusions 133Literature 134

3. Using isotope ratio mass spectrometry for assessing the metabolic potential of soil microbiota 135A.M. Zyakun and O. Dilly

Introduction 135Methods of analysis of microbial activity in soil 138Microbiological method 138Isotope ratio mass spectrometry in the study of substrate- induced respiration (SIR) 139Kinetics of CO2 production during substrate-induced respiration 140Characteristics of carbon isotopic composition of microbial products 141Amount of metabolic carbon dioxide and characterization of its origin in the soil 143Using the 13C/12C ratios to characterize the activity of the microbiota in arable soils 143Analyzed soil samples 145Mineralization of SOM and exogenous glucose 146Priming effect (PE) of glucose 154

Contents iii

Estimate of the duration of the effect of the exogenous substrate on the microbiota 158Conclusion 159References 160

4. Study of the isotopic composition of normal alkanes of continental plants 163N.A. Pedentchouk

Introduction 163The experimental part 166General provisions 166Methodological features of analysis 170Results and discussion 174Conclusion 180Literature 180

5. Using isotope ratio mass spectroscopy for analysis of tobacco 183A.B. Uryupin

Literature 187

6. Using isotope mass ratio spectrometry of carbon in doping control 188T. Sobolevski, I.S. Prasolov and G.M. Rodchenkov 188

Introduction 188The metabolism of steroid hormones 189The experimental part 192Equipment 192Reagents and materials 193Sample preparation 194Results and discussion 196Literature 204

7. Isolation methods in isotope geochemistry of noble gases 205A.I. Buikin

iv

Introduction 205Stepwise annealing 206Release of gases by heating with a laser beam 209The stratified oxidation method 210Stepwise fragmentation 211Conclusion 212Literature 212

8. Using laser spectroscopy for measuring the ratios of stable isotopes 213V.S. Sevastyanov

Introduction 213Absorption spectroscopy 215Infrared spectroscopy with Fourier transform 216Non-dispersive absorption spectroscopy 217Laser cavity ring down spectroscopy 217Laser on-axis integrated cavity output spectroscopy 223New methods and results 225Conclusions 228Literature 228

Index 231

Contents v

Preface

The book presents 8 reviews of the latest advances in isotope ratio mass spectrometry. The use of this important analytical method in various fields of science is described in detail: geology, geochemistry, biology, microbiology, archeology, criminology. In particular, the book deals with the isotopic ratios of stable light elements (H, C, N, O, S). DThe book discusses in detail the advantages and disadvantages of different methods of mass spectrometry in isotope analysis, including spark mass spectrometry (SMS), secondary ion mass spectrometry (SIMS), laser mass spectrometry (LMS), glow discharge mass spectrometry (GDMS), mass spectrometry of stable isotope (IRMS). Especially important is the development of local isotope mass spectrometric analysis using lasers (including excimer) and ion guns.

Already in 1926 V.I. Vernadsky pointed to isotopy as the requiring special attention of scientists. The development of analytical methods for the determination of stable isotopes started after the discovery of deuterium in 1934 by H. Urey, for which he received the Nobel Prize. After A. Nir constructed the mass spectrometer in the 40s of the last century isotope mass spectrometry started to be used extensively in research. However, at that time sample preparation took a lot of time, it was necessary needed to use relatively large charges of test samples – 10–100 µg, and the developed methods had relatively poor reproducibility and accuracy of the analysis results. Subsequently, there have been numerous attempts to simplify the methods of analysis and improve the accuracy of isotopic analysis. Currently the accuracy of analysis is ensured using international standard samples. Modern devices allow to analyze very small samples (10–100 ng) of organic and inorganic substances in the constant flow (CF) mode. Furthermore, using the online mode it has been possible to perform a combined analysis of isotopes in a single sample. For this purpose, the magnetic field of the mass analyzer is rapidly changed during the transition from registration of one m/z ratio to other. This is accompanied by the measurement of the peaks of the sample and from the standard, measurement of the background and computer processing of the results.

vi

It should be noted that in recent years Russian scientists published many papers devoted to the practical application of isotope ration mass spectrometry and the creation of new systems of sample preparation for isotopic analysis. Great contribution to the development of theoretical and experimental foundations of the isotopic analysis of light elements in Russi was made by Academician E.M. Galimov .

The number of Modern isotope mass ratio spectrometers in Russia is very small. It is therefore important not only to demonstrate achievements of a group of authors in the field of isotope mass ratio spectrometry but also systematize the latest achievements in this field and assess the prospects for their implementation in analytical practice for wider use of isotope mass ratio spectrometry. The book describes in detail new sample preparation systems and mass spectrometric methods for the determination of the isotopic composition of light elements in complex organic and inorganic compounds. The studies have important practical application. Described in detail are the most important results obtained in the isotopic analysis of objects of the environment, in biological, geochemical and archaeological research in criminology and doping control. All the discussed issues in the area of isotope ratio mass spectrometry can be used to create a complete picture of the developments in this area.

The book includes an introduction and seven chapters. The first chapter (V.S. Sevast’yanov ‘Isotope ratio mass spectrometry’) generalizes and systematize the recently proposed approaches and measures for the determination of the isotopic composition of light elements (H, C, N, O) in solid, liquid and gaseous samples of organic and inorganic substances. The designs of modern isotope mass spectrometers and sample preparation systems is described. Different mass spectrometric approaches for determining the isotopic composition of light elements are outlined. The new approaches and methodologies developed by the author for conducting isotopic analysis using sample preparation systems based on solid electrolytes are presented. Examples of the use of isotope ratio mass spectrometry for the study of isotope fractionation of isotopes in organic synthesis, for study of the isotopic composition of nanodiamonds in meteorites and in the collagen of bones of ancient tombs, for the determination of the isotopic composition of light elements in the oil and natural gas, for identifying sources of spirits, other drugs and explosives are given.

In the second chapter (T.A. Velivetskaya, A.V. Ignat'ev, S.I. Kiyashko ‘A universal method of preparation of liquid, solid and gaseous samples to determine the isotopic composition of carbon’)

Introduction

Contents vii

the authors propose a universal, economical, simple and rapid method of sample preparation of organic substances to measure the isotopic composition of carbon. The advantages of the method are discussed and its analytical characteristics are evaluated. The method can be used for isotope analysis of solid, liquid and gaseous substances. It is a good alternative of the classical method of combustion of samples in quartz ampoules .

The third chapter (A.M. Zyakun, O. Dilly ‘Using isotope ratio mass spectrometry for assessing the metabolic capacity of soil microbiota’) addresses the use of isotope mass ratio spectrometry for determining microbial mineralization of soil organic matter and organic products falling into the soil and the effect of exogenous substrates on the environmental sustainability of the soil and the environment. It is shown that the carbon isotope composition of metabolic carbon dioxide is the most sensitive parameter reflecting the strength and duration of the impact of the substrate on the geochemical and biological processes in the soil.

The fourth chapter (N.A. Pedentchouk ‘Study of the isotopic composition of normal alkanes of continental vegetation’) examines the new approach to the use of the isotopic composition of n-alkanes from the leaves of the continental vegetation in order to study the paleoclimate and plant physiology. The proposed method provides a unique information not available when using other methods. Discussed are the latest results of the author and his colleagues in the U.S. and in some countries of Western Europe in order to determine the effect of climatic conditions and plant physiology on the isotopic composition of hydrogen and organic carbon compounds from the leaves of current flowering and conifers.

In the fifth chapter (A.B. Uryupin ‘Using isotope ratio mass spectrometry for the analysis of tobacco’) the author proposes a systematic approach to the identification of areas of origin of tobacco and tobacco products on the basis of data on the isotopic composition of light elements. He discusses studies of the isotopic composition of not only nicotine but also of other classes of compounds belonging to the tobacco leaf (hydroxy acids, sugars, cellulose, optically active substances).

In the sixth chapter (T.G. Sobolevskii, I.S. Prassolov, G.M. Rodchenkov ‘Application of isotope ratio mass spectrometry of carbon in doping control’) the authors discuss methods for the detection of doping drugs which are close substitutes for hormones produced in the human body, or their predecessors (testosterone, dehydroepiandrosterone, androstenedione, androstendiol, etc.), as

Introduction

viii

well as proposing new criteria for intralaboratory evaluation of the results of analysis based on the determination of reference intervals for endogenous markers.

The seventh chapter (A.I. Buikin ‘Isolation methods in isotope geochemistry of noble gases’) is devoted to reviewing the methods of release of gases from inclusions of rocks and minerals for further implementation of isotope mass spectrometric analysis . The advantages and disadvantages of various methods are outlined and recommendations for their use are given.

The eighth chapter (V.S. Sevastyanov ‘Using laser spectroscopy for measuring the ratios of stable isotopes) examines the prospects for the use of optical isotope analyzers for determining the isotopic composition of the hydrogen and oxygen of water and isotopic composition of carbon of CO2. Although isotope mass ratio spectrometry (IRMS) is currently a well developed isotope method for determining the isotopic composition of greenhouse gases in the atmosphere, preference is given to the application of absorption infrared spectroscopy which has recently made great strides. Optical isotope analyzers are significantly more compact, less expensive and easier to manage than the isotope ratio mass spectrometers. In the case of infrared spectroscopy of isotopic ratios the large linear range and high signal to noise ratio are more important than the sensitivity. Thanks to highly accurate control of temperature and pressure in the optical cuvette and also strict control of the wavelength of the tunable laser it has been to significantly reduce the drift of the system. This allows to conduct calibration of the optical analyzer only once for a few days .

V.S. Sevastyanov

Introduction

1Isotope ratio mass spectrometry

1

Isotope ratio mass spectrometry:Devices, methods, applications

V.S. Sevastyanov

V.I. Vernadsky Institute of Geochemistry and Analytical Chemistry.Russian Academy of Sciences, Moscow

General characteristics of mass spectrometric methods for the determination of isotopic composition of light elements

Laser ionization mass spectrometry (LIMS) The most common devices when working with laser ion sources are double-focusing mass spectrometers, built on the basis of the Mattauch–Herzog ion-optical scheme. The main advantage of this system is the simultaneous recording of ions in a wide mass range. Ions are detected by a photoplate, which limits the accuracy of isotope measurements. The relevant accuracy is typically a few percent. In [1] the results are presented of determination in a magnetic mass spectrometer with a laser ion source of isotopic ratios 18O/17O and 18O/16O in samples of UO2 in layer analysis with an accuracy of 10%.

The scope of laser mass spectrometry (LIMS) with double focusing is about the same as of spark mass spectrometry (SMS) – multi-element analysis with high accuracy. An additional advantage of the laser method is the locality (up to 1 µm) that allows analysis of individual inclusions in geological samples without special sample preparation. The precision of the isotopic ratios obtained by the LIMS is much better than in the determination of the concentration of elements by the same method. This is due to the fact that the discriminatory effects of various kinds during the ionization process are much smaller for isotopes than for elements with different physical and chemical properties. Therefore, the main factors influencing the accuracy of

Isotope ratio mass spectrometry of light gas-forming elements2

isotopic analysis by LIMS are errors resulting in the detection and recording of the ions. Laser mass spectrometry is significantly inferior in the accuracy of determination of the isotope ratio to other mass spectrometric methods of isotope analysis of solids, such as mass spectrometry of secondary ions (SIMS) [2].

Spark mass spectrometry (SMS). Analytical capabilities of spark mass spectrometry are considered in [3, 4]. In contrast to the elemental analysis the isotopic analysis by spark mass spectrometry has not found wide application [3]. This is due primarily to the fact that this method has a fairly large relative error (more than 3–4%) of measurements of ion currents because of the photographic recording of the ions used in SMS. Isotopic analysis is expedient to carry out only when the isotopic relationship of determined elements is substantially (by more than 10%) different from the natural, and information is required simultaneously for several elements.

The use of the electrical recording mode (switching of peaks or position-sensitive detectors based on microchannel plates) significantly improves the accuracy of the measurement of currents but virtually eliminates the possibility of simultaneous recording of all the isotopes which is important under the pulsed nature of the spark discharge. Moreover, in the absence of opportunities for simultaneous recording of ions the use of SMS loses its meaning.

Glow discharge mass spectrometry (GDMS). We know of only a few papers devoted to the definition of isotope ratios using single-collector GDMS. Almost all studies refer to the determination of isotope ratios of elements Li, Tl, Pb, U. In [5] the authors studied the isotopic composition of oxygen in uranium oxide samples. The reproducibility of the isotopic composition was quite low, 0.5–4%.

In many cases, the elemental and isotopic analyses use the well designed and more reliable analytical methods such as SIMS or LA-ICP-MS.

Secondary ions mass spectrometry (SIMS). Secondary ions mass spectrometry allows high-precision isotope analysis with a resolution of 10–30 µm of both stable isotopes (H, C, N, O, S) and rock-forming elements (Li, B, Mg, Si). Electropositive elements (metals) are analyzed as positive ions by using a primary ion beam O–. At the same time, electronegative elements (non-metals) are determined in the form of negative ions by using a primary ion beam Cs+.

In [6] it is shown that the yield of negative ions for H, C, O exceeds the yield of positive ions 10–1000 times (nitrogen, on the contrary, has an abnormally low yield). The isotope ratios of elements in the beam of secondary ions are enriched by light isotopes relative to the


isotopic composition of elements of the target [7]. The nature of this enrichment has not yet been studied.

It is known that the sample matrix, its structure and the angular distribution of sputtered ions with different masses affect the isotopic fractionation. Therefore, measurement of the isotopic composition of light elements is performed relative to a standard sample of the same composition with the known abundance of isotopes. In carrying out isotopic analysis the instrumental parameters must be constant. In [8] extensive investigations were conducted of the influence of the matrix on the oxygen isotopic composition of 40 samples of silicate, phosphate minerals and glasses. A correlation was found with the average atomic number of the target, the cationic composition in olivines and glasses, and the structure of the minerals. Cracks and imperfections in the structure and foreign inclusions also affect the isotopic fractionation.

The instability of the primary beam directly affects the stability of the secondary beam, which can lead to isotopic fractionation. This process is also a function of the energy of secondary ions and depends on the width and position of the energy gap [9]. The change by 10 eV of the energy of secondary ions in the low-energy field causes a change in the fractionation by 7‰, while the shift in the high-energy region has almost no effect on fractionation.

Thus, the conditions of low mass resolution and transmission of high-energy secondary ions are the best for the stability of the instrumental isotope fractionation. The next source of instability is the charge compensation on the sample surface by etching with a beam of ions Cs+. For this reason the compensating electron beam must be well stabilized.

Secondary ions mass spectrometers Cameca ims-nf (n = 3, 4, 5, 6) allow using a diaphragm to separate secondary ions from the central part of the crater, for example, to exclude the ingress of OH– ions, formed on the edge of the spot as a result of migration of water on the sample surface. Mass spectrometer Cameca ims 1270 has higher sensitivity and higher mass resolution. Using a multicollector system allows high-precision isotopic analysis of oxygen and sulphur. A recently developed Cameca nanoSIMS instrument with double focusing with the Mattauch–Herzog geometry allows simultaneous detection of a wide range of masses. This device uses a narrow primary ion beam with the size of 50 nm and has improved efficiency of the extraction of secondary ions. Microprobe SHRIMP is very convenient for isotopic analysis. It has an improved extraction system in comparison with Cameca devices, but it does not perform energy filtering. Isolab 54 is a microprobe with the analytical characteristics similar to SHRIMP [2].


The main difficulty in measuring the isotopic composition of oxygen is the imposition of the 16OH molecular ion on the 17O peak. In general, oxygen is defined as the negative ion in bombardment with Cs+. At the same time accumulation of the charge on the surface of the sample surface is compensated by the charge of the electrons from the electron gun. The results of isotopic analysis of oxygen with the Cameca ims 1270 instrument are in good agreement in the ppm range with the results of the fluorination method. The correction to the matrix effect is also less than 1‰ [10]. Recently [11] the multicollector Cameca ims 1270 instrument was used for the isotopic analysis of the oxygen of zircon with the age of 4.3 billion years from Australia. The accuracy of the measurement of isotope ratios of oxygen was 0.1–0.2‰. In this case δ18O varied from 5.4 to 7.7‰ for different grains.

The isotopic composition of carbon is determined by measuring the secondary negative carbon ions in bombardment of the sample with primary ions Cs+. At the same time, the mass resolution required to separate 12CH– and 13C– ions is 3500. Nitrogen is a difficult element for SIMS analysis because it does not form negative ions, However, in the presence of carbon a very intense beam of CN– ions forms. This makes it convenient to measure the isotopic composition of carbon and nitrogen in biological objects [12]. The correctness of measurement of δ13C was 1‰. In [13–15] the authors studied the distribution of carbon and nitrogen isotopes in diamonds. When using the primary Cs+ ion beam in the microprobe Cameca 6f [14] the reproducibility of determination of δ13C was 0.3‰, and for δ15N 1‰. The locality of the method was 25 µm. This allowed the authors to study the distribution of the isotopic composition of carbon and nitrogen by volume in the single diamond from the kimberlite pipe Mir [15].

Thus, the SIMS is the best method for local measurement of isotope ratios of elements in heterogeneous samples.

Isotope ratio mass spectrometry (IRMS). Isotope mass spectrometry of light elements is an important method of analytical chemistry, having an extremely high sensitivity and accuracy of measurement of isotope ratios. It is widely used in many fields of science: chemistry, ecology, physics, biology, medicine, pharmacology, geology, archeology, forensics, helping to identify the nutritional components of food and finding of fraud [16].

The isotopic composition of light elements can be changed as a result of isotope fractionation during natural chemical and physical processes. These changes are proportional to the mass difference between isotopes. For example, evaporation of water leads to its enrichment with the isotopes 18O and 2H. The isotopic composition


of oxygen and hydrogen in the snow, deposited in the polar regions and in the mountains at high altitude above the sea level is mainly determined by temperature. Therefore, a systematic study of isotopic data variations of hydrogen and oxygen can be used for the study of glaciers, snow accumulation rates and climatic changes over the past 100 000 years [17]. Another interesting area of studies of isotopic variation is the method of calculation of paleotemperatures of ancient oceans by measuring the isotopic composition of oxygen in calcium carbonate and water, as proposed in [18]. In deposition under the equilibrium conditions the isotopic composition of oxygen in the calcium carbonate is different from the isotopic composition of oxygen in the water. The difference is due to the isotope exchange reaction between calcium carbonate and water the course of which is affected by the water temperature. Thus, by measuring the oxygen isotopic variations in the calcium carbonate, one can estimate the temperature of water at the time of its formation [17].

Carbon is one of the most abundant elements in the universe. It is located in the crust and mantle of the Earth, the atmosphere and in the hydrosphere. In organic compounds and in coal carbon is present in the reduced form, and in the oxidized state it is found mainly in the form of carbon dioxide, carbonate ions in aqueous solutions and in the form of carbonate minerals. Isotope fractionation occurs in various natural processes, including photosynthesis and isotope exchange reactions between compounds of carbon. Photosynthesis leads to the enrichment of synthesized organic compounds with the isotope 12C. On the other hand, the isotopic exchange reaction between gaseous CO2 and the carbonates dissolved in water leads to the enrichment of the carbonates with 13C. As a result, the prevalence of the 13C isotope in carbon on Earth’s surface varies within 100‰ [19].

The IRMS method allows to obtain quantitative information on the metabolism in the biological and ecological systems not available for other analytical methods. The isotopic composition of exhaled gases can serve as an indicator of processes occurring in the body, if isotopic labels have previously been introduced into the body. 13C/12C measurements in exhaled CO2 serve as a means of direct determination of metabolic disorders, the effects of anomalous sorption and allow us to investigate the condition of patients with diabetes. Pharmacology uses extensively the isotopic analysis data with the introduction of isotopic labels into the body, mainly to identify metabolites for quantitative analysis by isotope dilution, to study the uptake of drugs. The method of isotopic labels is used to control the biosynthesis of antibiotics, to


study the pharmacokinetic properties of the analgesic and anesthetic preparations of various drugs [19].

The isotopes of light elements are involved in chemical, biological and physical processes at different speeds and therefore the isotopic composition of the element, which is part of the given compound depends on the history and origin of the sample. Consequently, the IRMS method can also be used to establish the place of manufacture of drugs, narcotics and explosives on the basis of their isotopic composition [16].

At the present time a major producer of mass spectrometers is Thermo Fisher Scientific (Bremen, Germany). Mass spectrometers have similar analytical characteristics and have a large number of devices for sample preparation (Fig. 1.1). The isotope mass spectrometers are fitted with special sample preparation systems for the transfer of solid and liquid samples to the gaseous state, with the formation of simple gases H2, CO2, N2, NOx, H2O.

There are two types of feeding gas samples into the ion source of the mass spectrometer: a system of dual gas inlet (Dual Inlet) and a system of measurement in a constant flow of the carrier gas helium (Continuos Flow, in Fig. 1.1). The figure uses the following notations: Trace GC/Agilent GC – gas chromatographs of the brand Trace and Agilent; GC/C/TC – gas chromatographs coupled to an oxidation reactor and a pyrolyzer; GC/GP – a gas chromatograph connected to other devices for sample preparation; PreCon – a device for the concentration of gaseous impurities; Gasbench – a universal device for preparation and supply of gas samples to the isotope mass spectrometer; EA, TC/EA – the elemental analyzer and a pyrolyzer; ConFlo – an interface block for connection of devices with the isotopic mass spectrometer; Microvolume – a device for the concentration of CO2 and N2 on the cold trap; Multiport – multiport gas inlet system; Tube Cracker – a device for opening ampoules; Multiport Extention – multiport system with the ability to reduce the amount of sample gas; HDO Equilibrator – an isotopic equilibrator for water; Kiel Carbonate Device – device for decomposition of carbonates; H-device – a device to separate hydrogen from water; H/D Collector Dual Inlet – collectors for the registration of the hydrogen ions in gas dual inlet mode; H/D Collector Continuous Flow – collectors for the registration of hydrogen ions in a constant flow of carrier gas; Universal 3 (6) – the number of universal collectors; MEMCO 6 – a system of six collectors to register ions of different elements. The experimental setup with dual inlet (DI-IRMS) is presented in Fig. 1.2.


The gas dual inlet system may receive pure gases from glass vials after their destruction; H2, CO2, or may be supplied from an equilibrator after isotopic equilibration with water; CO2 can come from the device to decompose carbonates; H2 from the device HD-Device after the decomposition of water. The isotopic composition of light elements in the sample is measured relative to a standard gas of the known isotopic composition. Gas pressure is controlled by compression or expansion of containers of variable volume, made in the form of bellows which hold gases. From the bellows the gases are fed through a stainless

Fig.

1.1

. Th

e sc

hem

e of

the

isot

ope

mas

s sp

ectr

omet

er w

ith tw

o ga

s in

let s

yste

ms

The

mas

s spe

ctro

met

er

Det

ecto

rA

naly

ser

The

sour

ce

of io

ns

The

doub

le in

let s

yste

m

The

syst

em w

ith a

con

stan

t flo

w

Dua

l In

let

Syst

em

Ion

sour

ce


steel capillary about 1 m long with a pinched front end into the ion source mass spectrometer, located in a high vacuum. Narrowing of the capillary is necessary to reduce the gas flow in the vacuum, to provide for viscous flow in a capillary and thus prevent the fractionation of gases during their inleakage into the ion source.

The gases entering the ion source are ionized under the influence of the electron beam, the ions are then focused into a beam, accelerated and end up in a strong magnetic field where the beam is split into separate bundles according to the mass to charge ratio. Lighter ions are deflected in a magnetic field more appreciably than heavy ones. The formed beams fall on the respective collectors (Faraday cylinders) and form collector currents, which, passing through high-resistance resistors, are converted to voltage. Thanks to simultaneous recording of ionic currents it is possible to measure with high accuracy and reproducibility of measurement the isotopic ratios on the natural level.

Mass spectrometers with a constant flow system (Continuos Flow) were developed about 30 years ago. These devices have revolutionized isotope studies [20]. The scheme of the isotope mass spectrometer with the Continuos Flow system is shown in Fig. 1.3.

The test gas is introduced into a stream of helium, purified in the chromatograph and directly enters the ion source of the mass spectrometer. Originally, the gas chromatograph was used only for gas cleaning, and then began to be used for the rapid isolation of individual compounds and their subsequent oxidation and, if necessary, reduction of nitrogen oxides [21]. The concentration of the analyzed

The variable volume

The standard gas

Capillary

Valve with variable

directions

Mass spectrometer

ValvesCollec-

tors

The magnetic sector

Variable volume Pumps

ViseThe

source of files

Specimen CapillaryValves

PumpsPumps

??? ??? ???

Fig. 1.2. The scheme of the isotope mass spectrometer with dual inlet.

‘Cold finger’

Ion

sour

ce

Crimp

Changeovervalve


samples decreased by several orders of magnitude. However, this led to a deterioration in the accuracy of isotope measurements.

Currently, the system Continuos Flow consists of the gas chromatograph (GC-C-IRMS method), liquid chromatograph (LC-ISOLINK-IRMS method), the elemental analyzer (EA-IRMS method), the pyrolyzer (TC/EA-IRMS method), the installation for the decomposition of carbonates (Gas Bench) or a gas chromatograph combined with a pyrolyzer (GC-TC-IRMS method).

Since the relative isotopic composition can be measured with greater accuracy than the absolute isotopic composition, to represent the relative isotopic composition of light elements the quantity δ (delta) was introduced in 1950, which is calculated by the following formula [22]:

δ =−

R RRx std

std

. ,1000 (1.1)

where R is the ratio of the abundance of the heavy isotope to the light isotope, x is an index, indicating the measured sample, std is an index denoting the standard sample. The value of R for the stable isotopes

Fig. 1.3. The scheme of the isotope mass spectrometer with a continuous flow system.

Laser, elemental analyser, injector,

pyrolyser

Input of specimen

Gas chromatograph

Reduction reactor

Oxidation reactorNafion drier

Interface

Quartz capillary 0.1 mm The

source of ions

Magnetic sector

Faraday cylinders

44 45 46 Recording

system

Isotope mass spectrometer


of hydrogen can be written as D/H, for the carbon isotopes 13C/12C, for the nitrogen isotopes 15N/14N, for the oxygen isotopes 18O/16O, etc. The value of δ is expressed in thousandths (per mil), which are indicated by ‰. A positive value of δ means that the ratio of the heavy isotope to the light one in the analyzed sample is greater than in the standard sample.

The value of δ is calculated by measuring the intensity of ion signals in the isotope mass spectrometer and recorded, for example for carbon, as δ13C. Usually, for pure gases the reproducibility of the results of isotopic analysis is ±0.01‰ or better.

The main gases, measured by the IRMS method, and the masses of their isotopologues are presented in Table 1.1. For example, for CO2 gas measurements are taken of four isotopologue with the masses m/z 44(12C16O16O), 45(13C16O16O and 12C16O17O), 46(12C16O18O). The isotope ratio of oxygen is calculated from ratio of 46/44. Table 1.2 gives the international standard samples used in isotope ratio mass spectrometry, and their isotopic composition [22].

Using equation (1.1), for example, we can estimate the error of determination of the absolute isotopic composition of 18O/16O in samples relative to the VSMOW standard sample with a standard deviation of 0.01 ‰ or the error of determination of the absolute isotopic composition of carbon 13C/12C relative to PDB. This error is for oxygen is 2·10–8, and for carbon 1·10–7.

Ongoing isotope measurements are often carried out using laboratory reference materials, certified with respect to international reference materials issued by the International Atomic Energy Agency (IAEA) or the National Institute of Standards and Technology (NIST, USA). The cost of international standard samples is quite high, and the number of limited. To calculate the value of δ of the sample X, as measured in

Table 1.1. The main gases used in IRMS

Element Gas Masses of isotopologuesHydrogen H2 2, 3 (interference from H+

3)Carbon CO2 44, 45, 46Nitrogen N2 28, 29, (30)Oxygen CO2 44, 45, 46

O2 (fluorination) 32, (33), 34CO (pyrolysis) 28, 30

Sulphur SO2 64, 66SF6 146, (147), 148, (150)


relation to the laboratory standard sample A, relative to the international standard sample B, the following formula is used [23]:

δX–B = δX–A + δA–B + 0.001 δX–A δA–B (1.2)

At present, the isotope mass spectrometers have a multicollector system for the simultaneous and separate measurement of the current intensity of ions with different masses. The reproducibility of the results of isotope analysis has improved, but the chemical sample preparation and the process of separation of individual compounds contribute significantly to the total error of analysis. The main advantages of the modern isotope mass spectrometers are easy operation of the device, the analysis of samples with lower mass, reducing the time spent on processing the results of the analysis.

Table 1.2. The isotopic composition of the international standard samples

No. Name Isotopes Isotope ratio

Reproducibility (α = 0.95)

Atomic composition

1 Vienna Standard Mean OceanWater (VSMOW)

2H/1H 0.00015576 ±0.00000010 0.000155736

2 Vienna Standard Mean Ocean Water (VSMOW)

18O/16O 0.00200520 ±0.00000043 0.002000443

3 Vienna Standard Mean Ocean Water (VSMOW)

17O/16O 0.000373 ± 0.000015 0.000372115

4 Standard Light Antarctic Precipitation (SLAP)

2H/1H 0.00008909 0.000089082

5 Standard Light Antarctic Precipitation (SLAP)

18O/16O 0.00189391

6 Pee Dee Belemnite (PDB)

13C/12C 0.0112372 ± 0.0000090 0.011112329

7 Pee Dee Belemnite (PDB)

18O/16O 0.0020671 ± 0.0000021

8 Atmospheric nitrogen

15N/14N 0.0036765 ± 0.0000081 0.003663033

9 Canion Diablo Troilite meteorite

34S/32S 0.0450045


The minimum amount of hydrogen, required for the measurement, is 20 mol, carbon – 0.13 µmol, nitrogen – 0.5 µmol.

For pure gases in the method of dual gas inlet (Dual Inlet) the isotopic ratios 2H/1H, 13C/12C, 15N/14N and 18O/16O can be measured with relative standard deviations of 0.001, 0.001, 0.002, 0.003%, respectively [24].

Table 1.3 shows the reproducibility of the results of isotopic analysis of various elements when using the interface ConFlo III [www.thermo.com].

Comparison of the analytical characteristics of the mass spectrometric methods for determining the isotopic composition of light elements At the present time the isotopic composition of light elements is determined using mainly two methods: SIMS and IRMS. Depending on the task one or the other method is used. If the main task is to measure the isotopic composition of light elements in the local area with the size of 10–30 µm, the SIMS method is used. If high accuracy

Table 1.3. The reproducibility of the results of isotopic analyzes (n = 10)

Method Sample AmountStandard deviation, δ,‰

2H 13C 15N 18ODI-IRMS Gas CO2 Signal 3 V 0.06

Gas N2 Signal 3 V 0.06Gas CO Signal 3 V 0.15Gas H2 Signal 5 V 0.50

EA-IRMS Urea 50 µg С, N 0.15 0.15TC/EA-IRMS

Benzoic acid

50 µg О25 µg H

0.2 0.4

TC/EA-IRMS

Water 0.5 µl 2 0.2

GC-C-IRMS

Mixture in isooctane

0.03% n-C14, n-C15, n-C16, 0.8 nmol С

0.2

GC-C-IRMS

Caffeine 200 ng, 1.5 nmol N

0.5

GC-TC-IRMS

Vanillin 200 ng, 5 nmol О

0.8

GC-TC-IRMS

Mixture in isooctane

0,03% n-C14, n-C15, n-C16, 15 nmol H2

3.0


of analysis is required, it is recommended to use the IRMS method with an extensive sample preparation system.

The main task for stable isotopic analysis of elements by SIMS is to achieve stable analytical conditions and stable dependence of the isotope fractionation on the mass in the standard and the test samples. The accuracy of the isotopic analysis does not exceed 1‰. Mass spectrometers of secondary ions are very complex and expensive devices available to a limited number of users. At the same time, mass spectrometers of stable isotopes are cheaper and widely used in various research laboratories and universities.

Despite the outstanding analytical performance of mass spectrometers of stable isotopes, they can be further improved by improving sample preparation and by developing new, more improved methods of analysis. In addition, the need for new methods of isotopic analysis is associated with the expansion of the number of objects of analysis using IRMS in modern, rapidly developing fields of science.

Figure 1.4 shows the basic layout of the mass spectrometric analysis of light elements. It includes both the isotope and elemental analysis

Fig. 1.4. The basic scheme of mass-spectrometric analysis of light elements.

Specimen

Solid Liquid Gas

Etching of surface Thermal

desorption of gases

Laser desorption of gases

ComplexSpark discharge

Glow discharge

Ion etching

Laser effect

Ions Oxidation and reduction for simple gases Pyrolysis

Simple gases:CO2, CO, N2, H2

Elemental and molecular mass spectrometric

analysis

Plotting of distribution

curvesIsotope mass-

spectrometric analysis

Pyrolysis


of light elements of solid and liquid samples and also gases. Liquid samples and gases subjected to pyrolysis and oxidation (followed by reduction of oxides of nitrogen) transform into simple gases CO2, CO, N2, H2, and are then analyzed using element or isotope mass spectrometers. Solid samples can also be subjected to incineration or pyrolysis. In addition, the thermal or laser irradiation of the sample can desorb adsorbed gases from the surface, and surface etching using ion or laser beams or spark and plasma effects produce secondary ions of light elements, which are then recorded by the mass analyzers. The processes that occur during sample preparation and mass spectrometric determination of the isotopic composition of light elements are described in detail below.

Improvement of equipment for the determination isotope ratios of light elements

Experiments were performed on modernized equipment with an isotopic mass spectrometer DELTA Plus (Thermo Fisher Scientific); the block diagram is shown in Fig. 1.5.

The installation also included an HP 6890 gas chromatograph for the separation of organic compounds in the gas mixture, the standard oxidation reactor, the standard reduction reactor, the high-temperature solid-electrolyte reactor (SER), and the water trap Nafion. The manufactured solid-electrolyte reactor, as shown in Fig. 1.5, was placed between the standard oxidizing and reductive reactors and connected with a fused silica capillary with an outer diameter of 0.5 mm and inner diameter 0.32 mm. The high-temperature solid-electrolyte reactor (Fig. 1.6) was made on the basis of stabilized zirconium dioxide (0.9 ZrO2 · 0.1 Y2O3) as a thin ceramic tube with an inner diameter

Fig. 1.5. Block diagram of the DELTA Plus modernized isotope mass spectrometer.

Solid electrolyte reactor Reduction

reactorCu/600˚C

Source of

ions

Magnet

Oxidation reactor

NiO, CuOPt/940˚C

Injector

Gas chromatograph

Nafion drier

Faraday cylinder

Detection system



of 1 mm and a length of 10–12 cm. The size of the working area of the reactor was 6 cm. To create a working platinum electrode internal. To produce working Pt electrodes, the inner and outer surfaces of the tube were coated with platinum paste (Platinum INK 6082, Engelhard) and annealed in air at a temperature of 900°C. The metal gas capillaries and electric current leads of the SER were sealed with special glass and checked for vacuum tightness. The electric current, passing through the SER, was provided by the DC source B5-43A. Changes of the electric current were recorded using a Metra Hit 28 (Gossen Metrawatt) multimeter and recorded in the computer memory to demonstrate the spectrum obtained on the monitor screen and its subsequent mathematical processing.

Photographs of the solid-electrolyte reactor, located above the chromatograph, are shown in Fig. 1.7.

The SER and the capillaries carried high-purity helium carrier gas, grade 60, at a rate of 2 ml/min. The use of two oxidizing reactors allowed to carry out the oxidation of organic compounds in two modes, and compare the results. The first mode used the standard oxidation reactor and the SER was switched off, in the second mode,

Fig. 1.6. Schematic diagram of the high-temperature solid-electrolyte reactor.

ThermocoupleHeating element

Steel capillary

Power source

GlassConducting Pt-interlayer

Pt-electrodes

measuring device

Com-puter

a b

Fig. 1.7. Pictures of solid-reactor, located above the chromatograph: a – general view, b – increased size of the reactor.


the standard oxidation reactor was switched off and the SER, heated to operating temperature and providing the necessary supply of oxygen into the helium stream, was used as the oxidation reactor. The standard oxidation reactor was in the form of a thin ceramic based on Al2O3 and 32 cm long with an inner diameter of 0.5 mm, with three wires (metal oxides Cu, Ni, Pt) inserted into it. At the temperature of 950°C the surface of CuO and NiO was characterized by the oxidation of organic compounds, Pt serves as a catalyst.

Figure 1.8 shows a general view of the experimental setup for the decomposition of water to produce hydrogen and determine its isotopic analysis. The essence of the phenomenon is the catalytic decomposition of water on platinum without any significant fractionation of hydrogen

Fig.

1.8

. Blo

ck d

iagr

am o

f hi

gh-t

empe

ratu

re s

olid

-rea

ctor

for

the

deco

mpo

sitio

n of

wat

er, c

onne

cted

to th

e sy

stem

dua

l-in

let

isot

ope

mas

s sp

ectr

omet

er D

ELTA

Plu

s.

Vacu

um

line

Valv

e

Trap

of W

ater

Sept

aSe

alan

t

Hea

ter ~

800°

C

Pt-e

lect

rode

s

Ther

moc

oupl

e

1 μl

H2O

sy

ringe

Al 2O

3 cer

amic

sY

2O3-s

tabi

lised

zirc

oniu

m c

eram

ics

(sol

id e

lect

roly

te)

Dua

l in

let

of D

ELTA

Plu

s m

ass

spec

trom

eter


isotopes, and transfer of oxygen through the solid-electrolyte cell into the atmosphere. Equipment includes a ceramic tube (made of stabilized zirconia) conducting oxygen ions. Pt electrodes are fixed to the outer and inner surfaces of the tube. Conductive paths lead from the electrodes to the contact pads located in the chilled parts of the ceramic tubes. The contact paths receive voltage from the DC source. A device for measuring electric current is connected in series to the source. Part of the tube with the electrodes is located in a heating furnace at a temperature of about 800°C. The temperature inside the furnace is measured by a thermocouple. Inside the tube there is inserted a ceramic rod (aluminium oxide) with two channels. The rod goes from the bottom of the tube and protrudes by 2 cm. The gap between the edge of the tube and the rod is sealed with a heat-resistant sealant. The outer portion of the rod is inserted into a Teflon liner, which is fixed in a steel holder. The rod in the holder is locked using two screws not shown in Fig. 1.8. A heat-resistant rubber seal presses to the end of the rod with a metal flange with two holes, located against the two channels in the rod. The flange is fixed with four bolts to the steel holder. The first channel of the bar contains a steel capillary (inserted through the rubber seal) connected through a valve with the isotopic mass spectrometer DELTA Plus. Through the capillary the ceramic tube is evacuated to a vacuum of 50 µbar. A water sample with a volume of 1 µl is introduced into the second channel with a syringe through the rubber gasket. The temperature of 800°C results in the catalytic decomposition of water: 2H2O = 2H2 + O2. Oxygen in the form of O2–ions is transported through the ceramic, placed between two electrodes, from the cathode (negative electrode) to the anode (positive electrode). Thus, electric current is generated in the external circuit of the electrochemical cell. The voltage between the electrodes is 1.4 V.

The design of the SER for the decomposition of water was improved during the investigations. In this section sealing of the solid-electrolyte ceramics is sealed, as shown in Fig. 1.8, by the thermal compensation glaze. The flanges are sealed with a round screw gasket. All the flanges are pulled together with bolts. The water sample is introduced with a syringe through the rubber gasket into a thin central channel through which it travels into the heated zone of the SER (800°C) and decomposes on the platinum electrode. Hydrogen, after the decomposition of water in the cell, enters the isotope mass spectrometer through the channel between the metal insert and the ceramic cell. The same channel is used for evacuation (50 µbar) of the SER before the introduction of water samples (1 µl).


Calibration of the isotope mass spectrometers. Calibration of isotope mass spectrometry was performed on standard samples, and laboratory reference samples of the known isotopic composition. A list of these samples is shown in Table 1.4.

The isotope mass spectrometer and its sample preparation were tested used standard samples in liquid, solid and gas states. The isotopic composition of the light elements of the analyzed samples

Table 1.4. International standard samples used for calibration of gas isotope mass spectrometers

Name Material δDVSMOW, ‰ δ13SPDB, ‰ δ15NAIR, ‰ δ18OVSMOW, ‰

VSMOW Water 0 0SLAP Water –428.0 –55.5GISP Water –189.5 ± 1.2 –24.8 ± 0.1IAEA-OH-1 Water –3.9 ± 1.3 –0.1 ± 0.2

IAEA-OH-2 Water –30.8 ± 1.1 –3.3 ± 0.2

IAEA-OH-3 Water –61.3 ± 1.4 –8.7 ± 0.2

IAEA-OH-4 Water –109.4 ± 1.4 –15.3 ± 0.2

IAEA-CH-6 Sucrose –10.4 ± 0.2 (+36.4 ± 0.6)

IAEA-CO-8 Calcite –5.8 ± 0.1 –22.7 ± 0.2

(PDB)USGS 24 Graphite –16.0 ± 0.1IAEA- N-1 (NH4)2SO4

+0.4 ± 0.2 (+12.9)

IAEA- N-2 (NH4)2SO4 +20.3

± 0.2 (+12.3)

IAEA- NO-3 KNO3

+4.7 ± 0.2 (+25.3 ± 0.7)

IAEA- C-3 Cellulose –24.9 ± 0.5

NBS 22 Oil –118.5 ± 2.8 –29.7 ± 0.2Oztech TEX-892C Gas CO2 –3.64 –24.97

Oztech TEX-843C Gas CO2 –40.79 +10.39

Oztech Gas N2 0.67Oztech TEX-840H Gas H2 –2.59


Input of standard sample (solid, liquid)

Pyrolyser Introduction of standard gases H2, N2, CO, CO2

Input of standard sample (solid, liquid) Elemental

analyserInterface

ConFlow3

Introduction of standard sample (gas, liquid) Chromato-

graph

Introduction of standard gases N2, CO2

Source of ions


Introduction of standard gases H2, N2, CO2

Double inlet

system

Interface Combustion

Introduction of H2, CO2 Equilibrator

for water

Fig. 1.9. The scheme of the input standard samples into the ion source of the isotopic mass spectrometer.

in any aggregate state after converting them into simple gases was calculated using standard gases H2, N2, CO, CO2 of the known isotopic composition. The diagram of the input of the standard samples into the ion source mass spectrometer is shown in Fig. 1.9.

Prior to isotopic analysis the ion source of the mass spectrometer received injected pulses of portions of standard gases containing light elements, which were then measured in the sample. This was accompanied by the stabilization of the ion source parameters and verification of the reproducibility of the results of isotopic analysis. In most cases the reproducibility was not lower than 0.1 ‰. In the process of analysis immediately prior to the registration of the peak of the analyzed sample and after completion of analysis the ion source received in pulses portions of the standard gas used for the calculation of the isotopic composition of sample and control of the stability of the analysis conditions. A typical chromatographic spectrum of water with respect to ionic current m/z 28, 29, 30 is shown in Fig. 1.10. The standard gas is introduced into the ion source via ConFlow 3. The peak of the standard gas on the chromatogram is rectangular shape.

The calibration line for determining the isotopic composition of carbon, based on the international reference materials of different composition (NBS 22 – oil, Oil-V – oil, USGS 24 – graphite, IAEA-C-3


– cellulose, IAEA-CH-7 – sucrose), using elemental analyzer coupled with isotope mass spectrometer, is shown in Fig. 1.11. It is a straight line with a correlation coefficients equal to 0.999.

It is seen that regardless of the sample material with its full combustion in the oxidation reactor of the elemental analyzer, at proper alignment of the ion optics and in the absence of contamination of the isotope mass spectrometer, at the correct value of the isotopic composition of the calibration gas the systematic error of measurement of the carbon isotopic composition of the analyzed substance is practically zero.

Similar conclusions can be made for the calibration curve, obtained by determining the isotopic composition of oxygen in the samples by the TC/EA-IRMS method (a pyrolyzer, coupled with a mass spectrometer). This calibration curve is shown in Fig. 1.12. The straight line was constructed using standard waters, cellulose and KNO3. The tangent of the slope of the calibration curve is close to unity, and the correlation coefficient is 0.994.

The calibration curve for determining the oxygen isotopic composition of water, obtained by equilibration with the standard gas, is shown in Fig. 1.13. This graph has a correlation coefficient of 0.999 and the tangent of the slope of 0.96, close to unity. Balancing the oxygen isotopic composition of water with the isotopic composition of the oxygen of the standard gas CO2 occurred within 16 hours in the vessels (bottles) with water, placed in a thermostat with a temperature

Time, s

Inte

nsity

, V

R

atio

Fig. 1.10. Chromatogram of water, based on ionic current m/z 28, 29, 30, using a pyrolyzer, coupled with the isotope mass spectrometer (TC/EA-IRMS method).


of 25°C. The water in vessels stirred on a shaker with a frequency of 1.5 Hz.

The isotopic composition of the oxygen and hydrogen of water can be determined in the pyrolytic decomposition of water in a reactor at a temperature of 1450°C. Pyrolysis occurs on the surface of a glassy carbon reactor or glassy carbon granules by the reaction [25]

H2O + C → H2 + CO.

δ13C (Standard), %

Fig. 1.11. Calibration curve for determining the carbon isotopic composition of solids and liquids in the analysis of samples by EA-IRMS (the elemental analyzer coupled with the mass spectrometer),

δ18O (Standard), %

δ18O

(Mea

sure

d), %

Fig. 1.12. Calibration curve to determine the isotopic composition of oxygen in the solid and liquid substances in the analysis of samples by TC/EA-IRMS (a pyrolyzer connected to a mass spectrometer).

‰

‰

‰


Separation of the reaction products is carried out in a chromato-graphic column. The calibration curve for determining the hydrogen isotopic composition of water, as shown in Fig. 1.14, is a straight line over a wide range of the isotopic composition of hydrogen, from –990‰ (super light water) to 0‰. In order to continue this line in the region of positive δD it is necessary to have standard samples of heavy water. The tangent of the slope of the graph is close to unity, the correlation coefficient is 0.999.

Fig. 1.13. Calibration graph constructed to determine the isotopic composition of the oxygen of water, obtained by the method of equilibration of the isotopic composition of the oxygen of the standard gas CO2 and water.

δ18Os, %

δ18Om, %

Fig. 1.14. Calibration graph constructed to determine the isotopic composition of the hydrogen of water (obtained by pyrolysis of water, TC/EA-IRMS method).

δDs, %

δDm, %

‰

‰

‰

‰


For a more accurate construction of the calibration line in a wide range of the isotopic composition of hydrogen and development of the method of isotopic dilution, Tonkie Tekhnologii Company (Moscow) produced a series of samples with different contents of deuterium using very light water with a known deuterium content (CD = 1.1 ppm). According to formula (1.3), we can calculate the isotopic composition of the hydrogen of this water, which is δD = –992.9‰:

δsamplestand stand

stand

= ⋅ − +

⋅ ⋅ −

100 11

100100

R C R

R C

( )

( ), (1.3)

where Rstand is the ratio of the abundances of the heavy isotope to the light one, C is the concentration of the heavy isotope in at.%, δsample is the relative isotopic composition of the sample.

Light water was diluted with distilled water and the isotopic composition of hydrogen of distilled water was determined from the calibration curve, constructed in accordance with the international reference samples VSMOW, GISP, SLAP. The isotopic composition of the hydrogen of light and distilled water was used to calculate the concentration of deuterium in all intermediate samples. Diluted water samples were kept for 1 week at room temperature with periodic stirring. The resulting calibration curve is shown in Fig. 1.15. The correlation coefficient of the experimental data is 0.999.

Thus, it was shown that for laboratory standard water samples with the known isotopic composition can be prepared by the method of isotope dilution.

CD (True), ppm

CD (M

easu

red)

, ppm

Fig. 1.15. Calibration graph constructed to determine the isotopic composition of hydrogen of water (obtained by pyrolysis of water with isotopic dilution, TC/EA-IRMS).


Based on the analysis of the calibration curves constructed for determination of the concentration and isotopic composition of light elements, we can conclude that they all have a linear region with varying extension, depending on the specific conditions of analysis and the equipment used. Furthermore, it was shown that the calibration curves can be constructed using standard liquid and solid samples of organic and inorganic origin.

Mass spectrometric methods for determining the isotopic composition of light elements

In most cases the isotopic composition of light elements is determined using sample preparation systems (elemental analyzer EA, gas chromatograph GC, pyrolyzer TC/EA, a gas dual inlet system, water equilibrator EQ) connected through appropriate interfaces with the isotope mass spectrometer. Mass spectrometric methods for determining the isotopic composition of light elements were created for the mass spectrometer DELTA Plus and DELTA PLUS XP Thermo Fisher Scientific. Prior to isotopic analysis it is necessary to verify the accuracy of the instrument settings using the following procedures:

1) check the background spectrum and ensure there are no leaks;2) to conduct a procedure for determining the centre of the peak;3) autofocusing of the ion source;4) to test the reproducibility of measurement results;5) test the linearity of the results of isotopic measurements.1. The test for any leaks . The main peaks of the background

spectrum are formed by the residual gases in the vacuum system: H2O, N2, O2, Ar, CO2. High peaks of N2 and O2 indicate the leakage of air into the system. Typically, the high background of water is observed in the spectrum two days after starting the mass spectrometer, if the system was depressurised prior to this. If a leak is found the suspected locations are blown with argon and the magnitude of the peak with a mass of m/z 40 is recorded. A leak found in areas in which blowing increases dramatically the peak value of argon.

2. Finding the centre of the peak of the standard gas. The centre of the peak of the CO2 standard gas was determined on a middle detector (Faraday cylinder) which records ions with m/z 45, by changing the magnitude of accelerating high voltage of the ion source. The optimum value of the high voltage is fixed in the program and then used as an experimental parameter.

3. Autofocusing of the ion source. The autofocusing of the ion source is a standard feature of the mass spectrometer. Carrying out this


procedure increases the intensity of the ion beam and the accuracy of determination of the isotopic composition of light elements. In addition, the ion source can be set in the manual mode. The resultant parameters are recorded in a table entitled ‘configuration of gas’.

4. Checking the stability of isotopic analysis. The stability of isotopic analysis was checked by multiple pulsed gas inlet of portions of the standard gas into the ion source (ON–OFF mode) and by assessing the reproducibility of the results of isotopic analysis. A series of pulsed inputs of the standard nitrogen gas is shown in Fig. 1.16.

As a result, the gas inlet to the mass spectrometer forms a rectangular peak with a flat top on the chromatogram. The gas inlet pulse duration is typically 20 s. The height of the rectangle depends on the pressure of the standard gas fed into the ion source. The higher the gas pressure, the greater the height of the rectangle. Thus, by adjusting the gas pressure we can change the height of the rectangles h and study the influence of the height (amplitude of the peak) on the results of measurements of the isotopic composition of standard gas (δ). Typically the height of the rectangles is h = 3–6 V. One of the last rectangles is given the known value of the isotopic ratio δ of the light element of the standard gas. Further, with respect to this value we calculate the isotopic ratio of this element for other rectangular pulses. There is

Fig. 1.16. Chromatogram registered at ionic current with m/z 28, 29, 30, in the mode of the multiple inlet of the standard gas N2 at the same pressure in the ion source of the mass spectrometer (evaluation of the reproducibility).

Inte

nsity

, V

R

atio

Time, s


the gas inlet mode ON–OFF with the same amplitude (height) of the signals – (mode 1, Fig. 1.16) and with different amplitude – (mode 2, Fig. 1.17).

The ON–OFF-1 mode is used to control the stability of the isotope mass spectrometer and to evaluate the reproducibility of measurements of the isotopic ratio of the standard gas. Multiple repetition of this mode stabilizes the analysis conditions, thus improving the reproducibility. For the values δ13C, δ18O and δ15N the reproducibility of isotopic analysis results is usually no worse than 0.05‰.

Tim

e, s

Intensity, mV Ratio 30/28

Fig. 1.17. Chromatogram registered at ion current with m/z 28, 29, 30, in the mode of multiple inlet of the standard N2 gas at different pressures in the ion source of the mass spectrometer (evaluation of linearity).


5. Checking the linearity of the results of isotopic analysis. The linearity of the results of isotopic analysis is the dependence of the results of determination of the isotopic composition on the amplitude of the signal. To do this, the amplitude of the signal is manually increased from the minimum to maximum by increasing the gas pressure in the inlet system at the end of each pulse. Thus, the pulses of the standard gas of varying amplitude are produced, as shown in Fig. 1.17. Usually, the changes of the results of measurement of the isotopic composition are linear, equal to 0.06‰/V, i.e., when the amplitude of the signals changes by 1 V the isotopic ratio changes by 0.06‰. When using the EA-IRMS and TC/EA-IRMS methods to determine the linearity it is more efficient to use real samples with different masses.

In determining the isotopic ratio of hydrogen δ2N it is necessary first to determine the H+

3 factor by which the amendment is made to the linearity. The H+

3-factor was determined experimentally daily prior to isotopic analysis of hydrogen. In the ion source the hydrogen ions react with molecular hydrogen to form H+

3 ions and the hydrogen atom. The contribution of H+

3 in the ion current of mass 3, usually amounting to 5 ppm/nA, was quantitatively corrected using the H+

3-factor.Isotopic analysis required laboratory standard gases H2, N2, CO, CO2,

which were certified against standard gases using the gas dual inlet method. The isotopic composition of light elements was calculated using the program ISODAT NT 2.0 with respect to the isotopic composition of light elements of the laboratory standard gases. In addition, periodically analyzed were solid, liquid and gaseous standard samples, subjected to the same methodical procedure as the analyzed samples. This should be done periodically to confirm the results obtained. To determine the isotopic composition δ2H and δ18O, a pyrolyzer was used to construct a calibration graph for the isotopic composition of hydrogen or oxygen on the basis of the results of isotopic analysis of standard water samples VSMOW, GISP, SLAP, or other standards.

1. The gas dual inlet method (DI-IRMS). Figure 1.2 shows the scheme of the dual inlet system. The sample gas and the standard gas sample enter alternately the ion source mass spectrometer from variable volume bellows through steel capillaries, which in front of the ion source are mechanically compressed to ensure the viscous mode gas inleakage into the vacuum. Moreover, it is also necessary to ensure that the speed of the flows of the gases are almost identical. At the same time the gas pressure difference in the two bellows will remain constant.

The gas is fed into the dual inlet system with the gas from the tank through the inlet flange, sealed with a circular screw gasket. Prior to


this all systems are pumped to a pressure of 3·10–3 mbar. In addition, the gas to be analyzed can be supplied from a system of tube crackers or from a device that cracks one end of the ampoule.

In the viscous mode of inleakage of gas into the ion source the free path of molecules is less than one tenth of the inner diameter of the capillary (0.1 mm), and therefore the minimum pressure in the bellows must be 15–20 mbar. The minimum volume of the bellows is 40 ml, and the maximum 250 ml. The minimum amount of gas that can be analyzed is about 5 bar·µl or 220 nmol of pure gas. In the measurements the bellows is usually compressed to the pressure of 30–50 mbar, to obtain a signal of gas equal to 3–5 V.

Alternate measurements of the signals of the two gases make it possible to compensate for the effects of changing the sensitivity of the ion source and the strength of the magnetic field, to compensate for the effect of temperature on the operation of electronic units and the influence of the instrumental drift on the results of the determination of the isotopic composition of light elements. The reproducibility of measurements of the isotopic composition of carbon in the compliance with all of the above conditions is not worse than 0.1‰.

2. The method of isotopic equilibration of the standard gas and water (EQ-IRMS). Isotopic equilibration of a gas with a known isotopic composite of oxygen or hydrogen and water was carried out in an equilibrator, which allows to maintain a constant temperature of the water sample and also shake them with a frequency of 1–3 Hz. The water samples were poured into 24 special vessels which were placed in the equilibrator. Water was transported in containers made of borosilicate glass with a screw lid, with a silicone gasket and with a minimum amount of air in them.

Measurement of the isotopic composition of the oxygen of water. Approximately 5 ml of water was collected and placed in vessels of a special form having a volume of 20 ml for isotopic analysis. The vessels were then placed in the equilibrator in which the temperature was maintained at 25°C or more with an accuracy of 0.05°C. The air was evacuated from the vessels with a backing pump and the vessels were filled with the CO2 gas with the known oxygen isotopic composition δ18O to the pressure of ~0.4 bar. The vessels closed and held together with the standard gas for about 20 hours. Equilibration is accompanied by the following reactions:

CO2 (g) ⇄ CO2 (ad)(exchange of CO2 between the gas phase an0d water),

CO2 (ad) + H2O ⇄ H2CO3


(dissolved CO2 reacts with water to form carbonic acid).To construct the calibration graph, the samples were analyzed

together with the standard samples. Although there is isotopic fractionation between CO2 in the gas phase (CO2 (g)) and dissolved CO2 (CO2 (aq)), as well as between dissolved CO2 and H2CO3, this process will be the same for all samples. Therefore, in the calculation of the isotopic composition δ18O the fractionation effect can be ignored. After isotopic equilibration the gas travels through a water trap (–70°C, dry ice in ethanol) to the dual inlet system to measure the isotopic composition.

Measurement of the isotopic composition of the hydrogen of water. When measuring the isotopic composition of the hydrogen of water H2 is balanced with water at a temperature of 25°C, maintained with an accuracy of 0.05°C, for 1–2 h. At the same time, a plastic rod with platinum inclusions that serve as a catalyst of isotopic exchange is immersed in the water. Isotopic exchange occurs in the presence of platinum on the surface of the rod. To construct the calibration graph, the samples are analyzed together with the standard samples. The reproducibility of the isotopic analysis results is 0.5‰ and 0.04‰ for 2H and 18O, respectively.

3. The method of isotopic analysis using the elemental analyzer (EA-IRMS). The EA-IRMS method is used to determine the isotopic composition of carbon and nitrogen in solid and liquid substances. Samples weighing from 0.1 to 3 mg are placed in tip capsules were measuring 9 × 5 mm. If the solid samples are inhomogeneous, they are dried at a temperature of 60°C for 48 h, followed by grinding in a ball mill for 10 min. If the samples contain inorganic carbon in the form of carbonates, which interferes with the determination of the isotopic composition of organic carbon, these samples are treated with hydrochloric acid and then thoroughly washed with water. The optimal charge of the samples for carbon isotope analysis with the required accuracy should contain 200–600 µg of carbon. The carbon content should not exceed 5 mg of carbon, as the large mass of the sample is difficult to pack and completely combusted. The minimum sample charge should be an order of magnitude smaller. The optimal charge of the sample for isotopic analysis of nitrogen with the required accuracy must contain 100 µg of nitrogen.

Packed in a tin capsule samples were placed in the device for automatic input of samples (autosampler) of the elemental analyzer, and from, on the basis of a computer command, these samples were dumped into the oxidation reactor. At the same time, a stream of helium of 90–120 ml/min was fed with pure oxygen. The capsules with


the sample were immediately burnt, and the temperature was locally increased to 1800°C. Further, the combustion products were additionally oxidized in the oxidative reactor, heated to a temperature of 1020°C, and filled with reagents Cr2O3 and silver-plated beads of Co3O4. From the oxidation reactor the produced pure gases N2, CO2, nitrogen oxides and H2O travelled into the reduction reactor, heated to a temperature of 650°C and containing metallic copper and copper oxide. In this reactor, the nitrogen oxides were reduced to the molecular nitrogen and the remaining oxygen was absorbed. Water was removed from the stream of the He carrier gas by trapping in a trap with magnesium perchlorate. Next, nitrogen and carbon dioxide were separated in a capillary column PoraPlot Q at 50°C. The separated gases were injected through the device ConFlo 2 into the ion source of the isotope mass spectrometer for isotopic analysis. The received signals of N2 and CO2 should not be less than 1000 mV and not more than 9000 mV. To obtain good background spectra, it is first necessary to open the needle leak valve to supply helium through the fused silica capillary of ConFlo 2 into the ion source and blow it for 12 hours. A fuses silica insert for collecting sol (slag), formed from the mineral component of the samples, is placed in the oxidation reactor. The slag must be removed after analysis of 50 samples, as it prevents further analysis. To do this, the temperature of the reactor is lowered to 400–500°C, the oxidation reactor is opened and the insert with the slag is removed with forceps.

Prior to isotopic analysis it is rational to carry out a blank experiment and determine the idle running peak, which should be subtracted from the peak area of the sample. This should be followed by selecting a standard sample or a standard laboratory sample having the composition close to the analyzed material, and ensure the correct determination of its isotopic composition.

Usually the result of the first isotopic analysis is discarded and additional two or three parallel analyses are carried out and the results, which should be close in the value, are compared. After every 5–10 analyses of the samples the standard sample is analyzed for control of the consistent quality of analysis. Before the analysis the test samples with the same properties must be grouped as the transition to other models (depending on the sample size) may result in a negligible memory effect (less than 0.2‰).

In numerous analyses the oxidants lose their oxidizing and catalytic properties and the reduction reactor ceases to function. The measured value of the isotopic composition of the light elements of the standard samples no longer corresponds to its true value. Therefore, the reactors must be replaced.


If the reagents are contaminated with the compounds of sulphur and chlorine the ion chromatogram shows extraneous peaks (Fig. 1.18), which are clearly visible on the graph of the ratio of ion currents with the masses m/z 45 (46) and /z 44. The reagents are contaminated quite rapidly and fail in the analysis of marine sediments treated with hydrochloric acid vapours. Figure 1.19 shows how the deterioration of

T, c

Inte

nsity

, V

R

atio

46/

44

Fig. 1.18. Chromatogram of products of combustion of organic matter, based on the ionic current with m/z 44, with a contaminated oxidation reactor.

Time, s

Inte

nsity

, mV

Rat

io

Fig. 1.19. Chromatogram of products of incomplete combustion of organic matter, based on ionic current at m/z 44, 45, 46.

T, s


the oxidative properties of the reagents of the oxidation reactor leads to splitting of the analytical peak, which leads to distortion of the results of isotopic analysis.

4. The method of isotopic analysis using a high-temperature pyrolyzer (TC/EA-IRMS). This method is based on the pyrolytic decomposition of organic and some inorganic compounds at high temperatures (1450°C) on the surface of a glassy carbon reactor and glassy carbon granules. Samples can be solid, liquid and gaseous. Since the decomposition of substances do not require the presence of gaseous oxygen and oxygen oxidants in the system, this method has a great potential for isotopic analysis of hydrogen, oxygen and nitrogen. The advantage of this system is the small sample charge, which, however, requires high homogeneity of the samples. In the tradit ional decomposition of substances the sample charge is considerably larger. In this case, the inhomogeneity effect plays a substantially smaller role. The small length gas-chromatographic packed column Poraplot Q (Chrompack), installed after the pyrolytic reactor, allows separation of the resultant gases: H2, N2, CO. Pyrolysis of some simple compounds is described in the following scheme:

H2O + C → H2 + CO; 2KNO3 + 8C → N2 + 6CO + [2KC];

BaSO4 + 4C → 4CO + [BaS] + [AgS]; C12H22O11 → 11H2 + 11CO + C (sugar);

Cellulose → xH2 + yCO; Amino acids → xH2 + yCO + zN2 + balance.

A silver wire is placed at the bottom of the pyrolytic reactor to remove sulphur from the stream of the carrier gas helium (helium grade 60). Solid samples are wrapped in silver capsules (4 × 6) and placed in the device for automatic input of samples. Typically, when carrying out isotopic analysis the influence of the oxygen of the oxide film of Ag can be neglected. Liquid and gaseous samples are introduced with a syringe through a rubber gasket directly into the reactor (0.3–1 µl). The syringe needle must remain in the heated zone of the reactor during 10–16 s for the complete evaporation of the sample from the cavity of the needle. The magnitude of the analytical signal must more than 2000 mV. The temperature of the pyrolytic reactor is raised to operating temperature of 1450°C or lowered to the standby temperature of 950°C at a rate of 5°C/min.

Over time, the level of the background line of CO+ increases as the oxygen from the external ceramic tube (Al2O3) penetrates on the surface of the inner glassy carbon tube and interacts with carbon. After passing


through the pyrolyzer the pyrolysis products in a flow of helium (90 ml /min) are sent through a chromatographic column (90°C) to the interface ConFlo3 and then further into the ion source of the mass spectrometer where the pressure is 2 · 10–6 mbar. To improve the reproducibility of isotopic measurement results 2% of hydrogen is added to the flow of the carrier gas to maintain the reducing properties the system.

To calculate the isotopic composition of hydrogen and oxygen, the ionic currents with the masses m/z 2 and 3 in the measurement of the isotopic composition of hydrogen and ionic currents with the masses m/z 28, 29, 30 (in the form of CO+) are recorded using the program ISODAT NT 2.0. At the same time, the isotopic composition of light elements is calculated relative to the standard gas. To define the isotopic composition of oxygen, the sample should contain 2–6 µmol of oxygen (O). The reproducibility of the results of the isotopic analysis of hydrogen is 1–2‰, for oxygen 0.3–0.5‰. After analyzing 150 samples the remnants of the silver capsules accumulated in the reactor must be removed. After analyzing 300 samples the surface of the glassy carbon tube must be cleaned to remove contamination and replace the pellets, and after 2000 analyses the pyrolytic tube and the pellets should be replaced.

The main errors in determining the isotopic composition of hydrogen and oxygen in the samples result from the presence in them of moisture and air, as well as the presence of an oxide layer on the surface of Ag-capsules, in which the samples are introduced into the system. In addition, possible oxygen isotope exchange between CO and metal oxides [26]. Every day after analysis the chromatographic column was heated at a temperature of 150°C overnight. Prior to analysis the samples should be dried at a temperature of 40°C for remove moisture as its isotopic composition of oxygen and hydrogen can distort the results of isotopic analysis of the samples. The duration of a single determination of the isotopic composition was about 400 s. The measurements of each sample were repeated 3–4 times.

Unfor tunate ly, i t was found that the isotopic analys is of hydrogen and oxygen effect is accompanied by the memory effect of the pyrolyzer when passing to a sample with a different isotopic composit ion. Therefore, as shown in Fig. 1.20, the f irst two measurements of the next sample can be distorted because of the memory effect associated with the isotopic analysis of the previous sample.

5. The method of isotopic analysis using a capillary chromatograph (GC-C-IRMS). The essence of this method lies in the transformation of complex composition mixtures to simple, well-separated gases


required for the isotopic analysis of light elements. For this purpose a gas chromatograph with a capillary column is connected by the interface of the Combustion unit 3 to the isotopic mass spectrometer. The compounds separated in the chromatograph are converted in the oxidation reactor into simple gases, CO2, N2, nitrogen oxides, and H2O at a temperature of 940°C on the surface of a platinum wire, interwoven with the oxidized metallic wires made of Cu and Ni. Nitrogen oxides are reduced in the reductive reactor at a temperature of 600°C on the surface of the copper wire. The resulting waters are transferred from the flow of the carrier helium gas (grade 60, purity 99.9999%) through a semipermeable membrane Nafion. The flow rate of the carrier gas is 1–2 ml/min. Through the interface (open gap) the gases enter the ion source of the mass spectrometer where they are ionized. Further, the ions separated by mass (m/z) in the magnetic field of the analyzer, are recorded on three Faraday cylinders set to a certain mass. For example, for the molecular ion CO+

2 isotopologues m/z 44, 45, 46 are recorded, and for the N+

2 ion the isotopes with m/z 28, 29, 30 are recorded.The resultant values of the ionic current are used to calculate the isotopic composition of carbon in CO2 and nitrogen in N2. The advantage of this equipment over other devices is that it is possible to determine the isotopic composition of elements in each component of the complex mixture of gases.

The separation of complex mixtures of organic compounds in the HP 6890 chromatograph was carried out using two capillary columns PoraPlot Q (Chrompack) 25 m long and DB-5 (J & W Scientifics)

Water, 146%

Fig. 1.20. Memory effect of the pyrolytic reactor. Change in the isotopic composition of oxygen δ18O, depending on the number of input water samples N.

Water, 146 ‰


30 m long with a diameter of 0.32 mm. The operating conditions of the chromatograph were the following:

Conditions Gas analysis Analysis of solutions

Column PoraPlot Q DB-5Temperature of

injector100°C 250°C

Initial temperature of thermostat

40°C 150°C

Final temperature of thermostat

180°C 320°C

Heating rate 5°C/min 10°C/min

The analysis was carried out with heating of the ion source and the needle inlet valve. Samples in the form of liquid or gas were injected into the chromatograph with a syringe either manually or in the automatic mode using the device for the automatic input of samples CTC A200S. The volume of the liquid sample was less than 1 µl. The GC-C-IRMS method can be used to record 3–5 nmol of carbon. The strength of the analytical signals must be greater than 2000 mV. The volume of the gas sample was less than 300 µl. In determining the isotopic composition of minor components of the gas mixture the chromatograph is transferred to the backflush mode for a period of time required by the components with a high concentration to leave the column. This is done in order to prevent the passage of macrocomponent through the oxidative and reductive reactors and the introduction of large amounts of oxidation products into the mass spectrometer ion source, which causes an overload of electronic amplifiers. The overload of the oxidative reactor leads to rapid depletion and possible appearance if the memory effect which manifests itself in the wrong determination of the isotopic composition of the following components of the gas mixture.

After isotopic analysis of 100 samples it is necessary to additionally oxidize with gaseous oxygen the copper of the oxidative reactor for 12 h at 500–600°C. Deposition on the copper surface of the products of pyrolysis of organic compounds using oxygen for regeneration of the oxidative reactor leads to the formation on the copper surface the copper compound Cu2O (cuprous oxide), and not CuO. When a sufficient amount of Cu2O forms, the effectiveness of the oxidative reactor is reduced and it should be replaced. Degradation of the oxidation reactor


can cause the formation on the surface of copper of compounds with sulphur and chlorine. For example, the reactor could be damaged after 15 analyses of a mixture containing sulphur compounds [27]. If the flow of the carrier gas helium contains water vapour formed during combustion of organic compounds, they can cause protonation of the gas molecules of CO2 and lead to a shift in the results of isotopic analysis of carbon. Therefore, a water trap, made in the form of a tube with a Nafion membrane, must not be contaminated with the products of pyrolysis and solvent residues, causing its degradation up to the formation of discontinuities [27].

Metrological characteristics of mass spectrometry of isotope ratios

The effect of the strength of the analytical signal on the results of measuring the isotopic composition of light elements

The aim of research was to study the influence of the amplitude of the analytical signal on the results of measurements of the isotopic composition of light elements by using different sample preparation systems and inlet of the gases to the isotope mass spectrometers. This effect has not yet been studied comprehensively. In the literature there are references that at small values of the signal the measurement results of the isotopic composition of light elements are incorrect [28].The reasons for this phenomena are not well understood, so we decided to study in detail the influence of various factors on changes in the measurement results of the isotopic ratios of light elements.

The presence of water vapours in the ion source of the isotopic mass spectrometer leads to an error in the measurement of the isotopic composition of carbon due to a chemical reaction between the ions of carbon dioxide and water molecules [28]:

CO+2 + H2O → HCO2

+ + OH–.

Water vapours cause a change of the ratios of ionic currents of isotopologues with m/z 45 and 44 (45/44) and m/z 46 and 44 (46/44), since the signal of the H12S16O+

2 ions contributes to the main signal of the ions with mass m/z 45, and the signal of the H13S16O+

2 ions to the signal with a mass of m/z 46 [23, 29]. Similar reactions of protonation are possible for other gases, leading to the formation of H+

3 or N2H+

[23] ions. Depending on the ratio of signals from the sample and the standard gas the shift in the carbon isotopic composition of the sample can be both positive and negative. The influence of water vapours can


be reduced by lowering the potential of extraction lenses of the ion source. However, this is accompanied by a simultaneous deterioration of the sensitivity of the method [28].

Experiments were performed on two gas isotope mass spectrometers DELTA Plus and DELTA Plus XP using the gas dual inlet system, the elemental analyzer (EA) and the pyrolyzer (TC/EA), as well as using different gas inlet systems in the mass spectrometer – through gas interfaces Combustion 3, ConFlo 2, ConFlo 3. The characteristics of these systems were studied using standard gases CO, N2, CO2 with a known isotopic composition.

In carrying out the isotopic analysis of light elements the important aspect is the degree of evacuation and composition of the residual gases in the vacuum system of the mass spectrometer. Table 1.5 shows the intensity of the peaks of residual gases in the vacuum system of the DELTA Plus mass spectrometer. The most intense line is the peak of water (m/z 18).

With admission of helium through the HP 6890 chromatograph by the Combustion 3 system into the ion source of the mass spectrometer the intensities of all the lines of the residual gases increase by 3–5 times. Under these conditions, the influence of oxygen and water vapour on the measured isotopic composition of the sample should increase. The ions formed in the ion source and containing isotopologues CO2, N2, CO, etc., separate under the influence of magnetic fields and fall, depending on the mass-to-charge ration, on one of the three detectors, known as the Faraday cylinder. The ions trapped in the detector cause an electron current which is amplified by an electronic amplifier and converted into a voltage at the high-resistance resistor. The characteristics of high precision resistors and capacitors, actuators, presented in Table 1.6.

Thus, when recording the ions of the CO2 isotopologues the detector 3 detects ions with m/z 46 with a sensitivity of 3.3 · 102 and 3.3

Table 1.5. Intensities of the residual gases in the vacuum system of DELTA Plus mass spectrometer (Detector 3)

Pressure, mbar Ion mass, m/z, a.m.u 18 28 32 44

7.4·10–9 Signal, mV (needle valve is closed)

503 133 307 166

1.8·10–6 Signal, mV (passing helium through a gas

chromatograph)

2137 694 1360 514


times greater than the detector 1 (m/z 44) and detector 2 (m/z 45), respectively. This allows us to obtain analytical signals of similar strengths and thereby reduce the error in measuring the isotope ratios.

The dual inlet system (DELTA Plus) The study of the analytic characteristics of the gas dual inlet system was carried out on the standard CO2 gas with different isotopic composition in the conditions of inlet of the helium flow into the ion source of the mass spectrometer through an HP 6890 chromatograph and the Combustion 3 interface or without supplying helium (in this case the gas inlet valve to the mass spectrometer was closed). This made it possible, on the one hand, to study the effect of total pressure in the ion source on the results of isotope analysis, and on the other hand to study the effects of increased partial pressure of water vapour, which can penetrate into the mass spectrometer with a helium flow or is a microimpurity of the standard CO2 gas.

Table 1.7 presents the results of the influence of inlet of helium (P = 1.8·10–6 mbar) into the ion source of the mass spectrometer on the results of the determination of the carbon isotope composition in the CO2. Measurements were performed in the gas dual inlet mode, when the same gas is used as the sample and the standard. The ionic currents of the sample and the standard were identical Isample = Istand(δ

13Csample= δ13Cstand = –50.14‰).As shown in Table 1.7, the supply of helium and a simultaneous

decrease in the signal intensity of the sample and the standard gas reduce the background of the water vapour I (m/z 18). At the same time at the signal intensity I(m/z 44) < 265 mV the difference of the isotope ratios increases Δ13C > 0.2‰, and the standard deviation S starts to exceed 0.1‰. We believe that this is due to the interaction of CO2 with water vapours in the ion source of the mass spectrometer; the amount of the water vapours may increase with admission of gases (see Table 1.7). Since at small values of signal magnitude the values Isample and Istand after automatic alignment of the signals can vary quite significantly (up to 20% or more), then the conditions of isotopic analysis will differ. In

Table 1.6. Characteristics of resistors and capacitors used in electronic amplifiers of ion detectors, in determining CO2 isotopologues

Gas m/z Resistance, ohm Capacity of capacitor, pFCO2 44

4546

3·108

3·1010

1·1011

47052


addition, the geometric parameters of the capillaries 0.1 mm in diameter through which the gas is supplied and which are compressed at the ends in front of the ion source can also be different and this may contribute to the fractionation of isotopes [23].

The results obtained by measuring the isotopic composition of carbon of CO2 in measurements in the gas dual inlet mode with inleakage of helium into the ion source of the mass spectrometer were compared with the results of the analysis under standard conditions without helium inlet (Tables 1.7 and 1.8). It was found that under the condition Isample = Istand and δ13Csample = δ13Cstand = –50.14‰ the helium inlet into the ion source of the mass spectrometer does not lead to any significant change of the dependence of the measured isotopic composition of carbon on the value of the analytical signal.

When the sample is the CO2 gas with a large content of heavy carbon isotopes (δ13Csample = –3.64‰) the measured isotopic composition of carbon is too high at strong signals and underestimated at weak signals, regardless of the dependence on the helium pressure in the ion source of the mass spectrometer (Table 1.9). This can be explained by the occurrence of two competing processes.

One process involves the interaction of the sample with residual water vapours in the vacuum system of the mass spectrometer, which causes protonation of isotopologues and increases δ13C. The other process is due to deviation (relative to the baseline values) of the geometric parameters of the gas dual inlet system, which can lead to a decrease in the values of the isotope ratios in the CO2 sample.

In carrying out the isotope analysis of the carbon of CO2, when the signals of the sample and standard gas are different (Isample ≤ Istand), the

Table 1.7. The results of measurements of carbon isotopic composition of the CO2 sample as a function of on the intensity of ion current I(m/z 44) (with admission of helium in the ion source of the mass spectrometer, P = 1.8 × 10–6 mbar), Isample = Istand; δ

13Csample = δ13Cstand = –50.14‰

Helium Signal I

(m/z 44), VSignal I(m/z 18), mV / detector 3 Δ13С, ‰ S, ‰

No 3.8 490 0.01 0.02

Yes 3.8 2920 0.02 0.03Yes 0.586 1791 0.04 0.05Yes 0.265 1183 0.20 0.12Yes 0.057 940 1.65 0.40

Δ – the difference between the isotope ratios of the standard gas and sample, S – standard deviation.


difference in the isotope ratios exceeds | Δ13C |> 0.2 already at a signal I(m/z 44) = 870 V (see Table 1.9).

The data obtained by measuring the isotopic ratios of carbon of the CO2 sample and the change of the standard deviation, depending on the magnitude of the signal of the sample in the gas dual inlet mode and with no helium inlet, are shown in Figs. 1.21 and 1.22. The results of measurements of the carbon isotope composition of CO2 in the sample depending on the value of the signal in the standard gas dual inlet conditions and in the absence of inlet of helium are shown in Fig. 1.23. As can be seen from Fig. 1.23, in the absence of inlet of helium into the ion source the measured isotopic composition of

Table 1.9. The results of measurements of the carbon isotopic composition of the CO2 sample as a function of the ion current I(m/z 44) (with admission of helium into the ion source of the mass spectrometer, P = 1.8 · 10–6 mbar), Isample ≤ Istand = 3960 mV; δ13Csample = –3.64‰, δ13Cstand= –50.14‰

No. Signal of sample I(m/z 44), mV δ13C,‰ Δ13C,‰

(δ1– δi)S, ‰

1 3806 −3.53 0 0.062 2148 −3.40 −0.13 0.063 870 −3.33 −0.20 0.084 390 −3.22 −0.31 0.085 175 −4.68 1.15 0.206 77 −7.24 3.71 0.67

Δ = δ1–δ i is the difference of the isotope ratios between the standard gas and the sample.

Table 1.8. The results of measurements of carbon isotopic composition of the CO2 sample as a function of the ion current I (m/z 44) (without helium inlet into the ion source of the mass spectrometer), Isample = Istand; δ

13Csample = δ13Cstand = –50.14‰

Number SignalI(m/z 44), V Δ13C,% S, ‰

1 4.0 0.05 0.192 2.2 0.06 0.083 1.0 0.04 0.084 0.57 0.01 0.105 0.50 −0.11 0.14

6 0.30 −0.19 0.137 0.26 0.15 0.19

Δ is the difference between the isotope ratios between the standard gas and the sample.


I (m/z 44), mV

Fig. 1.21. The dependence of the results of measurements of the isotopic composition of carbon CO2 on the ion current I (m/z 44) (for admission of helium into the ion source of the mass spectrometer, P = 1.8·10–6 mbar), δ13Csample = δ13Cstand = –50.14‰, Isample = Istand (▲); δ13Csample = –3.64‰, δ13Cstand = –50.14‰, Isample < Istand (Table 1.9) (•).

I (m/z 44), mV

Fig. 1.22. The dependence of the standard deviation of measurement results of the isotopic composition of carbon from the CO2 sample on the ion current I(m/z 44) (with the admission of helium into the ion source of the mass spectrometer, P = 1.8·10–6 mbar), δ13Csample = δ13Cstand = –50.14‰, Isampl = Istand (▲); δ13Csample= –3.64‰, δ13Cstand = –50.14‰, Isamplle ≤ Istand (Table 1.9) (•).


carbon practically does not depend on the analysis conditions: Isample< Istand or Isample = Istand, where δ13Csample = –3.64‰, δ13Cstand = –50.14‰.

However, comparing the results of isotopic analysis of carbon of CO2, presented in Tables 1.7 and 1.10, it follows that if Isample ≤ Istand for the sample with the low value of δ13Cstand = –50.14‰ there is a stronger dependence of the results of isotopic analysis on the value of the analytical signal in the absence of inlet of helium into the ion source. This dependence is also shown in Fig. 1.23.

I (m/z 44), mV

Fig. 1.23. The dependence of the results of measurements of carbon isotope composition of CO2 on the ion current I(m/z 44) (without helium inlet into the ion source of the mass spectrometer), δ = δ13Csample = δ13Cstand = –50.14‰, Isample ≤ Istand (▲); δ13Csample = –3.64‰, Δ13Cstand = –50.14‰, Isample = Istand = (•), δ = –3.64‰, δ13Cstand = –50.14‰, Isample ≤ Istand (Table 10) (▪).

Number Signal of sample I(m/z 44) mV, δ13 C, ‰

Δ13C,‰ (δ1–δi)

S, ‰

1 3284 −3.72 0 0.012 2189 −3.47 −0.25 0.033 1381 −3.52 −0.20 0.044 912 −3.50 −0.22 0.075 495 −3.71 −0.01 0.086 210 −5.07 1.35 0.157 95 −7.11 3.38 0.29

Table 1.10. The results of measurements of carbon isotopic composition of the CO2 sample as a function of the value of the ion current I(m/z 44) (without helium inlet into the ion source of the mass spectrometer), Isample ≤ Istand = 3430 mV; δ13Csample = 13Cstand = –50.14‰


Fig. 1.24. The calibration graph of the dependence of the measured isotope composition of carbon of standard samples on the true value δ13C (standard) using the elemental analyzer, coupled with the isotope mass spectrometer (EA-IRMS).

Apparently, the presence of helium plays the role of a buffer reducing the number of collisions of gas molecules of the sample with the cathode of the ion source and thereby reduces the number of secondary reactions in the ion source of the mass spectrometer. At a higher value of δ13C of the sample the dependence of Δ13C on the signal amplitude decreases.

The elemental analyzer–isotope mass spectrometer system (EA-IRMS). Figure 1.24 shows the calibration graph, constructed for 5 standard samples of different composition (NBS 22 – oil, Oil-V – oil, USGS 24 – graphite, IAEA-C-3 – cellulose, IAEA-CH-7 – sucrose). This calibration curve is a straight line with a correlation coefficient equal to 0.999. In this way, regardless of the sample material, with its complete combustion in the oxidation reactor of the elemental analyzer, with proper adjustment of the ion source and in the absence of isotopic impurities the mass spectrometer systematic error in measuring the isotopic composition of the carbon of the analyzed substance is non-existent.

Table 1.11 shows the dimensions of the fused silica capillaries through which the gases are fed into the ion source of the mass spectrometer. Table 1.11 shows that the sample and the standard gas are fed into the mass spectrometer through the interfaces ConFlo Combustion 3 and 3 via the capillaries of different diameter. The interface ConFlo 2 uses a capillary with a length of about 1 m. One


end of the capillary is at about atmospheric pressure, and the other at a pressure of 1.8·10–6 mbar. Since the diameters of the quartz capillaries in the interfaces Combustion 3 and ConFlo 3 are different, gas streams that pass through them will also be substantially different.

Table. 1.12 shows the results of the comparison of the isotopic composition of carbon of oil-V, obtained at different ratios of the signal of the Isample and the standard gas Istand. As can be seen from Table 1.12, the measured isotopic composition of the oil changes with a decrease in the signal of the sample. At the signal smaller than 653 mV, the results of the isotopic analysis of δ13C (1) and δ13C (2), obtained at the ratio between the signals of Isample = Istand and Isample ≤ Istand = 3, respectively, start to differ. At Isample ≤ Istand the change of the observed values of δ13C is greater than at the equal signals Isample = Istand.

Thus, during isotopic analysis is necessary to strive to ensure that the signal of the standard gas was approximately equal to the signal of the sample. Full equality of the signals cannot be achieved, since

Table 1.11. The dimensions of the capillaries to supply a standard gas sample into the ion source of the isotope mass spectrometer

The mass spectrometer Interface Capillary diameter, mm

DELTA Plus Combustion 3 0.05 – standard gas0.10 – sample

DELTA Plus ConFlo 2 0.10 – sample or standard gas

DELTA Plus XP ConFlo 3 0.05 – standard gas0.10 – sample

Table 1.12. The results of measurements of the isotopic composition of carbon in oil in dependence on the analysis conditions by the EA-IRMS method: δ13 C(1) obtained at Isample = Istand; δ

13 C (2) – at Isample ≤ Istand = 3 V

Number Signal of sample I(m/z 44), mV δ13C (1), ‰ δ13C(2), ‰ Δ13C,‰

1 3031 −28.46 −28.46 02 2426 −28.05 −28.05 03 2438 −27.92 −27.92 04 1138 −27.16 −27.16 05 724 −28.53 −28.53 06 653 −27.42 −26.68 0.747 521 −29.43 −29.63 −0.208 227 −25.82 −24.22 1.609 115 −18.49 −17.98 0.51

Δ13C = δ13C(2) – δ13C (1)

–


it is technically difficult to select the charge of the sample with the required mass with the accuracy up to 1 µg.

The following Table 1.13 shows the need for correct selection of the boundaries of the analytical peak, as the chosen width of the peak (at the base) controls the correct determination of the isotopic composition. When extending the boundaries of the defined peak the measured isotopic composition tends to a value corresponding to a given isotope ratio. Therefore, the correct definition of the boundaries of the peak is a necessary procedure for the mathematical treatment of close standing peaks. The partial superposition of the peaks leads to an incorrect determination of the limits of the peak and, consequently, to the inaccurate determination of the isotopic composition, which will be shown below.

Table 1.14 shows that the results of measurements of the isotopic composition of oil change with increasing CO2 background, above which there is the peak of CO2 formed during the combustion of oil. It is believed that this effect is due to the presence of water vapour in the composition of the CO2 gas from which the background under the analytical peak forms. Figure 1.25 shows a plot of the dependence of the water vapour signals I(m/z 18) (detector 3) on the value of the signal I(m/z 44) formed by the admission of the CO2 standard gas via ConFlo 3 into the ion source of the mass spectrometer. This graph is a straight line. The increase of the inlet flow of the standard gas increases

Table 1.13. The dependence of the results of measurements of the isotopic composition of carbon of oil on the width of the region of processing the peak (under standard mathematical processing of the spectrum: the peak width 80 s, δ13C = –30.0‰)

Width of peak, s 11 16 27 47 90 130 154 163

Δ13C,‰ –30.7 –31.5 –32.0 –30.5 –29.7 –29.9 –30.0 –30.0

Table 1.14. The dependence of the results of measurements of the isotopic composition of carbon of oil on the value of I(m/z 44) of CO2 background under the analytical peak

Background I(m/z 44), mV 11 181 547 1189 3300 5900

δ13С,‰ −28.12 −28.25 −28.58 −28.72 −29.17 −29.99Δ13С,‰ 0 0.13 0.46 0.60 1.05 1.87

Δ = δ1–δ i is the difference in isotopic ratios between the first and subsequent determinations


the signal I(m/z 44) and the signal I(m/z 18), i.e., increasing the partial pressure of vapour water in the ion source of the mass spectrometer.

Figures 1.26 and 1.27 show the variation of measurement results of the carbon isotopic composition of a sample of Baikal sediment BIL-1 and oxygen of a codeine sample, depending on the value of the signal I(m/z 44) and I(m/z 28), respectively.

As can be seen from Fig. 1.26, the results of measurements of the isotopic composition of carbon begin to change significantly (Δ13C = 0.2‰) when the signal is less than 1000–1500 mV, and the results of measurements of the isotopic composition of oxygen (Fig. 1.27) – when the value of the signal is less than 2000 mV.

The isotopic composition of oxygen was determined by decompos-ition of organic matter with pyrolyzer TC/EA at 1450°C, and the isotopic composition of carbon – in combustion using the elemental analyzer EA. Thus, the area of analytical signals ΔI = 0 – IΔ (IΔ is the boundary of linearity), in which a significant change in the isotopic composition of light elements Δ13C takes place, depends on the conditions of analysis and on the element to be determined.

The gas chromatograph – isotope mass spectrometer system (GC-C-IRMS). The results of determination of the isotopic composition of carbon of the hydrocarbon gases and CO2 using a mass spectrometer DELTA Plus and the interface Combustion 3 showed that the isotopic composition of carbon remains constant in the range of the analyti-

I (m/z 44), mV

Fig. 1.25. The graph of the dependence of the value of the water vapour signal I(m/z 18) (detector 3) on the signal I(m/z 44) formed by the admission of the standard CO2 gas through ConFlo 3 into the ion source of the mass spectrometer.

I(m

/z 1

8), m

V


I (m/z 44), mVFig. 1.26. The dependence of the results of measurements of the isotopic composition of carbon (δ13C = –27.42‰) of the standard sample of Baikalian sediment BIL-1 on the value of the signal I (m/z 44).

I (m/z 44), mVFig. 1.27. The dependence of the results of measurements of the isotopic composition of oxygen of the sample of codeine (δ18O = 24.108‰) of the value of the signal I(m/z 28): • – Isample = Istand; ▲ – Isample < Istand.

cal signal I(m/z 44) from 0.5 to 9 V. At a stronger signal I(m 44) the isotopic composition of carbon is distorted and the amplifier goes into the saturation region. The maximum allowable signal is 13 V. At lower values of the signal I(m/z 44) the results of isotopic analysis are influ-ence by the processes occurring in the ion source. In addition, it was shown that for a small split injection of the gas flow (4:1) and using a syringe with a small volume (10 microliters, ILS, Germany, the inner


diameter of the needles 0.13 mm) the carbon isotopic composition of the analyzed gases is poorly reproduced and can be changed by 3‰. A possible explanation for this phenomenon is the isotope fractionation in the fine needle of the syringe and fractionation with a small divi-sion of the gas stream.

The study of isotope effects in the ON–OFF mode. As mentioned earlier, the ON–OFF mode is a mode of multiple pulse inlet of the standard gas through an interface into the ion source of the mass spec-trometer for some time, usually 20 s (see Fig. 1.16).

Using the ON–OFF-1 mode investigations were conducted into the effect inlet of other gases on the results of measurements of the isotopic composition of the standard gas, the peaks of which are formed at the joint admission of standard and secondart (impurity) gases. The results of these studies are shown in Table 1.15.

As shown in Table 1.15, the effect of nitrogen on the measurement results of the carbon isotopic composition δ13C is negligible. The intensities of the peaks I(m/z 44), I(m/z 45) and I(m/z 46) decreased as a result of ion scattering on the nitrogen molecules. The effect of CO2 on the measurement results of the nitrogen isotopic composition δ15N is more significant and can reach 30–60‰. The intensities of peaks I(m/z 28), I(m/z 29) slightly increase. Perhaps this is due to the formation in the ion source of CO+ ions by the reaction

CO2 → CO+ + O–.

As a result, the Faraday cylinders simultaneously record two various ions with the same mass, CO+ and N+

2.Figure 1.28 shows the peak of the standard gas in the form of a

rectangle located on the drop-down background of the preceding larger peak. The start of the leading edge of the standard peak was elevated by 800 mV relative to the trailing edge. Consequently, the background under the standard peak was incorrectly determined and the improper carbon isotope ratio δ13C = –49.19‰ was obtained instead of the known values of –49.88‰.

Table 1.15. Influence of impurity gases in the ON-OFF-1 mode on the results of isotopic measurements

The main N2 gas Impurity gas CO2

Δ15N, ‰ The main CO2 gas Impurity

N2 gas,Δ13C,

%I(m/z 28),

VI(m/z 29),

VI(m/z 44),

VI(m/z 44),

VI(m/z 45),

VI(m/z 46),

VI(m/z 28),

V

3.6 2.7 0 — 2.99 3.44 4.19 0 —3.8 2.9 3.0 30–

602.92 3.36 4.08 3.6 0.13


Thus, for a correct determination of the isotopic ratio of the peak we must have a sufficiently smooth background under the analytical peak. In addition, the results of measurements of the isotopic ratio are influenced by the location of the background which is used to calculate the area of the analytical peak, as shown in Fig. 1.29 (oil, δ13C = –28.6‰). In this case, the background could not be determined accurately due to its complex form. The applied program states that the area of the background signal under the peak is equal to the area of a rectangle, one side of which is equal to the width of the peak, while the other one to the background value, determined on a flat portion of the spectrum before (or after) the analytical peak [30, 31].

If the calculations are carried out using the straight background after the second peak from the standard gas CO 2, we obtain δ13C = –36.23‰. If calculations are carried out using the background value immediately before the analytical peak, then it will be δ13C = –82.50‰. And if we use the background value immediately the analytical peak, then it will be δ13C = –29.27‰.

Thus, the correct isotope ratio δ13C for oil could not be obtained because of the complex background under the analytical peak.

Time, s

Inte

nsity

, V

R

atio

Fig. 1.28. Chromatogram registered at ionic current m/z 44, 45, 46 in the EA-IRMS mode.


1. The isotopic mass spectrometer DELTAPlus. Operating regime: GC-C-IRMS and EA-IRMS. Figures 1.30 and 1.31 show the variation of the results of measurements of the carbon isotopic composition of standard gas CO2 (δ

13C = –50.260‰) depending on the analytical signal.In the GC-C-IRMS mode, where the standard CO2 gas is supplied

through the Combustion 3 interface, the change of the measured isotopic composition of carbon, equal to Δ13C = 0.2‰, was achieved at the signal I(m/z 44) = 400–600 mV. At the same time, when using EA-IRMS and the supply of CO2 standard gas through the interface ConFlo 2 the value Δ13C = 0.2‰ was achieved at the signal I(m/z 44) = 1400–1800 mV. Such significant differences in the values of the signals are associated with different design of interfaces (Table 1.11), and, therefore, with significantly different background of the water in the ion source of the mass spectrometer to connect these interfaces and additional equipment for sample preparation.

2. The isotope mass spectrometer DELTA Plus XP. Operating conditions: TC/EA-IRMS. Figures 1.32–1.35 show the changes of the

Time, s

Inte

nsity

, V

R

atio

Fig. 1.29. Chromatogram of oil recorded at ionic current with m/z 44, 45, 46 in the EA-IRMS mode.


results of measuring the isotopic ratio of carbon, nitrogen and oxygen in the standard gases depending on the value of the signal of the analytical peak I(m/z 44), I(m/z 28), respectively. These graphs show that the change in the results of measurements of the isotopic ratio

I (m/z 44), V

Fig. 1.30. The dependence of the results of measurements of the isotopic composition of carbon of the CO2 standard gas (δ13C = –50.26‰) on the strength of the analytical signal (CO2 gas was admitted into the ion source of the mass spectrometer through the interface Combustion 3).

Fig. 1.31. The dependence of the results of measurements of the isotopic composition of carbon of the CO2 standard gas (δ13C = –50.26‰) on the strength of the analytical signal (CO2 gas was admitted into the ion source of the mass spectrometer through the interface ConFlo 2).

I (m/z 44), V


equal to Δ13C = Δ15N = Δ18O = 0.2‰, is achieved when the value of the signal is less than the 1–1.2 V for carbon, 0.6 V for nitrogen and 1.2 V for oxygen. It also shows that a decrease of the signal leads to a change of the measurement results, corresponding to the increase in the magnitude of the isotopic ratio of carbon, oxygen, and to a

I (m/z 44), V

Fig. 1.32. The dependence of the results of measurements of the isotopic ratio of carbon of the CO2 gas standard on the strength of the analytical signal I(m/z 44); interface ConFlo 3, the amplitude of the signal is variable.

I (m/z 28), V

Fig. 1.33. The dependence of the results of measurements of the isotopic ratio of nitrogen of the N2 standard gas on the strength of the analytical signal I(m/z 28); interface ConFlo 3, the amplitude of the signal is variable.


reduction of this value for nitrogen. This change of the signals can be explained by the presence of water vapour in the ion source of the mass spectrometer, resulting in ion–molecule reactions at the cathode surface and near it [23, 29, 30]:

CO2 + H2O → CO2H+ + OH–;

CO + H2O → COH+ + OH–.

Since pure nitrogen contains almost no traces of water vapour, the surface of the cathode may be the area for other ion–molecule reactions leading to a change in the results of measurements of isotopic relations.

Figures 1.36 and 1.37 show that the change in the emission current of these electrons Iemis in the ion source leads to expansion the area in which the results of measurements of the carbon isotope composition of CO2 begin to depend on the strength of the analytical signal I(m/z 44). For an emission current of Iemis = 1.5 mA Δ13C is higher than 1‰ at I(m/z 44) < 1, and at an electron emission current Iemis = 1.0 mA is higher than this value of Δ13C already at the signal I(m/z 44) < 2.4 V. Similar changes in the results of measurements of the isotopic composition of carbon can also be observed in contamination of the ion optics elements of the mass spectrometer or at changes of their potentials.

The results of these studies are presented in Table 1.16. The table shows that in the dual inlet mode (DELTA Plus) the additional supply of helium via Combustion 3 leads to an increase of signal ΔI = (0 – IΔ) V corresponding to changes of the isotopic composition of the standard gas by 0.2‰ and more. In isotopic analysis in the EA-IRMS mode, where the flow of helium through the EA is significantly larger, the area ΔI becomes even wider, 1400–1800 mV. A similar situation exists for the device DELTA Plus XP. Comparison with the results of measurements at admission of standard gas N2, where the area ΔI is much smaller, shows the value of 600 mV. In this case the gas does not contain water vapour. This confirms the strong effect of water vapour on the width of the region ΔI.

Study of the completeness of combustion of difficult to oxidise compounds

The oxidation of various organic compounds in the elemental analyzer is carried out in an inert gas atmosphere with a certain amount of oxygen [32]. The limited oxygen content in the combustion zone of the sample is offset by the use of catalysts that produce oxygen when heated. However, the burning of difficult to oxidize polycyclic, aromatic


I (m/z 28), V

Fig. 1.35. The dependence of the results of measurements of the oxygen isotope ratio of CO standard gas on the strength of the analytical signal I(m/z 44), Interface ConFlo 3, the amplitude of the signal is constant: • – the results of measurements of the isotopic composition of oxygen, ▲– the standard deviation.

I (m/z 28), V

Fig. 1.34. The dependence of the results of measurements of the oxygen isotope ratio of the CO standard gas on the strength of the analytical signal I(m/z 44). Interface ConFlo 3, the amplitude of the signal is variable.


Fig. 1.36. The dependence of the results of measurements of the carbon isotope composition of CO2 on the strength of the analytical signal I(m/z 44) for emission current Iemis = 1.0 mA.

I (m/z 44), mV

Fig. 1.37. The dependence of the results of measurements of the carbon isotope composition of CO2 on the strength of the analytical signal I(m/z 44) for emission current Iemis = 1.5 mA.

I (m/z 44), mV


and heterocyclic compounds may cause difficulties relating to the completeness of their oxidation [33]. The completeness of combustion of the sample determines the accuracy of determination of the isotopic composition of light elements [31].

In order to select the best conditions for the oxidation of these compounds, a series of well-known catalysts, listed in Table 1.17, was tested. The combustion sample was represented by the asphaltene fraction of oil, containing polyaromatic compounds.

The principle of operation of the elemental analyzer EA 1110 (CE Instruments) is shown in Fig. 1.38. A sample of an organic compound, packaged in a tin capsule, is subjected to oxidative decomposition in the reactor. The resulting gaseous products of decomposition are oxidized completely in the additional oxidation zone, which contains various catalysts: Cr2O3, silver coated Co3O4, CuO. In the case of incomplete oxidative decomposition of complex polycyclic organic substances energetic oxidizers, easily producing oxygen, and catalysts are added to the tin container. Then, the gaseous products in the stream of helium (100 ml/min) passed through the reduction zone heated to a temperature of 650°C. This zone absorbs the excess of oxygen introduced into the

Table 1.16. The effect of the strength of the analytical signal I on the change of the results of measurements of the isotopic composition of gases Δ at various modes of feeding the gas sample into the ion source of the mass spectrometer and the -sample preparation methods

Mass spectrometer

Instrument configuration, operation Δ,‰ IΔ, mV

DELTA Plus

Dual inlet, δ13Csample≠ δ13Cstand,

Isample = Istand 13С: 0,2 220

Dual inlet, δ13Csample= δ13Cstand,

Isample = Istand, He inlet 13С: 0,2 270

Dual inlet, δ13Csample≠ δ13Cstand,

Isample ≠ Istand, He inlet 13С: 0,2 390–870

EA-IRMS, oil 13С: 0,2 1000–2000GC-C-IRMS, ON–OFF 13С: 0,2 400–600EA-IRMS, ON–OFF 13С: 0.3–0.5 1400–1800

DELTA Plus XP

EA-IRMS, Baikal sediment 1000–1500TC/EA, ON–OFF 2000TC/EA, ON–OFF 1000–1200TC/EA, ON–OFF 1200TC/EA, ON–OFF 600


Quartz wool

Quartz wool

Quartz wool

Quartz wool

Quartz wool

Quartz wool

Quartz wool

Chromium oxide Copper

Copper oxide

Copper oxide

Water trap

Chromatographic column

Ag/oxideCo

10 mm

120 mm

10 mm

50 mm

40 mm

10 mm

50 mm

10 mm

280 mm

10 mm

50 mm

40 mm

Autosampler

O2He

IRMS

Fig. 1.38. Scheme of the elemental analyzer and the filling of the catalyst with oxidative and reductive reactors.

Asphaltene samples with additional oxidizer charge

δ13CPDB, ‰

Standard deviation,

‰

Number of

analysesWithout addition of oxidizers; depleted reactor –29.03 0.35 3

Without addition of oxidizers; new reactor –28.48 0.24 8

Depleted reactor with added oxidizer

CeO2 –28.72 0.19 9Cr2O3 –28.55 0.12 5Co3O4 –28.39 0.26 3CuO –28.31 0.26 3

Table 1.17. Results of the determination of carbon isotope composition of samples of asphaltenes with additional oxidants

reactor, and nitrogen oxides are reduced to molecular nitrogen. The temperature of the oxidation zone is 1020°C.

A charge of asphaltenes was sufficient to obtain the analytical signal stronger than 1 V, and was 0.2 mg. Oxide catalysts – pure reagents


CeO2 of the Aldrich company, CuO, Cr2O3, Co3O4 of Thermo Fisher Scientific were packed with the sample in the tin container and their oxidative properties were studied.

It was found that 1 mg of oxidants was sufficient for complete combustion of the asphaltenes using the depleted oxidative reactor. Without additional oxidants, the measured concentration 13C in the asphaltene in samples was less than in the case of the reactor with fresh reactants. The results of determination of the isotopic composition of carbon are shown in Table 1.17. It is evident that the use of additional oxidants, together with the sample, yields the correct value of the isotopic ratio of carbon. However, the best accuracy and reproducibility were obtained with the use of cerium and chromium oxides as additional oxidants. Therefore, these catalysts have been recommended as the best to ensure complete combustion of difficult to oxidize compounds.

Developing a new method of sample preparation based on solid electrolytes for isotope mass spectrometric analysis

Oxidation of organic compounds with a solid electrolyte based on zirconium dioxide. At the present time special attention is paid to the study of high-temperature electrochemical reactions of gases in systems with solid electrolytes. This is associated with the development of fuel cells, gas electrolyzers, electrochemical pumps and gas sensors [34–36]. Traditionally, electrochemistry studies the reactions of anodic and cathodic transformations of oxygen, the decomposition of water, as well as the oxidation of hydrogen and carbon monoxide [34]. In recent years there has been an increased interest in the possibility of using electrochemical devices with oxygen-conducting solid electrolytes as catalytic reactors [37]. This allows, in contrast to the case of electrochemical systems with solutions, to carry out a sufficiently large number of chemical reactions between gaseous reagents. Solid-electrolyte ceramics has a high mechanical strength and chemical resistance, and can work in the temperature range 500-1000°C. Amperometric sensors based on this ceramic material possess qualities such as durability, the linear dependence of the signal on the concentration, the high accuracy of analysis [35, 38]. The electrodes for sensing devices often use porous platinum coatings with high catalytic activity in the working temperature range [39].

The accuracy of the results of isotopic analysis considerably depends on the properties of the oxidation reactor, capable of oxidizing difficult to oxidize compounds. Incomplete combustion leads to a distortion of


the isotopic data. This is especially important for light hydrocarbons, containing in their composition no more than four carbon atoms. For their complete oxidation it is necessary to provide a high temperature and a sufficient amount of oxygen. Therefore, we attempted to create a simple and convenient oxidation reactor for complete oxidation of organic compounds.

The experiments were performed with the setup shown in Fig. 1.5. The developed solid-electrolyte oxidation reactor had two-electrode and three-electrode connection circuits. The scheme of the two-electrode high-temperature solid-electrolyte reactor is shown in Fig. 1.6, and the three-electrode reactor is shown in Fig. 1.39.

Experiments were carried out with a standard gas mixture in argon, which contained the following concentrations of components: nitrogen 14.8 vol.%, CO2 6.1vol.%, CH4 9.5 vol.%, C2H4 6.8 vol.%, C3H8 5.6 vol%., n-C4H10 3.0 vol.%, iso-C4H10 7.0 vol.%, and other gases.

The conditions for gas chromatographic separation of the gas standard mixtures were as follows:

Column PoraPlot Q (25 m×0.32 mm);

carrier gas helium 60;helium flow 2 ml/min;injector temperature 100°C;heating rate 5°C/min;final temperature 200°C.The investigated gas mixture is injected into a gas chromatograph

with a syringe (sample volume 100 µl, dilution 20:1). In the capillary column PoraPlot Q the introduced gas mixture is divided into separate components that consistently pass through standard oxidation reactor and the solid-electrolyte reactor. The resulting oxidation products of all components are gaseous, therefore they freely leave the zone of the

FurnacePotentiostat

T °C

Helium flow with oxidation

products

ThermocoupleYSZ ceramics

Steel capillary

Helium flow with gas

GlassPt-electrodes

RE AE WE

Fig. 1.39. Scheme of the three-electrode solid-electrolyte reactor: WE – the working electrode, RE – reference electrode, CE – counter electrode.

CE


reactor and are transported in a stream of helium into the reduction reactor, and then pass a water trap and are fed into the ion source of the isotopic mass spectrometer to measure the isotopic composition of carbon in CO2.

If the partial pressures of oxygen in the volumes separated by the solid-electrolyte volumes are different, then the oxygen ions diffuse through the solid electrolyte, and the corresponding steady-steady stationary diffusive flux jd (s

–1 cm–2) in the first approximation can be described by the equation

jd = –D grad cO, (1.4)

where D is the diffusion coefficient, cm2 s–1, cO is the oxygen concentration, cm–3. Diffusion of oxygen ions leads to the accumulation of the positive charge on the outer electrode and the negative charge on the inner electrode and the potential difference of these electrodes (E) is expressed by the Nernst equation:

E RTnF

pp

= lncathodeanode)

O

O

2

2

( )(

, (1.5)

where R is the universal gas constant, T is absolute temperature, n is the electronic equivalent of oxygen, F is the Faraday constant, pO2

is the partial pressure of oxygen at the cathode or the anode. When connecting these electrodes to the circuit a constant current flows through the circuit. When changing the external voltage U, applied to the electrodes of the solid-electrolyte reactor (SER) with the opposite sign with respect to E, it is possible to control (dose) the oxygen ion current flowing into a stream of the carrier gas. Oxidation of organic compounds entering into the reactor in a stream the carrier gas takes place on the surface of the inner electrode.

For example, the oxidation of methane will be accompanied by the following reaction:

CH4 + 4O2–→ CO2 + 2H2O + 8e–.

Similar equations can be written for other hydrocarbon gases. The scheme of processes occurring at the three-phase boundary (solid

electrolyte – platinum electrode – gaseous medium) of the three-electrode SER in the oxidation of hydrocarbon gases is shown in Fig. 1.40. The oxygen contained in air is adsorbed and then dissociated on the outer platinum electrode. The three-phase boundary, formed by the gaseous medium, the electrode and solid electrolyte, is characterized by the formation of oxygen ions O2–, which migrate through the crystal lattice of the solid electrolyte. At the three-phase boundary with a working


Auxiliary electrode 1

Air Reference electrode

Auxiliary electrode 2

Pt

O2– O2–

YSZ

Oads

Working electrode

CH4

H2 OCO2

He flow

Fig. 1.40. The scheme of processes occurring during the oxidation of hydrocarbon gases at the three-phase boundary of the three-electrode solid-electrolyte reactor.

Ioni

c ox

ygen

cur

rent

, μa

Voltage, mV

Fig. 1.41. The dependence of the oxygen ion current flowing in the two-electrode SER on the voltage applied to the electrodes of the SER.

platinum electrode, the oxygen ions give their charge and transform into adsorbed atoms, which can then be desorbed in the form of molecules. In addition, oxidation of hydrocarbon gases takes place at the three-phase boundary.

Figure 1.41 shows a plot of oxygen inlet into the helium stream in relation to the voltage applied to the electrodes of the solid-electrolyte reactor at a temperature of 900°C. In the section of the curve from –150 to 0 mV, the electric current flowing through the SER, varies from 240 to 0 µA. The nature of the curve is described in the first approximation by a linear function. The anode is the inner electrode of the SER along which the helium flow moves, and the cathode works in air (outer electrode) with a high content of oxygen.

At low current densities the anodic and cathodic polarization of the electrodes is negligible and, therefore, the slope of the curve describes

Counterelectrode 1

Counterelectrode 2

µA


the total active ohmic resistance of the SER (current leads, electrodes and the solid electrolyte). A change of the direction of the electric current flowing through the SER, dramatically increases the cathodic polarization related to the difficulty in transferring the residual oxygen from He (anodic polarization in this case can be neglected, since evolution of oxygen on the outer electrode in air takes place without difficulties). This is reflected in a break of the curve at –150 mV (in fact it is the electrochemical potential of oxygen in helium).

For small values of negative current the curve has the form close to a linear dependence, and the slope of the straight line characterizes the sum of the active resistance of SER and cathodic polarization of the inner electrode. Further, with increasing current density the curve must assume the character of the power law associated with the polarization processes on the electrodes.

Thus, by changing the voltage applied to the electrodes of the SER, it is possible to change the flow of oxygen coming into the stream of the carrier gas. Similarly, one can change the flow of oxygen coming into the flow of the carrier gas helium, when the potential of the working electrode in the three-electrode SER changes. Figure 1.42 shows this dependence, obtained at a temperature of 940°C. This dependence is a power function and can be described by the equation y = y0 + axb.

The oxygen ion current for the three-electrode SER is about 3–5 times more than that for the two-electrode SER for the same potential of the working electrode. This discrepancy in the magnitude of the oxygen ionic current can be attributed to the polarization of the working electrode, which prevents the movement of oxygen ions. When using

Ioni

c ox

ygen

cur

rent

, mA

Potential of the working electrode, mV

Fig. 1.42. The dependence of the oxygen ion current flowing in a three-electrode SER on the potential of the working electrode at a temperature of 940◦C.


the PS 8 potentiostat for a three-electrode reactor, the polarization of the working electrode is eliminated by introducing a counter electrode.

Figure 1.43 shows the dependence of the oxygen ion current (mass m/z 32), measured with a detector of the isotope mass spectrometer and converted to the voltage of the oxygen ionic current, passing through the SER, the magnitude of which is regulated by the voltage, applied to the electrodes of the reactor. This graph is a straight line. Based on this graph, we can calculate the amount of oxygen entering the ion source of the mass spectrometer in the absence of organic gases in the flow of the carrier gas.

With the introduction to the system of a sample of hydrocarbon gases the inner porous platinum electrode of the SER is the area on which the catalytic reaction of deep oxidation of organic compounds with the formation of carbon dioxide takes place. Measurement of the amplitude of the signal of ionic current of CO+

2 with the detector of the isotope mass spectrometer at the temperature of the SER of 950°C showed that the maximum signal amplitude for the two-electrode SER is achieved at the oxygen ionic current passing through the SER greater than 80 µA. The corresponding signal, detected on the Faraday cylinder, is I32 = 850 mV. In the isotopic analysis of the hydrocarbons the value of I32 should not be significantly higher than the obtained value as an excess of oxygen in the ion source of the mass spectrometer can lead to poor analytical performance of the method and reduce the life of the cathode.

The oxidation of the hydrocarbon gases is characterized by the linear dependence between the oxygen ionic current, passed through the solid electrolyte, and the magnitude of the ionic current of CO+

2 registered

I32, V

ISER, μA

Fig. 1.43. The dependence of the voltage produced by the oxygen ionic current at the output of the amplifier of the mass spectrometer I32 with m/z 32 on the oxygen ionic current ISER, passing through the solid electrolyte.

I32, V

ISER, µA


with the detector of the isotope mass spectrometer. Figure 1.44 shows such a linear dependence obtained in the oxidation of propane (three-electrode SER). Similar lines can be constructed for other hydrocarbon gases.

Thus, the amount of oxygen, consumed for oxidation of hydrocarbon gases, is proportional to the amount of the formed gas CO2. This means that the SER can be used to determine the content of compounds of organic gases in a mixture with the following formula [40]:

C VnFV

QM

s

= ⋅ ⋅4

100%, (1.6)

where C is the volumetric concentration of the compound in the gas sample, VM is the volume of one mole of gas under normal conditions, n is the number of oxygen molecules required to oxidize one molecule of the defined reductant, F is the Faraday constant, Vs is the sample volume, Q is the charge passed through the solid electrolyte.

Figure 1.45 shows the change in voltage at the output of the amplifier of the mass spectrometer detector when recording oxygen ions in the oxidation of hydrocarbon gases in a three-electrode solid-electrolyte reactor. Despite the different levels of oxygen in the stream of the carrier gas, the signal strengths of the solid-electrolyte detector, obtained during the oxidation of hydrocarbon gases (HCG), virtually do not change. This proves that the complete oxidation of the gases may be due to the oxygen located at the three-phase boundary [41] and the influence of gaseous oxygen or the oxygen adsorbed on the surface of the platinum electrode is negligible. The chromatogram of a mixture of

Fig. 1.44. The dependence of the voltage produced by the ion current of CO+2 at the

output of the amplifier of the isotope mass spectrometer on the oxygen ion current ISER, passing through the SER during the oxidation of propane.

ISER, mA

ICO2, V

ISER, mA

ICO2, V


Signal, mV

Signal, mV

Time, s

Time, s

a

b

Fig. 1.45. Changes of the voltage produced by the oxygen ionic current at the amplifier output of the mass spectrometer detector in oxidation of hydrocarbon gases at different values of the working electrode potential: a) –100 mV, b) –175 mV.

iso-C4H10

iso-C4H10

n-C4H10

n-C4H10


hydrocarbon gases, registered on the basis of the oxygen ionic current, transmitted through the solid electrolyte, is shown in Fig. 1.45.

For a correct measurement of the isotopic composition of the carbon of organic compounds it is necessary to ensure that oxygen is not fed into the flow of the carrier gas, as later stages it can interact with the carbon of the cathode of the ion source and lead to distortion of the δ13C values. On the basis of experimental data it was found that the concentration of oxygen in the stream of the carrier helium gas should not exceed 60 ppm.

The optimal mode of operation of the solid-electrolyte reactor is the regime that ensures complete oxidation of organic compounds at the three-phase boundary and at the same time almost does not allow oxygen to travel from the external medium into the flow of the carrier gas (Fig. 1.40). Based on these criteria, the experiments were conducted with the working electrode potential equal to φw = –175 mV, and the temperature of 940°C.

The completeness of oxidation of organic compounds in the reactor is confirmed by the following arguments:

1) the value of the analytical signal of the SER in the oxidation of organic compounds does not depend on the amount of oxygen in the stream of the carried gas (Fig. 1.45);

2) the measured isotopic composition of the carbon of organic compounds (within the error range), obtained by oxidation with the SER and the standard oxidation reactor, is the same.

Criterion 2 can be used to control the correctness of the standard oxidation reactor, which requires periodic recovery of the oxidizing capacity by reduction of copper oxide (Table 1.18).

Table 1.18 shows that the weakening of the oxidizing ability standard the reactor led to an error in the measurement of the isotopic ratio of carbon δ13C compared with the results obtained by use of teh SER and the reduced standard oxidation reactor. Reproducibility of measurements of δ13C is 0.3‰. In addition, the partial oxidation of the hydrocarbon gases under the constant analysis conditions led to a decrease in the amplitude of the analytical signals, particularly affecting the amplitude of the signal of ethylene (C2H4).

Studies have shown that the areas of signals, obtained during the oxidation of the hydrocarbon gases, except methane, depend only slightly on the temperature of the solid-electrolyte reactor in the temperature range from 820°C to 940°C. For methane, this dependence of the area of the signal on temperature is more pronounced (Fig. 1.46).

Lowering the reactor temperature below 900°C results in partial oxidation of methane. In this case the measured isotope ratio of the


The

area

of t

he o

xyge

n pe

ak, m

A •

s

Temperature, °C

Fig. 1.46. The dependence of the peak areas of oxygen passing through the solid-electrolyte reactor in the oxidation of methane on the temperature of the reactor.

Table 1.18. The amplitudes of the analytic signals of the components of the gas mixture for SER (I1, mA), for ‘depleted’ (I2, V) and reduced standard oxidation reactor (I3, V), as well as the values of isotope ratios of carbon δ13C using the appropriate reactor

Compound I1, mА δ13С1, ‰ I2, V δ13С2,‰ I3, V δ13С3, ‰CH4 0.40 −39.33 4.7 −38.50 5.8 −38.99C2H4 0.38 −32.97 0.9 −41.96 7.6 −33.21C3H8 0.47 −32.62 4.1 −32.51 6.6 −32.66iso-C4H10 0.57 −37.14 4.2 −38.36 6.5 −37.14n-C4H10 0.48 −34.12 3.7 −34.97 6.4 −34.93

carbon of methane changes slightly. This means that incomplete oxidation of methane in the solid-electrolyte reactor does not always lead to marked fractionation of carbon isotopes (Table 1.19).

For propane the fractionation of carbon isotopes begins at temperatures below 850°C, for butane at temperatures below 820°C, and in the oxidation of ethylene the fractionation is not observed in the whole temperature range from 820 to 940°C. Such a behaviour of carbon isotopes can be explained by the different sizes of gas molecules and the different mobility of these molecules in the porous electrode. On the basis of the experiments we selected the sufficiently high optimum temperature of operation of the SER, equal to 940°C, providing the complete oxidation of the organic compounds studied.

Figures 1.47 and 1.48 show the calibration curves, representing the dependence of the peak area of oxygen used for the oxidation of


the carbon of methane and iso-butane on the mass of carbon in these compounds in the sample.

The experiments showed that the peak areas of CO+2, formed during

the oxidation of hydrocarbon gases with a standard oxidation reactor and the SER, were equal in magnitude. In addition, it is known [20] that the standard oxidation reactor of the isotope mass spectrometer allows for complete oxidation of hydrocarbon gases at a temperature of 950°C. Consequently, in the linear region of the calibration graph for hydrocarbon gases (with a small amount of injected substance) the complete oxidation of the compounds in the sample using the SER takes place.

Figures 1.49 and 1.50 show the chromatogram obtained from the current of CO+

2 ions with masses m/e 44, 45, 46 in the oxidation of gaseous components of the mixture, and coulometric spectra obtained using the two-electrode (Fig. 1.51) and three-electrode (Fig. 1.52) detectors for the same gas sample. A comparison of these spectra shows

Table 1.19. The dependence of the measured carbon isotope ratio on the temperature of SER

T, oC13СPDB,‰

Methane Ethylene Propane iso-Butane n-Butane940 −39.81 −32.97 −32.70 −37.04 −34.92910 −39.58 −32.99 −32.40 −37.19 −34.67880 −39.54 −32.85 −32.12 −36.88 −34.71850 −39.36 −32.95 −26.62 −37.00 −34.63820 −39.30 −32.69 −21.56 −36.70 −34.17

S, mA • s

M, μg

Fig. 1.47. The dependence of the peak areas of oxygen going to the oxidation of carbon of methane on the mass of carbon oxidized in the SER (T = 940°C, φw = –175 mV).


that the solid-electrolyte reactor operating in the coulometric mode as a chromatographic detector with high sensitivity allows to determine the content of the organic gases in the sample and can be used as a chromatographic detector of organic gases.

A comparison of the chromatograms in Figs. 1.51 and 1.52 shows that the use of the three-electrode scheme of the SER leads to increased sensitivity of the method with a reduction of the background level. Introduction of the additional SER into the standard gas chromatographic-mass-spectrometric system had no effect on the form and the width of the chromatographic peaks due to the high temperature of the reactor and its small volume comparable to the standard volume of the reactor. Therefore, there is little need to reduce the inner diameter of the SER.

S, mA • s

M(C), μg

Fig. 1.48. The dependence of the peak areas of oxygen, used for the oxidation of carbon of iso-butane, on the mass of carbon oxidized in the SER (T = 940°C, φw = –175 mV).

Inte

nsity

, V

Time, s

iso-C4H10n-C4H10

Fig. 1.49. Chromatogram of a standard gas mixture, obtained for ion current CO2+ at m/z 44, 45, 46.


Inte

nsity

, V

Time, s

Fig. 1.50. Separated section of the chromatogram shown in Fig. 1.49.

ISER, mA

t, s

iso-C4H10 n-C4H10

Fig. 1.51. Chromatogram of the gas mixture obtained using the two-electrode circuit including SER ( = 970oC, V = 100 µl, split injection 20:1).

The results of isotopic analysis obtained in the two modes of oxidation of the sample (using the standard SER and the oxidation reactor) are presented in Table 1.20. A comparison of these data shows that for all gases, except CO2, the value of δ13C obtained using the two-electrode SER, was on average 0.4‰ more positive than that obtained using the standard reactor. In addition, for the SER the reproducibility (standard deviation) of isotopic analysis was better and ranged from 0.11 to 0.25‰.

The results of isotopic analysis obtained using the three-electrode SER and the standard oxidation reactor, were close to the value of


δ13CPDB (Table 1.20). This confirms the conclusion about the complete oxidation of hydrocarbon gases with the SER. The reproducibility of the results of isotopic analysis for the two methods did not differ and ranged from 0.13 to 0.36‰.

In conjunction with the isotopic analysis the SER can be used to control the purity of the carrier gas and determine the concentration of organic gases in the sample. The limit of detection of the developed solid electrolyte conversion oxygen-conducting detector (OCD) was evaluated using the following formula [42]:

Ioni

c pe

ak o

f oxy

gen,

mA

Time, s

iso-C4H10

n-C4H10

Fig. 1.52. Chromatogram of hydrocarbon gases, registered fo the oxygen ionic current passed through the solid electrolyte using a three-electrode circuit of the SER (T = 940oC, V = 100 µl, split injection 20:1, φ = –175 mV).

Table 1.20. Results of the determination of carbon isotope composition of hydrocarbon gases and CO2

Oxidation reactor CH4 CO2 C2H4 C3H8

iso-C4H10

n-C4H10

Standardδ13С, ‰ −38.73 −20.27 −32.88 −32.45 −37.04 −34.84

S, ‰ 0.31 0.22 0.26 0.13 0.21 0.31n 8 7 8 9 7 8

Solid electrolyte 2-electrode

δ13С, ‰ −38.29 −20.68 −32.47 −32.13 −36.71 −34.50S, ‰ 0.15 0.22 0.29 0.25 0.15 0.11

n 4 7 7 6 6 4

Solid electrolyte 3-electrode

δ13С, ‰ −39.00 −20.76 −32.75 −32.53 −36.65 −34.31S, ‰ 0.35 0.21 0.24 0.18 0.23 0.36

n 12 10 9 8 10 10


m xAmin ,=∆2 (1.7)

where mmin is the minimum determined amount of material, Δx is the noise level, A is the sensitivity of the detector. The minimum detectable amount of hydrocarbons was calculated from the resultant spectra (Figs. 1.51, 1.52). Comparison with the characteristics of a flame-ionization detector (FID) was performed using the same chromatograph and under the same analysis conditions. The results obtained are shown in Table 1.21.

Table 1.21 shows that the flame ionization detector has better analytical characteristics than the developed chromatographic detectors based on the solid electrolyte. However, studies have shown that these characteristics can be significantly improved.

The calibration curves were used to calculate the sensitivity of the chromatographic method with the solid-electrolyte detector. It was found that the sensitivity of the method using three-electrode solid-electrolyte detector is about 3–5 times higher than the sensitivity of the method with two-electrode solid-electrolyte detector. The reproducibility of measurements of the area of the oxygen peaks consumed for oxidation of the hydrocarbon gases, is shown in Table 1.22. The table shows that the reproducibility of the results of chromatographic analysis of hydrocarbon gases using the three-electrode solid-electrolyte detector is much better than using the two-electrode detector.

The electrochemical decomposition of water using a solid electrolyte based on zirconium dioxide to determine the isotopic composition of hydrogen The results of measurements of the isotopic composition of the hydrogen of water are used widely in geochemistry, hydrology, to identify fake food, in physiology. The isotopic analysis of the hydrogen of water is carried out using different ways of its decomposition:

Table 1.21. Comparative characteristics of solid-electrolyte and flame-ionization detectors

ComponentThe limit of detection, g

OCD (2-electrode) OCD (3-electrode) FIDCH4 6.4 · 10−10 1.5 · 10−8 7.0 · 10−11

C2H6 8.2 · 10−10 1.4 · 10−8 4.8 · 10−11

C3H8 8.0 · 10−10 1.9 · 10−8 1.4 · 10−10

iso-C4H10 1.1 · 10−9 3.6 · 10−8 2.1 · 10−10

n-C4H10 6.41 · 10−10 1.8 · 10−8 1.3 · 10−10


electrolysis, pyrolysis, chemical interaction with zinc, uranium or chromium, isotopic equilibration of hydrogen with water. Benefits and disadvantages of these methods are presented in Table 1.23. The isotope ratios of hydrogen and oxygen of water are measured in the double inlet gas mode or in a continuous flow.

Despite the large number of methods for determining the isotopic composition of hydrogen in water, the industry produces only one device HD device (Thermo Fisher Scientific) whose principle of operation is based on the decomposition of water during its interaction with a chromium powder.

We have developed a new simple analytical tool for sample preparation for the mass spectrometric isotopic determination of hydrogen in the gas dual inlet mode (see Fig. 1.8), based on the principle of electrochemical decomposition of water on the surface of a platinum electrode with subsequent excretion of oxygen from the reaction zone into the surrounding medium through the wall of the solid electrolyte reactor, which has oxygen-ionic conductivity at elevated temperatures [34].

To determine the hydrogen isotopic composition of the analyzed water a calibration curve was constructed according to international standards for water with the known isotopic composition of hydrogen using samples IAEA-OH-4 with the isotopic composition δD = –109.4 ± 1.4‰, GISP with δD = –189.5 ± 1.2‰, SLAP with δD = –428.0‰. The calibration graph is a straight line y = 0.899x – 69.1. Using this chart, we can determine the hydrogen isotopic composition of water with the unknown isotopic composition. The duration analysis is 10–15 min. To eliminate the memory effect in the transition from analysis of one sample to analysis of another sample it is first necessary to carry out experiments with the introduction of a water sample without measuring the isotopic composition of hydrogen. The reproducibility of measurements of the isotopic composition hydrogen is 0.5‰ (n = 7).

The shape of the curve of decomposition of water using the high-temperature solid-electrolyte reactor based on zirconium dioxide is

Table 1.22. Reproducibility of result (Sr) of chromatographic analysis of hydrocarbon gases using conversion solid-electrolyte detector

Detector CH4 C2H4 C3H8 iso-C4H10 n-C4H10

Solid electrolyte, Sr, 0.10 0.32 0.32 0.25 0.302-electrode 6 6 9 7 7Solid electrolyte, Sr, 0.11 0.12 0.15 0.12 0.193-electrode n 10 13 9 8 9


shown in Fig. 1.53. Since the ionic current of oxygen passing through the electrolyte corresponds to the number of decomposed water molecules in unit time, then the area under the curve is proportional to the total number of decomposed molecules. Based on these curves it is possible to calculate the degree of decomposition of water during the analysis, which is equal to 90%. It is shown that in the process of decomposition of water there is no isotopic fractionation. A trap with liquid nitrogen was used to prevent penetration of residual water vapour into the mass spectrometer.

An inventor’s certificate (No. 2003137448) was granted for the design of the instrument. Currently, the upgraded commercial version of the device, with improved analytical performance and high reliability, is being developed.

Thus, i t was easy to set up easy to manufacture uni t for decomposition of water, allowing to obtain good reproducibility of the results of isotope analysis, which has a small energy requirement, electrical safety, does not require chemical reagents for its work, and allows to carry out continuous monitoring of the amount of decomposed

No. Method Advantages Shortcomings Literature

1 Electrolysis Error 0.2‰

Complete dissociation of water requiredAnalysis time 10 minSample volume 15–20 µl

[43]

2 Pyrolysis

Sample volume 0.5–1 µl,Analysis time several minutes

Memory effectHigh temperature 1400oCError 2–3‰

[44][45]

3Chemical decomposition using Cr

Error 1‰

Toxicity of reagentsHigh price of reactor (100 analyses)Analysis time of sample 7 min

[46]

4 Equilibration

Error 0.8‰No chemical reagentsNo memory effect

Thermostat is requiredSample volume 1–5 mlAnalysis time 1 h

[47][48]

Table 1.23. Advantages and disadvantages of different methods to determine the hydrogen isotopic composition of water


water. The service life of such a device according to the literature is 2 years or more.

In addition to the reduction reactor for the decomposition of water to be used with the isotope mass spectrometer operating in the gas dual inlet mode, we have also developed a reduction three-electrode solid-electrolyte reactor (see Fig. 1.39), working in the constant flow of the carrier gas (helium). This reactor was used in conjunction with the standard oxidation reactor in the system with the isotope mass spectrometer (see Fig. 1.5). Between these reactors there was the capillary column Pora Plot Q (30 m × 0.32 mm), placed in a thermostat with a separation column at a temperature of 40°C. It was designed to separate the products of combustion of hydrocarbon gases in the oxidation reactor. The temperature of the reduction solid-electrolyte reactor was 940°C, and the potential of the working electrode φ = –1.2 V. The running time of the sample through the reactor was a few seconds.

It was found that under the selected reduction conditions the CO2 molecules are not reduced, while the water molecules are fully reduced. Oxidation of CH4 results in the formation of water and CO2. The area of the oxygen peak formed due to the decomposition of water molecules exceeds more than 10 times the oxygen peak area formed by decomposition of CO2 molecules with the solid electrolyte reactor. The chromatogram, resulting from the decomposition using the solid electrolyte reactor of the water molecules, formed during the oxidation of mixtures of hydrocarbons in a flow of carrier gas in the standard oxidation reactor, is presented in Fig. 1.54.

Calculations performed using the following formulas were used to estimate the number of oxygen molecules used in the oxidation of

t, s

Fig. 1.53. The curve of the decomposition of water using the high-temperature solid electrolyte reactor.

t, s


hydrocarbon gases, and the number of oxygen molecules formed in the reduction of water with the SER:

n K CPVNS RT

A

ptheor = ; (1.8)

n Qeexp ,=4

(1.9)

where n theor, nexp is the number of oxygen molecules used for the formation of water molecules during the oxidation of hydrocarbon gases and produced in the reduction of the water molecules, respectively, Q is the integral of ionic current of oxygen O2–, passed through the solid electrolyte, e is the electron charge, C is the volumetric concentration of the compound in the gas sample, V is the volume of the sample under normal conditions, P is normal pressure, NA is the Avogadro number, R is the molar gas constant, Sp is the sample division coefficient, K is the number of oxygen molecules required for the formation of water molecules during the oxidation of one molecule of hydrocarbon gas or other fuel compound.

The experiments with the decomposition of injected water with the water formed during the oxidation of hydrocarbon gases, as well as alcohols, have shown that within the experimental accuracy, the equality ntheor ≈ nexp holds. This proves that in the solid-electrolyte reduction reactor complete decomposition of water in a stream of carrier gas takes place in a time of about 3 s. The chromatographic peak of the

Fig. 1.54. Chromatogram obtained in the passage of oxygen ions through the solid electrolyte, resulting from the decomposition of water molecules which are formed during the oxidation of hydrocarbon mixtures in a standard oxidation reactor in a flow of carrier gas.

I, mA

Time, s

iso-

C4H

10n-

C4H

10


reduction of the water molecules, introduced into the capillary column, is shown in Fig. 1.55. The results obtained for the hydrocarbon gases are given in Table 1.24. The table shows that the discrepancy between the values of Δ = ntheor– nexp is small, equal to 10–20%. The number of measurements for each gas was equal to three.

Thus, the developed solid-electrolyte reduction reactor allows almost complete decomposition of water, without changing its reduction characteristics during a long time. These qualities of the reactor play an important role in carrying out the isotope mass spectrometric analysis of water. In addition, the solid-electrolyte reduction cell as well as and the solid-electrolyte oxidative cell can serve as chromatographic detectors that do not require any calibration, as opposed to other detectors.

The distribution of the isotopes of light elements in various objects Finding the source of drugs and explosives

The problem of determining the geographical origin of narcotic (NS) and explosive (ES) substances, in particular derived from natural sources, is still relevant in the world. One way of solving it is based on the measurement of isotope ratios of carbon and nitrogen of the investigated compounds.

It is known that the isotopic composition of plants is formed in the biochemical processes and, therefore, the observed isotopic variations reflect not only species differences but also differences in the metabolism of plants, related to environmental conditions. The isotopic composition of ambient gases, as well as parameters such as humidity, temperature, the duration of daylight, determine the isotopic ratios of elements in plants. Plants grown at high humidity or in soils with

Fig. 1.55. Ionic current signal, formed by the passage of oxygen ions through the solid electrolyte, in the decomposition of water molecules in the solid-electrolyte reactor in a stream of carrier gas.

I, mA

Time, s


high water content, can have values of isotope ratios δ13C 4–5‰ more negative than plants growing in dry climates. The isotopic composition of nitrogen in plants can vary depending on soil composition and microbial activity of the nitrogen fixers by 10‰ or more [46]. Processes of biological fractionation of isotopes are discussed in detail in the book [50].

Narcotics. For many years, interest has been shown in solving the problem of establishing the geographical origin of drugs derived from natural source materials [49, 51, 52]. Basically we are talking about the isotopic analysis of carbon and nitrogen of heroin, morphine and cocaine. Heroin is a semi-synthetic product obtained by acetylation of morphine. As the isotopic composition of carbon in acetic anhydride may vary depending on the conditions of its production, refining heroin by carbon 13C may be due to both the geographic origin of the sample and a source of acetic anhydride used by manufacturers of drugs. Measurement of the carbon isotope composition of acetic anhydride is of great importance in determining the origin of samples in one batch. The degree of enrichment of morphine with carbon 13C may indicate the geographical origin of the samples. The difference in the composition of the carbon of heroin and corresponding deacetylated heroin can range from –4.99‰ to 0.17‰ [53].

Cocaine is one of the most common drugs together with heroin. Determination of the isotopic composition of carbon and nitrogen in samples of cocaine and coca bush leaves from Bolivia, Peru, Colombia was carried out by the authors of [54]. In coca leaves δ13C values varied in the range from –32.4‰ to –25.3‰, and the values δ15N varied over a wide range from 0.1 to 13‰. It should also be noted that in the drugs the values of δ13C and δ15N were 3–8‰ more negative than for the plants from which they were obtained [49]. The most complete and

Table 1.24. The results of the decomposition of water formed during the oxidation of hydrocarbon gases (50 µl sample volume, split injection 20:1), methanol (0.1 µl, 1:40) and water (0.1 µl, 20:1), using a solid-electrolyte reactor

Compound ntheor, 1016 mol. O2 nexp, 1016 mol. O2 Δ, 1016 mol. O2

CH4 1.1 1.2 −0.1C2H4 0.7 0.8 0.1C3H8 1.3 1.4 −0.1

iso-C4H10 2.2 2.1 0.1n-C4H10 1.1 0.9 0.2

H2O 8.0 9.3 −1.3СН3ОН 2.6 2.3 0.3


serious analysis of the isotopic composition of carbon and nitrogen of heroin and morphine from South America, Southeast Asia, Southwest Asia and Mexico, as well as cocaine from South America, was performed in [49]. It turned out that the heroin from the four regions had the maximum difference in the isotopic composition of carbon 2.4‰ nitrogen 3.1‰, and morphine 1.4‰ and 3.5‰, respectively. For cocaine, the maximum difference in the isotopic composition of carbon and nitrogen was 0.6‰ and 7.1‰ respectively.

Most analyzed drugs have doubtful origin, as the samples are taken from batches of seized drugs. Obviously, the creation and expansion of the corresponding data bank is crucial for reliable identification of the geographic source of the drugs.

The isotopic ratios 13C/12C and 15N/14N of solid and liquid samples were determined on an isotopic mass spectrometer Thermo Fisher Scientific DELTA Plus, connected with an HP 6890 gas chromatograph through the Combustion 3 interface (configuration GC-C-IRMS) and an elemental analyzer EA 1110 (CE Instruments) through the ConFlo 2 interface (configuration EA-IRMS).

In the GC-C-IRMS configuration the dissolved samples of drugs were divided in a capillary column DB-5 (J & W Scientific, 30 m × 0.32 mm). The analysis conditions were as follows: carrier gas helium, temperature of the injection 250°C, the initial temperature of the thermostat 150°C, the final temperature of the thermostat 320°C, heating rate 10°C/min.

The leaves of cannabis, heroin, morphine and cocaine were analyzed.The degree of purification of the samples heroin and cocaine was 60-100%. One part of the samples in powder form was analyzed using the elemental analyzer, and the other part was dissolved in methanol (Merck company) and analyzed using the capillary gas chromatograph.

Samples of cannabis leaves were selected in different regions of Russia (Bryansk, Kursk region, Mordovia, Tatarsk Republic, Kabardino-Balkar Republic, Primorsky Krai, Stavropol’ region) and Ukraine (Sumy region). Prior to analysis the cannabis leaves were dried at a temperature of 50°C and powdered in an agate mortar to produce a homogeneous mass. Sample charges for analysis were weighed on a Mettler AT 261 analytical balance. The isotopic composition of these samples was analyzed using the EA-IRMS method.

In all samples the carbon concentration was significantly greater than the nitrogen concentration. For example, for one nitrogen atom in the molecule of cocaine and morphine there are 17 carbon atoms, and in heroine 21 carbon atoms. Therefore, in determining the isotopic composition of nitrogen the required charge of cannabis leaves and


powders of drugs was 0.5–1.5 mg, and in determining the isotopic composition of carbon the sufficient sample weighed 0.5 mg. The concentration of drugs, dissolved in methanol, was 2 mg/ml in determining the isotopic composition of carbon and 18–20 mg/ml in the determination of the isotopic composition of nitrogen. A sample with a volume of 1 µl was injected into a chromatograph using the A200S automatic sample injector. Every sample was analyzed at least three times. Identification of peaks in the chromatograms, recorded on an isotopic mass spectrometer DELTA Plus in the configuration GC-C-IRMS (detection of ions with a mass of 44 and 28), was performed using retention times obtained using a gas chromatograph/mass spectrometer HP 5973 under the same chromatographic conditions.

15 samples of cocaine, 5 samples of heroin, and a sample of Colombian morphine, three heroin samples from Korea, a sample of heroin from Afghanistan, and 8 samples of the cannabis leaves from Russia and Ukraine were analyzed. The results of the isotopic composition of carbon and nitrogen in the cannabis leaves are presented in Table 1.25.

As the table shows, the carbon isotope ratios vary in a narrow range from –28.38 to –26.43‰, and isotopic ratios of nitrogen change in a wider range of values from –3.17‰ up to 9.65‰. The standard deviation of measurement results constituted 0.12–0.25‰ for carbon and 0.14–0.37‰ for nitrogen. The cannabis leaves from Mordovia were more heterogeneous in structure than the other samples, which led to

Table 1.25. The results of the isotopic composition of carbon and nitrogen samples of leaves (S is standard deviation)

No. Region Type δ13СPDB, ‰ S, ‰ n δ15Nair,

‰ S, ‰ n

1 Bryansk region Glukhovsk zelenets

−27.72 0.21 4 1.83 0.27 4

2 Mordovia YuSO-27 zelenets

−26.54 0.16 5 −3.17 1.60 5

3 Kursk region Caucasus zelenets

−27.19 0.25 4 1.93 0.30 5

4 Tatarsk Republic Caucasus zelenets

−26.50 0.21 5 2.04 0.23 5

5 Sumy region YuSO-19 zelenets

−27.65 0.23 5 5.70 0.14 5

6 Primorsky Krai −26.43 0.16 5 5.17 0.14 47 Stavropol' −28.38 0.12 4 9.65 0.25 48 Kabardino-Balkaria −28.12 0.14 4 4.13 0.34 3


a greater scatter of the measurement results. The data on the isotopic composition of carbon and nitrogen of the cannabis leaves, grown in different regions of Russia and Ukraine, were received for the first time. Changes in the isotopic composition were probably related to regional changes in the soil properties and natural–climatic conditions of plant growth. Thus, the isotopic nitrogen ratio of the cannabis leaves from the Stavropol’ region showed the highest value δ15Nair = 9.65‰, and from the Mordovian Republic was the lowest δ15Nair = –3.17‰, for the isotope ratios of carbon the dependence was opposite: δ13CPDB = –28.38‰ and δ13CPDB = –26.54‰, respectively.

Table 1.26 shows the isotopic ratios of carbon and nitrogen of heroin, morphine and compounds present in these samples. Using an HP 5973 gas chromatograph/mass spectrometer, the samples of heroin were found to contain acetylcodeine and monoacetylmorphine, and cocaine – codeine and lidocaine. A number of impurities could not be identified. It is seen that the carbon isotope ratios of heroin vary in the range from –38.25‰ to –32.74‰, and the isotopic nitrogen ratio varies in a wider range of –6.69‰ to 7.15‰. As a rule, the isotopic composition of carbon of monoacetylmorphine compared with the carbon isotopic composition of heroin is enriched with the heavy isotope 13C, and the isotopic composition of carbon of acetylcodeine with the light isotope of carbon. The absolute standard deviation of the results of the determination of the carbon isotope ratios was 0.09-0.25‰, for nitrogen it was slightly higher 0.17–0.21‰, and when measuring the traces the standard deviation reached 0.45‰.

As shown in Table 1.27, the heroin from Korea has more negative values of δ13CPDB (–38.25 ÷ –35.11‰) than the heroin from Colombia (–35.94 ÷ –32.74‰), and more positive δ15Nair values (–1.95 ÷ 7.15‰) and (–6.69 ÷ 1.29‰), respectively. When comparing the obtained carbon isotope ratios of heroin with the literature data [51], we can conclude that the isotope ratios of carbon of the heroin from Colombia, Korea, and Afghanistan (see Table 1.26) differ from the heroin produced in Nigeria, Thailand, Pakistan and India, but not from the heroin from Turkey.

The presence of specific impurities in drugs can increase the reliability of the identification and determination of their origin. For example, for the heroin from Afghanistan (No. 10) and Columbia (No. 3) the difference between the δ13CPDB values for the heroin compounds is 0.59‰ and increases for monoacetylmorphine to 1.96‰ and up to 1.92‰ for acetylcodeine. To draw more serious conclusions it is necessary to expand the database for the isotopic composition of carbon and nitrogen. In addition, the carbon isotopic composition


of heroin depends on the isotopic composition of carbon in acetic anhydride used in the production of heroin. In the future we plan to carry out deacetylation of heroin, which will more accurately define the geographic area of the origin of heroin and compare the results obtained by different researchers.

Since the measurement of nitrogen isotope ratios of narcotic substances by the GC-C-IRMS method requires a large sample volume, this leads to overloading of the capillary column and the distortion of the shape of the analytical peak. Incomplete combustion of the separated components in the oxidation reactor may also take place. The influence of these factors on the accuracy of measurements of the isotopic composition is not clarified in the literature. Therefore, parallel measurements of cocaine samples were taken by the EA-IRMS and GC-C-IRMS methods, as it is known that the elemental analyzer allows to burn samples to 10 mg or more. The isotopic analysis of nitrogen by EA-IRMS requires samples of drugs weighing 1 mg. The purity of the samples was tested for in the HP 5973 chromatograph–mass spectrometer. The results of the determination of the isotope ratios of carbon and nitrogen of cocaine by the EA-IRMS and GC-CIRMS methods are shown in Table 1.27.

The numbers in Table 1.27, marked with an asterisk, correspond to samples of cocaine contaminated with impurities. The values of isotopic relations, marked in Table 1.27 with the sign ' were not used in the calculation of averages. As shown in Table 1.27, for pure cocaine from

Table 1.26. The results of the isotopic composition of nitrogen and carbon of heroin, morphine and the compounds present in the samples

No. Drug substance Analysis method

Heroin Monoacetyl-morphine

Acetyl-codeine

δ13СPDB, ‰

δ15Nair, ‰

δ13СPDB, ‰

δ13СPDB,‰

1Heroin (Columbia) GC-C-IRMS –34.95 0.78 –32.73 –34.53



4Heroin (Columbia) EA-IRMS –34.61 1.29

5 Heroin (Columbia) EA-IRMS –33.26 –6.69


Table 1.27. The results of the determination of isotope ratios of carbon and nitrogen samples of cocaine by the EA-IRMS and GC-C-IRMS methods (tα is the Student’s coefficient for confidence level α, S is the standard deviation, n is the total number of measurements)

No. The origin of cocaine

δ13CPDB, ‰ Δ13CPDB, ‰

δ15Nair, ‰ Δ15Nair, ‰EA-

IRMSGC-C-IRMS

EA-IRMS

GC-C-IRMS

1 Columbia, district Koara –35.47 –35.76 0.29 –2.19 –1.73 –0.46

2 Colombia, dep. North. Santander –36.14 –36.00 –0.14 –3.39 –2.62 –0.77

3 Columbia, dep. North. Santander –34.81 –34.54 –0.27 –5.60 –5.32 –0.28

4 Columbia, dep. North. Santander –35.82 –36.93 1.11 –6.10 –6.23 0.13

5 Columbia, dep. North. Santander –36.04 –35.86 –0.18 –3.70 –3.22 –0.48

6 Columbia, dep. North. Santander –35.72 –3.70

7* Columbia, dep. Caquetá –30.85' –37.31 6.46' –9.93' –5.90 –4.03'

8* Columbia, dep. Caquetá –35.46' –36.49 1.03' –3.69' –1.64 –2.05'

9 Columbia, Dep. Huila –35.51 –36.31 0.80 –4.88 –4.33 –0.55

10 Columbia, Dep. Cundinamarca –34.96 –34.84 –0.12 –9.71 –10.50 0.79

11 Columbia, Dep. Valle –36.05 –36.08 0.03 –5.15 –7.14 1.99

12* Columbia, Dep. Boyaca –34.47' –36.30 1.83' –10.36' –8.93 –1.43?

13 Columbia, Dep. Guaviar –34.88 –4.53

14* Columbia –35.84 –36.38 0.54 –3.09' –1.96 –1.03'15* Columbia –39.92 –35.53 –4.39' –5.24 –4.63 –0.61Average ±tαS/√n (α = 0.95)

−35.48±0.32

−36.03±0.46 0.55 −4.77

±1.2−4.93±1.7 0.16

'– Values are not taken into account when calculating the average values due to the presence of impurities in the sample; * – samples containing impurities

Δ13CPDB = δ13CPDB (EA-IRMS) – δ13CPDB (GC-C-IRMS); Δ15Nair = δ15Nair (EA-IRMS) – δ15Nair (GC-C-IRMS).


Colombia, the average values of δ13CPDB determined by different methods differ slightly, within 0.55‰. In this case, the isotopic composition of carbon as determined by the EA-IRMS method was shifted toward more positive values compared with those obtained by GC-C-IRMS. The difference between the mean values of nitrogen δ15Nair, determined by the two methods, was even smaller, 0.16‰. The results given in [55] for diacetylmorphine were worse: the difference in the values of δ15Nair for the two methods was 0.49‰. Such a difference in the results of measurements of the nitrogen isotopic composition by the two methods is not explained in the article. Furthermore, Table 1.27 shows that in determining the isotopic ratios of nitrogen the application of higher concentrations of drug substances (20 mg/ml) leads to an overload of the capillary column, but this does not cause fractionation of the nitrogen and carbon isotopes. The amount of carbon in the sample at the same time was 3·10–6 g.

According to Table 1.27 at a confidence probability of 0.95 the cocaine samples from different regions of Colombia have the average isotope ratio δ13CPDB = –35.48±0.32‰ and δ15Nair = –4.77±1.2‰. Comparing the results with those of [49] one can see that the difference as regards the isotopic composition of carbon is approximately 1‰, and there is almost no difference in the isotopic composition of nitrogen. The resulting differences in the results of measurements of isotopic ratios of carbon can be explained either by different sample preparation, resulting in the fractionation of carbon isotopes, or by the insufficient purity of the analyzed samples. In [49] it is also shown that despite the similar climatic conditions of growth of coca in different regions of South America, the samples of cocaine from Colombia, Bolivia, Peru and Ecuador can be separated on the basis of the isotopic composition of carbon and nitrogen.

The carbon isotopic compositions of six opium alkaloids in six samples of opium, obtained from different Afghanistan provinces, are presented in Table 1.28.

The isotopic composition of carbon for different alkaloids varies in a relatively large range of values of δ13C from –39.6 to –29.91‰. The smallest variations of the values of δ13C were observed for morphine and thebaine, and the greatest range of variations of δ13C is typical for meconin from different Afghanistan provinces. For the Badakhshan Provinces the presence or absence of irrigation affects only the carbon isotopic composition of papaverine and noscapine. Although it is known [49] that abundant watering leads to a decrease in the δ13C values of plants.


These results of determination of the isotopic ratios of carbon and nitrogen of the leaves of cannabis, cocaine, heroin and morphine indicate that when creating a sufficient database of sufficient the comparative isotopy δ13C and δ15N can be used to identify the geographic source of drugs.

The isotope ratios of carbon and nitrogen in pure drugs can be determined by either of the two methods EA-IRMS and GC-C-IRMS, and for the isotopic analysis of contaminated samples it is recommended to use only the GC-C-MS method, which allows to isolate and analyze specific compounds.

Explosives. Under the present conditions an urgent task is to identify explosives in connection with the problem the theft of explosives from plants and different structures of the Ministry of Defense, as well as due to the need for forensic investigation of crimes involving explosives.

Of the many compounds capable of explosion, explosives and components of explosive mixtures are made of only 20–30 substances. The main ones are nitrocompounds (TNT, tetryl, hexogen (RDX), octogen (HMX), tetranitropentaerythrite (TEN), nitrocellulose, nitromethane, etc.) and salts of nitric acid, especially ammonium nitrate. As a rule, these substances are not used in pure form but as mixtures, e.g. mixtures of HMX, RDX and TEN with TNT, nitroglycerin with nitroglycol, diethylene glycol dinitrate and nitrocellulose, TNT with ammonium nitrate, a mixture of ammonium nitrate with liquid (e.g. straw oil) and powder (for example, wood flour, powdered aluminium) flammable substances.

In many cases, only the determination of the isotopic composition of light elements (C, N, O) of the basic compounds that make up

Table 1.28. The isotopic composition of carbon δ13CPDB (‰) of the individual components of the opium

Alkaloid

Afghanistan provinces

Nangarhar Farah Helmand NimruzBadakhshan

With irrigation

No irrigation

Meconin -34.87 -29.89 -29.91 -35.01 -36.19 -36.53Codeine -36.46 -33.77 -34.44 -33.82 -36.67 -36.41Morphine -31.05 -32.16 -31.89 -31.56 -32.20 -32.26Thebaine -31.99 -31.46 -31.59 -32.09 -32.53 -32.27Papaverine -38.28 -38.72 -38.68 -38.10 -39.60 -37.67Noscapine -34.97 -34.45 -33.39 -32.61 -34.99 -33.76


the explosives allows us to identify their place of production. The results of isotopic analysis of individual compounds are not affected by contaminants and additives to explosives. At the same time, the nitrogen isotopic composition provides the largest amount of information for explosives containing nitro groups.

Modern methods of analytical chemistry used in criminalistic laboratories (mainly chromatographic, GC mass spectrometric methods, ion mobility spectrometry), can identify the presence of a wide range of explosives with a fairly high sensitivity of 10–8–10–6 g in the sample [56]. However, these methods usually do not provide information on possible sources of explosives, or do not permit correct identification of the samples belonging to the same batch. Many laboratories around the world (Defence Science Technology Laboratory – DSTL, England) have long been using isotope-ratio mass spectrometry (IRMS) for isotopic analysis of explosives. It has been proved that this method has the great potential for obtaining unique information about the origin, purity and the conditions of production of both the explosive substances and their components. At present, studies are carried out of the isotopic variations of the light elements of explosives manufactured in the industrial and underground facilities, and also of the fractionation of elements depending on the production conditions. On the basis of their explosive ability all explosives can be divided into several groups and the isotopic composition of light elements must be determined for each group.

The study of commercial hexogen (RDX) and underground nitroglycerin (NG) confirms that the results of isotopic analysis of 13C/12C, 15N/14N, 18O/16O can be used to identify explosives substances. The IRMS method can be successfully applied to the PETN explosives and peroxides. Experts at the University of Reading (England) deem it necessary to monitor the dissipation of drugs and explosives by the establishment of a data bank of the isotopic composition of light elements drugs and explosives from different parts of the world.

Thus, extensive isotopic studies of the light elements of explosives are being carried out in the developed countries. In Russia, such work has not been conducted. ‘Forensic Science in Isotope Mass Spectrometry’ conferences are regularly organized in England to discuss in detail the results of forensic examinations with the use of isotope studies. The organizers of these conferences propose cooperation with all interested organizations.

Using the DELTA Plus device with the EA 1110 elemental analyzer, we analyzed samples of trinitrotoluene, made in four Russian plants. We


measured the isotopic composition of nitrogen 15N/14N and carbon 13C/12C. It has been shown (Table 1.29) that all TNT samples have similar isotopic compositions of carbon and the nitrogen isotopic composition varies in a wide range from –8.41 to 4.54‰. Therefore,the isotopic composition of nitrogen of the TNT samples is the most informative value for the identification of the samples as belonging to one of the manufacturing plants. The chromatograms of air samples with vapours of the TNT samples, listed in Table 1.29, are shown in Fig. 1.56. The chromatograms were produced in ECHO-M chromatograph [57] with a multi-channel column. As can be seen from Fig. 1.56, the form of the chromatograms of four TNT samples is very similar to each other and they can not be separately identified. However, the samples are clearly distinguishable on the basis of the isotope composition.

Thus, the available experimental data and the world experience suggest that it is essential to develop techniques of the isotopic analysis

Table 1.29. The isotopic composition of carbon and nitrogen of TNT

Sample number δ13CPDB,‰ δ15NAIR,‰

1 -29.86 +4.542 -29.74 −2.483 -29.72 −8.414 -29.51 −5.65

Am

plitu

de, V

Time, s

THT, sample No. 1-4, column A 2047-62, T¬i = 180°C, Tc = 180°C, Td = 200°C, Q = 50 mL/min

Fig. 1.56. Chromatograms of air samples with vapours of TNT samples, presented in Table 1.29. Ti, Tc, Td – temperature of the injector, column and detector, respectively.

TNT, sample No. 1–4, column A 2047-62, Ti = 180oC, Tc = 180oC, Td = 200oC, Q = 50 ml/min


of explosives and a databank of these isotope ratios of light elements in explosives from different manufacturers.

Alcoholic beverages. The urgency of the identification of ethyl alcohol, produced from raw food material, is due to the fact that it is often substituted by synthetic ethyl alcohol, forbidden for use by ‘The list of strong and toxic substances’ of the Standing Committee on Drug Control (from 01.07.2002). Currently the only standard for determining the falsification of vodka and spirits is GOST R 51786-2001 ‘Vodka and ethyl alcohol from raw food raw materials. Gas chromatographic method for the determination of authenticity’. However, this method does not allow make an unambiguous conclusion about the nature of ethyl alcohols when alcohols are well purified. Identification of impurities is even more complicated in the case of mixing of alcohols of different nature (economically viable because of the low cost of synthetic alcohol). One effective method to determine the nature of ethyl alcohol is liquid scintillation spectroscopy. The natural abundance of radiocarbon, which is formed in the upper layers of the atmosphere from atmospheric nitrogen under the influence of the neutron component of cosmic rays, is variable. By participating in the cycle of exchange with the biomass of the Earth, 14C is absorbed by living plants. The ethanol, made from the plant material of a new crop, is contained a fixed amount of 14C. This reference value, called the annual reference value, is determined each year by results of analyzes conducted by the Bureau of the reference data of the EUC together with the Research Centre in Ispra. It is obvious that in the synthetic alcohols derived by synthesis from petroleum or from natural gas, due to the complete decay the concentration of 14C is equal to zero. The radioactivity of spirits was determined in a liquid scintillation ultra-low-background spectrometer Quantilus 1220 (manufactured in Finland). The investigated alcohol sample with a concentration of not less than 85% was mixed with the liquid scintillator Optifase Highsafe-3, is which light flashes appear in passage of β-particles as a result of decay of 14C. The flashes were recorded with a photoelectronic multiplier (PEM). Studies were carried out on samples of ethyl alcohol of different nature – synthetic grade A, O, technical, grain rectified by different manufacturers and different brands – Lux, Extra. The samples were prepared with different ratios of parts of the grain and synthetic alcohols. Table 1.30 shows the results of analysis of the samples performed by liquid scintillation spectrometry and GC-C-IRMS.

The results obtained by means of liquid scintillation spectroscopy and IRMS are in good agreement with each other. These results


confirm not only the origin of the analyzed samples, but the ratio of the proportion of grain and synthetic alcohols in them. The reproducibility of measurement of the radioactivity of carbon did not exceed 0.03. Analysis time was 3–4 h.

As seen in Fig. 1.57, the ratio of the stable isotope content 13C and radioisotope 14C for the ethyl alcohols of different nature have the form of a linear dependence. An important property of the IRMS method is the possibility of establishing not only the origin of ethyl alcohol, but also the source of its production (wheat, corn, beetroot, sugar cane and others). Table 1.31 shows the results of measurements of 13C in the samples of ethyl alcohol of various origins, performed by IRMS.

For example, the analysis of the samples No. 1 and 2 in Table 1.31 shows that the raw materials in these samples are grains from various climatic zones since the content of 13C in these samples differs in

Table 1.30. Results of analyses of alcohol samples made by liquid scintillation spectrometry and GC-C-IRMS

Sample No. Name of sample Radioactivity,

Bq/g (carbon) δ13CPDB,‰

1 Rectified grain alcohol Lux 6.3 –25.30

2 Synthesis alcohol grade A, GOST R 51999 0 –34.77

3A mixture of alcohols: grain-rectified Lux and synthetic grade A in the ratio 1:1

2.9 –29.95

4A mixture of a lcohols : gra in-rectified Lux and synthetic grade A in the ratio 3:1

4.4 –28.28

Radioactivity 14C, Bq/g

Fig. 1.57. Relationship between the isotopic composition of carbon δ13C and radioactivity of alcohols.


absolute value. The content of 13C isotope in the samples of commercial alcohol (samples No. 5 and 6) indicates that they do not belong to synthetic alcohols. The use of isotope mass spectrometry allowed to determined on the basis of the isotopic composition of the carbon of ethyl alcohol of various raw materials to identify the fact that the synthetic ethyl alcohol has the lowest value of δ13C, and ethanol from sugar cane the highest value.

The results are in good agreement the data obtained by A.M. Zyakun [58], indicating the values of 13C for grain alcohols based on rye equal to –22.1 ± -21.9‰, wheat-based equal to -25.6 ± -23.8‰, corn -15.6 ± -15.1‰.

The origin of ethanol can be successfully determined by nuclear magnetic resonance spectroscopy on deuterium nuclei (2H-NMR), which allows to quantify the total and selective deuterium content in each of the positions in the molecule where it is located [58]. The results for quantitative determination of the deuterium content in CH2D- and CHD-groups are presented in Table 1.31. The table shows that the alcohols of different origin can be efficiently separated not only by the isotopic composition of carbon but also the content of deuterium in various functional groups.

Thus, the nature of ethyl alcohol and the relative content of each of the alcohols (synthetic, grain) in the mixtures can be determined with sufficient reliability and validity using the methods of liquid scintillation spectrometry, mass spectrometry of stable isotopes and the spectrometry

Table 1.31. The results of determination of the isotopic composition of carbon and deuterium of ethanol of alcohols of different origin

Number Name of test samples δ13CPDB,‰ (D/H)2, ppm (D/H)3, ppm

1 Rectified grain ethyl alcohol Extra –23.15 129.9 100.5

2 Rectified grain ethyl alcohol Lux –23.88 126.2 96.5

3 Crude synthetic alcohol grade O –33.98 147.4 132.9

4 Synthetic alcohol, higher purification –32.86 144.4 129.7

5 Technical alcohol GOST 17298 –24.33 135.0 108.0

6 Technical alcohol GOST 18200–87 –24.30 134.6 106.2

7 Cane alcohol –12.67


of nuclear magnetic resonance of deuterium nuclei. However, the most sensitive and express method is the mass spectrometry of stable isotope with the duration of analysis of approximately 10 min.

The effects of isotope fractionation, accompanying organic synthesis

Natural hydrocarbons are mainly formed during the thermal destruction of organic matter ( thermogenesis) or by microbial processes (bacteriogenesis). Abiogenic hydrocarbons are formed in the reduction of carbon dioxide. It is believed that these processes may occur during cooling of the magma [59], in the geothermal systems in the interaction of water with rocks and the serpentinization of ultramafic rocks [60, 61]. The main criterion for abiogenic hydrocarbons, synthesized from hydrogen and carbon oxides (by the Fischer–Tropsch reaction) is considered, according to [62–64], to be the specific (reversed) distribution of the carbon isotopic composition in light saturated hydrocarbons: δ13CC1> δ13CC2> δ13CC3> δ13CC4. Secondary processes, including migration, mixing, and oxidation of biogenic gases, may also lead to a reversible distribution of carbon isotopes in hydrocarbon gases.

It was found that the carbon isotopic ratios of the hydrocarbons extracted from the Khibiny alkaline rocks and the hydrocarbons synthesized in an electric discharge in methane (kinetically controlled polymerization reaction) decreases with the increase of the number of carbon atoms in them [63], i.e. δ13CC1> δ13CC2> δ13CC3 = δ13CC4. It is due to the fact that in polymerization the 12CH4 molecules react more easily with the formation of the hydrocarbon chains than the 13CH4 molecules. A similar distribution of the carbon isotopes has been found in light hydrocarbons extracted from meteorites [62]. More recently, a similar distribution of the isotope was found in the gases from the deep wells drilled in the Canadian Shield [62]. The hydrocarbons of gas and oil deposits (C1–C4) have always the opposite distribution of δ13C, i.e. an increase in the isotope ratio with increasing carbon number [62, 65]. The same carbon isotopic distribution is typical of the gases obtained at low- and medium-temperature pyrolysis of high molecular organics. This is because weaker bonds 12C–12C fracture more readily than the 12C–13C bonds.

As far as we know, the isotope effects in the reaction of the Fischer–Tropsch catalytic synthesis have not as yet been investigated in detail [66]. It is assumed that the hydrocarbons (HCG) form in this reaction as follows:


CO + H2 → HCG + H2O + CO2.

The study of isotope effects of the Fischer–Tropsch reaction is important not to only the isotopic geochemistry of carbon, but may be useful for the study of the catalysis mechanisms in petrochemical synthesis.

The experimental section. The catalysts were prepared by melting magnetite and promoting additives in an arc discharge. The promoting additives were represented by aluminium oxide, potassium oxide and calcium oxide. Samples of the catalysts differed in the relative content of these components. In some cases, chromium oxide or kaolin were added to the catalyst composition. Before use in synthesis the catalysts were reduced with hydrogen at 5 MPa and 450°C for 12 hours. After reduction the catalysts were pyrophoric so all subsequent operations with them were carried out in a protective atmosphere of CO2. The synthesis was carried out in flowing plants with reactors of 100 ml filled with catalyst (grain size of iron 2-3 mm) at a pressure of 3 MPa, a temperature of 250-270°C and a flow rate of the N2 + H2 + CO gas mixture (2.5:1.5:1) of ~40 l/h at the input of the reactor. This composition simulated the gas produced by methane conversion in the air. The reaction reaches a steady level after about 20 h after the start of the experiment. Under these conditions, the conversion of the original CO amounts to 90-95%. Two samples of the gaseous products in the transient mode and two or three samples in the steady state were selected. At the end of the experiment samples of water and liquid hydrocarbon products, formed in the experiment, were also taken. As a rule, water contains up to 5 wt.% of soluble organic compounds, mainly alcohols. The composition of light hydrocarbons was analyzed in the CHROM-5 chromatograph by the standard method using a flame ionization detector, nitrogen as the carrier gas and the column with the modified Porapak Q. The isotopic composition of the carbon of initial CO and the products of the Fisher–Tropsch reaction CO2, CH4, C2H4, C2H6, C3H6, C3H8, C4N8 and C4N10 was determined in the DELTA-Plus isotope mass spectrometer, connected with the HP 6890 capillary chromatograph. A sample with a volume of 100 µl was injected into a gas chromatograph with a syringe and separated using a PoraPlot Q capillary column (30 m × 0.32 mm). The analysis conditions were as follows: carrier gas helium, the temperature of the injector of samples 100°C, initial temperature of the thermostat 40°C, final temperature of the thermostat 180°C, heating rate 5°C/min. Samples for analysis were taken in sealed tubes with a capacity of 10 ml with solid rubber stoppers. The reproducibility of the isotopic composition of carbon was


0.3-0.5‰. The composition of the gas phase in the steady state to C8 is shown in Table 1.32. Generally speaking, at a relatively high degree of conversion of CO the chemical composition of the gas phase practically does not depend on the time when the sample is taken. However, at the very beginning of the process until the system does not yet contain a sufficient amount of water, CO2 is the first to appear in the gas due to recombination of CO on the catalyst surface. The experimental series 1 was carried out with a catalyst the use of which results mostly in the synthesis of olefins. Series 2 used a catalyst on which paraffins were predominantly synthesized. In these experiment series the concentration of light hydrocarbons monotonically decreases with increasing carbon number.

As shown in Table 1.33, the Fischer–Tropsch reaction produced CO2, saturated and unsaturated hydrocarbons in which the isotopic composition of carbon depends on the reaction time in the stationary mode for series 1 and is almost independent of time for series 2. In addition, as a rule, for the compounds of series 1 the saturated hydrocarbons have lower values of the carbon isotope ratios than the unsaturated hydrocarbons with the same number of carbon atoms.

The initial carbon isotopic composition of CO is δ13CVPDB = -25‰. Table 1.33 shows the carbon isotopic composition of CO2 and

hydrocarbons to C4 in the products of Fischer–Tropsch synthesis at different stages the synthesis process, and Table 1.34 gives the average isotopic composition of carbon for the compounds with the same number of carbon atoms. The value of δ13C decreases with an increase the number of carbon atoms. After 70 h of operation of the catalyst in series 1, apparently the catalyst becomes contaminated that leads to a decrease of δ13C for methane and increase of this value for other compounds (Fig. 1.58). In particular, the value of δ13C increases greatly for carbon in the composition of CO2 it varies by 10‰.

Figure 1.59 shows the change in the time-averaged isotopic composition of carbon for the compounds with the same number of carbon atoms in relation to this number. For the compounds formed in series 1, there is the following pattern:

δ13CCl ≈ δ13CC2 > δ13CC3 > δ13CC4.

For the compounds formed in series 2 there are virtually the same relationships, except for the carbon isotopic composition of the C4 compounds:

δ13CCl ≈ δ13CC2> δ13CC3 < δ13CC4.


A similar change in the isotopic composition of carbon is observed for deep gas [67]. The distribution of the carbon isotopic composition in deep hydrocarbon gases in the Yen-Yakhinsk field (well depth 7163 m) is shown in Fig. 1.60.

The concentration of hydrocarbon gases and the carbon isotopic composition of gases from the Yen-Yakhinsk fields are shown in Table 1.35. As the table shows, the carbon of methane has a very large value of δ13CPDB = -19.4‰. All other hydrocarbon gases have

Table 1.32. Chemical composition (vol.%) of gaseous products synthesized by the Fischer-Tropsch reaction using modified iron catalysts: T = 230°C, P = 30 atm

CompoundSeries

1 2

N2COH2

CO2CH4C2H4C2H6C3H6C3H8∑C4∑C5∑C6∑C7∑C8

65.10.84

8.981.640.470.200.310.110.320.320.150.100.07

63.33.90

7.162.620.1

0.540.080.260.220.210.110.080.08

Number of C atoms

Fig. 1.58. The dependence of the carbon isotopic composition of the synthesized compounds on the number of carbon atoms and synthesis time.


Table 1.33. Carbon isotopic composition of CO2 and hydrocarbons up to C4 (‰ PDB) in products of Fischer–Tropsch synthesis at different stages of the synthesis process

CompoundsSampling time, h

10 20 30 40 50 70Series 1

CO2CH4C2H4C2H6C3H6C3H81-C4H82-C4H8n-C4H10iso-C4H10

−6.3 −11.7−41.6−43.2−46.8−46.2−41.1

−−44.5−48.4−47.2

−19.8−39.4−42.7−44.7−46.1−41.3

−−46.3−48.7−47.0

−11.1−42.2−38.5−44.7−44.5−44.6

−−44.5−46.4−44.1

−1.3−43.3−35.2−42.6−40.7−44.4−46.3−42.1−44.7−50.9

+1.1−44.9−40.1−46.0−41.7−46.2−44.9−44.9−45.0−44.1

Series 2CO2CH4C2H4C2H6C3H6C3H81-C4H82-C4H8n-C4H10iso-C4H10

−7.4 −13.3−43.6

−−43.8

−−44.7−45.5−42.6−44.7

−

−13.0−43.7

−−43.3

−−45.9−46.2−40.3−45.0

−

a significantly lower value of δ13CPDB. Thus, it was shown that in abiogenic synthesis of the hydrocarbons by the Fischer–Tropsch reaction the values of the isotopic ratios of hydrocarbons increase with an increase in the number of carbon atoms in the compounds. In addition, changes in the carbon isotopic composition of the synthesized compounds can be used to evaluate the processes occurring in the reactor during synthesis.

Isotope effects in carbonaceous chondrites

One of the ways in addressing the prevalence of chemical forms of abiogenic organic matter in the protoplanetary cloud is the study of variations in the isotopic composition of carbon in bituminoid and


kerogen constituents of organic matter of meteorites. In these studies, it is important to know the primary isotope composition of carbon in the protoplanetary cloud. As an object for its determination, we used the nanodiamond of meteorites that represent the least modified material of the solar system, which a is source of information about nuclear and chemical processes in the vicinity of stars [68]. The average size of diamond grains in meteorites is 2.6 nm. For the investigations isolated

Table 1.34. The average carbon isotopic composition of CO2 and hydrocarbons (δ13CPDB, ‰) in the products of Fischer–Tropsch synthesis at different stages of synthesis

CompoundsSampling time, h

10 20 30 40 50 70Series 1

CO2C1C2C3C4

−6.3 −11.7−41.6−44.3−44.7−47.2

−19.8−39.4−43.3−44.8−47.7

−11.1−42.2−41.0−44.5−45.2

−1.3−43.3−35.2−41.7−46.3

+1.1−44.9−42.6−42.2−44.7

Series 2CO2C1C2C3C4

−7.4 −13.3−43.6−43.8−44.7−44.6

−13.0−43.7−43.3−45.9−44.7

Series 1

Series 2

Number of C atomsFig. 1.59. The dependence of the time-averaged carbon isotopic composition of the synthesized compounds on the number of carbon atoms for the two series of experiments and different catalysts.


1 – methane2 – ethane3 – propane4 – iso-butane5 – n-butane

Compound

Fig. 1.60. Changing the carbon isotopic composition with an increase in the number of carbon atoms in compounds in the hydrocarbon gases of the Yen-Yakhinsk field.

nanodiamonds of the carbonaceous meteorite Boriskino CM2, which has undergone a minimal degree of thermal metamorphism, and of the thermally metamorphosed meteorite Efremovka CV3. The Efremovka meteorite belongs to the reduction group of meteorites with a small content of nanodiamonds (0.004 wt.%) compared with the content of nanodiamonds in non-metamorphized meteorites (0.14 wt.%).

Isolation of nanodiamonds from meteorites included the following steps: a) the removal of silicate and metal phases by dissolution in HCl acid and the HCl + HF mixture; b) the destruction of poorly soluble phases containing carbon and sulphur, alkali treatment, treatment with potassium dichromate, hydrogen peroxide and perchloric acid, c) the removal of mineral phases soluble in phosphoric acid, such as SiC and spinels. The last stage was separation of the nanodiamond grains from other preserved acid-resistant phases. For this purpose, the unique capacity of such diamond grains to be in the colloidal state in the alkali medium was utilized. This capacity is determined by the presence of carboxyl groups on the surface of the nanodiamond grains. For this reason the deposit produced in the final stage of chemical etching was suspended in the ammonia solution by ultrasound treatment. The resulting slurry was then centrifuged, resulting in the formation of a

Table 1.35. The concentration of hydrocarbon gases and carbon isotopic composition of the gases from the Yen-Yakhinsk field

Methane Ethane Propane iso-butane n-butane

Concentration, vol.%δ13CPDB,‰

92.93–19.4

0.67–22.1

0.20–30.8

0.03–30.9

0.17–30.5


colloidal solution of nanodiamonds, depleted in other acid-resistant phases. The diamond colloidal solution was dried producing a light brown deposit characteristic of nanodiamonds of meteorites.

Analysis of the isotopic composition of carbon in nanodiamond aliquots was performed in the EA 1110 elemental analyzer, connected with the DELTA Plus isotope mass spectrometer. Samples with a mass of approximately 0.2 mg were placed in tin capsules. To ensure oxidation of the sample, 1–3 mg of CeO2 was added to tin capsules with the samples. The method of isotope analysis of the carbon of nanodiamonds was developed using the standard sample of USGS 24 graphite with the known isotopic composition of carbon δ13CPDB = -16.0‰ and the powder of ultrafine detonation diamonds UDA-6 with a crystallite size of 4–10 nm. Since these samples do not burn at a sufficiently high rate in the elemental analyzer, they are burnt with an additional oxidant. It was experimentally proved that 1–2 mg of CeO2 was enough to ensure complete combustion of these samples. The isotopic composition of carbon of graphite USGS 24 defined in our studies coincided within the error range with a given value of δ13C. The reproducibility of the results of isotopic analysis of carbon was high enough and equalled 0.1‰. The results of determination of the carbon isotopic composition are given in Table 1.36.

As the table shows, the isotopic composition of carbon for the nanodiamond of the Boriskino CM2 meteorite has the value δ13C = (-33.67 ± 0.1)‰, and for the nanodiamond of the Efremovka CV3 meteorite δ13C = (-31.89 ± 0.1)‰. The data in the table show that there is a clear increase in δ13C values of the nanodiamond in the transition from the weakly thermally metamorphized meteorite (Boriskino) to the strongly metamorphized meteorite (Efremovka). This trend confirms the conclusion of studies [69, 70] regarding the change of the carbon isotopic composition of the nanodiamond of meteorites in relation to

Table 1.36. The isotopic composition of carbon of the nanodiamonds of meteorites and the international standard of graphite at different temperature of the oxidation reactor of the elemental analyzer

Sample Combustion temperature, oC δ13CPDB, ‰ S, ‰

Nanodiamond of CM2 Boriskino meteorite

1020900

-33.67-29.17

0.100.16

Nanodiamond of Efremovka CV3 meteorite

1020900

-31.89-27.69

0.100.39

UDA-6 synthetic diamond 1020 -25.96 0.10USGS 24 graphite 1020 -15.90 0.13


their degree of metamorphism. It is most likely that these changes are due to the presence in the substance of parent bodies meteorites of several populations of nanodiamond grains having different isotopic composition of carbon and differing in thermal stability.

The yield of carbon of the nanodiamonds was measured on the basis of the results of combustion of atropine (C17H23NO3). The yield of carbon in the oxidation of nanodiamond samples was at least 80 wt.%. The absence of 100% yield of carbon in combustion of the nanodiamond samples is determined in all likelihood by factors such as the presence of significant amounts of hydrogen in the nanodiamonds (up to 5 wt.%) and also by the presence of different chemical functional groups on the surface of the grains.

Lowering the temperature of the oxidation reactor of the elemental analyzer to 900°C leads to partial combustion of the nanodiamond grains. The central part of the grain burns later. The results of carbon isotope analysis of nanodiamonds at reduced temperature show that either the peripheral part of the nanodiamonds, which burns faster, has a heavier carbon isotopic composition than the central portion, or the nanodiamond films are heterogeneous in their composition and structure which affects the rate of their combustion. The conclusion that the nanodiamond films are inhomogeneous in their physico-chemical properties and carbon isotopic composition seems more likely. In addition, we can not exclude that the nanodiamond is composed of several populations of grains, one of which is presolar, while others have a solar origin.

Determination of the isotopic composition of hydrogen and oxygen of water, isotope effects during evaporation

The isotopic composition of oxygen and hydrogen of water can significantly change during the phase transition of water [17, 71]. The physical cause of this phenomenon is the difference in the vapour pressure of the isotope components of water, which leads to the fractionation of 18O and 2H during evaporation, condensation, melting and freezing of water. Such a separation of isotopes becomes more extensive with the reduction of the phase transition temperature. Thus, the vapour, formed at higher temperatures, will have relatively higher values of isotope ratios. In evaporation the water is enriched in heavy isotopes in the liquid phase, and condensation and freezing are accompanied by a decrease of the isotopic composition of water vapour or water. The isotopic dependences, accompanying the evaporation and condensation of water vapour, are determined by


two factors: isotopic equilibrium separation at the phase boundary and isotopic distribution due to diffusion and convection in the boundary layer of one or both phases [72].

Since the isotopic composition of oxygen and hydrogen in water vapour depends on the evaporation conditions, we studied the effect of fractionation of oxygen isotopes in the presence of water on the surface of a porous sponge or a multi-channel polymer film. Such a system simulates evaporation from porous rocks.

Evaporation was simulated in the experimental setup shown in Fig. 1.61. This installation included an Mdrive 23 step motor, which moves a piston in a cylinder with an inner diameter of 2 cm. Under the action of excess pressure the water (water volume 10–12 cm3) saturates a stationary sponge 1–2 cm thick, having pores with a diameter of about 0.5 mm. When the pressure is increased the water rises through the pores of the sponge until it reaches its surface. Due to the continuous piston pressure the water remains on the surface of the sponge in spite of its evaporation.

Rotation of the motor shaft by one step leads to the displacement of the piston by 2.5 µm. Prior to the experiments the water and the sponge were thoroughly degassed using a backing pump so that the air bubbles could not block the movement of fluid in the sponge and generate uncontrolled partial pressure in the gas phase. The sponge was pre-soaked with water. At room temperature the pressure above the sponge was equal to the pressure of saturated water vapour (about

Fig. 1.61. Schematic of equipment for the evaporation of water.

ВентильВакууметр

Фор-вакуумныйнасосВодяная

ловушка

Шаговыйдвигатель

Поршень

Губка

Вода

Конденсат

M

Vacuum gauge Valve

Sponge

WaterPiston

Water trap

Condensate

Step motor

Rough vacuum pump


20 mm Hg). Water vapour was condensed in a water trap cooled by dry ice. The volume of water equal to 0.25 ml condensed during 20 min. The experiment lasted from 30 to 60 min. A similar system was established to study the evaporation of water through a multi-channel film 10 µm thick with pores with a diameter of 0.25 µm.

Measurements of the isotopic composition of oxygen and hydrogen of water were taken in a DELTA Plus XP mass spectrometer connected to a TC/EA pyrolyzer at 1450°C. The reproducibility of the results of measurements of the isotopic composition of oxygen and hydrogen was 0.3–0.4‰ and 1‰, respectively. The results of the study of the fractionation of the oxygen of water during evaporation under various conditions are shown in Table 1.37. The table shows that the presence of the sponge on the surface of water leads to a decrease in the values of the oxygen isotope ratios of the condensate (Δ2), compared with evaporation without the sponge. Apparently this is due to difficult evaporation of water. Pressing the sponge and exit of water on its surface lead to a small increase in the oxygen isotope ratios of the condensate compared with the results for the uncompressed sponge, as the evaporation from the compressed sponge is somewhat simplified. The results of the isotopic analysis of oxygen practically do not depend on the thickness of the sponge, since the measurements are statistically indistinguishable. In the process of evaporation the isotopic composition of oxygen of water in the sponge also changes, δ18O increases. This is especially noticeable in the case of a free sponge. The condensate formed in evaporation through a free, uncompressed sponge has the smallest the value of δ18O.

In the presence of a porous multichannel film on the surface of water the latter is evaporated almost without fractionation. The isotope composition of the oxygen of the condensate is lowered by only

Table 1.37. Oxygen isotopic composition of water and condensate under different conditions of evaporation

Experiment No.

Sponge thickness, cm

δ18OSMOW, ‰

∆1=δ1–δ2, ‰

∆2=δ1–δ3, ‰

Initial water,

δ1

Water from

sponge, δ2

Condensate, δ3

12345

2, compression1, compression2, no compression1, no compressionno sponge

−14.91−14.91−11.39−11.39−15.23

−14.47−14.07−9.80

−24.38−24.76−21.77−21.68−23.63

−0.44−0.84−1.59

9.479.85

10.3810.29

8.4


1.3‰ (Fig. 1.62). This is due to the fact that the small diameters of the channels and the rapid evaporation of water are responsible for this situation in which the diffusion and convective processes in the surface layer of water do not have time to equalize the change in the isotopic composition of water molecules, according to Stanton’s criterion [73].

Thus, it was found that in the evaporation of water the change in the isotopic composition of the condensate is strongly dependent on the state of its surface.

The effect of decreasing values of δ18O and δD of the condensate in evaporation of water is widely used for producing water with an isotopic composition similar to that of the water from mountain springs [74]. In addition, there is evidence that water with low isotope ratios has a number of useful properties: it has high biological activity, confirmed by experiments on plants [75], and also has antitumor properties [76, 77]. There has been a significant increase in the duration and quality of the lives of cancer patients, the improvement of working of the major body systems. Drinking water containing 105 ppm deuterium is sold as a prophylactic anti-tumor fluid in the U.S. and some European countries.

To evaluate the isotopic composition of the oxygen and hydrogen of table water produced by foreign firms and of the water from natural sources, we measured the isotopic composition of oxygen and hydrogen of different water samples. The results are shown in Table 1.38.

The isotopic composition of samples of table water of foreign production, presented in the Table 1.38, corresponds to the data of [78]. In addition, the obtained values of the isotopic composition of

Membrane Water Sponge compressed

Sponge free

Fig. 1.62. Changing the oxygen isotopic composition of water condensate during evaporation through a sponge and a multichannel membrane.


oxygen and hydrogen of the water samples are similar to the isotopic composition of tap water in these countries [79]. At the same time, the table water from natural mountain springs of Bashkiria has the lightened isotopic composition. It means that the useful qualities of table water can be determined not only by its chemical composition and fame of the manufacturer but also by the isotopic composition, which is often not specified in the certificate.

The distribution of carbon isotopes in complex organic compounds of biological origin (oil, hydrocarbon gases)

The problems of identifying the source of crude oil and identification of genetic linkages between deposits are very rele-vant in organic geochemistry. Isotope geochemical studies, in combination with the physico-chemical methods of analysis, are widely used for identifying the genesis of oil deposits, areas of migration and the formation of deposits.

In studying the problem of sources of oil of multilayer deposits of the north-east Russian Platform (Shumavskoe Verkhnekamskoye basin, Batyrbayskoe and Bardymskoe Bashkir arches) and Preduralsk deflection, oils of the Kama region were studied by isotopic–geochemical methods. For this purpose, the carbon isotopic composition of oils, isolated fractions of rising polarity and the concentrates of normal alkanes were measured. Isotopic analysis of the carbon of the oils and their fractions was performed on a DELTA Plus isotopic mass spectrometer, connected to the 1110 EA elemental (EA-IRMS), and the isotopic analysis of the carbon of normal alkanes was performed using a DELTA Plus mass spectrometer, coupled with an HP 6890 capillary chromatograph (GC-C-IRMS).

Table 1.38. Results of isotopic analysis of oxygen and hydrogen of water samples

No. Number of water sampleIsotopic composition of water

δDSMOW, ‰ δ18OSMOW, ‰

1 Perrier, France -6.87 -41.962 Vittel, France -6.91 51.963 Selters, Germany -53.80 -6.574 Premier, Ufa district -102.18 -12.425 Kurgazak, Salavat district -100.73 -12.656 Red Key, Nurimanovsky district -96.52 -10.86


The chromatographic analysis conditions were as follows: column HP-5 (30 m × 0.32 mm), injector temperature 300°C, the initial temperature of the thermostat 40°C (3 min exposure), the final temperature of the thermostat 310°C (30 min exposure), temperature programming at 4°C/min, carrier gas – helium grade 60. Before analyzing the samples were dissolved in hexane and heated to a temperature of 40°C for the full dissolution of the fractions. Using a syringe with a volume of 1 µl the samples were injected into the chromatograph. The reproducibility of the results of isotopic analysis carbon for the EA-IRMS method was 0.1–0.2‰, and for the GC-C-IRMS method it was 0.2–0.3‰.

The research results were used to determine the specific features of the oil from the Pashiysky Upper Devonian clastic sediments of the Bardymsk deposits, characterized by a maximum content of the 13C isotope in the hexane–benzene fraction and slighty higher – in the benzene–ethanol fraction (Fig. 1.63). Similarities between the of isotope fractionation curves of the oil from the Visean deposits of the Shumovskyi and Batyrbaysk deposits and the oil in the Middle Carboniferous deposits of the Shumovskyi field can be seen.

The distribution of the stable isotopes of carbon fractions with higher polarity of the oils lower carboniferous terrigenous oils and devonian carbonate deposits of the Belopashinsk area is almost identical, which corresponds to the similarity of other geochemical parameters and their genetic uniformity (Fig. 1.64).

Figure 1.65 shows that the graphs of the dependence of the carbon isotopic composition of normal alkanes on the number of carbon atoms for the two carboniferous Shumovskaya deposits are virtually identical and differ from the charts for the oil of the Batyrbaisk carboniferous

Fig. 1.63. Carbon isotopic composition of oils and fractions of higher polarity Bashkirsk arch and Verkhnekamskaya basin.

H HB B BE Asph

Bashkirsk arch and Vernekamskaya basin

BatyrbaiskC1tl

BardymskD3tmShumovskC2vr

ShumovskC1tl

Oil fractions


and Batyrbaisk Devonian fields. The graphs of the isotopic composition of the carbon of normal alkanes for two deposits of the Belopashinskaya region are also similar.

Therefore, a comparison of the carbon isotope composition of the normal alkanes allows us to refine the results of the correlation for the fractions of higher polarity.

Table 1.39 shows the composition of hydrocarbon gas fields of Eastern Siberia. The carbon isotopic composition of these compounds was measured by the GC-C-IRMS method. The chromatographic analysis conditions were as follows: Pora Plot Q column (30 m×0.32 mm), injector temperature 100°C, the initial temperature of the thermostat 40°C (holding time 3 min), the final temperature of the thermostat 180°C (holding time 15 min), temperature programming 4°C/min, carrier gas – helium grade 60. The gas sample with a volume of 50-300 ml was injected into the chromatograph using a gas-tight

Fig. 1.64. Carbon isotopic composition of oils and fractions of higher polarity of the Solikamsk basin of the Predural'sk deflection. Petroleum fractions: H – hexane, HB - hexane–benzene, B – benzene, BE – benzene–ethanol, Asph – asphaltenes.

Fig. 1.65. Carbon isotopic composition of n-alkanes of oils of the Kama area.

Solikamsk basin of the Predural'sk deflection

Belopashninsk, C1tl

Belopashninsk, D3fm

H HB B BE AsphOil fractions

Shumovsk deposit, C1tlBardymsk, D3tmBelopashinsk, D3fm

Shumovsk deposit, C2vrBatyrbaisk, C1tlBelopashinsk, C1tl–

––––––––

δ13C

, ‰

Isotope ratio mass spectrometry of light gas-forming elements106Ta

ble

1.39

. C

ompo

nent

of t

he g

as sa

mpl

es

No

Fiel

dW

ell

Laye

rD

rilli

ng

rang

e, m

Con

tent

, mol

.%

CO

2C

H4

C2H

6C

3H8

iso-

C4H

10n-

C4H

10is

o-C

5H12

n-C

5H12

1B

olot

noe

1Yu

1 124

44–2

447

0.92

40.4

16.

0419

.55

11.3

910

.69

4.61

3.18

2Va

khsk

oe

646

Yu12 –

324

34–2

452

1.35

48.6

99.

4320

.75

3.99

10.0

91.

002.

103

Ger

asim

ovsk

UPN

1.00

82.1

65.

045.

611.

181.

880.

350.

334

Gur

arin

sk18

221

30–2

134

4.86

68.1

93.

8210

.85

1.75

3.29

0.58

0.47

5Za

padn

o-

Kliu

chev

skoe

68Yu

11 –2 +

Yu

131.

3663

.72

6.79

12.0

24.

206.

002.

051.

58

6Za

padn

o-

Poly

denn

oeU

PN0.

5888

.30

2.10

1.57

1.12

1.61

0.80

0.92

7Lo

mov

oe

356

Yu1

2667

–267

22.

0263

.17

11.3

013

.00

2.47

4.41

1.09

1.17

8Pr

igra

nich

noe

6B

119

18-1

922

0.30

69.4

72.

548.

803.

487.

931.

871.

81

9Se

vero

-Va

styg

ansk

oe18

Yu12 –

323

06-2

316

0.73

86.4

85.

272.

510.

410.

320.

030.

01

10Se

vern

oe30

5B

30.

0896

.25

0.80

0.36

0.22

0.19

0.07

0.05

11So

vets

koe

UPS

V-9

0.26

84.9

52.

675.

231.

352.

390.

440.

45

12Sr

edne

-N

yuro

l'sko

e13

1Yu

13 –4

2400

-241

92.

2265

.42

6.75

11.1

83.

175.

211.

461.

13

13C

hkal

ovsk

oye

23Pz

4.47

63.9

78.

6111

.24

3.04

4.28

1.18

1.00

14C

hkal

ovsk

oye

91Yu

127

48-2

757

4.62

61.9

36.

9711

.53

1.85

6.15

1.51

2.06

15Yu

zhno

- M

ylid

zhin

skoy

e22

1B

1021

28-2

132

2.38

79.7

15.

635.

731.

740.

780.

190.

09

16Yu

zhno

- C

here

msh

ansk

oye

on F

ND

0.65

61.7

64.

3513

.16

3.31

8.78

1.98

2.31


syringe. The reproducibility of the results of isotopic analysis of carbon was 0.2-0.3‰.

Figure 1.66 shows the correlation of the isotopic composition of carbon of the individual hydrocarbons. The correlation ratio for the CH4–C2H6 pair is R2 = 0.32, for C2H6–C3H8 R

2 = 0.57, for C2H6-C4H10 R2 = 0.78 (● – n-butane, ■ – iso-butane), for C3H8– C5H12 R2 = 0.49 (n-pentane) and R2 = 0.73 (♦ – iso-pentane), for n-C4H10– n-C5H12 R

2 = 0.83. The correlation between the isotopic composition of the carbon of methane and ethane is not very high, whereas the correlation between heavier hydrocarbons is quite high (from 0.73 to 0.83). However, if we exclude the points with the low δ13C value for ethane (δ13C = -44

Fig. 1.66. Relationship between δ13C of individual hydrocarbons in associated oil gases.


÷ -50‰), then there is an increase in the correlation ratio. Similar results were obtained for the hydrocarbon gases of the Perm region in the Urals in [62].

Thus, there is a correlation between the carbon isotopic composition of the hydrocarbon gases which can be disrupted by the unpredictable value of the carbon isotopic composition of ethane in some fields. In addition, it was found that with an increase in the number of carbon atoms in the hydrocarbon molecule is disrupted for ethane from some deposits.

Determination of the isotopic composition of carbon in the collagen of bones of ancient tombs

The absolute chronology of the Bronze Age cultures of the Eurasian steppe is based on analysis of radiocarbon data. Various carbonaceous materials taken from clearly stratified complexes – animal and human bones, wood and charcoal, plant remains (seeds, pieces of litter, textiles) and organic soil materials are dated by the radiocarbon method to obtain historical intervals of archaeological cultures. However, there are many factors that influence the radiocarbon age of the samples of various carbonaceous materials. One of them is the influence of the so-called reservoir effect [80, 81]. Most samples, taken from the same archaeological context, have different radiocarbon ages.

The reservoir effect is related to variations in the isotopic composition of carbon inside the reservoir from which the organism derives its carbon [80]. The effect of the marine reservoir has been well studied [82–84]. But still influence the effect of the freshwater reservoir on the radiocarbon age of consumers of river products has not bee investigated sufficiently [80, 84, 85]. Studies in recent years [80, 81, 86] have shown that consumption of food of the water origin – fish, shellfish – leads to the fact that the bone collagen of humans and animals turns out to be much older as a result of the influence of the reservoir effect. Correction of the data on the effect of the freshwater reservoir is extremely difficult because of its large regional and temporal variability [80].

The objectives of this study is to investigate the influence of the reservoir effect on the radiocarbon age of the collagen of human bones of Early Catacomb, Eastern Manych and Yamnaya Catacomb cultures.

According to existing international agreements on which the radiocarbon dating is based [82], the age of samples is calculated taking into account the value of the half-life of 14C, an amendment is made for isotopic fractionation, and to the radiocarbon age is then converted


to the calendar age by calibration of the 14C data. The INTCAL04 program is recommended for this [87].

The isotope fractionation implies that the radiocarbon age of the samples with the same calendar age but different δ13C values will also vary, with a deviation of 16 years per each 1‰ of δ13C. The content of radiocarbon 14Cc is calculated from the measured amount of 14Cm considering isotopic fractionation by the formula:

14 1413

C C 1 2 C 251000c m= − +

( ) .δ (1.10)

Factor 2 accounts for the doubling of the isotope fractionation for δ14C compared with δ13C [82].

With the development in 1970–1980 of the instrumental methods of analysis of the chemical composition of the collagen extracted from bones of ancient humans [88], it became possible to identify the main components of the diet by studying the stable isotope ratios of nitrogen and carbon in collagen. The isotopic composition of bone collagen changes little with time and it completely updated in 10-30 years [89]. Measurement of the value of δ13C is a mandatory procedure for all radiocarbon laboratories. But only a few laboratories now measure δ15N. Since the values of δ13C and δ15N of the collagen of human bones and animals are determined by the feed system, then their isotopic composition can be used to identified a group of people that are largely fed on fish. The radiocarbon age in this case will be correctly identified due to the influence of the reservoir effect.

As shown in Table 1.40, the δ15N values are more positive for people who eat fish than for humans who consume foods of terrestrial origin. The difference between the ratio of the isotopes in the collagen of human bones and animal bones, constituting the source of their food, for carbon is about 5‰ in the direction of enrichment with the 13C isotope in the human bones [90]. For nitrogen, this difference amounts to about 4‰ in the direction of enrichment with the 15N isotope [89, 91].

The first reservoir effect in archaeological samples was studied in [92]. The bones of arctic animals and humans whose time of death was known and their diet was dominated by seafood (100%), were dated by the radiocarbon method. All dates shown the reservoir effect with the correction ΔR = 400 years. In [86] studied were conducted of parallel dating of marine and terrestrial organisms originating from the cultural layers of early Eskimo villages and Dezhnevo and Ekven in Chukotka. They identified the reservoir effect and differences in the


calendar dates of the marine and terrestrial organisms from 140 to 607 years old. The most illustrative example related with the study of the feed system, the reservoir effect and its impact on the radiocarbon age, was conducted by archaeologists who studied a mesolithic memorial in the Balkans – in the Iron Gates Gorge [84].They determined the values of δ13C, δ14C and δ15N in the bone collagen of people were buried after a battle, which took place in this area 7000 years BC. In the bodies of people who died in battle there were tips of arrows, made from bones of ground hoofed animals. Both the human bones human and the ungulate bones were dated by the radiocarbon method. The bones of the ancient man were 300-500 years older. It is believed that the alleged age of the samples of the human bones was the result of the reservoir effect. A significant place in the food supply system of the mesolithic

Table 1.41. The results of mass spectrometric studies of bone collagen of individuals of Yamnaya Catacomb and polyritual groups of North West Caspian region

Mound (m), burial (b), sex, age, δ13C, ‰ δ15N, ‰Zunda-Tolga-V: m. 1, b. 7, a woman 50-55 years old -17.77 14.93

Zunda-Tolga-V: m. 1, b. 7, pin from animal bones -21.01 5.36

Zunda-Tolga-2: m. 2, b. 2, a man ≥ 50 years -17.24 +16.69

Mu-Sharett-1: m. 8, b. 3, a woman 17-19 years old -17.40 15.54

Ulan-Zukha: m. 3, b. 8, adult -17.56 14.45East Manych, Left bank, I, 1966, sheep -18.52 9.02Zunda-Tolga-V: m. 1, b. 7, a woman 50-55 years old -17.77 14.93

Table 1.40. The mean values of δ13C and δ15N and the reservoir effect expected in the collagen of human and animal bones at theoretical 100% of the diet in each of the listed categories

The food supply system δ13C,‰ δ15N,‰ Reservoir effect, years

Group C3 plants -21 +5 +0Meat of herbivores (C3 group plants) -18 +8 0

C4 group plants -7 +5 0Seafood -13 +18 400Freshwater fish -24 +16 1500-2500Lake fish -20 +16 500-1500


Table 1.42. The results of mass spectrometric studies of bone collagen of individuals of the Early Catacomb culture of North-West Caspian Sea

Cemetery, burial mound (m), burial (b), sex, age, δ13C,‰ δ15N,‰Peschanyi-V: m. 1, b. 1, a woman aged 25–35 -18.25 14.13Peschanyi-V: m. 2, b. 3, man 40–45 -18.15 +15.79Peschanyi-V: m. 3, b. 1, man 45–50 -17.02 17.16Peschanyi-V m. 3, 2, man 40–45 -21.54 15.24Peschanyi-V: m. 1, a sacrificial place I, a horse -21.16 4.66Temrta-III: m. 1, b. 1 woman 30–35 -18.34 14.95 Temrta-III: m. 1, b .4, man 40–45 -18.15 14.73 Temrta-III: m. 2, b. 1, man (?) 45–50 -17.85 14.12Temrta-III: m. 1, discovery 7, sheep -19.47 9.57Baga-Burul: m. 5, b. 6, man 30–35 -18.79 +12.64Baga-Burul: m. 5, b. 19, a bull -18.5 9.92Mandzhikiny-2: m. 42, b. 1, woman 40–45 -17.16 17.28 Mandzhikiny-2: m. 42, b. 4, child 3–4 years -15.29 18.13 Mandzhikiny-2: m. 45, b. 2, man 17–20 -17.10 17.50 Mandzhikiny-2: m. 54, b. 6, a woman aged 25–35 -17.59 15.04Zunda-Tolga-2: m. 1, b. 1, man adultus -17,46 +16,13Zunda-Tolga-2: m. 2, b. 3, a man about 35 years, -18.62 13.92Zunda-Tolga-5: m. 1, b. 5, a man senilis -18,65 +14,03Temrta-V: m. 1, Section 2, a woman 17–20 -17.97 12.49Temrta-V: m. 1, b. 2, man 20–25 years -20.60 -Temrta-V: m. 1, b. 3, woman of 40–50 -17.98 10.73Temrta-V: m. 1, b. 3, a woman of 40–50 -17.49 15.40Temrta-V: m. 1, b. 3, man 40–50 years, -17.74 12.79 Temrta-V: m. 1, b. 3, man 40–50 years -17.18 15.61Temrta-V: m. 1, b. 2, pin from animal bones -18.28 8.75Har-Zuha-1: m. 5, b. 3b, the woman senilis -15,42 +18,10Har-Zuha-1 m. 7, b. 4, a man of 30–35 -18.10 14.15Har-Zuha 1: m. 1, b. , a man of 45–50 -17.62 15.66

population living in the Iron Gates Gap, was occupied by the fish from the Danube. The δ15N values for the bone collagen of humans were +15‰, for the bones of land animals 5‰.

The collagen from the bones was chemically separated as a residue insoluble as described in [76]. The isotopic composition of carbon and nitrogen of the bone collagen was measured by the EA-IRMS method in a DELTA Plus isotope mass spectrometer, connected to the EA 1110 elemental analyzer. To do this, samples were taken samples weighing 0.3–0.8 mg, which were packaged in tin capsules and placed in a device for supplying samples to the elemental analyzer. Each sample was analyzed three times and the results averaged. The standard deviation


Table 1.43. The results of radiocarbon dating of wood samples and collagen of human bones from Early Catacomb burial grounds at Khar-Zukha

Mound (m)/burial (b) Material

14C-age in years of

our days (BP)δ13C, ‰ δ15N, ‰

m. 1, b. 1 Wood 3561 ± 308m. 5, b. 3A Mature woman 3940 ± 70 -15.42 +18.1

m. 1, b. 5 Male 45–50 years 4059 ± 152 -17.62 +15.70

of measurement results of δ13C was ±0.2‰, and in measurement of δ15N ±0.3‰.

The results of isotopic analysis of the bone collagen of humans and animals found in the mounds and graves, are presented in Tables 1.41 and 1.42. The tables show that in the collagen of human bones the δ13C values varied from -21.54 to -15.29‰ and δ15N - from +10.73 up to +18.13‰. Accordingly, in the bones of herbivores δ13C values range from -21.16 to 18.28‰ and δ15N from +4.66 up to +9.92‰. Mass spectrometric data strongly suggest that the fish component occupies an important place in the food supply system of the analyzed individuals. This has caused a shift to the older part of many data (ΔR), obtained from the human bones.

The confirmation of the permanent fish component of the food system of the humans is provided by the microblade scales of river and lake fish, and shells of river mussels found in the graves.

For the early catacomb culture, the conjectural value of ΔR can be determined at present by comparing the 14C data for the collagen of the human bone and the wood from the Khar-Zukha cemetery (Table 1.43). Graves in this cemetery are characterized by similar burial rites number and, most likely, were left by an independent group of the early catacomb population.

The data in Table 1.43 shows the reservoir effect in the bones of a deceased person equalling 400 ± 100 14C years. More precise definition is impossible because of the significant error in the dating of wood. The difference of dates obtained for the bones of humans and sheep of the Ostrovnoy burial ground of the East Manych culture was ΔR = 230 ± 10014C years. Further implementation of the parallel dating of different carbon-bearing samples from burials of the Early Catacomb culture should clarify the correction ΔR.

Thus, these data allow us to introduce a regional and a temporal amendment ΔR to the reservoir effect and correct the historical periods of existence of Bronze Age cultures of the North-West Caspian Sea.


Conclusions

Studies have shown ways to improve the analytical characteristics of the isotope mass-spectrometric method of analysis of stable isotopes. One promising avenue is the use of solid electrolytes in devices for sample preparation for the isotope analysis of light elements. This allows us to replace the cumbersome and not very reliable standard oxidizing and reduction reactors by reactors that use solid-electrolyte oxygen-and hydrogen-conducting ceramics. Thus, it will be possible not only to carry out complete oxidation of complex organic compounds, but also reduce oxides of light elements. In the future it will be possible to abandon pyrolytic attachments to IRMS, working at high temperatures (1500°C) and also standard systems for the decomposition of water.

Thus, the further development of isotope ratio mass spectrometry of light elements is associated with the optimization of the method of isotope analysis, with new, more sensitive electronic equipment, and with new sample preparation devices.

References

1. A. Eloy J. F. Analyses chimiques semi-quantitative par spectrographie de mass a ionization par bombardement photonique, Int. J. Mass Spectr.and Ion Phys. 1971, V. 16, No. 1/2, pp. 101–115.

2. Ireland T. R., SIMS measurement of stable isotopes, Handbook of stable isotope analytical techniques, V. 1. Ed. by P. A. de Groot. Chapter 30, pp. 652–691.

3. Sysoev, A.A., Artayev V.B., Kashcheev V.V., Isotope mass spectrometry, Moscow: Energoatomizdat, 1993, 288.

4. Chupakhin M.S., Probe methods in spark mass-spectrometry, Moscow: Energoato-mizdat, 1985.

5. Pajo L., Tamborini G., Rasmussen G., Mayer K., Koch I., A novel isotope analysis of oxygen in uranium oxides: comparison of secondary ion mass spectrometry, glow discharge mass spectrometry, and thermal ionization mass spectrometry, Spectrochim. Acta, 2001, V. B56, pp. 541–549.

6. Wilson R. G. SIMS quantification in Si, GaAs, and diamond – an update, Int. J. Mass Spectrom. Ion Proc., 1995, V. 143, pp. 413–417.

7. Williams P., On mechanisms of sputtered ion emission, Appl.Surface. Sci., 1982, V. 13, 8. 241–259.

8. Eiler J.M., Graham C.W., Valley J.M., SIMS analysis of oxygen isotopes: matrix effects in complex minerals and glasses, Chem. Geol., 1997, V. 138, pp. 221–244.

9. Riciputi L. R., A comparison of extreme energy filtering and high mass resolution techniques for measurements of 34S/32S ratios by ion microprobe, Rapid Com-mun. Mass Spectrom., 1996, V. 10, pp. 282–286.

10. Leshin L. A., Rubin A. E., McKeegan K. D., The oxygen isotopic composition of olivin and pyroxene from CI chondrites, Geochim.Cosmochim. Acta,1997, V. 61, pp. 835–845.


11. Mojzsis S. J., Harrison T.M., McKeegan K. D., Oxygen isotope evidence from ancient zircons for liquid water at the Earth’s surface 4 300 Myr ago, Nature, 2001, V. 409, pp. 178–181.

12. Larras-Regard E., Mony M. C., Improvements in biomedical imaging by SIMS (IMS 3F, 4F, and nanoSIMS), In: SIMS 11. G. Gillen, R., L.Lareau, J. Bannett, F. Stevie (eds.), 1997, pp. 119–122.

13. Fayek M., Harrison T. M., Glove M., McKeegan K. D., Coath C. D., Boles J. D., In situ isotopic evidence for protracted and complex carbonate cementation in a petroleum reservoir, North Coles Leveem, San Joaquin Basin, California, USA, J. Sedim. Res., 2001, V. 71 (3), pp. 454–478.

14. Hauri E.H., Wang J., Pearson D.G., Bulanova G. P., Microanalysis of δ13C, δ15N, and N abundances in diamonds by secondary ion mass spectrometry, Chemical Geology, 2002, V. 185, pp. 149–163.

15. Bulanova G. P., Pearson D.G., Hauri E.H., Griffin B. J., Carbon and nitrogen iso-tope systematics within a sector-growth diamond from the Mir kimberlite, Yakutia, Chemical Geology, 2002, V. 188, pp. 105–123.

16. Tokarev M.I., Fainberg V. S., Khodeev Yu. S.m Modern possibilities and the pros-pects for mass spectrometry of light elements, Mass Spectrometry, 2004, Volume 1 (3), pp. 179–190.

17. Vasilchuk Yu. K., Kotlyakov V. M., Fundamentals of isotope geocryology and glaciology, M.: Publishing House of University, 2000, 616 p.

18. Urey H. C., The thermodynamic properties of isotopic substances, J. Chem. Soc., 1947, V. 47, No. 3, pp. 562–581.

19. Fogel M. L., Cifuentes L.A., Isotope fractionation during primary production. In Organic Geochemistry / Ed. by M.H. Engel, S.A. Macko, New York and London: Plenum Press, 1993, pp. 73–220.

20. Matthews D. E., Hayes J. M., Isotope-ratio-monitoring gas chromatography-mass spectrometry, Analytical Chemistry, 1978, V. 50,pp. 1465–1473.

21. Hayes J. M., Freeman K. H., Hoham C. H., Popp B., N.. Compound-specific isoto-pic analyses: a novel tool for reconstruction of ancient biogeochemical processes, Organic Geochemistry, 1989, V. 16, pp. 1115–1128.

22. Slater C., Preston T., Weaver T., Stable isotopes and the international system of units, Rapid Communications in Mass Spectrometry, 2001, V. 15, pp. 1270–1273.

23. Werner R. A., Brand W. A., Referencing strategies and techniques in stable iso-tope ratio analysis, Rapid Communications in Mass Spectrometry, 2001, V. 15, pp. 501–519.

24. Hachey D. L., Wong W. W., Boutton T. V., Klein P. D., Isotope ratio measure-ments in nutrition and biomedical research, Mass Spectrom. Rev., 1987, V. 6, No. 2, pp. 289–328.

25. Sharp Z. D., Atudorei V., Durakiewicz T., A rapid method for determination of hydrogen and oxygen isotope ratios from water and hydrous minerals, Chemical geology, 2001, V. 178, pp. 197–210.

26. Kornexl B., Gehre M., Hofling R., Werner R. A. On-line δ18O measurement of organic and inorganic substances, Rapid communications in mass spectrometry, 1999, V. 13, pp. 1685–1693.

27. Merritt D. A., Freeman K. H., Ricci M. P., Studley S. A., Hayes J. M., Performance and optimization of a combustion interface for isotope ratio monitoring gas chro-matography/mass spectrometry, Analytical Chemistry, 1995, V. 67, pp. 2461–2473.

28. Whiticar M. J., Eek M., Challenges of 13C/12C measurements by CF-IRMS of biogeochemical samples at sub-nanomolar levels, New approaches for stable isotope ratio measurements: Proc. Advisory Group meeting held in Vienna, 1999,


Sep. 20–23, pp. 75–95. International Atomic Energy Agency. IAEA-TECDOC-1247. 29. Meier-Augenstein W., Hess A., Hoffman G. F., Rating D., Evaluation of water

removal and memory effect in 13CO2 breath tests by isotope ratio mass spectrom-etry, Environ. Health Stud., 1994, V. 30, pp. 349–358.

30. Ricci M.P, Merritt D. A., Freeman K. H., Hayes J. M., Acquisition and processing of data for isotope–ratio–monitoring mass spectrometry, Org. Geochem., 1994, V. 21, No. 6/7, pp. 561–571.

31. Merritt D. A., Hayes J. M., Factors controlling precision and accuracy in isotope–ratio–monitoring mass spectrometry, Anal. Chem., 1994, V. 66, pp. 2336–2347.

32. Gel'man N. E., Terent'yev E. A., Shanina T. M., Kiparenko L. M., Rezl B., Meth-ods for the quantitative elemental microanalysis, Moscow: Khimiya, 1987, 296.

33. Miroshin V. P., Dubin V. M., The catalyst for the oxidative decomposition of polycyclic organic compounds under analysis CHN–automatic Hewlett-Packard, Zh. Analit. Khim., 1991, V. 46, No. 3, pp. 493–499.

34. Perfiliev MV, AK Demin, BL Kuzin, A. Lipilin Vysosokotemperaturny electrolysis gases, Moscow: Nauka, 1988, 232.

35. Talanchuk P. M., Shmatko B. A., Zaika L. S., Tsvetkova O. A., Semiconductor and solid electrolyte sensors, Kiev: Tekhnika, 1992, 222 p.

36. Vecher A. A., Vecer D. V., Solid electrolytes, Minsk: Universitetskoe, 1998, 109 p. 37. Zuev B. K., Olenin A. Yu., Solid-electrolyte sensor as a detector for gas chro-

matographic determination of combustible contaminants in air, Zh. Analit. Khim., 2006, V. 61,No. 2, pp. 157–163.

38. Somov S. I., Reinhardt G., Guth U., Gopel W., Multi-electrode zirconia electrolyte amperometric sensors, Solid State Ionics, 2000, V. 136–137, pp. 543–547.

39. Shkerin S. N., Perfil'ev M. V., Kinetics of electrode reactions on the model electrode in the system Pt, O2/O2–. The dependence of the electrode impedance on the tem-perature and composition of the electrolyte, Elektrokhimiya, 1990, pp. 1461–1467.

40. Kunin L. L., Fedorov M. S., Solid electrolytes based on zirconium dioxide in the gas analysis,Zh. Analit. Khim., 1990, V. 45, No. 3, pp. 421–433.

41. Metcalfe I. S., Electrochemical promotion of catalysis at metal surfaces, Proceed-ings of the 26th Risø International Symposium on Materials Sciences: Solid State Electrochemistry, 2005, pp. 30–50.

42. Brazhnikov V. V., Detectors for chromatography, Moscow: Mashinostroenie, 317 p. 43. Iqbal M. S., Rashid F., Javed N. A., An electrolytic device for preparation of hydrogen

and oxygen from water for isotopic analysis, Talanta, 1991, V. 38, pp. 603–605. 44. Sharp Z.D., Atudorei V., Durakiewicz T., A rapid method for determination of

hydrogen and oxygen isotope ratios from water and hydrous minerals, Chemical geology, 2001, V. 178, pp. 197–210.

45. Gehre M., Geilmann H., Richter J., Werner R. A., Brand W.A.Continuous flow 2H/1H and 18O/16O analysis of water samples with dual inlet precision, Rapid com-munications in mass spectrometry, 2004, V. 18, pp. 2650–2660.

46. Gehre M., Hoeffing R., Kowski P., Strauch G., Sample preparation device for quantitative hydrogen isotope analysis using chromium metal, Anal. Chem., 1996, V. 68, pp. 4414–4417.

47. Horita J., Ueda A., Mizukami K., Takatori I., Automatic δD and δ18O analyses of multi-water samples using H2- and CO2-water equilibration methods with a com-mon equilibration set-up, Appl. Radiat. Isot., 1989, V. 40, pp. 801–805.

48. Brand W. A., Avak H., Seedorf R., Hofmann D., Conrad Th., New methods for fully automated isotope ratio determination from hydrogen at the natural abundance level, Isotopes Environ. Health Stud., 1996, V. 32. pp. 263–273.

49. Ehleringer J. R., Cooper D. A., Lott M. J., Cook C. S., Geo-Location of heroin


and cocaine by stable isotope ratios, Forensic Science International, 1999, V. 106, pp. 27–35.

50. Galimov E. M., The nature of biological isotope fractionation, Moscow: Nauka, 1981, 247 p.

51. Desage M., Guilluy R., Brazier L. J., Gas chromatography with mass spectrometry or isotope-ratio mass spectrometry in studying the geographical origin of heroin, Analytica Chimica Acta, 1991, V. 247, pp. 249–254.

52. Hays P. A., Remaud G. S., Jamin E., Martin Y.-L., Geographic origin determination of heroin and cocaine using site-specific isotopic ratio deuterium NMR, J. Forensic Sci., 2000, V. 45, pp. 552–562.

53. Besacier F., Chaudron-Thozet H., Lascaux F., Rousseau-Tsangaris M., Application of gas chromatography-nitrogen isotopic mass-spectrometry to the analysis of drug samples, Analusis, 1999, V. 27, pp. 213–217.

54. Ehleringer J. R., Casale J. F., Lott M. J., Ford V. L., Tracing the geographical origin of cocaine, Nature, 2000, V. 408, pp. 311–312.

55. Besacier F., Guilluy R., Brazier J. L., Chaudron-Thozet H., Girard J., Lamotte A., Isotopic analysis of 13C as a tool for comparison and origin assignment of seized heroin samples, J. Forensic Sci., 1997, V. 42, pp. 429–433.

56. Rudenko B.A., Chromatography on terrorism, partners and competitors, 2005, No. 9, pp. 30–34.

57. Heavily V. M., Filonenko V. G., Shishmarev A. T., Baldin N. M., Naumenko I. I., Express analysis of objects of the environment with portable gas chromatographs and polycapillary columns, Zh. Analit. Khim., 1999, V. 54, No. 9, pp. 957–956.

58. Zyakun A. M., Identify the sources of raw material of ethanol distilleries in the product, Problems of identification of alcohol-bearing products, Moscow: Gos-standart, 2001, pp. 110–114.

59. Kelley D. S., Methane-rich fluids in the oceanic crust, J. Geophys. Res.,1996, V. 101, pp. 2943–2962.

60. Berndt M. E., Allen D. E., Seyfried W. E. J., Reduction of CO2 during serpen-tinization of olivine at 300°C and 500 bar, Geology, 1996, V. 24. pp. 351–354.

61. Horita J., Berndt M. E., Abiogenic methane formation and isotopic fractionation under hydrothermal conditions, Science, 1999, V. 285, pp.1055–1057.

62. Galimov E. M., The isotopes of carbon in petroleum geology, Moscow: Nedra, 1973.63. DeMarais D.J., Donchin J.H., Nehring N. L., Truesdell A. H., Molecular carbon

isotopic evidence for the origin of geothermal hydrocarbons, Nature,1981, V. 292, pp. 826–828.

64. Schoell M. Multiple origins of methane in the earth, Chemical Geology,1988, V. 71, pp. 1–10.

65. Sherwood Lollar B., Westgate T. D., Ward J. A., Slater G. F., Lacrampe-Coulume G., Abiogenic formation of alcanes in the Earth’s crust as a minor source for global hydrocarbon reservoirs, Nature, 2002, V. 416, pp. 522–524.

66. Lancet H. S., Anders E.. Carbon isotope fractionation in the Fischer-Tropsch synthesis of methane, Science, 1970, V. 170, pp. 980–982.

67. Prinzhofer A., Huc A. Y., Genetic and post-genetic molecular and isotopic frac-tionations in natural gases, Chemical Geology, 1995, V. 126, pp. 281–291.

68. Ott U. Interstellar grains in meteorites, Nature, 1995, V.364,pp. 25–33. 69. Sephton M.A. Organic compounds in meteorites, Nat. Prod.Rep., 2002, V. 19,

pp. 292–311. 70. Russell S. S., Arden J.W., Pillinger C. T., A carbon and nitrogen isotopic study of

diamonds from primitive chondrites, Nature, 1989, V. 31, pp. 343–355. 71. Alley R. B., Cuffey K. M., Oxygen- and hydrogen-isotopic ratios of water in pre-


cipitation: beyond paleothermometry, Ed. by J.W. Valley, D.R. Cole, Reviews in mineralogy and geochemistry. Stable isotope geochemistry, 2001, V. 43, pp. 527–553.

72. Polyakov V. B., Isotope effect, limited by diffusion in the flow of heterogeneous reactions in the kinetic region. Theory, Fiz. Khim., 1989, V. 8. No. 11, pp. 1539–1541.

73. Polyakov V. B., Maltsev K. A., The isotope effect limited by diffusion in the flow of heterogeneous reactions in the kinetic region. Experiment, Fiz. Khim., 1989, V. 8, No. 12, S. 1648–1652.

74. Ferronskii V. I., Polyakov V. A., Isotopy of the hydrosphere, Moscow: Nauka, 1983, 280 p.

75. Lobyshev V. N., Kalinichenko L.P., Isotope effects of D2O in biological systems, Moscow: Nauka, 1978.

76. Bild W. et al., Research concerning the radioprotective and immunostimulating effects of deuterium-depleted water, Rom. J. Physiol., 1999, V. 36, No. 3–4, pp. 205–218.

77. Somlyai G. The biological effect of deuterium depletion, Budapest: Academiai Klado, 2002, 149 p.

78. Bowen G. J., Winter D. A., et al., Stable hydrogen and oxygen isotope ratios of bottled water of the world, Rapid communications in mass spectrometry, 2005, V. 19, pp. 3442–3450.

79. Forstel H., Hoube J., Hutzen H., Use of trap water samples for monitoring the geographical variation of stable isotopes used in authenticity studies, Z. Leb-ensm. Unters. Forsch., A, 1997, V. 204, pp. 103–108.

80. Lanting J.N., van der Plicht J. Wat hebben Floris 5, skelet Swifterband en visotters gemeen? / / Palaeohistoria, 1995/1996, V. 37/38, pp. 491–519.

81. Cook G. T., Bonsall C., Hedges R. E.M., McSweeney K., Oronean V. B., Pettitt P. B.. A freshwater diet-derived 14C reservoir effect at the stone age sites in the Iron Gates gorge, Radiocarbon, 2001, V. 43, pp. 448–453.

82. Arslanov Kh. A., Radiocarbon: geochemistry and geochronology, Leningrad Uni-versity Press, 1987, 298 p.

83. Khasanov B. F., Savinetsky A. B., Dating of marine and terrestrial organisms from the northern Bering Sea, OPUS: Interdisciplinary studies in archeology, 2002, No. 1–2, pp. 188–196.

84. Cook G. T., Bonsall C., Hedges R. E. M., McSweeney K., Oronean V. B., Pettitt P. B., A freshwater diet-derived 14C reservoir effect at the stone age sites in the Iron Gates gorge, Radiocarbon, 2001, V. 43, pp. 448–453.

85. Olsson I.U., Dating of non-terrestrial materials, Proc. Groningen Symp. 14C and Archaeology / Ed. by W. G. Mook, H. T. Waterbolk, PACT Publications 8, 1983, pp. 277–294.

86. Olsson I. U., The radiocarbon contents of various reservoirs / Ed. by R. Berger, H. E. Suess, Radiocarbon dating, Los Angeles: University of California Press, 1979, pp. 613–618.

87. Mook W. G., van der Plicht J., Reporting 14C activities and concentrations, Ra-diocarbon, 1999, V. 41, pp. 227–239.

88. Pate F. D., Bone chemistry and paleodiet, Journal of Archaeological Method and Theory, 1994, V. 1, pp. 161–209.

89. Muldner G., Richards M. P., Fast or feast: reconstructing diet in later medieval England by stable isotope analysis, Journal of Archaeological Science, 2005, V. 32, pp. 39–48.

90. Chistolm B. S., Nelson D. E., Schwarcz H. P., Stable-carbon isotope ratios as a measure of marine versus terrestrial protein in ancient diets, Science, 1982, V. 216, No. 4550, pp. 1131–1132.

91. Schwarcz H. P., Melbye J., Katzenberg M. A., Knuf M., Stable isotopes in human


skeletons of southern Ontario: Reconstructing palaeoiet, Journal of Archaeological Science, 1985, V. 12, No 2, pp .187–206.

92. Tauber H., 14C activity of arctic marine mammals / Ed. by R. Berger, H. E. Suess, Ra-diocarbon dating, Los Angeles: University of California Press, 1979, pp. 447–452.

119Universal method for preparation of samples

2

Universal method for preparation of liquid, solid and gaseous samples for

determining the isotopic composition of carbon

T.A. Velivetskaya1, A. Ignatev1, S. Kiyashko2

1Far East Geological Institute, Far East Branch of the Russian Academy of Sciences, Vladivostok

2Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, ul. Palchevskogo, Vladivostok

Introduction

The measurements of isotope ratios 13C/12C of carbon are widely used to study various natural processes, including global climate changes and ecological and geochemical study of environmental changes. Variations in the isotopic composition carbon present valuable information to determine the source of carbon reservoirs, understanding the physical and biochemical processes that lead to the displacement of carbon flows between the reservoirs, which plays an important role in studying processes in the modern biosphere and atmosphere, as well as for the construction of paleoecological and paleoclimatic reconstructions. To this end, measurements are taken of the carbon isotopic ratios 13C/12C of various natural materials having various forms of solid, liquid and gaseous substances. The carbon isotope analysis of solid, liquid and gaseous samples is carried out using appropriate methods and specialized equipment. The quantitative conversion of the carbon of samples to the form of CO2 for mass spectrometric analysis is usually carried out using the solid oxidizer – copper oxide.


This method and its implementation were described for the first time in [1, 2] and is now already a classic method. According to this method, a milligram amount of the sample with an oxidizing agent is sealed in an evacuated quartz ampoule. The ampoules with the samples are calcined at 850–900°C for a long time (5–6 h) and slowly cooled (~12 h). Each ampoule is then opened in a high-vacuum line with a special device [3, 4] followed by cryogenic purification of CO2 to remove other combustion products. Sofer [5] modified this method by proposing to replace expensive quartz ampoules by more economical ampoules made of borosilicate glass (Pyrex). In this case, the sample is calcined at lower temperatures of 550–590°C which are not always sufficient for complete oxidation of carbon [6]. Study [7] describes a method of low temperature (500°C) conversion using oxidants: potassium peroxodisulphate and silver permanganate. It is shown that complete oxidation of the carbon of organic compounds is achieved in 24 h.

After a number of improvements [8, 9], the method of combustion in a quartz ampoule has been widely used for determining the carbon isotopic composition of various solid organic substances, soil and diffuse forms of carbon in rocks and sediments. The classical method of burning carbon in ampoules provides high accuracy and reproducibility of measurements of the isotopic composition δ13C of solid samples. However, the application of this method for the combustion of volatile liquid and gaseous samples is associated with considerable technical problems and impairs the reproducibility of the results of isotopic analysis. This method is also time-consuming, costly and unproductive.

In the early 1980s, a method was proposed for measuring δ13C in a stream of helium, based on application of a CN-elemental analyzer coupled with a mass spectrometer via a ConFlo interface (EA-IRMS method) [10–12]. Samples of an organic material, packed in tin capsules, are placed in the reactor of the elemental analyzer heated to 900–1050°C, with continuous stream of helium passed through the reactor. The sample is combusted in the ‘flash’ mode with the injection of oxygen into a helium stream of helium. The combustion products pass gradually through a series of columns with oxidizing and reducing reagents. Water is removed using a trap with an absorbent (typically magnesium perchlorate), CO2 and N2 are separated in the chromatographic column and fed with a helium stream helium into the mass spectrometer through the ConFlo interface. In such systems, the processes of conversion, the chromatographic separation of reaction


products, the travel of the gas flow in the ion source of the mass spectrometer and the measurement of δ13C are integrated into a single automated process that takes 8–10 min for one sample.

With this technique, the size of the charges of analyzed samples is reduced by tens of times and productivity is significantly increased. The reproducibility of the measurement results of δ13C for routine analyses is 0.1–0.5‰. It should be noted that the EA-IRMS method is usually used for organic samples, which have a high carbon content (plant and animal tissue). If the samples have a low carbon content (carbon is dispersed in the rocks, sediments, soil) the charge must be increased by several times in order to obtain an optimal signal for measurement. In this case, an excessive amount of the carbon-containing matrix can cause incomplete combustion of carbon and reduce the accuracy of analysis [13]. To solve these problems, Liu and coworkers [14] proposed to apply an Nd-YAG laser for the isotopic analysis of organic carbon in sedimentary rocks and obtained the reproducibility of the results of measurement of 0.1‰.

Isotopic studies of gaseous forms of carbon, such as CH4 in the atmosphere and volcanic gases, require very high precision analysis. The technique of quantitative extraction and conversion of CH4 to CO2 is not trivial. Sample preparation systems include a high-vacuum line for cleaning CH4 and combustion in the presence of a platinum catalyst. The resulting CO2 is cryogenically separated from other reaction products and analyzed in a mass spectrometer in the dual-inlet system configuration [15]. The reproducibility of the results of isotopic analysis was 0.05‰. Application of the new method for measuring δ13C in a helium stream allowed more quickly and efficiently to determine the carbon isotopic composition of methane in the samples with an extremely low concentration [16].

Thus, solid, liquid and gaseous samples are processed using the corresponding methods of sample preparation and techniques. In this study we propose a universal method of sample preparation, which applicable for both solid and for liquid and gaseous samples. Implementation of the method is simple and requires no expensive equipment to perform isotopic analysis with high accuracy for the samples of solid organic matter, soil, scattered forms of carbon in rocks and sediments, oils, volatile liquids and gases substances.


Fig.

2.1

. A u

nive

rsal

met

hod

for

the

prep

arat

ion

of l

iqui

d, s

olid

and

gas

eous

sam

ples

to

dete

rmin

e th

e is

otop

ic c

ompo

sitio

n of

car

bon

The experimental part

Equipment and materials

Solid and liquid samples of organic carbon and gaseous forms of carbon (CH4 and CO2 atmosphere, and gas hydrates) were prepared for isotopic analysis using the high-vacuum installation in Fig. 2.1). The installation consists of the pumping line, including the backing pump and a

Turb

omol

ecul

arpu

mp

Con

nect

ors Va

lves

Port

s

Bac

king

pum

p Sam

pler

w

ith a

mpo

ules

Bac

king

pum

p

V7

V1 V2

V1

V3

V4 V5

V5

V6

V5 V9

V10

Hea

ters

Hea

ters

Rea

ctor

for

liq

uid

sam

ples

Rea

ctor

for

sol

id s

ampl

es

Hea

ters

Sept

um

P1 P2 P3 P4 P5 P6

Ther

mor

egul

ated

tra

p

U-t

rap


turbomolecular pump, six parallel ports (inputs) for the simultaneous connection of six lines for the extraction CO2; a sampler for CO2. The vacuum lines are made of molybdenum glass and connected together by metallic joints with a screw seal. The extraction line includes a quartz reactor for burning samples and a cryogenic U-trap. Each reactor is heated by two separate heaters. The installation has an additional temperature-controlled cryogenic trap for gas purification.

The design and construction of this trap are simple (see Fig. 2.2). A Pyrex glass ‘finger’ with a nichrome spiral around and a thermocouple are placed inside a cylindrical casing. Between the finger and the upper edge of the casing there is a sealing rubber ring, with the bottom cover of the casing open. A valve is installed in the top of the casing. The trap is cooled with liquid nitrogen and the temperature of the ‘finger’ is regulated by heating the coil to a predetermined value.

The Finnigan MAT 252 mass spectrometer, working in a dual-inlet system configuration, is used to measure isotope ratios of carbon in the sample gas.

Fig. 2.2. The cryogenic temperature-controlled traps: a – a trap in the mode of freezing samples at –196°C (heater is turned off, the valve is open); b – a trap in the mode of distillation of the samples (the heater is turned on, the valve is closed).

Temperature regulator

Rubber ring

Finger

Jacket

Heater

Thermocouple

Liquid nitrogen

Dewar vessel

Valve

a b


Combustion of solid and low-volatility liquid samples to determine the isotopic composition of carbon

Preparation of the reactor and loading of the sample. The reactor for burning solid and viscous liquid samples is produced from a quartz tube 30 cm long and 0.6 cm inner diameter, one end of the tube is sealed (Fig. 2.3a). The reactor is filled in the following order. At the bottom of the reactor there is a sample (1–2 mg), mixed with the CuO powder in the ratio 1:10 by weight. This is followed by placing aa layer of silica wool (1–2 mm) and an oxidation column, consisting of copper oxide granules with the addition of platinum wire; the height of the oxidation column

Fig. 2.3. The scheme of the reactor for the oxidation of organic carbon samples: a – reactor for solid and low-volatility liquid samples; b – reactor for volatile liquid samples; c – reactor for gaseous samples in conjunction with a temperature-controlled trap.

Heaters

Quartz reactor

Oxidation column CuO granules + Pt wire Sample

Heater

Septum

Quartz reactor

Oxidation column CuO granules + Pt wire

Silica wool

Quartz reactor

Oxidation column CuO granules + Pt wire

Heater

Silica diaphragm

TeeV1

V2

Septum

a

b

c


is 15 cm, granules 1–2 mm. Copper oxide must be prebaked at 900°C to remove trace amounts of contamination with organic carbon.

The filled reactor is connected to a U-trap of one of the ports in the vacuum apparatus, such as port P1 (Fig. 2.1). Two heaters are installed on the reactor. Heater H1 is placed at the reactor so that the heating zone included only part of the column (about two thirds of its length). The rest of the column and the sample enter the heater zone 2.

Burning and clearing procedures. The reactor is evacuated and the temperature of H1 is set to 850°C. This provides preheating of the oxidation column, with the sample and part of the CuO column remaining at room temperature. When the temperature of the H1 reaches 850°C, the U-trap is cooled with liquid nitrogen and pumping is stopped by valve V1. Heater H2 is switched on set to a temperature of 850°C. This heater provides heating of the sample and the remainder of the CuO-column. During heating the volatile fractions (products of incomplete oxidation of organic matter) in the zone with the sample start to sublimate and become fully oxidized to CO2 and H2O in the preheated CuO-column and immediately frozen out in the U-trap. Once the temperature of the H2 reaches 850°C, 10 min is sufficient for complete combustion of the sample and the freezing of all the reaction products in the U-trap. The valve V8 between the U-trap and the reactor is then closed, the valve B1 is open for pumping of non-condensed gases from the U-trap. After that, pumping is stopped by the valve V7 and CO2 is cleaned. The Dewar vessel with liquid nitrogen in the U-trap is replaced by dry ice and the pure CO2 is frozen in one of ampoules.

This cleaning method allows to divide only CO2 and H2O and is effective in the analysis of organic compounds of simple composition CxHyOz, but is not effective in the analysis of multicomponent organic substances (e.g., organic soils and sediments) as the products of oxidation NxO and SO2 can be formed in sufficient quantity. In the analysis of complex component organic substances, we propose to use a differential cryogenic trap for cleaning CO2 (Fig. 2.1, the port number 5). The trap is placed in a Dewar vessel with liquid nitrogen, and the valve on the top of the trap should be open to the atmosphere for nitrogen to fill the internal volume of the jacket and fast cool the finger (Fig. 2.2a). All reaction products are frozen in the finger. After that the valve in the trap is closed and the heater on the finger switched on. The nitrogen vapour pressure increases, and liquid nitrogen is squeezed out from the internal volume of the jacket. The finger is heated to the required temperature (Fig. 2.2b). Stepwise heating of the finger allows to separate cryogenically different types of gases. At a temperature of –120°C the vapour pressure of CO2 increases and CO2 is frozen in the


sampler ampoule. Heating up to –75°C increases the pressure of SO2 vapour, which is ejected from the system. It should be noted that it is very difficult to cryogenically split N2O and CO2, as the boiling points of N2O and CO2 are very close. However, the amount of N2O is very small, since the oxidation reaction on the platinum catalyst shifts the process in the direction of formation of NO, which is slightly frozen out at the liquid nitrogen temperature, and is easy to pump out.

A sampler with pure CO2 is connected to the dual inlet system of the mass spectrometer for analysis of δ13C.

The reactor and the oxidation column are used repeatedly for the analysis of a new sample. The reactor is disconnected from the vacuum line, the granular CuO is poured into a clean container and the reactor is mechanically cleaned. A new sample is placed in the reactor, as described above. This type of reactor and the process of CO2 production are applicable for both solid organic samples and viscous liquids (oil). The operating life of the oxidation reactor and the column is sufficient for 100 analyses.

Combustion of volatile liquids to determine carbon isotopic composition

Preparation of the reactor and input of the sample. The reactor for burning highly volatile liquids is constructed from a quartz tube 20 cm long with the inner diameter of 0.6 cm (Fig. 2.3b). The tube is vacuum sealed with a rubber septum at one of the ends. Part of the reactor is filled with an oxidizing column of granulated copper oxide with the addition of platinum wire (length of the oxidation column 8 cm, granules 1–2 mm). The column is located at a distance of 3 cm from the edge of the reactor plugged with the septum. The heater is placed on the reactor and shifted close to the septum. The column is in the central part of the heater. The reactor is connected to a U-trap of one of the ports of the vacuum system, for example, P3 (see Fig. 2.1).

Burning and cleaning procedure. The reactor is evacuated and the heater temperature set to 850°C. The heater preheats the oxidizing column and heats (to a temperature of 150°C) the volume between the septum and the oxidation column. When the temperature of the heater reaches 850°C, the trap is cooled with liquid nitrogen and pumping is stopped with the valve V3. A sample of a highly volatile liquid (1–1.5 ml) is added into the reactor through a septum using a microsyringe. Sample vapours are burned in the oxidation column and the reaction products CO2 and H2O are frozen immediately in the U-trap. The time of complete combustion and collection of reaction


products is not more than 3 min. After that the valve V9 is closed and the valve V7 opens for evacuating non-condensed gases. The CO2 is cleaning procedure is the same as that described for solid samples. The purified CO2 is collected in the sampler for subsequent mass spectrometric analysis. The system is ready for the next analysis.

The above reaction conditions of the complete oxidation of carbon of solid and non-volatile liquid samples (T = 850°C, t = 10 min), and also volatile liquid samples (T = 850°C, t = 3 min) are optimal for this method and were determined experimentally [17, 18].

Combustion of methane for the determination of the isotopic composition of carbon

Preparation of the reactor. The reactor for methane combustion is made of a quartz tube 20 cm long with an inner diameter of 0.6 cm (Fig. 2.3c). A quartz diaphragm (quartz disk 0.2 cm thick, bore 0.5 mm) is soldered to the reactor at a distance of 5 cm from the edge of the tube. Part of the reactor is filled with an oxidation column of granular copper oxide with the addition of platinum wire (length of the oxidation column 8 cm, the granules of 1–2 mm). A layer of silica wool (2–3 mm) is placed between the oxidation column and the silica diaphragm. The heater is placed on the reactor so that the column is the central part of the heater. A T-piece and a thermostatically controlled cryogenic trap are connected sequentially to the reactor (on the size of the silica diaphragm). One end of the T-piece is plugged with septum. The reactor is connected to the U-trap of one of the ports of the vacuum apparatus, such as P6 (Fig. 2.1).

Burning and clearing procedure. The reactor and the thermostatically controlled trap are evacuated and the heater is set to a temperature of ~850°C. The heater preheats the oxidation column. When the temperature of the heater reaches 850°C, the temperature-controlled trap and U-trap are cooled with liquid nitrogen and pumping is interrupted. Valve V1 is closed and a sample (1–2 cm3) is introduced through the septum by means of a gas syringe (Fig. 2.3c). CO2 and H2O, contained in the sample, are completely frozen out in the trap. Methane is partially frozen out at the liquid nitrogen temperature. The heater of the differential trap is switched on and set to a temperature of –180°C. The vapour pressure of methane increases, methane passes through the diaphragm to the oxidation column. The reaction products are immediately frozen out in the U-trap. The time of complete combustion and collection of the reaction products is less than 10 min. Thereafter the non-condensed gases are pumped out and cryogenic


cleaning of CO2 is carried out, as described above. Pure CO2 is collected in a sampler.

For the distillation of CO2, frozen in the temperature-controlled trap, the heater temperature is set at –80°C. The thawed CO2 passes through the oxidation column and freezes out in the sampler ampoule.

The described procedure for the preparation of gaseous samples is applicable to samples with medium and high CH4 content.

Results and discussion

The accuracy and reproducibility of the proposed method were assessed using international standards IAEA-CH-7 (polyethylene), IAEA-CH-6 (sucrose), NBS-22 (oil), IAEA-C-6 (sucrose), IAEA-C-3 (cellulose) (Table 2.1). The reproducibility of the results was < ±0.1‰ for the certified standards. The difference between the measured δ13C values and the corresponding data in the catalogue of the International Atomic Energy Agency (IAEA) ranges from 0.01‰ to 0.20‰.

The reliability of the δ13C results of different solid organic compounds, obtained by the proposed method, was evaluated by comparison with the corresponding results for δ13C obtained by the classical ampoule method. For this purpose, samples of organic matter, organic carbon from the soil, current modern marine sediments and sedimentary rocks (Table 2.2) were analyzed. The results of analysis of δ13C for organic materials such as polyethylene, cellulose, sugar, starch, obtained by the method of combustion in the column and the method of combustion in ampoules, are characterized by equally good repro- reproducibility (≤0.1‰). The values of δ13C, obtained by different

Table 2.1. δ13C (‰) values of the IAEA international standards, obtained by incineration in the oxidation column

δ13CV-PDB (mean ±σ)

Sample Combustion method for in oxidizing column Accepted values ΔC

Polyethylene IAEA-CH-7 -32.12 ± 0.02 (n = 7) -32.15a 0.03

Sucrose IAEA-CH-6 -10.50 ± 0.03 (n = 5) -10.45a 0.05Oil NBS-22 -30.04 ± 0.03 (n = 6) -30.03a 0.01Sucrose IAEA-C-6 -10.60 ± 0.09 (n = 5) -10.8b 0.20Cellulose IAEA-C-3 -24.63 ± 0.03 (n = 5) -24.72b 0.09aPublished in [19].bIAEA data: http://wwwnaweb.iaea.org/nahu/nmrm/nmrm2003/browse.htm.cThe difference between measured and accepted values.


Table 2.2. Comparison of δ13C (‰) analyses, obtained by combustion in the oxidation column and the method of combustion in quartz ampoules

Sample

δ13CV-PDB (mean ±σ)The method of

combustion of a sample in the oxidation column

The method of combustion in quartz

ampoulesΔC

Zolyethylene IAEA-CH-7 -32.12 ± 0.02 (n = 7) -31.95 ± 0.08 (n = 5) 0.17Sellulose IAEA-C-3 -24.63 ± 0.03 (n = 5) -24.47 ± 0.03 (n = 3) 0.16Sugar beet -23.90 ± 0.10 (n = 5) -23.93 ± 0.12 (n = 3) 0.03Sugar cane -11.88 ± 0.08 (n = 11) -11.79 ± 0.05 (n = 3) 0.09Cellulose -24.48 ± 0.05 (n = 20) -24.47 ± 0.02 (n = 4) 0.01Corn starch -10.88 ± 0.05 (n = 14) -10.76 ± 0.08 (n = 4) 0.12Podzolic soila -24.81 ± 0.07 (n = 3) -24.74 ± 0.05 (n = 3) 0.07Brown forest soila -25.91 ± 0.08 (n = 4) -25.70 ± 0.03 (n = 4) 0.21Humus -24.56 ± 0.02 (n = 3) -24.63 ± 0.03 (n = 4) 0.07

Marine sediments No. 4b -22.67 ± 0.11 (n = 3) -22.43 ± 0.09 (n = 3) 0.24

Marine sediments No. 5c -24.91 ± 0.10 (n = 3) -24.77 ± 0.14 (n = 3) 0.14Mudstone (Precambrian) No. 1d -22.26 ± 0.11 (n = 6) -22.14 ± 0.07 (n = 2) 0.12

Mudstone (Precambrian) No. 2d -23.19 ± 0.06 (n = 5) -23.01 ± 0.04 (n = 2) 0.18aSamples provided by the Institute of Biology and Soil Science, Far East Branch of the Russian Academy of Sciences.bCoarse-grained sand-shelf of the Sea of Okhotsk, the content of organic 0.02% carbon.cMuddy sediments, Sea of Okhotsk, the organic carbon content 1.4%.dSedimentary rocks, the Russian platform, the upper Vendian, the samples provided by M.B. Burzin (Institute of Paleontology, Russian Academy of Sciences). eDifference between methods.

methods agree within the experimental error and show the difference between the methods Δ on average less than 0.1‰. Good agreement between the data obtained by the method of combustion in the column and the traditional method of combustion in ampoules indicates a high reliability of the results of the proposed method.

An example of using our method for the analysis of various natural plant tissue samples is shown in Table 2.3. The reproducibility of measurements of the isotopic composition of carbon is on average ±0.1‰.

For the purpose of testing the proposed method for the analysis of the organic carbon of soil , soil samples of different types were analyzed: podzolic soil with the organic carbon content


of 0.58% (the weight of the sample 120 mg), brown forest soil with the organic carbon content of 3.53% (weight of samples 20 mg) and humus with the organic carbon content of 7.8% (9 mg sample weight) (Table 2.2) . These samples were also analyzed by combustion in quartz ampoules. Comparison of the results of measurements of δ13C by the two methods shows that the reproducibility of the results obtained by the proposed method is not inferior to classical analysis.

The possibility of high-precision analysis of samples with a low carbon content was demonstrated on samples of marine sediments, consisting mainly of mineral particles with the organic carbon content from 0.02 to 1.4% (Table 2.2). The samples were analyzed using two methods. The weight of the analyzed samples was from 25 to 1000 mg, depending on the content of organic carbon and was identical for both methods. The results obtained by burning in the oxidation column are consistent with the corresponding data obtained by the ampoule method. The reproducibility of the results of both methods was on average ±0.11‰, the discrepancy between the values of δ13C methods is less than 0.25%. The data presented in Table 2.2 allow us to conclude that the method of burning in the column is a good alternative of the classical method of combustion in quartz ampoules.

Some samples of marine sediments were also analyzed by the EA-IRMS method (the elemental analyzer coupled to a mass spectrometer of isotope ratios) with a CN-elemental analyzer Flash1112 Thermoquest (Table 2.4). The weight of the analyzed samples was 50 mg for the method of burning in the column and from 1 to 4 mg for the EA-IRMS method. Comparison tests showed significant discrepancy between δ13C values obtained in individual methods. The results obtained by the EA-

Table 2.3. δ13C values (‰) of C3 and C4 plant types, obtained by combustion in an oxidizing column

Type of plant (sample) δ13CV-PDB (mean ± σ)

Salsola sp. (leaves) -13.53 ± 0.09 (n = 3)Salsola sp. (stems) -22.35 ± 0.07 (n = 3)Leymuz mollis (leaves) -26.51 ± 0.11 (n = 3)Leymuz mollis (stems) -24.31 ± 0.10 (n = 3)Arundinella sp. -11.10 ± 0.12 (n = 3)Pucrenr nilagiricus -10.52 ± 0.09 (n = 5)Fimbristysis ochotensis -6.20 ± 0.04 (n = 6)Panax ginseng (root) -32.14 ± 0.08 (n = 9)Zea mays (corn) -11.09 ± 0.12 (n = 8)Schisandra chinensis (climber) -28.26 ± 0.05 (n = 12)


IRMS method have low reproducibility, of the order of 1‰. The values of δ13C are on average 3‰ lower compared with the corresponding results of the method of burning in the column (Table 2.4).

Perhaps the EA-IRMS method does not provide the complete oxidation of organic carbon in the sediments and leads to a substantial difference of the δ13C values in these methods. It should be noted that Table 2.4 gives the results, specially selected from a large number of our routine analyses to demonstrate the greatest deviations of the results from the true values. In most cases, the reproducibility of the results obtained in the EA-IRMS method is 0.2-0.5‰, sometimes 0.05‰; the deviation of the results in the method of burning in the oxidation column is usually in the range 0.1–0.7‰. The large deviations, shown in Table 2.4, can be explained by the specifics of the samples (e.g., the content of readily combustible organic compounds or strong heterogeneity). The results presented in Table 2.4 are examples that relate to problems of the small yield of the reaction in the EA-IRMS method.

To extend the capabilities of the proposed method, we analyzed low-volatility liquids such as mineral and plant oils, using a reactor for burning of solid samples (Table 2.5). A high reproducibility of the results of determination of δ13C ≤ ± 0.05‰ was obtained.

The method of combustion in the oxidation column was tested for analysis of the organic carbon of volatile compounds and their liquid aqueous solutions. To do this, 96% and 40% samples of ethanol, some examples of organic solvents and alcohol products of industrial production were analyzed (Table 2.6). The volume of the analyzed samples was 0.5–1.5 µl. This volume is sufficient for obtaining the

Table 2.4. Comparison of δ13C values (‰) obtained by the combustion method in the column and the EA-IRMS method using the elemental analyzer

Sample

δ13CV-PDB (mean ± σ)The method of

combustion in the oxidation column

EA-IRMS method Δa

Marine sediments No. 3 -24.73 ± 0.24 (n = 3) -27.29 ± 0.84 (n = 3) 2.56

Marine sediments No. 4 -26.26 ± 0.19 (n = 3) -29.51 ± 1.22 (n = 3) 3.25

Marine sediments No. 5 -26.66 ± 0.08 (n = 3) -29.93 ± 1.04 (n = 3) 3.27aDifference between the methods.


optimum amount of CO2 to measure the isotopic composition by the standard method in the MAT-252 mass spectrometer with a two-channel inlet system. Reproducibility was ≤±0.04‰. The method was tested for the presence of ‘memory’ by analyzing alternately the samples of ethyl alcohol which differed significantly in the isotopic composition of carbon (δ13C = –29.21‰ and –12.45‰). The measured values of δ13C showed deviation from the mean value within the error of the method that indicates the absence of ‘memory’.

Table. 2.7 demonstrates the results of the analysis of gaseous samples as an example of spontaneous gas emissions from the gas drainage of the Tavrichanskoe brown coal deposit, Far East Russia. The composition of the samples consists mainly of CO2, CH4, N2, and O2. Depending on the content of CO2 and CH4, the volume of the analyzed samples ranged from 1 to 100 cm3. The reproducibility of the results was on average ±0.04‰ and ±0.06‰ for methane and carbon dioxide, respectively.

The proposed method of burning in the oxidation column has been successfully working in our laboratory for over 5 years. The

Table 2.5. δ13C values (‰) for samples of different oils and the values obtained by combustion in an oxidizing column

Sample δ13CV-PDB (mean ±σ)NBS 22 (oil) -30.04 ± 0.03 (n = 7)Forevacuum mineral oil -28.87 ± 0.05 (n = 5)Olive oil -29.45 ± 0.04 (n = 6)Corn oil -16.54 ± 0.05 (n = 6)Soybean oil -29.87 ± 0.04 (n = 6)

Table 2.6. δ13C values (‰) of volatile liquid samples, the values obtained by combustion in an oxidizing column

Sample δ13CV-PDB (mean ± σ)Ethanol, the sample No. 1 -23.89 ± 0.02 (n = 19)Ethanol, the sample No. 1 (40% aqueous solution) -23.90 ± 0.04 (n = 4)

Alcohol Lux (Russia) -24.15 ± 0.03 (n = 3)Alcohol (made in China) -12.45 ± 0.03 (n = 6)Vodka Tatarstan -25.86 ± 0.04 (n = 3)Balsam Ussuri -23.47 ± 0.05 (n = 3)Whisky (U.S. production) -13.47 ± 0.04 (n = 3)Acetone -26.97 ± 0.04 (n = 5)Hexane -29.21 ± 0.03 (n = 5)


Table 2.7. δ13C of methane and CO2 in the samples of natural gas. The values were obtained by combustion in an oxidation column

SampleConcentration,% δ13CV-PDB,‰

CO2 O2 N2 CH4 C2H6 C3H8 CO2 CH4

2 0.59 16.44 72.65 10.30 0.0048 – -50.6±0.08 -72.8±0.053 16.53 3.47 21.91 57.99 0.0824 0.0027 -32.9±0.03 -57.8±0.037 16.63 3.39 28.45 51.50 0.0365 0.0011 -34.3±0.06 -59.3±0.049 14.23 1.51 15.54 68.66 0.0474 0.0015 -33.6±0.05 -59.5±0.03

characteristics such as versatility, simplicity and efficiency determine make this method preferable when performing high-precision isotopic analyzes of organic carbon.

Conclusions

The idea of converting organic carbon to carbon dioxide in the preheated oxidation column allowed us to offer a versatile, economical, simple and rapid method of sample preparation of organic substances for the measurement of δ13C with high reproducibility. The method can be used for isotopic analysis of solid, liquid, and gaseous samples.

Due to the high efficiency of oxidation of organic carbon in the CuO-column, the time required for total carbon conversion is not longer than 10 min. Repeated use of reactors greatly reduces the consumption of expensive quartz material. The accuracy of the proposed method has been tested on international standards and confirmed by comparison with the results obtained by the method of combustion of samples in quartz ampoules.

The method of oxidation in a column with CuO shows reliable results and high reproducibility of δ13C values for samples of various organic substances, soil organic carbon, the scattered carbon in sedimentary rocks, viscous and highly volatile liquids, samples of natural gas with high methane content and medium. The proposed method is a good alternative to the classical method of combustion of samples in quartz ampoules.


Literature

1. A. Buchanan D. L., Corcoran B. J., Anal. Chem., 1959, V. 31, pp. 1635–1638.2. Frazer J.W., Mikrochim. Acta, 1962, pp. 993–999.3. . DesMarais D. J., Hayes J.M., Anal. Chem., 1976, V. 48, pp. 1651–1652.4. Coleman D.D., Anal. Chem, 1981, V. 53, pp. 1962–1963.5. Sofer Z., Anal. Chem, 1980, V. 52, pp. 1389–1391.6. Boutton T.W., Wong W. W., Hachey D. L., Lee L. S., Cabrera M. P., Klein P. D.,

Anal. Chem., 1983, V. 55, pp. 1832–1833.7. Ertl S., Spitzy A., Isotop. Environ. Health Stud., 2004, V.40, pp. 163–170.8. Le Feuvre R. P., Jones R. J., Analyst, 1988, V. 113, pp. 817–823.9. Macko S.A., Lee W. Y., Parker P. L., J. Exp. Mar. Biol. Ecol., 1982, V. 63,

pp. 145–149.10. Matthews D. E., Hayes J. M., Anal. Chem., 1978, V. 50, pp. 1465–1473.11. Preston T., Owens N. J. P., Biomed. Mass Spectrum, 1985, V. 12, pp. 510–513.12. Gehre M., Strauch G., Rapid Commun. Mass Spectrom., 2003, V. 17, p. 1497.13. Fujimoto T., Nishimura C., Omori H., Matsuda J., J. Mass Spectrom. Society

Japan, 2004, V. 52, pp. 196–204.14. Liu Y., Naraoka H., Hayashi K., Ohmoto H., Geochem. J, 2000, V. 34, pp. 195–205.15. Lowe D. C., Brenninkmeijer C. A. M., Tyler S. C., Dlugokencky E. J., J.

Geophys. Res., 1991, V. 96, pp. 15455–15467.16. Merritt D. A., Hayes J.M., Des Marais D. J., J. Geophys. Res., 1995, V. 100,

pp. 1317–1326.17. Velivetskaya T. A., Ignatyev A. V., Reize M. V. and Kiyashko S. I., Rapid

Commun. Mass Spectrom., 2007, V. 21, pp. 2451–2455.18. Velivetskaya T. A., Ignatiev A. V., Reize M. V., Kiyashko S. I, Mass spektrometriya,

2006, V. 3, pp. 169–174.19. Coplen T. B., Brand W.A., Gehre M., Groning M., Meijer H. A. J., Toman B.,

Verkouteren R. M., Anal. Chem., 2006, V. 78, pp. 2439–2441.

135Isotope ratio mass spectrometry of the metabolic potential

3

Using isotope ratio mass spectrometry for assessing the metabolic potential of

soil microbiota

A.M. Zyakun1 and O. Dilly2

1G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino,

Moscow region2Department of Soil Conservation and Reclamation,

Brandenburg Technical University, Cottbus, Germany

Introduction

Most national programs that control the ecological condition and quality of soil use microbial indicators that reflect their biological activity and microbial diversity. Soil microorganisms play key roles in the biodegradation of natural and anthropogenic organic compounds involved in mineral food cycles, directly responding to changes in the environment and reflecting the sum of all the factors that regulate the trophic cycles. Currently, the European Union (EU) and many countries around the world are working on bills for the protection of soil quality and soil biodiversity, using information trends in environmental changes and expansion of the scale of land use in the future.

Soil quality is ‘the ability to function for a long time as a conservative component of terrestrial eco-system, ensuring its biological productivity and maintaining the quality of air and water, as well as the health of plants, animals and man’ [1]. The actual biological productivity of quality soil of agricultural lands together fertility is determined by its environmental sustainability. The latter manifests itself as a self-sustaining, self-regulatory and self-purification capacity of the soil against stress factors harmful to the ecosystem


– biotic and abiotic. In particular, the environmental sustainability of the soil provides its harmlessness for man, the soil and terrestrial biota, ecological features of bioproduction, uncontaminated water and air environments associated with the soil and also, importantly, the safety and quality of crops, protecting them against infections harmful organisms.

High-quality soil provides a potential biological productivity of terrestrial ecosystems, their environmental sustainability and continuous functioning as a global source of biophylic elements. The potential biological productivity and ecological sustainability of soils determine not only the optimum amount and quality of bioproduction of agricultural and forest ecosystems but, in the first instance, the conditions of ecological well-being of the man.

At the present time, the loss of fertile soil and its degradation is the major socio-economic problem, creating a threat to the ecological, economic and national security of Russia [2]. The greatest damage to soils is caused by the pollution with carcinogenic oil products, persistent pesticides, man-made radionuclides, heavy metals, dehumidification, contamination with toxinogenous phytopathogens and other harmful organisms, waste management of the societies and livestock, as well as soil erosion, local waterlogging and salinity [3]. Degraded soils have lost much of the natural microflora, its characteristic structure and function. Such soils are not capable of self-cleaning, nitrogen fixation, immobilization of nutrients. The consequence of land degradation is the loss of recreational features of the land and forest land, the inability to use them to create and improve residential areas, a significant shortage of crop production of such lands, deterioration of its quality, and also pollution of coupled environments (atmosphere, areas, surface water, groundwater and artesian water).

Modern problems of scientific research is to constantly orient the scientific community to develop effective methods for obtaining representative biological data on the quality of soil and the role of microorganisms in maintaining and preserving the ecological state [4]. This is an extremely difficult task, since the soil microorganisms rapidly respond and adapt to environmental conditions. In addition, the impacts on the environment, caused by human activities, can be separated from the natural changes only when they are defined in comparison with historical data or when an adequate permanent monitoring of ecologically important processes and places in the environment is carried out.

Therefore, the immediate task of environmental studies is the selection of specific indicators of microbial processes in soil, which


would reflect both short- and long-term changes in the condition and quality of soil. The major criteria for these indicators should be: 1) the usefulness in the characterization of certain ecosystem processes, 2) the possibility of integrating physical, chemical and biological properties, and 3) the sensitivity to land use and climate change [4].

Subsequently, indicators were proposed to characterize the metabolic capacity of the soil microbiota, which can be divided into four main groups according to the type of information they can provide.

1. Microbial biomass in soil and the amount of microbial cells.2. The activity of soil microorganisms and the substrate-induced

respiration (SIR).3. A variety of soil microbiota and microbial community structure.4. The interaction of plant organisms.It is known that microorganisms are the main factors providing

the necessary plant nutrients because they participate in the processes of immobilization and mineralization of labile mineral and organic matter in the soil [5]. Microbial biomass can serve as an indicator of the effectiveness of different patterns of land use (e.g. choice of crop rotation, use of fertilizers, tillage, use of herbicides against weeds, etc.). In addition, the amount and specificity of the microbial biomass are an important indicator, indicating the extent of soil contamination by toxic pollutants [6–9].

The major modern and advanced methods to obtain reliable values of these parameters include the method of molecular and isotope ratio mass spectrometry. Molecular mass spectrometry reveals the presence of different microbial representatives in the soil on the basis of certain specific cellular components and products of their life, and isotopic mass spectrometry provides a unique opportunity to evaluate the microbial metabolic activity on the basis of the rate and magnitude of consumed substrates.

Application of isotope ratio mass spectrometry has been demonstrated on examples of definitions of the metabolic potential of microbiota in arable [10] and forest [11] soils. The sources of production of microbial catabolites – carbonic acid – were separated by using test organic substrates, which differed in the abundance ratios of stable isotopes of carbon 13C/12C to the native soil organic matter (SOM).

Indicators of activation of the microbiota, using SOM, include the effect of glucose priming, or the priming effect (PE), and respiratory quotient (RQ), which is quantitatively equal to the ratio of molar concentrations of the resultant CO2 an the oxygen consumed in this process [12]. The results of the study of microbial mineralization of


organic products in the soil showed that the addition to the soil of an easily metabolizable product (e.g. glucose), along with the activation of soil microbiota contributes to the destruction of soil organic matter and increases the flow of CO2 from the soil into the atmosphere.

The purpose of this communication is to demonstrate the analytical capabilities of isotope ratio mass spectrometry in the determination of metabolic activity of soil microorganisms and the impact of exogenous substrates on the environmental sustainability of the soil and environment.

Methods of analysis of microbial activity in soil

Microbiological method

Traditional methods of determining the number of microorganisms, living in soil, are based on the total evaluation of the metabolic activity or direct count of microbial cells [13–16]. The procedure for determination of viable cells requires their cultivation and includes two approaches: a) the method of counting in cups containing selective growth media, and b) the method of counting the most probable number of living cells. Some uncultivated soil microorganisms can potentially be cultivated, if the adequate food conditions are provided for their growth. However, many microorganisms remain uncultivated because they are dormant (latent) and require a special ‘reanimation’ for the beginning of their growth, or, as non-viable, but still intact, can be determined by microscopy [17].

Using the appropr ia te cul ture media , i t i s in some cases possible to identify and count specific functional groups of micro-organisms. Nevertheless, even with universal media for ensuring the overall growth of microorganisms, the determined number of the microorganisms is usually an order of magnitude lower than the number obtained directly by microscopy. It should be noted that by adding the appropriate isotope-labeled substrates and using isotope ratio mass spectrometry it is possible to identify the metabolic activity of soil microorganisms not cultivated in the laboratory conditions.

The special significance of determining microbial biomass in the evaluation of soil quality is reflected in continuing the current efforts to develop and improve the methodology for the determination of quantitative indicators [18]. There are three methods used for the quantitative characterization of microbial biomass: a) fumigation with chloroform vapours which affects the growth of microorganisms [19], b) fumigation with chloroform vapours – extraction of microbial products


and their mass spectrometric characteristics [20, 21], c) substrate-induced respiration (SIR) of the soil microbiota [22]. When using the SIR method for the determination of the metabolic potential of the microbiota there are problems with determining the substrate, which is used by microorganisms in the production of biogenic carbon dioxide. To solve this problem successfully it is necessary to use the methods of isotope ratio mass spectrometry.

Isotope ratio mass spectrometry in the study of substrate-induced respiration (SIR)

At present, SIR is the most frequently used physiological method for measuring the activity of microbial biomass in soil. So, after adding an easily metabolizable substrate (e.g. glucose) to the oil, there is an immediate increase in the rate of formation of the metabolic carbon dioxide, which is assumed to be proportional to the number of microbial cells and their biomass. Using an exogenous substrate, which differs in the isotopic composition of carbon from native soil organic matter, and using techniques isotope ratio mass spectrometry it is possible to quantify the sources of metabolic carbon dioxide and identify its key producers. It is assumed that the number of bacteria and fungi in the soil can be determined by the selective inhibition of the rate of SIR with antibiotics and fungicides [23–26].

In all of these original methods, the rate of CO2 production is recorded in the first few hours and also for some time after the introduction of the substrate. The data obtained in this way can be attributed to different phases of microbial metabolism in soil: a) the basal production of CO2 prior to the introduction of the substrate, b) the initial stage of the substrate-induced respiration (SIR) immediately after making the substrate, c) the lag period, and d) the exponential growth of the specific respiratory rate, quantitatively measured by the slope of the linear dependence, which is obtained in the case of representing the respiratory rate in the logarithmic scale with respect to the time coordinate.

Separation methods for bacterial and fungal biomasses, using antibiotics and fungicides in quantifying substrate-induced logarithmic growth may be applicable, provided that all microorganisms will grow immediately after introducing the substrate, or at least growing microorganisms are representative of microorganisms that had showed no growth [25].


Kinetics of CO2 production during substrate-induced respiration

The carbon isotopic characteristics of the metabolic carbon dioxide are used to determine the mineralized substrate in the growth process of the microbiota in the tested soils. Based on the fact that the rate of formation of microbial products dp/dt is proportional to the specific activity (q) and the concentration of viable microorganisms (N) [27] we can write the differential equation

dpdt

qN= . (3.1)

The exponential growth of microorganisms (Nt) in time can be described by the equation

= N0eμt, (3.2)

where N0 is the initial concentration of microorganisms, μ is the specific rate of their growth, t time. The rate of formation of products during the exponential growth rate of microorganisms is obtained after the joint solution of equations (3.1) and (3.2):

dpdt

qN e= 0µt . (3.3)

The product concentration at a given time t is found by integrating (3.3), provided that p0 corresponds to the concentration of the product at t = 0:

p p q N e= + −0

0 1( ) .µ

µ

t

(3.4)

Obviously, equation (3.4) is applicable in the case of formation of products by individual cultures of microorganisms. However, it can also be used to describe the formation of products by a mixed community of soil microbiota on the condition that the microorganisms exhibit an exponential growth as a result of introduction of the substrate, such as has been demonstrated in the evaluation of the potential rate of microbial denitrification [28].

Earlier [29, 30] it was argued that microbial mineralization in soils of easily metabolizable substrates, such as glucose, is realized by two groups of microorganisms: the microorganisms that perform exponential growth after the introduction of this substrate in the soil (r-strategy), and the microorganisms that increase the rate constant of CO2 production without increasing the number of their cells (K-strategy or non-growing microorganisms) [31]. Denoting the rate constant of CO2 formation or CO2 respiration, realized by the non-growing


microorganisms, by the value K, and replacing the initial rate of respiration of the growing microorganisms growing qN0 by r, the summary rate of production of carbon dioxide of the soil microbiota after introducing the substrate can be represented by the equation

dpdt

re Kt= +µ . (3.5)

After integrating (3.5) we obtain an expression that reflects the formation of microbial products of this metabolite:

p p e r Ktt

= + − +01( ) .

µ

µ (3.6)

The rate of substrate-induced respiration (SIR), defined as the rate of CO2 production, which occurs as a result of mineralization of the introduced substrate (t ≈ 0), is represented by the expression

SIR = r + K. (3.7)

If it is true that the number of K-strategists does not increase with the introduction of an exogenous substrate, but only its mineralization activity increases, there must exist an optimal amount of the substrate above which the production of CO2 is not increasing. It is assumed here that at the initial time after the introduction of the substrate to the soil the activity of r-strategists is minimal, but over time they grow exponentially with a corresponding increase of the metabolic CO2 production.

In principle, a significant indicator in this case is the unambiguously determined amount of the substrate entered in the soil and used as a factor that activates the growth of soil microbiota and the formation of CO2. The difficulty in quantifying carbonic acid in these measurements is that the sources of metabolic carbonic acid can be both the soil organic matter (SOM) and the easily metabolizable substrate (e.g. glucose) introduced into the soil. When using the substrate, characterized by the 13C-isotope content differing from that of the SOM, it is possible, based on the material–isotope balance, to determine the amount of metabolic carbonic acid, which was formed by mineralization of the substrate and SOM, respectively.

Characteristics of carbon isotopic composition of microbial products

The natural abundance of stable carbon isotopes in the carbon-bearing products makes up about 1.1% of 13C and 98.9% of 12C. To provide the necessary accuracy of measuring the ratios of the abundance of 12C and 13C, the carbon-bearing mineral and organic products are


converted to CO2, and this is followed by determination the ratio of peak intensities of ionized molecules with different isotopic carbon in the mass spectrum of CO2.

The values of 13C/12C for organic products in the soil, glucose as an exogenous substrate and BaCO3, obtained by deposition of metabolic CO2, are measured using a specialized isotope ratio mass spectrometer. The quantitative characteristic of the prevalence of the 13C isotope in the analyzed samples is the relative value δ13C, which is determined according to the expression

δ13C = 1000%,RRsa

st

−

1 . (3.8)

where Rsa = [13C] / [12C] and Rst = [13C] / [12C] are the ratios [13C] / [12C] for isotopically different molecular ions of CO2, obtained from the sample and the international standard PDB (Pee Dee Belemnite), respectively.

The material–isotopic balance for mixtures of SOM and glucose, added to the soil, is carried out using the expression

δ13CSOMQSOM + δ13CGluQGlu = δ13CGlu+SOM (QSOM+QGlu), (3.9)

where the values of δ13CSOM and δ13C(Glu + SOM) reflect the content of the 13C isotope in soil organic matter before and after the introduction of glucose into the soil; δ13CGlu is the carbon isotopic composition of glucose; QSOM and QGlu is the amount of SOM and glucose in the soil samples, respectively. Assuming that the isotopic characteristics of the metabolic carbon dioxide inherit SOM and the soil glucose with the accuracy up to the isotope effects in their microbial consumption, and using (3.9), we can compute, using (3.10), the fraction of CO2 (F), which was formed by mineralization of SOM after adding the glucose in soil:

F = C C

C C

13(G u + SOM)

13G u

13SOM

13G u

δ δδ δ

l l

l

−−

, (3.10)

where δ13CSOM and 13C(Glu + SOM) reflect the amount of the isotope 13C in CO2 produced in the soil before and after the introduction of glucose into the soil; δ13CGlu is the isotopic composition of the carbon of the glucose.

Usually, in the experimental conditions associated with the definition of the substrate-induced respiration (SIR) we use glucose as an easily metabolizable substrate, the carbon which is different from the value of δ13C of soil organic matter (SOM) [10]. Due to this the total amount of


CO2, released from the soil during microbial mineralization of glucose and SOM, using expressions (3.9) and (3.10) can be divided into CO2, which is formed from glucose and from SOM, respectively. Thus, the difference between the amounts of the carbon of CO2, formed in mineralization of the SOM in the soil samples with glucose and in the reference sample (without adding glucose), related to the amount of CO2 in the reference sample (in percentage), can be used to assess the degree of additional mineralization of native SOM as a result of microbial use of the exogenous substrate – glucose. In the literature this phenomenon (i.e. the activation of microbial mineralization of the SOM by the exogenous glucose) was named the priming effect (PE) of glucose [10, 11, 32, 33]. It was shown [10] that the use of glucose and SOM with different carbon isotopic composition allows us to estimate the degree of activation of microbial mineralization of SOM in the presence of glucose and calculate the value of PE in the soil, using expression

PE = C

C (G u + SOM) SOM

SOM

FC l −

. %,100 (3.11)

where CGlu + SOM is the total amount of carbon of CO2, which is formed in mineralization of glucose and SOM in the soil, flavoured with glucose; FCGlu + SOM is the amount of carbon of CO2, produced in the mineralization of SOM in the soil, flavoured with the addition of glucose and calculated using (3.10).

Amount of metabolic carbon dioxide and characterization of its origin in the soil

Using the 13C/12C ratios to characterize the activity of the microbiota in arable soils

As noted above, the microorganisms in the soil play an important role in the transformation and circulation of soil organic matter (SOM) [34, 35]. The activation of microbial mineralization of SOM in arable, steppe and forest soils is the subject of research for the agricultural chemists interested in obtaining maximum yield of agricultural lands. The information obtained in such studies is of interest also for ecologists involved in finding sources that can cause continually increasing concentration of carbon dioxide in the atmosphere potentially contributing to changes in global temperature and climate of the Earth as a whole [36].

Adding to the soil organic and mineral substanes has some impact on microbial processes associated with the use of SOM [37–


39]. Acceleration or deceleration of the microbial degradation of organic products (humates) in arable soil compared with the processes without the addition of substrates shows positive or negative activation (priming) of these processes by the added products and is called the positive or negative priming effect (PE), respectively [32, 40–42]. The currently available results on the role of organic additives in the soil in order to activate the microbial mineralization of SOM are associated with contradictory opinions, both sceptical [43–45] and very optimistic [46]. The positive PE, amounting to 336% of the initial CO2 production of the soil, was observed after the introduction of green plant mass to the soil. This example showed that the microbial mineralization of SOM can be activated by entering organic products in the soil and its extent may be very high [46].

Most of the exogenous substrates, entering the soil, stimulate microbial degradation of compounds containing aromatic structures of humates (positive PE). However, in the case of a simulation system for the study of microbial mineralization of lignin-like substances, the addition of glucose as an additional substrate was not accompanied by the PE, and the addition to the soil of oxalic acid and catechol was companied by a negative PE, i.e. microorganisms switched to consumption of the added substrates [38]. The question arises: is it possible (and if so, to what extent) to intensify the microbial processes that contribute to the transformation and mineralization of SOM in soils by adding easily recyclable substrates?

It is known that the consumption of plant products by the micro-organisms results in the formation of a microbial biomass, carbon dioxide and organic metabolites with the isotopic composition of carbon, following the composition of the consumed products with the accuracy up to the isotopic effect. The values of δ13C of the carbon dioxide released from the soil in the process of destruction of plant material can be used to trace the mineralization of plant residues and other substances that enter the soil [47–50]. Glucose as a plant product is often used as an exogenous substrate in experiments with the biodegradation of organic products [51–54]. It is known that the isotopic composition of carbon of the SOM of arable soils, in which C3 plants were grown, and of the produced CO2, formed in microbial mineralization, is characterized by the values of δ13C in the range –30 ÷ –25‰ [55, 57]. In turn, the carbon dioxide formed during the mineralization of glucose as a product of photosynthesis of C4 plants with the carbon isotopic composition which has the δ13C value in the range –13 ÷ –11‰, differs from the SOM in the soil after cultivation of C3 plants in them. Consequently, the difference between the δ13C


values of exogenous glucose and endogenous SOM in such cases can be used as the isotopic label to determine the origin of the CO2 formed during the microbial mineralization of organic products in the soil.

It was shown [54] that the microbial mineralization of glucose added to the soil in laboratory experiments, was followed by the generation of CO2, the amount of which was not balanced with respect to the molecular oxygen used in this process, as it followed from theoretical calculations. The ratio of molar amounts of the produced CO2 and the consumed amount of O2, which is called the respiratory quotient (RQ), was higher than 1, ie, RQ > 1, while the RQ for the quantitative oxidation of glucose to CO2 must be equal to 1. The observed fact is seen as evidence of the presence, together with oxygen, of additional electron acceptors for microbial mineralization of glucose in the soil. Given the scientific and practical significance of the issues in this case, it was required to carry out special studies to understand the observed phenomenon.

Modelling experiments were based on the quantitative and carbon isotopic characteristics of the metabolic CO2, SOM, and the substrate added to the soil (glucose), creating an opportunity to assess the effect of this easily metabolizable substrate on the activation of microbial mineralization of organic products in agricultural soil, as well as clarify the features of molecular oxygen as an electron acceptor in the process of mineralization of native organic products (humic substances).

Analyzed soil samples

Modelling experiments to study mineralizing microbial activity were carried out using samples of soils taken in a field (arable land) in the Lake Bornheved region, Germany (54°06'N, 10°14'E), where the mean annual temperature and annual precipitation amounted to 8.1°C and 697 mm respectively, [39]. Each of the soil samples (AB1, AB2 and AB3), collected in three locations of the upper layer from 0 to 20 cm, was composed of 10 separate samples taken at a distance of about 20 m from each other. The remains of the plants were removed and the soil sifted through a sieve (2 mm) and stored for no more than 4 weeks at 4°C. The organic matter was about 15 000 µg C/g dry soil, and pH ~ 5.5 [39]. The soil samples were held for 6 days at 22°C, prior to measurement of the respiration of the soil microbiota which was activated by adding exogenous glucose.

In the first stage, experiments were conducted, differing in the amount of introduced glucose (C-Glu) for 1 g of dry soil (DS) (µg C-Glu/g DS): 2000 (I), 500 (II), 50 (III) and 0 (IV, reference). Each


experiment was performed on three soil samples using, respectively, AB1, AB2 and AB3. Prior to addition to the soil, the appropriate amounts of glucose were mixed with 0.5 g of talc powder. The soil samples (100 g of DS) were placed in 250 ml flasks which contained 10 ml glass cups with 2–6 ml of 1 M NaOH to trap the evolved CO2. The resulting CO2 was determined by the volume of 1 M HCl, used in titration of the alkali absorbent until discolouration in the solution of the pH indicator – phenolphthalein. The carbon dioxide, recorded in the form of sodium carbonate, was then precipitated as BaCO3 using BaCl2. Then, 3 mg of the dry sediment, containing about 200 µg C, was used to measure the characteristics of the 13C/12C ratio using isotope ratio mass spectrometer Finnigan MAT Delta Plus (Germany).

Addition to the tested soil samples of different amounts of substrate (glucose) could obviously caused adaptation to varying degrees of some groups of soil microorganisms to this substrate. Consequently, to evaluate the adaptation of the microbiota to the added substrate, glucose was again added to the soil (the second stage of experiments).

In the second phase of experiments, in addition to the samples of arable soil 500 µg C-Glu/g of DS was repeatedly added in the variants II, III, and IV after 8-day mineralization of organic products in soils.

In both experimental series the microbial activity of the soil samples was evaluated using quantitative parameters of the production CO2 and the O2 consumed by the microbiota (the values of RQ). Consumption of oxygen was monitored by a respirometer (Sapromat, Germany). The respiratory quotient (RQ), defined as the rat io of the molar concentrations of the released CO2 and O2 consumption, was used as an indicator of the mineralizing activity of microorganisms.

Mineralization of SOM and exogenous glucose

If the number of active microbial cells in the tested soil samples is constant, the increase and the duration of mineralizing activity of the soil microbiota depend on the amount of glucose introduced into soil (Fig. 3.1): the maximum microbial production of CO2 was observed after adding 2000 µg C-Glu/g DS (experiment I) and the minimum – 50 µm C-Glu/g DS (experiment III). In the first phase of the experiments within 8 days of incubation, in the experiments I and II the total amount of released CO2 was more 7 and 2.7 times greater than the amount of CO2, respectively, detected in the reference samples (Table 3.1). According to the material balance, about half of the deposited glucose remained in the soils of the experiments I and II at the end of the first stage. In experiment III, CO2 production during this period


Rate of CO2 formation, μg C-CO2/g DS • h Respiratory coefficient RQ

ba

Time, days

Isotope characteristic 13C, ‰

c

Time, days

Time, days

Fig. 3.1. Characteristics of microbial mineralization of organic products in soil: a – the rate of CO2 formation; b – respiratory quotient (RQ); c – δ13C-CO2 prior to (experiment IV), and after adding glucose to the soil (experiment I–III) within 8 days of incubation.

Respiratory quotient RQ

was close to the reference value (experiment IV), which evidences the exhaustion of glucose introduced into the soil. The maximum difference in the amounts of CO2, formed in the experiments I–III, was recorded in the initial period of incubation. As shown by Fig. 3.1, after the introduction of glucose to the soil the maximum rate of formation of CO2 was observed after 24 h in the experiments II and III and 48 h in the experiment I. Table 3.2 shows the rate of O2 consumption, formation of CO2 and the corresponding values of RQ in the first days after the addition of glucose in the soil. In all experiments the value of RQ was highest compared with the reference sample and significantly greater than 1.0. In experiments I and II, the maximum rates of release of CO2 from the soil were relatively close and equalled, respectively, 6.61 and 6.15 µg C-CO2 · g

–1 DS · h–1 (Table 3.2), exceeding by more than 20 times the rate of release of CO2 prior to adding glucose tpo the soil and the reference sample. In experiment III in the first 23 h after the addition of glucose the rate of CO2 formation increased by 3 times compared with the rate of mineralization SOM prior to adding glucose (Table 3.2). The rate of mineralization of organic products in the latter case (experiment III) gradually decreased with time, almost reaching the rate of release of CO2 in the reference sample (Table 3.1).

As noted above, in the first days of observation after the introduction of glucose to the soil, the rates of formation of CO2 in the experiments


Table 3.2. The rate of O2 consumption and the formation of CO2, the value of the respiratory quotient (RQ) in the mineralization of glucose in the soil microbiota of the soil within 24 hours after application of glucose to samples of arable soil

Glucose, µg C-Glu/g DS O2 consumption, µg O2/g DS·h

Generation of CO2, mg C–CO2/g DS · h RQ

0.0 (reference) 1.12 (0.09) 0.36 (0.06) 0.83 (0.01)50.0 (experiment III) 2.02 (0.11) 1.07 (0.29) 1.17 (0.05)500.0 (experiment II) 12.08 (0.32) 6.15 (0.42) 1.36 (0.07)2000.0 (experiment I) 14.86 (1.37) 6.61 (0.60) 1.24 (0.01)

Comment. In parentheses are the standard deviations of three parallel measurements in each of the experiments.

Table 3.1. The rate of formation of CO2 and the amount of CO2 released from the soil during the observation period (experiments I–III), and without adding glucose (experiment IV)

Experiment Observation period, h

Rate of CO2 emissions,

µg C-CO2/(g DS · h)

*The formation of CO2, µg C-CO2/g DS

I

0–23 6.61 (0.60) 152.0 (14)23–48 7.12 (0.59) 178.0 (15)48–73 6.08 (0.41) 154.0 (10)

73–145 4.67 (0.03) 327.0 (2)145–177 4.08 (0.19) 131.0 (6)

0–177 — 952.0 (47)

II

0–23 6.15 (0.42) 142.0 (10)23–48 2.47 (0.37) 62.0 (9)

48–145 1.35 (0.05) 132.0 (4)145–177 0.98 (0.03) 31.0 (1)

0–177 — 367.0 (24)

III

0–23 1.07 (0.29) 25.0 (7)23–145 0.79 (0.05) 97.0 (4)

145–177 0.42 (0.02) 13.0 (1)0–177 — 135.0 (12)

IV0–145 0.83 (0.07) 121.0 (10)

145–177 0.41 (0.06) 13.0 (2)0–177 — 134.0 (12)

* The amount of CO2 formed during the observation period in the experiment. Values in the parentheses are the standard deviations of measurements for three parallel


Fig. 3.2. Characteristics of microbial mineralization of organic products in the soil after the introduction of 500 µg C-Glu/g DS in the experiments II–IV after 8 days incubation: a) the rate of formation of CO2, b) respiratory quotient (RQ); c) δ13C–CO2 released from soil.

Rate of CO2 formation, μg C-CO2/g DS • h Respiratory coefficient RQ

ba

Time, days

Isotope characteristic 13C, ‰

c

Time, days

Time, days

I (2000 µg C-Glu/g DS) and II (500 µg C-Glu/g DS) had similar values, despite the significantly different amounts of glucose introduced into soil (Fig. 3.1). According to the kinetic concepts, the similar independence of the rate of CO2 production on the amount of added substrate evidences the ‘saturation’ with the substrate of the active part of the soil microbiota, which is represented as K-strategists. Given the results of the above observations, the metabolic potential of K-strategists in the samples of arable soils in the Lake Bornheved (Germany) did not exceed 500 µg C-Glu/g DS. In case of exceeding the specified amount of the introduced glucose (experiment I) there was a further increase in the rate of production through the development of microorganisms belonging to r-strategists. It should be noted that the relatively rapid exhaustion of the amount of the deposited glucose by the K-strategists in the experiments II and III did not allow the r-strategists to develop which led to a large reduction of the rate of CO2 production. As noted above, an additional amount of the substrate was added in the experiments to assess the degree of adaptation to the soil microbiota to the exogenous substrate.

According to Fig. 3.2 (second stage of the experiments), repeated adding of 500 µg C-Glu/g DS in the experiments II, III and IV was also

Respiratory quotient RQ


accompanied by an increased rate of formation of CO2 in the first days after application of glucose. The subsequent significant decrease in the production of CO2 show a metabolic advantage of K-strategists with respect to r-strategists in the competition for the substrate. Table 3.3 presents the rate of formation of CO2, O2 consumption, and also changes of RQ within 24 hours after application of glucose to the soil in the second phase of the experiments. The peculiarity of the experiments II, III and IV at this stage was the different degree of adaptation of the soil microbiota to exogenous glucose. Thus, there was no adaptation indicated in the experiment IV, it was minimal in the experiment III (pre-incubation with 50 µm of C-Glu/g DS) and maximum in the experiment II (pre-incubation with 500 µg C-Glu/g DS). As shown in Table 3.3, the maximum rate of formation CO2 and the value of RQ, significantly greater than 1, were recorded in the experiments with low adaptation of the soil microbiota to glucose (experiments III and IV). This is regarded as the evidence of the dominant role of K-strategists in the initial stages of consumption of the easily metabolizable substrate – glucose.

δ13C as a specific feature of the carbon of substrates. The isotopic composition of carbon of initial SOM in AB1, AB2, and AB3 is characterized by the δ13C value equal to –27.01 ± 0.32‰ (Table 3.4). The resulting δ13C value is typical of soil organic matter after growth C3-plants in it for a long time [28]. The isotopic composition of the carbon of glucose, used in the experiments, is characterized by the value of δ13C = –11.4‰, typical of the C4-plants. Thus, the glucose doses added to the soil differed by 15.6‰ in the values of δ13C from the isotopic composition of the carbon of SOM and were regarded isotopically labelled substrates.

According to calculations, made using the expression (3.9), it was expected that the value of δ13C of total organic matter in the soil after

Table 3.3. The rate of O2 consumption, CO2 formation and the value of RQ in the process of mineralization of soil microbiota of glucose for 24 hours after application of 500 µg C-Glu/d DS and 8-day pre-incubation with different amounts of glucose

Experiments Glucose, µg C/g DS

O2 consumption, µg/g DS·h

Generation of CO2, mg C/g

DS · hRQ

II 500.0 11.73 (0.93) 5.10 (0.43) 1.16 (0.01)III 50.0 12.55 (0.68) 6.21 (0.22) 1.32 (0.03)

IV 0.0 (reference) 11.58 (1.32) 5.90 (0.79) 1.35 (0.03)

µ


application of glucose (the first and second stages experiment) should be equal to –25.2‰ in the experiment I (15 000 µg C-SOM/g DS + 2000 µg C-Glu/g DS), approximately –26.03‰ in the experiment II (15000 µm C-SOM/g DS + 1000 mg C-Glu/g DS), approximately –26.46‰ in the experiment III (15 000 µg C-SOM/g DS + 550 µg C-Glu/g DS) and about –26.5‰ in the experiment IV (15 000 µg C-SOM/g DS + 500 µg C-Glu/g DS). As shown in Table 3.4, the addition of glucose to the soil (the first and second stages of the experiment) in experiments II, III and IV after 19 days of incubation did not change the content of 13C in the total organic matter (within the measurement error ±0.2‰). Therefore, it was assumed that the amount of glucose added to the soil, up to 1000 µg C-Glu/g DS, was mineralized by the soil microbiota to CO2 within 19 days. A marked difference of about 0.9 ± 0.2‰ in the values of δ13C for organic matter in the soil relative to its initial value was detected after 19 days of incubation in experiment I, where 2000 µg C-Glu/g DS was added (Table 3.4). The expected difference between the δ13C value of organic matter in the soil after application of glucose and the original content of 13C in the SOM was 1.8‰, and the experimentally observed difference of 0.9±0.2‰ showed that in the experiment I the amount of glucose mineralized to CO2 did not exceed 50% or no more than 1000 mg C-Glu/g DS. Thus, the analysis of the quantitative and carbon isotopic composition of organic matter in the experiments I, II, III and IV allowed us to determine the overall metabolic activity of the microbiota in the soil samples which was characterized by an average rate of mineralization of exogenous glucose of around 50 µg C-Glu/g DS daily. This agrees with the degree of glucose uptake in experiment III, where the daily rate of CO2 production was similar to the reference sample.

Table 3.4. Carbon isotopic characteristics of organic matter in arable soil samples (δ13C,‰) prior to (initial) and after 19 days of incubation with different amounts of glucose introduced into soil

Sampleδ13 C, ‰

Initial 2000* 500* 50* 0.0*AB1 −27.60 −26.10 −26.83 −26.57 −26.82AB2 −26.49 −26.10 −27.49 −26.42 −27.09AB3 −26.94 −26.12 −26.53 −26.92 −26.68

−27.0(0.6)** −26.1(0.01) −27.0(0.5) −26.6(0.3) −26.9(0.2)*The amount of glucose added to soil samples (µg C-Glu/g DS). **Average δ13C values for soil samples, the values in the in parentheses are standard deviations of three parallel measurements.


Possible fractionation of 12C and 13C when SOM is consumed by the microbiota was estimated on the basis of the difference between the isotopic composit ion of the carbon of SOM and CO2, formed during the mineralization of SOM only. The isotopic composition of CO2 evolved in the experiments I, II, III and IV prior to the introduction of exogenous glucose, calculated as the mean of the data in Table 3.5, was about –27.15 ± 0.2‰. Within the measurement error the carbon isotope composition of CO2 followed the SOM characterized by δ13CSOM = –27.01 ± 0.32‰ (Table 3.4). Consequently, it may be assumed that the fractionation of the carbon isotopes in microbial mineralization of soil organic matter did not take place or was insignificant. This finding is consistent with the literature data, which also shows a slight fractionation of the isotopes of the carbon of substrates metabolized by soil microbiota [10, 49, 58, 59]. Thus, the value of δ13C of CO2 in the experiments inherits the δ13C value of organic products used by the microorganisms as a substrate.

Within 7 days after the introduction of glucose into the soil (experiments I, II, III) the δ13C value of metabolic CO2 significantly differed compared with the reference value (the first stage of the experiments) as a result of mineralization of glucose and SOM (Table 3.6). The largest enrichment of 13C of the generated CO2 in comparison with the original value was recorded in experiment I in the first 7 days of incubation. In experiment III, the value δ13C–CO2 was difficult to control already six days after introduction of glucose into the soil. Changes in the values δ13C–CO2 in the first 24 h after application of different doses of glucose in the experiments I, II, III are shown in

Table 3.5. The isotopic composition of organic matter in soils and CO2, formed during its mineralization by soil microbiota for 3 days (prior to the introduction of glucose in the soil)

Sampleδ13C, ‰

SOM (initial) CO2 (initial)I*

CO2 (initial)II*

CO2 (initial)III*

CO2 (initial)IV*

AB1 −27.60 −27.1 −27.3 −28.0 −27.0AB2 −26.5 −27.4 −26.6 −26.7 −27.0AB3 −27.0 −27.2 −27.1 −27.2 −27.3

−27.0(0.6)** −27.24(0.2) −27.0(0.4) −27.3(0.7) −27.1(0.2)

*I, II, III, IV – experiments in which glucose was subsequently added in the amounts of 2000, 500, 50 and 0 µg C-Glu/g DS, respectively.**Average δ13C values for soil samples, the values in parentheses are standard deviations of three parallel measurements.


Fig. 3.3. Variations in the isotopic composition of carbon dioxide (δ13C,‰) formed during the microbial mineralization of organic products one day after application of glucose into the soil, depending on the amount of glucose (µg C-Glu/g DS): 1 – 2000; 2 – 500; 3 – 50; 4 – 0.Fig. 3.4. The rates of mineralization of organic products in the soil within 1 day after application of glucose, depending on the amount of exogenous glucose (µg C-Glu/g DS): 1 – CO2 from SOM; 2 – CO2 from glucose.

Isotope characteristic δ13C, %

Rate of formation of CO2 μg C-glu (×10)/g DS

Rate of mineralization, μg C-CO2/(g DS • h)

Amount, μg C-glu (×10)3/g DS

(‰)

Table 3.6. Change of the value of δ13C for CO2 released during the incubation of soil samples depending on the amount of glucose added to the soil

Time, daysδ13C,‰

2000** 500** 50** 0.0* (reference)

0 −27.1 (0.5) −27.0 (0.3) −27.1 (0.5) −27.1 (0.2)1 −15.3 (0.3) −14.3 (0.2) −17.9 (0.2) −26.8**2 −13.5 (0.3) −13.3 (0.4) − −26.4**3 −12.8 (0.2) − − −24.4**6 −13.0 (0.6) −20.1 (0.2) −24.6 (0.6) −25.3 (0.3)7 −13.9 (0.8) −20.1 (0.1) −23.8 (0.3) −24.2 (0.7)

*The amounts of glucose introduced into the soil (µg C-Glu/g DS). **The values of δ13C (‰) were calculated by extrapolating between initial values and values obtained after seven days of incubation δ13C values in soil samples (without making glucose). The values in the parentheses are the standard deviations for three parallel measurements in each of the experiments.

Fig. 3.3. These data and results of preliminary measurements of the δ13C value of glucose and SOM have been used to estimate the amount of the carbon of glucose included in the total pool of CO2 on the basis of the material–isotopic balance (3.9). Analysis of the dynamics of change in the rate of glucose mineralization to CO2 (Fig. 3.4) showed that the metabolic potential of the microbial pool in the soil at the initial stage after the introduction of glucose (presumably K-strategists) has a limit, and ‘saturation’ with the exogenous glucose is reached when adding more than 500 µg C-Glu/gDS to the soil.


To understand the catabolic processes, it was essential to determine the contribution of glucose carbon and SOM to the production of CO2 at the stage of development of microorganisms in soil, related to K-strategists. Taking into account the fact that CO2 with δ13CGlu+SOM= –15.34‰ formed in experiment I during 24 hours (Fig. 3.3), and taking into account the characteristics of the carbon isotopic composition of SOM (δ13CSOM = –27.1‰) and glucose (δ13CGlu = –11.4‰), the equations (3.9) and (3.10) were used to calculate the amount of carbon of SOM and glucose mineralized by the microbiota. It was found that the carbon fraction of SOM in the total CO2 pool is 25% and that of glucose 75%. This indicates that one carbon atom from SOM is associated with three carbon atoms from glucose or 1 mol of glucose activates the use of one mole of the two-carbon organic unit from SOM. This unit is likely to be acetyl-CoA, which was formed from the SOM by activating this process by microbial oxidation of glucose.

Priming effect (PE) of glucose

The characteristics of the isotopic composition of carbon of SOM (δ13CSOM), glucose (δ13CGlu) and total CO2 (δ13CGlu+SOM) were used to determine the rate of formation of carbonic acid in mineralization of SOM and exogenous glucose, respectively (see Fig. 3.5). Microbial mineralization of SOM after the addition of glucose to the soil was significantly higher than in the reference sample throughout the 19-day observation period. The maximum mineralization of SOM was noted only in the first 48 h after glucose addition. After 3 days of exposure the rate of mineralization of SOM significantly decreased, despite the

Fig. 3.5. The rates of microbial mineralization of soil organic matter (SOM), depending on the incubation time: 1 – reference sample, 2 – activation of mineralization of SOM, by glucose introduced into soil (priming effect); 3 – mineralization of glucose in the soil.

Rat

e of

min

eral

izat

ion,

μg

C-C

O2/(

g D

S • h

)

Time, Days


activity of soil microbiota, as evidenced by a high rate of glucose mineralization.

As noted above, the soil microbiota is composed of two groups of microorganisms: K- and r-strategists [31, 60–62]. The microorganisms K-strategists are the waking part of the soil population which is capable of mineralizing SOM. The microorganisms r-strategists are the dormant part which can develop rapidly in penetration into the soil of the easily metabolizable substrate (e.g. glucose); r-strategy does not mineralize the SOM or mineralizes it in small quantities [10, 62].

With this in mind, it was assumed that in the experiment I (Fig. 3.5) immediately after the introduction of glucose to the soil the rate of mineralization of glucose and SOM is determined by the activity of microorganisms belonging to K-strategists [61]. It may be seen (Fig. 3.5) that the r-strategists have an advantage in competition for glucose as a substrate for 3 days, because at a high mineralization rate of glucose the degree of mineralization of SOM was low. After incubation for 8 days the glucose and/or essential mineral substances, ensuring a high rate of metabolism of the r-strategists, were exhausted. As a result, some of the cells died away, and, accordingly, the rate of production of CO2 decreased [61]. Part of the biomass of dead cells served as a substrate for K-strategists, so that the rate of mineralization SOM again increased (Fig. 3.5).

The maximum value of PE equalling 357% was observed within 1 day after the introduction of glucose into the soil in experiment I, and decreased to 84% after 8 days of incubation (Table 3.7). The value of PE also decreased with decreasing dose of glucose introduced into the soil: in experiment II the value of PE in the first day was 213%, while in the experiment III it decreased to 22%.

In the second phase of experiments 500 µg C-Glu/g DS was added repeatedly to soil samples (experiments II, III and IV) after 8-day incubation with different amounts of glucose. The results of these experiments show a definite correlation between the values of PE and RQ. So, within 8 days of incubation the PE changed from positive values greater than 100% to negative values (Table 3.8), and RQ – from the values greater than one to the initial values. Therefore, the increase in the microbial mineralization of SOM, activated by glucose, is associated with the outflow of part of the electrons produced during the oxidation of glucose, to acceptors other than O2.

I t i s known tha t SOM cons i s t s bas ica l ly o f d i ff i cu l t - to metabolize products (humic substances), which contain aromatic structures. Transformation of SOM to the substrates available for microorganisms provides for transformation of these structures


(e.g. hydroxylation or oxygenation) with the flow rate of reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotidephosphate (NADPH) [63, 64]. Microbial mineralization of organic products is associated with the cycle of tricarboxylic acid (TCA). The main factors that control the functioning of the TCA are: 1) the rate of arrival of acetyl groups; 2) the presence of oxaloacetate; 3) the rate of oxidation of NADPH to its oxidized form (NAD+) in the electron transport chain [65]. It is obvious that the consumption by the microorganisms of the substrate such as glucose can provide the energy intake required for the transformation of SOM to the product which can then be used as a substrate. In addition, glucose is a source of pyruvate, which is converted into a key metabolite of the TCA – oxaloacetate. In the given experimental conditions the amount of O2 in the experiments was maintained at its atmospheric concentration and the possibility of appearance of the anaerobic or microaerophilic conditions was completely excluded. Therefore, the reason determining the increase in RQ (Table 3.8) could be the emergence, in addition to O2, of other electron acceptors, for

Table 3.7. The mineralization of soil organic matter (CO2 from SOM), exogenous glucose (CO2 from glucose) and PE after adding glucose in soil samples

Experience *Incubation time, days

CO2 from SOM, mg C–CO2/g

DS· h

CO2 from glucose C–

CO2/gSP mg · h

PE, %

I 0 0.38 (0.07) — —II 0 0.37 (0.06) — —III 0 0.37 (0.06) — —IV 0 0.36 (0.04) — —I 1 1.69 (0.15) 4.92 (0.5) 357 ± 20II 1 1.15 (0.03) 5.00 (0.41) 213 ± 4III 1 0.45 (0.13) 0.62 (0.16) 22 ± 17I 2 1.00 (0.04) 6.12 (0.62) 170 ± 6II 2 0.30 (0.01) 2.16 (0.38) −19 ± 6I 3 0.59 (0.06) 5.48 (0.44) 60 ± 8I 7 0.68 (0.21) 3.40 (0.10) 84 ± 28II 7 0.55 (0.02) 0.43 (0.01) 49 ± 3III 7 0.22 (016) 0.06 (0.04) —IV 7 0.33 (0.03) 0.08 (0.03) —

*The incubation time is the time interval measured from the moment of introduction of glucose into the soil.The values in the parentheses are standard errors of measurements of three parallel experiments.


example the expenditure of energy on the transformation of aromatic structures contained in the SOM [37]. Therefore, we can assume that part of the energy generated during the metabolism of glucose was used by the microbial cells in the hydroxylation of SOM. This hypothesis is confirmed by the increase in RQ from the value of 0.83, recorded in the mineralization of SOM, to 1.36, observed after the introduction of glucose into the soil (Table 3.8). The two-carbon unit is detached from the hydroxylated intermediate SOM products, probably in the form of the acetyl derivative of acetylation coferment (acetyl CoA), which is oxidized to CO2 in the TCA. Thus, it can be assumed that the additional increase of microbial mineralization of SOM after adding glucose to the soil compared with the reference sample (PE) is due to the corresponding SOM transformation and formation of products, which can then be used by soil microorganisms.

Table 3.8. The mineralization of soil organic matter (CO2 from SOM), exogenous glucose (CO2 from glucose), RQ and PE before (t = 0) and after the introduction of glucose to the soil

Experiment Incubation time, days.

CO2 from SOM, µg C–CO2/g

DS· h

CO2 from glucose C–CO2/g DS

mg · h

RQ PE, %

I 0 0.38 (0.07) — 0.83(0.02) —II 0 0.37 (0.06) — 0.85(0.04) —III 0 0.37 (0.06) — 0.83(0.05) —IV 0 0.36 (0.04) — 0.85(0.01) —I 7 0.46 (0.12) 1.57 (0.13) 1.05 (0.04) 21 ± 16

*II 7 1.09 (0.07) 4.01 (0.36) 1.16 (0.01) 181 ± 9*III 7 1.28 (0.06) 4.94 (0.20) 1.32 (0.03) 246 ± 8*IV 7 1.47 (0.14) 4.43 (0.65) 1.35 (0.03) 287 ± 18

I 8 0.46 (0.09) 1.27 (0.02) 1.03 (0.05) 24 ± 12*II 8 0.37 (0.04) 3.05 (0.19) 1.12 (0.02) 0 ± 5*III 8 0.34 (0.02) 2.84 (0.29) 1.11 (0.01) −8 ± 2*IV 8 0.41 (0.15) 3.27 (0.46) 1.15 (0.01) 8 ± 20

I 11 0.39 (0.06) 0.94 (0.09) 1.01 (0.02) 2.5 ± 8*II 11 0.21 (0.04) 0.67 (0.16) 0.96 (0.02) −43 ± 5*III 11 0.21 (0.03) 0.37 (0.02) 0.90 (0.0) −43 ± 4*IV 11 0.27 (0.04) 0.40 (0.03) 0.87 (0.13) −29 ± 5

I 14 0.33 (0.02) 0.70 (0.08) 0.93 (0.03) −11 ± 2*II 14 0.25 (0.01) 0.30 (0.08) 0.88 (0.04) −32 ± 2*III 14 0.22 (0.02) 0.28 (0.01) 0.88 (0.01) −41 ± 2*IV 14 0.25 (0.05) 0.28 (0.04) 0.85 (0.05) −34 ± 6

*500 µg C-Glu/g DS of glucose was added to the soil samples. The values in the parentheses are standard errors of measurements of three parallel experiments.


Estimate of the duration of the effect of the exogenous substrate on the microbiota

In the early 80s of the last century a criterion was proposed for assessing the degree of influence of anthropogenic pollutants on soil microbiota [66]:

a) if the changes of microbial indicators, due to both natural factors and pollutants, are returned to normal within 30 days, it is believed that a substance released to the soil has no toxic effect and does not violate the natural fluctuation processes;

b) if the parameter changes are observed up to 60 days after the pollutant has entered the soil, its effect is considered tolerant;

c) if the changes in these indicators remain for more than 90 days after penetration of the pollutant into the soil, the presence of the pollutant in the soil is qualified as a factor of stress exposure.

In our case, the examples of different amounts of the glucose added in the soil (experiments I, II, III) and having different isotopic composition in comparison with the SOM, demonstrated a different degree of activation of mineralizing ability of the microorganisms directly using both the indicated exogenous substrate and further oxidation of the native soil material (priming effect). The rate of CO2 production and consumption of the exogenous substrate, determined by the isotopic characteristic of carbon, are considered as specific indicators of the effect of this substrate on the soil microbiota. Thus, the single application of 2000, 500 and 50 µm of C-Glu/g ??? to the soil, which is 13.3%, 3.3% and 0.33% of the amount of SOM in the original soil, was accompanied by different rates of CO2 production and different duration of the period during which its amounts in comparison

Table 3.9. Time of return to the initial state of microbial activity in soil after introduction of an exogenous substrate (glucose), determined by the rate of CO2 production and the characteristics of carbon isotopic composition

Experiment*The relative

amount of substrate, %

Time return of microbial activity in the soil to initial value, days

**The rate of CO2 production

***The magnitude δ13C-CO2 (‰)

I 13.3 18.4 103II 3.3 5.4 20III 0.33 0.74 14

*(Amount of C-glucose/amount of C-SOM)·100%.**Data from Table 3.1 were used in the calculations.***Data from Table 3.6 were used in the calculations.


exceed those in the natural processes up to that time. As shown in Table 3.9, the rate of mineralization of the total organic matter in soil (glucose + SOM) in the experiments I–III returned to the initial value (up to glucose addition) after 1 to 18 days. Since the amount of the exogenous substrate (glucose) up to 13.3% of the SOM amount influences the rate of CO2 production for less than 30 days, according to [66] it can be assumed that the impact of this substrate (glucose) on the mineralizing activity of the soil microbiota does not violate natural microbial processes in soil.

However, according to the balance calculations (see Table 3.1), at a large reduction of the rate of CO2 production after 7 days of observation (experiment I), the soil retained over 50% of the introduced glucose. This indicator is in agreement with the δ13C values (see Table 3.6), which show that the substrate added to the soil continues to participate in the processes of microbial metabolism, despite the significant decrease in the rate of CO2 production. As shown in Table. 3.9, according to the isotopic indicators of metabolic CO2, the time required by the microbial metabolism to return to the initial parameters is more than 100 days for a glucose addition of 13.3% glucose and around 20 and 14 days at much smaller loads of glucose (3.3 and 0.33%) of the amount of SOM. According to these indicators, glucose entry into the soil in the amount greater than 13% of the SOM is characterized as a stress effect on the microorganisms in the soil. Thus, the parameter of the isotopic composition of carbon of the metabolic carbon dioxide, formed during mineralization of total organic substances (glucose + SOM), is a more sensitive parameter, which reflects the impact of the exogenous substrate on soil microbiota.

Conclusion

Isotopic ratio mass spectrometry can serve as a highly informative method for the determination of microbial mineralization of soil organic matter and organic products in the soil. Using the easily metabolizable substrate, which differs in the carbon isotopic composition from SOM (e.g. glucose), it is possible to evaluate the effect of the exogenous product released to the soil, on the mineralization of SOM compared with the native soil (priming effect). The large increase in metabolic activity in the soil after adding glucose indicates the limit of the growth of soil microorganisms imposed by the easily metabolized substrates. It is shown that the mineralization of SOM by the soil microbiota requires a source of energy, contributing to the transformation of the SOM to products that are available as substrates for microorganisms. The


introduction of a molecule of exogenous glucose provides transformation of SOM by microorganisms, resulting in the mineralization of the two-carbon product from SOM. Assuming that the initial transformation of the SOM takes place by hydroxylation or oxygenation of aromatic structures SOM, the magnitude of the respiratory quotient (RQ) is lagging behind in time in relation to the PE as an indicator of priming-production of CO2. On the basis of the results it may be concluded that the fact that the values RQ > 1 are exceeded is due to the difference in the time of recording O2 consumption and the subsequent formation of CO2 by microbial mineralization of transformation products of SOM.

In assessing the impact of the exogenous substrate on the soil microbiota it was found that the isotopic composition of the carbon of metabolic carbon dioxide, formed as a result of microbial mineralization of SOM and the exogenous substrate, is the most sensitive parameter reflecting the degree and duration of the effect of the substrate on biogeochemical processes in the soil.

References

1. Doran J. W., Sarrantonio M., Liebig M. A., Advances in Agronomy, 1996, V.56, 1–54.

2. Dobrowolski G. V., Nikitin E. D., Soil conservation as an indispensable component of the biosphere, Moscow, Nauka, 2000, 184.

3. Domsch K. H., Interpretation and evaluation data. Recommended Test for Assessing the Side-effects of Pesticides on the Soil Microflora, Weed Research Organization Report, No. 59, 1980, 6–8.

4. Doran J. W., Appl. Soil Ecol., 2000, V. 15, 3–11.5. Smith J. L., Paul E. A., The significance of soil microbial biomass estimation,

Soil Biochemistry, Ed. by J.M. Bollag and G. Stozky, New York, Marcel Dekker, 1990, 357–398.

6. Brookes P. C., McGrath S. P., J. Soil Sci., 1984, V. 35, 341–346.7. Nordgren A., Baath E., Soderstrom B., Soil Biol. Biochem., 1988, V. 20, 949–954.8. Chander K., Brookes P. C., Soil Biol. Biochem., 1991, V. 23, 927–932.9. Fliessbach A., Martens R., Reber H.H., Soil Biol. Biochem., 1994, V.26, 1201–1205.10. Zyakun A. M., Dilley A., Prikl. Biokhim Microbiol., 2005, V. 41, No 5, pp. 582-

591.11. Dilly O., Zyakun A., Geomicrobiology J., 2008, V. 25, 425–431.12. Dilly O., Soil Biol. Biochem., 2001, V. 33, 117–127.13. Zuberer D. A., Recovery and enumeration of viable bacteria, Methods of soil

Analysis, Part 2: Microbial and Biochemical Properties / Ed. by R.W. Weaver, S. Angle, and F. Bottomley, Soil Science Society of America Book Series, No. 5, Medison, Wisconsin, 1994, 119–144.

14. Alef K., Nannipieri P., Methods in Applied Soil Microbiology, New York, Academic Press, 1995, 370.

15. Dobereiner J., Isolation and identification of nitrogen fixing bacteria from soil and plants, Methods in Applied Soil Microbiology / Ed. by K. Alef and P. Nannipieri,


New York, Academic Press, 1995, 134–135.16. Lorch H.J., Benkieser G., Ottow J. C. G., Basic methods for counting microorgan-

isms in soil and water, Methods in Applied Soil Microbiology / Ed. by K. Alef and P. Nannipieri, New York, Academic Press, 1995, 136–161.

17. Madsen E. L., A critical analysis of methods for determining the composition and biogeochemical activities of soil microbial communities in situ, Soil Biochemis-try / Ed. by G. Stotzky and J.M. Bollag, New York, Marcel Dekker, 1996, V. 9, 287–370.

18. Martens R., Biol. Fertil. Soils, 1995, V. 19, 87–99.19. Jenkinson D.S., et. al., Soil Biol. Biochem,, 1976, V. 8, 209–213.20. Voroney R.P., Paul E.A., Soil Biol. Biochem, 1984, V. 16, 9–14.21. Vance E.D., Brooks P.C., Jenkinson D.S., Soil Biol. Biochem., 1987, V. 19, 703–707.22. Anderson J.P.E., Domasch K.H., Soil Biol. Biochem., 1978, V. 10, 213–221.23. Anderson J.P.E., Domasch K.H., Arch. Microbiol, 1973, V. 93, 113–127.24. Anderson J.P.E., Domasch K.H., Ann. Microbiol. Enzimol., 1974, V. 24, 189–194.25. Anderson J.P.E., Domasch K.H., Can. J. Microbiol., 1975, V. 21, 314–322.26. Sus'yan E. A., Ananieva N, D , Blagodatskaya E. B., Microbiology, 2005, V. 74,

No. 3, pp. 394–400.27. Stenstrom J., Hansen A., Svensson B., Swed. J. Agric. Res., 1991, V. 2, 55–62.28. Pell V., Stenberg R., Stenstrom J., Forstenstrom I., Soil Boil. Biochem., 1996, V.

28, 393–398.29. Drobnik K., Plant Soil, 1960, V. 12, 199–211.30. Gerson U., Chet I., Rev. Ecol. Biol. Sci., 1981, V. 18, 285–289.31. Pianka E. R., The American Naturalist, 1970, V. 102, 592–597.32. Kuzyakov Y., Friedel J. K., Stahr K., Soil Biol. Biochem., 2000, V. 32, 1485–1498.33. Fontaine S., Mariotta A., Abbadie L., Soil Biol. Biochem., 2003, V. 35, 837–843.34. Zvyagintsev D.G., Beyond the Biomass / Ed. by K. Ritz, J. Dighton, K. E. Giller,

Chichester, Willey Press, 1994, 29–37.35. Mamilov A. Sh., Bysov B. A., Zvyagintsev D.G., Dilly O., Appl. Soil Ecology,

2001, V. 16, 131–139.36. IPCC 2001 / Report of Working Group I of the Intergovernmental Panel on Climate

Change, Climate change 2001 the scientific basis, Cambridge, Univ. Press, 2001.37. Wu J., Brookers P. C., Jenkinson D. S., Soil Biol. Biochem., 1993, V. 25, 1435–1441.38. Hamer U., Marschner B., Plant Nutr. Soil Sci., 2002, V. 165, 261–268.39. Dilly O.J., Plant Nutr. Soil Sci., 2004, V. 167, 261–266.40. Tuomela M., Hatakka A., Karjomaa S., Itovaara M., Biodegradation, 2002, V. 13,

131–140.41. Fontaine S., Mariotti A., Abbadie L., Soil Biol. Biochem., 2003, V. 35, 837–843.42. Fontaine S., Bardoux G., Benest D., Verdier B., Mariotti A., Abbadie L., Soil Sci.

Soc. Am. J., 2004, V. 68, 125–131.43. Dalenberg J.W., Jager G., Soil Biol. Biochem., 1981, V. 13, 219–223.44. Dalenberg J.W., Jager G., Soil Biol. and Biochem., 1989, V. 21, 443–448.45. Shen J., Bartha R., Appl. Environ. Microbiol., 1996, V. 62, 1428–1430.46. Kuzyakov Y., Raehmann J., Geyer B., Gartenbauwissenschaft, 1997, V. 62, 151–157.47. Mary B., Mariotty A., Morel J.L., Soil Biol. Biochem, 1992, V. 24, 1065–1072.48. Rochette P., Flanagan L. B., Gregorich E.G., Soil Sci. Soc. Am. J., 1999, V. 63,

1207–1213.49. Ekblad A., Hogberg P., Plant and Soil, 2000, V. 219, 197–209.50. Kristiansen S.M., Brandt M., Hansen E.M., Magid J., Christensen, B.T., Soil Biol.

Biochem., 2004, V. 36, 99–105.51. Vasconcellos C. A., Pesquisa Agropecaria Brasil, 1994, V. 29, 1129–1136.


52. Degens B., Sparling G., Soil Biol. Biochem., 1996, V. 28, 453–462.53. Aoyama M., Angers D.A., N‘Dayegamiye A. N., and Bissonnette N., Soil Biol.

Biochem., 2000, V. 32, 295–300.54. Dilly O., FEMS Microbiol. Ecology, 2003, V. 43, 375–381.55. Smith B. N., Epstein S., Plant Physiol., 1971, V. 47, 380–384.56. Teery J. A., Stowe L. G., Oecologia, 1976, V. 23, 1–2.57. Cheng W., Plant and Soil, 1996, V. 183, 263–268.58. Ekblad A., Nyberg G., Hogberg P., Oecologia, 2002, V. 131, 245–249.59. Stenstrom J., Svensson K., and Johansson M., FEMS Microbiol. Ecol., 2001, V.

36, 93–104.60. Tate R. L., Soil Microbiology, New York, Wiley Press, 1995, 398.61. Paul E. A., Clark F. E., Soil Microbiology and Biochemistry, San Diego, Acad.

Press, 1989, 273.62. Fontaine S., Bardoux G., Abbadie L., Mariotti A., Ecology Lett., 2004, V. 7,

314–320.63. Metzler D. E., Biochemistry, in: Chemical reactions in living cells, Moscow, Mir,

1980, V. 2, pp. 306–360.64. Newsholme E. A., Start C., Regulation in metabolism, New York, Wiley Press,

1973, 124–145.65. Lovley D. R., Coates J. D., Blunt-Harris E. L., Phillips E. J. P., Woodward J.C.,

Nature, 1996, V. 382, 445–448.66. Domsch K.H., Jagnow G., Anderson T.H., Residue Rev., 1983, V. 80, 65–105.

163Study of the isotopic composition of normal alkanes

4

Study of the isotopic composition of normal alkanes of continental plants

N.A. PedentchoukSchool of Environmental Studies, University of East Anglia,

Norwich, UK

Introduction

The isotopic composition of various biological substances is one of the most popular objects of studies by paleoclimatologists and ecologists who study the relatively recent climatic changes in the Holocene, as well as in previous geological epochs [1–5]. The wide popularity of this type of research is based on a relationship between the isotopic composition of carbon, hydrogen, and oxygen in the environment (e.g. ocean, sea, and lake waters, rain and snow precipitation, carbon dioxide in the atmosphere) and the isotopic composition of these elements in organic and inorganic components of the flora and fauna (e.g. cellulose and lipids of plants, animal bones, phosphates, carbonates, shells) found in continental and marine environments. Since the organically bound hydrogen, carbon and oxygen participate in the global hydrological and carbon cycles, climate changes affecting these cycles have a significant impact on their isotopic composition. Thus, having a clear understanding of the degree of fractionation of isotopes of a particular element in the biosynthesis processes as well as the level of diagenetic history of the sample, the researcher has the opportunity to study the climate-induced changes in these cycles in the past.

Methodologically, the study of the isotopic composition of organic components of plants and animals can be at two levels. The sample of an organic material can be studied in bulk or after separation into separate organic fractions/compounds. For example, the isotopic composition of the carbon included in the organic compounds of tree leaves can be measured on a small amount of crushed leaves,


without prior separation of lignin, cellulose, lipids and other organic compounds that make up the whole leaf. On the other hand, the separation of the sample into different organic fractions makes it possible to measure the carbon isotopic composition of each of these fractions separately. The first approach is popular because of its relative ease of sample preparation for analysis and shorter time required for isotopic measurements on the mass spectrometer. However, despite the long time and considerable effort, the second method has several important advantages.

Because different biochemical components, even in the same organism, have different isotopic composition because of the varying degrees of fractionation during biosynthesis, the study of individual organic compounds allows us to establish the most direct link between the isotopic composition of organically bound elements (e.g. C, H, or O) and the isotopic composition of this element in the environment. Another important factor is the different degree of diagenetic stability of various organic compounds. Therefore, the isotopic change of a particular item, tested at the level of individual compounds, makes it possible to avoid potential errors when the isotopic composition of element measured in bulk, undergoes a major change due to the loss of a fraction during diagenesis, and not due to changes of the palaeoclimatic conditions.

Normal alkanes (n-alkanes), consisting of the elements C and H and originating mainly from the waxy coating of leaves and needles, together with lignin, cellulose and fatty acids, are among the most common organic compounds synthesized by terrestrial vegetation. However, high stability during diagenesis, as well as a simple sample preparation procedure (compared with cellulose and fatty acids) make the n-alkanes ideal candidates for research of isotopic composition.

Several recently published review articles were devoted to the preparation and use of the isotopic analysis data of individual organic compounds in various fields of geosciences [6–9].

Recently, the paleoclimate [10, 11] has been widely studied using stable hydrogen isotopes. Shefus et al [12] explained the increase in δ2H by 20% of n-alkane C29, extracted from marine sediments, by the presence of dry conditions in the equatorial Africa during the Younger Dryas, i.e. from 12800 to 11500 years ago. Schuman et al [13] interpreted the two positive changes of δ2H (up to 40%) of normal fatty acid C28 from lake sediments as an indicator of the increase of the amount of precipitation in the summer period, compared to other seasons in the north-western part of the United States approximately 8200 years ago. Pagani et al [14] explained the increase in δ2H values


of n-alkanes C27/29 by 55‰ as a result of the reduction in rainfall in the tropics, resulting in an increase of the relative amount of moisture reaching the Arctic in the early stages of the Late Paleocene thermal maximum. Moreover, understanding the magnitude of the negative change in δ13C values (from –4.5 to –6‰) of n-alkanes C27/29 at this time is essential for calculating the potential amount of methane, suddenly released from the deposits of methane hydrates under the seabed [15].

The relative proportion of isotopically different conifers and angiosperm plants, the main components of the continental plant biomass, depends on the regional climate [16]. This relative proportion may be subjected to considerable changes, even in a relatively short period of time, less than 1000 years [17, 18].

Thus, the correct interpretation of the δ13C and δ2H data of n-alkanes from sediments and sedimentary rocks is related largely on the detailed information about possible changes in the composition and isotopic values of the local flora. The importance of these factors was emphasized by Smith et al [19]. The authors of this paper confirm that the transition from the mixed conifer/angiosperm flora to mainly angiosperm flora has led to more significant changes in δ13C values (–4 to –5‰, n-alkanes from the soil, Wyoming, USA), as compared with the values of δ13C obtained in studied of marine carbonates during the Late Paleocene thermal maximum. The role of this factor in calculating the amount of methane was highlighted above.

In addition to palynology, the simultaneous study of data on δ2H and δ13C of n-alkanes from the leaves of plants can help to understand the dynamics of coniferous and angiosperm plants. Compared with deciduous angiosperm, evergreen angiosperm plants and conifers usually have a waxy solid coating on the leaves/needles, resulting in stomata of these two types, usually smaller in size and surrounded by a thick layer of wax [20]. This structure leads to a more limited conduction of the stomata with respect to CO2 and H2O vapour of some evergreen and coniferous plants [21]. More limited access to gas CO2 during biosynthesis leads to 13C-enrichment of organic compounds. At the same time, the smaller stomata size limits the evaporation of H2O molecules from the cavity of the leaf/needle and this has an impact on the degree of D-enrichment of the leaf water used during biosynthesis.

Thus, it is highly likely that there is a relationship between the physiology of different types of plants, expressed in the structure and biochemical composition of the leaves/needles, and the isotopic parameters of these plants.

Until 2008, only two groups of authors published results of a collaborative study of the isotopic composition of δ2H and δ13C


n-alkanes, which showed no significant difference in these two types of plants. Investigation of conifers and angiosperm plants of several species in locations in Japan and Thailand showed no correlation between isotopic values of δ2H/δ13C [22]. On the other hand, despite the fact that plants with different productivity of transpiration showed little correlation between δ2H/δ13C, this correlation did not reveal any specific differences between conifer and angiosperm species [23].

Thus, the main objectives of work are: 1) overview of the techniques required for the selection, sample

preparation and isotope analysis of n-alkanes of biosynthesized continental vegetation;

2) discussion of the results of isotopic analysis of angiosperm plants and conifers, and the possibility of using the developed techniques for the study of paleoclimate.


General provisions

Organic compounds of interest to paleoclimate can be extracted from sediment, oil, coal, marine, lake and river sediments, as well as from the soil and the various organs of the lower and higher plants (mainly leaves and needles). In several recent publications special attention has been given to discussing in detail the traditional methods of extraction, as well as the advantages and disadvantages of these methods in relation to the above examples of different kinds [9, 24–27]. The basic principle is the most effective separation of organic compounds of interest to the geochemist from the sample matrix. Solid and consolidated samples (sandstone, slate, limestone, coal) typically require initial grinding to a powder. Depending on the type of sample and laboratory resources available to the researcher, the total organic fraction can be extracted using ultrasound in Soxhlet apparatus or in an ASE 300 automated extractor. Subsequent phases of the separation of certain organic fractions and derivatization of the individual compounds depend on the specific purposes of scientific research. The non-polar hydrocarbon fraction containing n-alkanes is separated from the total organic fraction by liquid column chromatography. The column is filled with silica gel (particle diameter 0.063–0.200 mm). The eluent is volatile solvents (hexane, methylene chloride, methanol); n-alkanes are present in the non-polar fraction and are extracted with hexane. The degree of ‘contamination’ of n-alkanes by other compounds in the non-polar fraction depends on the nature of the sample. For example, the


hexane fraction, isolated from the total organic fraction after extraction of the leaves of current plants, is in most cases is pure enough for subsequent isotopic analysis. However, the non-polar fraction released from the sediments and soils usually requires an additional separation of n-alkanes from iso- and cyclo-alkanes with urea or molecular sieves [28].

The isotopic composition of the elements hydrogen and carbon is expressed using the value of delta (δ), which is determined by the following formula:

δ = − ⋅R RRx stand

stand

1000,

where R is the ratio of heavy to light isotope, the indices x and std refers to the sample and the standard, respectively. In the case of elements hydrogen and carbon atoms, R is the ratio of 2H/1H and 13C/12C, respectively.

Determination of the isotopic composition of carbon and hydrogen of individual compounds, in this case the n-alkanes, requires the GC-IRMS system (gas chromatograph coupled with an isotopic mass spectrometer), equipped with an oxidation reactor and a high-temperature converter. The oxidation reactor is used for transfer of individual n-alkanes into carbon dioxide followed by measurement of carbon isotopes, the high-temperature converter is used for the transfer of individual n-alkanes to hydrogen gas with subsequent measuring of its isotopic composition. Modern mass spectrometers, equipped with a gas chromatograph, can achieve the following error of measurement of the isotopic composition of carbon and hydrogen of organic compounds: 0.1–0.3‰ for compounds containing 0.1–5 nmol of C, 2–5‰ for compounds containing 10–50 nmol H [8].

Figure 4.1 shows a simplified diagram of the instrument for GC-IRMS, equipped with an oxidation reactor. The hexane fraction containing n-alkanes is injected with the help of a microsyringe into the gas chromatograph in which the n-alkanes are separated in a capillary column. Then, in the oxidation reactor in the presence of a catalyst (CuO/NiO/Pt, 940°C [29]) they are transformed into H2O and CO2. The nitrogen oxides formed during the process are reduced to N2 in the reduction reactor (Cu, 600°C [30]). H2O is removed from the flow of carrier gas in a water trap Nafion. ‘Purified’ CO2 is fed into the mass spectrometer equipped with Faraday collectors which detected ions with masses 44, 45 and 46, relating to ions of three isotopomers 12C16O2,

13C16O2 and 12C18O16O, respectively. The value of δ13C for the


Oxi

datio

n re

acto

r C

uO/N

iO/P

t/940

°CIn

terf

ace

cont

aini

ng re

duct

ion

reac

tor

(Cu/

600°

C) a

nd w

ater

sepa

rato

r

Inje

ctor

Solu

tion

with

sam

ple

Ion

sour

ceM

ass s

pect

rom

eter

Cap

illar

y co

lum

nFa

rada

y co

llect

ors

(m/z

44,

45,

46

Mag

netic

sect

or

anal

yser

Gas

ch

rom

atog

raph

CO

2A

mpl

ifier

Stan

dard

gas

Com

pute

r with

pro

gram

me

for c

ontro

lling

m

ass s

pect

rom

eter

Fig.

4.1

. Th

e ge

nera

l sch

eme

of a

gas

chr

omat

ogra

ph o

f a m

ass

spec

trom

eter

for m

easu

rem

ent o

f the

car

bon

isot

opic

com

posi

tion

(δ13

C).


sample is calculated relative to the standard gas with a known isotopic composition or relative to the internal standard. Figure 4.2 shows a typical chromatogram of analysis by the GC-IRMS method. The chromatograph shows six peaks of the standard gas and several peaks of n-alkanes with a pronounced predominance of compounds with an odd number of carbon atoms – a typical distribution of n-alkanes from leaves of higher plants. The chromatogram also shows good chromatographic separation of individual compounds, which is one of the most important factors for obtaining high-quality data for δ13C.

The main difference between methods of determination of the isotopic values of δ2H and δ13C for individual organic compounds is the way obtaining the sample gas. To convert organic compounds into the gas H2, the oxidation reactor is replaced by a high-temperature converter operating at a temperature of 1450°C. At this temperature the organic compounds emanating from the GC are converted to the gases H2, CO and the corresponding isotopomers [31]. In this case there is no formation of H2O. H2 gas in the flow of the carrier gas is supplied to the mass spectrometer equipped with Faraday collectors to register ions with the masses of 2 and 3, for the determination of 1H2 and 1H/2H, respectively. Another feature of this technique is the need to consider the effect of protonation in the ion source on the value δ2H. The protonation reactions lead to the formation of H3

+ ions. Since the number of H3

+ is proportional to the square of the pressure of hydrogen gas, it is recommended to use the correction H3-factor which is determined on the basis of measurements of standard gas peaks of different sizes [32, 33].

This article presents the results obtained by the author and his colleagues in Washington State, USA, and some countries in Western

Fig. 4.2. GC-IRMS chromatogram of n-alkanes extracted from the non-polar hydrocarbon fraction of the leaf of the English oak (Quercus robur), Newcastle, UK; n-alkanes with low concentrations with the even number of carbon atoms are not indicated.

Sign

al in

tens

ity,

mV

Standard gas Holding time, s Standard gas


Europe. The influence of climatic conditions on the values of δ13C and δ2H of organic compounds from the leaves of modern angiosperm plants and conifers and the influence of the physiology of these plants on their isotopic composition were studied.

Methodological features of analysis

The central part of the Washington state, USA. The climate at the study site is dry, with four pronounced seasons, and with less 10 cm of rain from May to October [34]. Water from a well 95 m deep was the main source of moisture for the plants examined during sample collection. Leaves were collected from three angiosperms (Betula pendula - birch, Populus tremuloides – aspen poplar, and Syringa vulgaris – lilac common) and two conifers (Pinus sylvestris – Scots pine and Picea pungens – blue spruce) plants. All the five plants of approximately the same age (7–8 years old in 2005) grow in open conditions close to each other (<10 m) and receive the same amount of sunlight. The plants were watered at the same time (2–3 hours) once or twice a week depending on the weather. Each tree received about 70 liters of water during watering. Relative humidity and temperature were measured every two hours, using VERITEQ SP-2000-20R device, set in the shade at a height of 2 m above the ground. Fourteen samples of water from wells and from 7 to 10 leaves (or 3 branches of conifers) of each tree were selected for isotopic analyses from May to October 2005

The leaves were extracted with hexane using ultrasound (30 min, twice). Lipid fractions were separated by in a chromatographic column (particle size 70–230 mesh) in the following sequence: hexane, hexane /methylene chloride (9:1 by volume) and the mixture of methylene chloride/methanol (2:1 by volume). The hexane fraction was used to obtain the δ13C and δ2H values in the isotope mass spectrometer MAT 253 (Thermo Finnigan, Bremen, Germany), connected to a gas chromatograph with a combustion microfurnace Thermo Finnigan Trace GC/C III (for δ13C) and a high-temperature converter (for δ2H). The data on δ2H of the water samples from wells were determined on the same isotopic mass spectrometer using the device H/Device (Thermo Finnigan). Individual n-alkanes were separated on a capillary column J & W Scientific DB-1 (60 m × 0.25 mm × 0.25 µm film thickness). The GC column temperature was raised from 60°C (1 min) to 320°C (25 min) at 6°C per minute. The δ13C data are shown in relation to the standard Vienna Pee Dee belemnite (VPDB). The reproducibility of δ13C measurements was ±0.6‰ or better. High-temperature pyrolysis of organic compounds for the formation of hydrogen H2 was carried out


at a temperature of 1400°C. The δ2H values of n-alkanes are indicated with respect to the Vienna Standard Mean Ocean Water (VSMOW). The reproducibility of the results of δ2H measurements was generally better than ±5‰, reaching ±11‰ only in several cases. The δ2H values of water from the wells are shown in relation to VSMOW and normalized on the basis of the water standards from the Greenland ice (GISP) and Antarctic ice (SLAP). The reproducibility of δ2H measurements of water from the wells was ±1‰ or better.

Western Europe. In contrast to the project described above, the study of the isotopic composition of C and H n-alkanes of the angiosperm plants and conifers plants in Europe was carried out in several different climatic and ecological zones: the Atlantic North (Newcastle-upon-Tyne, UK, and Bremen, Germany), Mediterranean Mountains (Porano, Italy), and Mediterranean South (Donatan National Park, near Seville, Spain) zones. Figure 4.3 shows the amount and isotopic composition of precipitations (which were only source of moisture for the plants under study) and also the relative humidity typical of the studied sites. The largest amount of rainfall was recorded in Bremen and Porano, while the smallest amount in Newcastle and Sevilla. Sevilla is characterized by the highest values of δ2H, while Newcastle and Bremen by the lowest values. The difference in relative humidity between these four places depends on the time of the day, but most are similar Newcastle and Bremen. In the studied aspect Seville is more similar to Newcastle and Bremen in the morning, Porano – in the evening.

Samples of leaves were collected from three types of plants: deciduous angiosperm, evergreen angiosperm and coniferous trees. The plants differed in age and height, but all samples were collected on the shady side of trees. The distribution of certain types of plants at each site is shown in Fig. 4.4. All three types of plants grow in Newcastle, and Bremen and Porano. However, due to the hot and dry climate, the angiosperm deciduous plants are absent in Seville. The procedure for the extraction of n-alkanes was identical to that used when processing the leaves from the Washington state. The isotope data δ2H and δ13C of the n-alkanes were obtained in the isotopic mass spectrometer Delta V Plus (Thermo Finnigan, Bremen, Germany) with a gas chromatograph and a combustion microfurnace Thermo Finnigan Trace GC/C III and a high-temperature converter. The reproducibility of δ13C measurements was not less than ±0.5%, δ2H not less than ±6%. n-C29 alkane was selected for comparison between different plant species.

A feature of this project is the fact that in addition to study of the isotopic composition of plants, measurements were also taken maximum stomatal conductance in relation to CO2 and H2O transpiration of


Бореальная

4050

6070

8090

IV

III

II

I

(cm

)

Ann

ual P

reci

pita

tion2

5060

7080

9010

0(%

)

IV

III

II

I

Ann

ual R

ange

of R

elat

ive

Hum

idity

(mor

ning

)3

3040

5060

7080

9010

0

IVIIIIII

Ann

ual R

ange

of R

elat

ive

Hum

idity

(eve

ning

)3

(%)

IVII

I

II

I-8

0-6

0-4

0-2

00

IV

III

III

δ��(�

��

��

)

Ann

ual R

ange

of •

D o

f Pre

cipi

tatio

n1

Alp

ine

Nor

thB

orea

lN

emor

alA

tlant

ic N

orth

Alp

ine

Sout

hC

ontin

enta

lA

tlant

ic C

entra

lPa

nnon

ian

Lusi

tani

anA

nato

lian

Med

iterr

anea

n M

ount

ains

Med

iterr

anea

n N

orth

Med

iterr

anea

n So

uth

Env

iron

men

tal S

trat

ifica

tion

of E

urop

eE

nvir

onm

enta

l Zon

eδ2 H

δ2 H

500

km

Fig.

4.3

. Dis

tribu

tion

of v

eget

atio

n in

Eur

ope:

I an

d II

- A

tlant

ic N

orth

, III

- M

edite

rran

ean

Mou

ntai

ns, I

V -

Med

iterr

anea

n So

uth.

Sou

rces

of

clim

ate

data

: 1) δ

2 H o

f pre

cipi

tatio

n da

ta w

ere

dete

rmin

ed u

sing

the

onlin

e ca

lcul

ator

ww

w.w

ater

isot

opes

.org

, bas

ed o

n la

titud

e, lo

ngitu

de a

nd a

ltitu

deab

ove

sea

leve

l; 2)

dat

a fr

om 1

961

to 1

990

[43]

, and

3) d

ata

from

ww

w.w

eath

erba

se.c

om. T

he b

ase

map

of E

nviro

nmen

tal S

tratif

icat

ion

of E

urop

eis

a m

odifi

ed m

ap fr

om [4

4].

Fig.

4.3

. Dis

trib

utio

n of

veg

etat

ion

in E

urop

e: I

and

II -

Atla

ntic

Nor

th, I

II -

Med

iterr

anea

n M

ount

ains

, IV

- M

edite

rran

ean

Sout

h. S

ourc

es o

fcl

imat

e da

ta: 1

) δ2

H o

f pr

ecip

itatio

n da

ta w

ere

dete

rmin

ed u

sing

the

onlin

e ca

lcul

ator

ww

w.w

ater

isot

opes

.org

, bas

ed o

n la

titud

e, lo

ngitu

de

and

altit

ude

abov

e se

a le

vel;

2) d

ata

from

196

1 to

199

0 [4

3],

and

3) d

ata

from

ww

w.w

eath

erba

se.c

om.

The

base

map

of

Envi

ronm

enta

l St

ratif

icat

ion

of E

urop

e is

a m

odif

ied

map

fro

m [

44].


the trees studied in Newcastle. These measurements were performed on device CIRAS-1 Portable Photosynthesis System for at least two leaves on the shadow side of a tree, only the angiosperm deciduous and angiosperm evergreen trees as the special cuvette, required to work with pine trees, was not available at the beginning of this project.


The central part of the Washington state, USA

Detailed measurements of the isotopic composition of carbon and hydrogen of n-alkanes from leaves collected in the Washington state are shown in [35]. Since the alkane n-C27 was the only compound available

Fig. 4.4. The isotopic composition δ13C and δ2H of alkane n-C29, isolated from plants collected in the UK and Germany. Shaded-in black signs indicate the types of coniferous trees, gray – evergreen angiosperms; unshaded symbols – deciduous angiosperm.

I – Newcastle-upon-Tyne,Great Britain

II – Bremen, Germany

2δH

/‰2δ

H/‰


in sufficient quantity for reliable measurement of the δ13C/δ2H data, further discussion will be limited only to this compound. The data for n-C27 alkanes are clearly distinguished between two coniferous and two types of angiosperm trees (Fig. 4.5). From May to October the values of δ13C and δ2H for Syringa vulgaris and Populus tremuloides ranged from –31.9 to –32.7‰ and from –168 to –186‰, respectively, showing a slight tendency to a decrease in the δ2H values late in the season of plant growth (Fig. 4.6). However, the δ13C and δ2H data for Pinus sylvestris and Picea pungens ranged from –28.8 to –30.6‰ and from –190 to –212‰. As with other types of angiosperm plants, Betula pendula had more positive values of δ2H (from –162 to –178‰) with respect to the coniferous species. The δ13C data for Betula pendula were consistent with those of Pinus sylvestris and Picea pungens in May,

Fig. 4.4. (continued). The δ13C and δ2H data of alkane n-C29 from plants collected in Italy and Spain. Shaded in black signs indicate the types of coniferous trees, gray – evergreen angiosperm; unshaded symbols – deciduous angiosperm.

III – Porano, Umbria,Italy

IV – Seville, Spain

2δH

/‰2δ

H/‰


and changed by +4‰, thus becoming even more 13C-enriched relative to other angiosperm species at the end of the season.

Since all five species of plants were in the same climatic conditions and irrigated with water with the same isotopic composition, the most likely explanation of the data are differences in the physiology and biochemistry of these plants. The relative 13C-enrichment of Pinus sylvestris and Picea pungens may be explained by the low conductance of the stomata of these two species in relation to the Syringa vulgaris and Populus trenuloides and, as a consequence, lowering of the concentration of CO2 in the intercellular space within the leaf and a decrease in the 13C isotope discrimination during biosynthesis [36]. The data on the stomatal conductance of plant species under study in the Washington state, is currently not available, but the previously published material – either for the same species (Pinus sylvestris and Populus tremuloides) or for plants belonging in the same genus (Picea sitchensis and Betula alleghaniensis), – shows that the conifers do have a reduced stomatal conductance [37] (Fig. 4.7). However, the reduced conductivity stomata of conifers can not explain the lower data for δ2H of the alkanes in these plants, as the reduced flow rate of water molecules in the process of evaporation would lead to more positive δ2H values of the water inside the leaves [38]. The difference between the δ2H data of the alkanes between the conifers and angiosperm tree species, apparently, can not be explained without additional

Fig. 4.5. Data on isotopic composition of δ13C and δ2H of n-C27 alkane of five species of plants collected in Washington state, USA. Shaded symbols represent coniferous tree species, unshaded – flowered ones.

-33 -32 -31 -30 -29 -28 -27 -26

-210

-200

-190

-180

-170

-160

Populus tremuloides(Poplar)

Betula pendula (Birch)

Picea pungens(Spruce)

Pinus sylvestris(Pine)

Syringa vulgaris(Lilac)

δ2 H /

‰

δ13C / ‰


information about the internal structure of the leaves of these species and fractionation processes of hydrogen atoms during biosynthesis.

The results of the degree of the apparent isotope fractionation, obtained in the course of this project from –120 to –65‰ (Fig. 4.8) differ significantly from previously published data [39–41]. Previous data on the extent of the apparent hydrogen isotope fractionation are

a Relative humidity

Sampling days May June July Aug Sep Oct

May June July Aug Sep Oct

Water from wellb

Fig. 4.6. Seasonal changes in the isotopic composition of δ13C (a) and δ2H (b) of n-C27 alkane of five species of plants and δ2H of water from the wells during the summer and autumn of 2005, central Washington state, USA.

2δH

/‰

a

b


Pinus sylvestris, AustriaPicaesitchensis,Oregon,USAPopulustremuloides, Michigan,USABetulaalleghaniensis, New Hampshire,USA

CO2

H2O vapour

CO2

H2O vapour

H2O vapour

CO2

CO2

H2O vapour

Conductivity of stomata, cm/s0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40

Fig. 4.7. The maximum conductance of the stomata. Data from [37]. Gray columns – coniferous vegetation types, diagonal lines – flowered species.

ε n-C

27/W

ater

, %

Sampling days May June July Aug Sep Oct

Fig. 4.8. The degree of the apparent fractionation (ε) between the δ2H data of alkane n-C27 of five species of plants and δ2H of water from wells; εn-C27/water = (Rn-C27/Rv–1) × 1000‰, where R = D/H. At any given time the same value of δ2Hv was used for the calculation of ε of each of the five plant species.


usually relatively constant and more negative (~120%) for conifers and angiosperm plants. The reason for such strong difference can be dry climate in which the plants grew in the central part of the Washington state. So the generalizations based on the results of this project should be treated with caution.

Another unexpected finding of this study is the significant difference between the δ13C data for Betula pendula in relation to other angiosperm species. A possible explanation for this observation is a strong climatic stress (especially in July and August when the relative humidity is lowest), which the plant, usually growing in more humid conditions, experienced. This stress may impact on the isotopic composition of carbon in n-alkanes of Betula pendula, preserved throughout the season. An indirect confirmation of this is the fact that Betula pendula is the only plant, which has experienced a significant increase in the relative amount of alkane n-C31 in comparison with other n-alkanes having a shorter carbon chain. The relative increase of the amount of the alkanes with a longer carbon chain and a higher melting point can be a response to the arid climatic conditions [42].

Western Europe

Despite significant differences in the climatic conditions, the δ13C/δ2H data of the n-C29 alkane of different types of trees in the Atlantic North, Mediterranean Mountains and Mediterranean South areas are relatively similar (see Fig. 4.4). However, if we consider each site separately, the isotopic data δ13C/δ2H in Newcastle, Bremen and Porano time show a weak negative relationship between δ13C and δ2H. Moreover, the angiosperm deciduous species are basically more D-enriched and more depleted in 13C compared to the evergreen angiosperm and coniferous species. No correlation was observed in the Seville area, where the isotope experimental data for n-C29 are presented only for the evergreen and coniferous species. Significant differences in the δ2H data for the precipitation in Porano and Seville, in comparison with Newcastle and Bremen, have not led to a similar difference in the δ2H data of n-C29 alkane. The range of δ2H isotope data of this compound is virtually identical for all four areas.

Maximum stomatal conductance measurements require special equipment, which should be available in all areas of vegetation study. Since this was not possible in the init ial phase of the project, measurements were taken only in Newcastle. The maximum stomatal conductance in relation to CO2 and H2O vapour is shown in Fig. 4.9. The maximum stomatal conductance in relation to CO2


shows a negative correlation with the δ13C data for alkane n-C29 of two deciduous and two evergreen angiosperm species. In turn, the maximum stomatal transpiration in relation to H2O vapour shows a positive correlation with the δ2H data of the same four types of trees. The positive correlation between the transpiration and the δ2H data of the alkanes is difficult to explain because, as noted above, the reduced transpiration should lead to more positive δ2H values of the water inside the leaf [38]. The isotopic composition of H water molecules undergoes significant changes during biosynthesis and in all probability, the difference in the inner structure of the leaves and/or features of the biosynthesis between the angiosperm and coniferous species has a more significant effect on the δ2H of the alkanes than the original D/H of water.

By analogy with the explanation of the data from the Washington state, the above presented data on the isotopic composition C among the angiosperm plants and conifers in Newcastle, Bremen and Porano can be explained in terms of the physiology of the studied plants. The relative 13C depletion of the n-alkanes of the deciduous angiosperm plants is probably associated with a higher maximum stomatal conductance in relation to CO2. Explaining the difference between the δ2H data of the alkanes of the coniferous species of trees and angiosperm plants requires additional information on the isotopic composition of water within the leaf, the internal structure of leaves, as well as on the fractionation of H in the biosynthesis of organic compounds that make up the waxy coating of the leaves of the studied plants.

Conclusion

In this chapter, we considered a new approach to the use of the isotopic

Con

duct

ivity

CO

2, m

mol

e m

-2 s-1

Tran

spira

tion

of H

2O,

mm

ole

m-2 s-1

Fig. 4.9. The maximum conductivity of the stomata. The data were obtained in Newcastle 12:00 and 13:00 July 8, 2008; – angiosperm evergreen kinds of trees; – angiosperm deciduous trees.


composition of n-alkanes from the leaves of the continental vegetation to study the paleoclimate and plant physiology. Preparation samples for the study of the δ13C and δ2H data of the n-alkanes requires some effort and the availability of highly specialized equipment.

However, the data obtained at the level of individual organic compounds can potentially provide unique information not available when using bulk isotope methods.

We also considered the most recent results of the author and his colleagues in the Washington state, USA and some Western European countries in order to determine the effect of climatic conditions and physiology on the values of δ13C and δ2H of organic compounds from the leaves of the angiosperm plants and present-day conifers. Preliminary results of these projects are essential for the interpretation of the isotopic data of n-alkanes used for the study of the paleoclimate. Noticeable changes in the values of δ13C and δ2H of the organic compounds extracted from sediments or sedimentary rocks, can not only be the consequence of changes in climatic factors, but can also be due to the change of types of plants. Moreover, the degree of difference between the isotopic values between different types (and in each individual type) of the plants may be due to different climatic conditions and the physiology of these plants.

Literature

1. Parrish J. T., Interpreting Pre-Quaternary Climate from the Geologic Record. New York, Columbia University Press, 1998, 338.

2. Burroughs W. J., Climate Change: A Multidisciplinary Aproach. Cambridge, Cam-bridge University Press, 2001, 298.

3. Mackay A., Battarbee R., Birks J., Oldfield F., Global Change in the Holocene. London, Arnold, 2003. 528.

4. Cowie J., Climate Change: Biological and Human Aspects. Cambridge, Cambridge University Press, 2007. 504.

5. Dawson T. E., Siegwolf R. T.W., (Eds.) Stable Isotopes as Indicators of Ecological Change. London, Academic Press, 2007. 417.

6. Meier-Augenstein W., Aplied gas chromatography coupled to isotope ratio mass spectrometry, Journal of Chromatography A, 1999. V. 842, 351–371.

7. Glaser B., Compound-specific stable-isotope (δ13C) analysis in soil science, Journal of Plant Nutrition and Soil Science, 2005. V. 168, 633–648.

8. Sessions A. L., Isotope-ratio detection for gas chromatography, Journal of Separa-tion Science, 2006, V. 29, 1946–1961.

9. Evershed R. P., Bull I. D., Corr L. T., Crossman Z. M., Van Dongen B. E., Evans C. J., Jim S., Mottram H. R., Mukherjee A. J., Pancost R.D. Compoundspecific stable isotope analysis in ecology and paleoecology, In: Stable Isotopes in Ecology and Environmental Science, Ed. by R. Michener, K. Lajtha. Blackwell Publishing Ltd, 2007, 480–540.


10. Pancost R.D., Boot C. S., The palaeoclimatic utility of terrestrial biomarkers in marine sediments, Marine Chemistry, 2004, V. 92, 239–261.

11. Tiple B. J., Pagani M., The early origin of terrestrial C4 photosynthesis, Annual Reviews of Earth and Planetary Sciences, 2007, V. 35, 435–461.

12. Schefuß E., Schouten S., Schneider R. R., Climatic controls on central African hydrology during the past 20,000 years, Nature, 2005, V. 437, 1003–1006.

13. Schuman B., Huang Y., Newby P., Wang Y., Compound-specific isotopic analyses track changes in seasonal precipitation regimes in the Northeastern United States at ca 8200 cal year BP, Quaternary Science Reviews. 2006, V. 25, 2992–3002.

14. Pagani M., Pedentchouk N., Huber M., Sluijs A., Schouten S., Brinkhuis H., Sinninghe Damste‚ J. S., Dickens G. R., The IODP Expedition 302 Expedition Scientists (2006). Arctic hydrology during global warming at the Palaeocene / Eocene thermal maximum, Nature, 2006, V. 442, 671–675.

15. Dickens G.R. Methane oxidation during the Late Palaeocene thermal maximum, Bulletin De La Societe Geologique De France, 2000, V. 171, 37–49.

16. Woodward F. I., A review of the effects of climate on vegetation: Ranges, com-petition, and composition, In: Global Warming and Biological Diversity / Ed., by R. L. Peters, T. E. Lovejoy. New Haven & London, Yale University Press, 1992. 105–123.

17. Tinner W., Lotter A. F., Central European vegetation response to abrupt climate change at 8.2 ka., Geology, 2001. V. 29, 551–554.

18. Williams J.W., Shuman B. N., Webb T. III., Bartlein J., Leduc L. Latequaternary vegetation dynamics in North America, Scaling from taxa to biomes, Ecological Monographs, 2004, V. 74, 309–334.

19. Smith F.A., Wing S. L., Freeman K. H., Magnitude of the carbon isotope excursion at the Paleocene-Eocene thermal maximum, The role of plant community change, Earth and Planetary Science Letters, 2007, V. 262, 50–65.

20. Esau K., Anatomy of Seed Plants, 2nd ed., John Wiley & Sons, Inc., 1977, 57621. Körner C., Scheel J. A., Bauer H., Maximum leaf conductance in vascular plants,

Photosynthetica, 1979, V. 13. 45–82.22. Chikaraishi Y., Naraoka H., δ13C and δ2H relationship among three n-alkyl com-

pound classes (n-alkanoic acid, n-alkane, and n-alkanol) of terrestrial higher plants, Organic Geochemistry, 2007, V. 38. 198–215.

23. Hou J., D‘Andrea W. J., MacDonald D., Huang Y., Evidence for water use ef-ficiency as an important factor in determining the δ2H values of tree leaf waxes, Organic Geochemistry, 2007, V. 38, 1251–1255.

24. Smith R.M., Before the injection modern methods of sample preparation for sepa-ration techniques, Journal of Chromatography A, 2003, V. 1000, 3–27.

25. Zygmunt B., Namiennik J., Preparation of samples of plant material for chromato-graphic analysis, Journal of Chromatographic Science, 2003, V. 41, 109–116.

26. Peters K. E., Walters C. C., Moldowan J. M., The Biomarker Guide. Second Edition. I. Biomarkers and Isotopes in the Environmental and Human History, Cambridge, Cambridge University Press, 2005, 471.

27. Péres V.F., Saffi J., Melecchi M. I. S., Abad F. C., de Assis Jacques R., Martinez M.M., Oliveira E. C., Caramão E. B. Comparison of soxhlet, ultrasound-assisted and pressurized liquid extraction of terpenes, fatty acids and Vitamin E from Piper gaudichaudianum Kunth, Journal of Chromatography A. 2006, V. 1105. 115–118.

28. Grice K., de Mesmay R., Glucina A., Wang S., An improved and rapid 5A molecular sieve method for gas chromatography isotope ratio mass spetrometry of n-alkanes (C8–C30+), Organic Geochemistry, 2008, V. 39, 284–288.

29. Merritt D. A., Freeman K. H., Ricci M. P., Studley S.A., Hayes J.M., Performance


and optimization of a combustion interface for isotope ratio monitoring gas chro-matography/mass spectrometry, Analytical Chemistry. 1995, V. 67, 2461–2473.

30. Brand W.A., Tegtmeyer A.R., Hilkert A., Compound-specific isotope analysis: ex-tending towards 15N/14N and 18O/16O, Organic Geochemistry, 1994, V. 21, 585–594.

31. Burgoyne T.W., Hayes J. M., Quantitative production of H2 by pyrolysis of gas chromatographic effluents, Analytic Chemistry, 1998, V. 70, 5136–5141.

32. Sessions A. L., Burgoyne W. W., Hayes J.M., Correction of H3+ contributions in

hydrogen isotope ratio monitoring mass spectrometry, Analytical Chemistry. 2001a, V. 73, 192–199.

33. Sessions A. L., Burgoyne W. W., Hayes J.M., Determination of the H3 factor in hydrogen isotope ratio monitoring mass spectrometry, Analytical Chemistry, 2001b, V. 73, 200–207.

34. Kittitas Country, 2004, Kittitas County 2004 Road Atlas.Apendices. Accessible from: http://www.co.kittitas.wa.us/publicworks/atlas/Atlas2004_Apendices.pdf.

35. Pedentchouk N., Sumner W., Tiple B., Pagani M., δ13C and δ2H compositions of n-alkanes from modern angiosperms and conifers: An experimental set up in central Washington State, USA, Organic Geochemistry, 2008, V. 39. 1066–1071.

36. Farquhar G. D., O‘Leary M. H., Berry J. A., On the relationship between carbon isotope discrimination and the intercellular carbon dioxide concentration in leaves, Australian Journal of Plant Physiology, 1982, V. 9, 121–137.

37. Körner C., Scheel J. A., Bauer H., Maximum leaf conductance in vascular plants, Photosynthetica, 1979, V. 13, 45–82.

38. Farquhar G. D., Cernusak L. A., Barnes B., Heavy water fractionation during transpiration, Plant Physiology, 2007, V. 143, 11–18.

39. Chikaraishi Y., Naraoka H., Compound-specific δ2H–δ13C analyses of n-alkanes extracted from terrestrial and aquatic plants, Phytochemistry. 2003, V. 63, 361–371.

40. Bi X., Sheng G., Liu X., Li C., Fu J., Molecular and carbon and hydrogen isotopic composition of n-alkanes in plant leaf waxes, Organic Geochemistry, 2005, V. 36, 1405–1417.

41. Sachse D., Radke J., Gleixner G., δ2H values of individual n-alkanes from ter-restrial plants along a climatic gradient implications for the sedimentary biomarker record, Organic Geochemistry, 2006, V. 37, 469–483.

42. Kozlowski T. T., Pallardy S.G., Physiology of Woody Plants, 2nd ed., Academic Press, 1997, 411.

43. New M., Lister D., Hulme M., Makin I., A high-resolution data set of surface climate over global land areas, Climate Research, 2002, V. 21, 1–15.

44. Metzger M. J., Bunce R.G.H., Jongman R.H.G., Mucher C. A., Watkins J.W., A climatic stratification of the environment of Europe, Global Ecology Andes Bio-geography, 2005, V. 14, 549–563.

183Using isotope ratio mass spectroscopy for tobacco analysis

5

Using isotope ratio mass spectroscopy for analysis of tobacco

A.B. UryupinA.N. Nesmeyanov Institute of Organoelement

Compounds, Russian Academy of Sciences, Moscow

Modern methods of mass spectrometric studies are widely used in the study of organic substances of plant origin, particularly of raw tobacco and tobacco product [1, 2]. The most popular methods for routine identification of mixture components are hyphenated methods based on a combination of mass spectrometers with chromatographic equipment [3]. Creating a database by equipment manufacturers – the mass spectra of individual standard compounds – aroused the illusion that chromatic mass spectrometers can be used to ‘decode’ the composition of any complex mixture of substances of tobacco with a high level of probability. In actual practice, the results of this analysis should be approached with sufficient caution.

Another direction of mass-spectral studies is the isotope ratio mass spectrometry (IRMS) [4], the development of which has been possible as a result of development of high-precision equipment of a new generation on the basis of its original methods of analysis with direct practical interest [5].

The absolute magnitude of the isotope ratios can vary in a very narrow range, which is manifested in the case of ‘light’ chemical elements that make up the substance of tobacco (hydrogen, carbon, nitrogen, oxygen and sulphur). These elements, in contrast to the minor, ‘heavy’ elements found in tobacco (e.g. lead, uranium, etc.) are generally enriched in nature by the lighter isotope. It should be emphasized that because of the specific features of the discussed method the necessary step of analysis of any substance, including the components of tobacco, it its combustion or conversion into simple

Isotope mass spectrometry of light gas-forming elements184

gases. There is an alternative method that does not require combustion or thermal transformation of the sample – NMR spectroscopy [6, 7], but in its implementation it is necessary to adhere to a variety of experimental conditions.

At present IRMS is increasingly recognized as a method useful for applications in the food industry to monitor and confirm the authenticity of the place of production of foods and their ingredients. The previously existing methods for identification of counterfeit products have been based on identifying in the food products individual chemicals that are not in the natural product, or on the determination of non-compliance of the content of the components of comparable products. The IRMS method with measurements of δ can be used to distinguish chemically identical substances. A necessary condition for the successful practical application of IRMS for identification of raw tobacco and tobacco products is the preliminary collection of data on isotope ratios of individual objects of identification and statistical analysis of the collected data. This allows to take into account the impact on δ of the specificity of the tobacco plant, its variety, growing area, climate and seasonal conditions.

At the moment, there are no international standard methods for determining isotope ratios designed to address tobacco issues. Most of the techniques used in study of other objects of food production (which also include tobacco) involve the measurement of isotope ratios for a single element, although the reliability of the conclusions substantially increases by the use of isotope ratios of two elements (δ13C and δ18O or δ13C and δ15N). For professionals, engaged in the identification of tobacco and tobacco products, of great interest is the isotopic analysis aimed at clarifying the origin of plant products in the same way as is done in the case of wine [5] (for example, by correlation of the values of δ13C of alcohol and δ18O of water in samples).

The complex chemical composition of tobacco makes it necessary to select individual components for which the effect of biological fractionation of the isotopes would be expressed in degrees, corresponding to the sensitivity of the existing methods. Tobacco alkaloids belong to nitrogen-containing metaboli tes of plants whose origin (e.g. caffeine) was previously also determined by the multielement variant of the IRMS method (the determination of δ13C, δ15N and δ18O). As for nicotine, the main alkaloid of tobacco, the most informative in terms of identifying its source is the paired determination of the isotope ratios of nitrogen and hydrogen [8].

The results of GC-C-IRMS determination of the isotope ratios of carbon, hydrogen and nitrogen in nicotine samples, separated in 6 min


by microwave extraction from tobacco, were used to assess the country of origin of smuggled cigars [9]. Similarly, multielement isotope analysis was used to differentiate between Canadian and Chinese contraband cigarettes. The method of measuring the 15N/14N and 2H/1H ratios included extraction of the nicotine of the tobacco of Canadian (47 brands) and Chinese cigarettes (23 brands) with methanol, followed by direct sample introduction into the chromatograph coupled with an isotopic ratio mass spectrometer. The nicotine in the Canadian cigarettes had higher values of isotope ratios for hydrogen δ2H = –232.7 ÷ –203.4‰ than the values for Chinese cigarettes δ2H = –262.6 ÷ –219.9‰ and lower values of the isotopic ratios for nitrogen δ15N = –7.7 ÷ –6.3‰ and –7.6 ÷ –5.7‰ for the Canadian and Chinese cigarettes, respectively [10].

Evaluation of variations in the natural content of 15N was carried out not only for nicotine, but also for other alkaloids – its analogues, as well as its metabolites (e.g. N-methyl-2-phenylpyrrolidine and products demethylation) [11]. The alkaloids were extracted from the biological matrix by solid-phase extraction. The measurement results of δ15N for different samples differed by 4‰, reproducibility was better than 0.5‰. In addition, nicotine, together with acetanilide and urea, was a convenient standard substance (reference material) for the IRMS method in the interval values of the isotope ratios δ13C = –30.05 ÷ +7.72‰, δ2H = –162 ÷ –45‰, δ15N = –6.03 ÷ +35.62‰ [12].

It is also important to study the isotopic composition of not only nicotine, but also other classes of compounds belonging to the tobacco leaf (hydroxy acids, sugars, cellulose); differences in the values of isotope ratios δ authors are attributed to the different regional conditions of the biosynthesis of these components [13].

A certain ‘gross feature’ of cigarettes may be the total content of the 13C isotope in the gaseous products of combustion and decay contained in cigarette smoke (CO2 and CO), analyzed using the GC-C-IRMS method [14].

With more stringent requirements for the composition of ingredients added to the raw tobacco and tobacco products (sauces, flavourings) the problem of confirmation of their natural origin becomes very important.

Many of the ingredients are optically active substances and their molecules contain one or more centres of asymmetry. Optically active compounds can exist in several isomeric forms, differing in the configuration of these centres. Ingredients of natural origin are usually one of the optical isomers, and those produced in industry are a mixture of optical isomers. It was found that the isotopic ratios

Isotope mass spectrometry of light gas-forming elements186

of the optical isomers may be different. For example, in the case of natural flavouring linalool the isotopic composition of hydrogen is δ2Hsmow = –265 ÷ –307‰, and for synthetic linalool, for which the ratio of optical isomers depends on the source, δ2Hsmow ranges from –185 to –209‰ [15, 16]. Similar studies were carried out for menthol, one of the most important aromatizers of tobacco [17] and also for (E)-methyl cinnamate [18].

The synthetic simulator of raspberry aroma, used in the production of some tobacco products, contains the alpha-ionol for which δ13C and δ2H were determined. The ‘raspberry’ aromatizer of natural origin was characterized by δ13C = –30.3 ÷ –35.1‰ and δ2H = –176 ÷ –221‰, and the synthetic one by δ13C = –24.5‰ and δ2H = –184‰ [19]. Therefore, using IRMS we can detect non-natural ingredients in tobacco flavours and sauces.

However, to solve a set of ‘tobacco issues’ it is necessary to create a database of isotope ratios with the use of raw tobacco and auxiliary materials supplied by well-known manufacturers, guaranteeing the authenticity of the samples. Then, on the basis of international standard analysis methods it will be possible to obtain reliable results.

Of additional interest are the chemical changes of the ingredients deposited on by tobacco in smoking, in particular, the assessment of the possible contribution of the resultant products to the yield of so-called ‘Hoffmann analytes’ [20]. The prediction of modes of transformation of ingredients on the basis of their direct pyrolysis cannot be regarded as reliable, because it can not simulate the conditions of combustion of substances deposited on the tobacco. A good alternative is the IRMS method using compounds labelled with the radioactive isotope 14C or 13C to trace the fate of volatile and non-volatile components and products of combustion in the main and side streams of tobacco smoke. Test cigarettes, with labelled 13C vanillin, benzaldehyde, or D-glucose, added to the tobacco, were smoked using a special ‘fish tail’ nozzle for sampling in the lateral stream and smoke in a variety of traps. The 13C/12C ratio, were measured in CO, CO2, and volatile hydrocarbons of the smoke and also in the butts and ashes, and the contribution of labelled compounds was evaluated. In this way, it was shown that significant amounts of the volatile ingredient (benzaldehyde)are transferred into the smoke without decomposition, and the semi-volatile and non-volatile ingredients (vanillin and glucose) contributed significantly to the concentration of 13C in CO and CO2. Similar conclusions were drawn on the basis of the results of studies using IRMS of the transformations standard tobacco humectants (glycerin, propylene glycol), geranyl acetate and tetramethyl pyrazine [21].


In general, it should be noted that isotope ratio mass spectrometry is still not used widely for analysis of tobacco.

Literature

1. Tobacco Encyclopedia, Mainz: Verlagsgruppe Rhein Mainz GmbH & Co.KG, 2000, 192

2. Rodgman A., Beitraege zur Tabakforschung Int., 2002, V. 20, 83–103, 277–297.3. Khmel'nitsky R. A., Brodsky E. S., Methods of analytical chemistry, Chromatog-

raphy-mass spectrometry, Moscow, Khimiya, 1984.4. Handbook of Stable Isotope Analytical Techniques / Ed. by P.A., de Groot, Elsevier

B.A. 2004.5. Rossmann A., Food Reviews International, 2001, V. 17, 347.6. Hays P. A., Remaud G. S., Jamin E., Martin Y. L., J. Forensic Sci., 2000. V. 45,

552.7. Freeman R., Magnetic resonance in chemistry and medicine. Moscow: URSS, 2009,

331.8. Jamin E., Naulet N., Martin G. J., Plant. Cell. Environ., 1997, V. 20, 589.9. Binnet M. -J., Hupe M., Lafontaine P., Pittcon 2005, Abstr. N 920-5, Open-Vessel

Microwave Assisted Extraction of Nicotine from Tobacco for Stable Isotope Mea-surements.

10. Binette M. J., Lafontaine P., Vanier M., Ng L. K., Journal of Agricultural and Food Chemistry, 2009, V. 57, 1151.

11. Molinie R., Ferchaud-Roucher V., Lebreton J., Robuns R. J., Rapid Commun. Mass Spectrometry, 2005, V. 19, 2039.

12. Schimmelmann A., Albertino A., Sauer P. E., Qi H., Molinie R., Mesnard F., Rapid Commun. Mass Spectrometry, 2009, V. 23, 3513.

13. Jamin E., Naulet N., Martin G. J., Phytochemical Analysis, 1998, V. 8, 105.14. Russow R.W., Intorp M., Mueller C., Stangl C., Isotope Environ. Health Study,

2007, V. 43, 257.15. Bentley R., Chemical Reviews, 2006, V. 106, 4099.16. Goryaev M., Pliva I., Methods of research of ester oils. Alma-Ata: Publ. Kazakh

Academy of Sciences, 1962, 751.17. Sybilska D., Asztemborska M. J., Biochem. Biophys.Methods, 2002, V. 54, 187.18. Fink K., Richling E., Heckel F., Schreier P., Journal of Agricultural and Food

Chemistry, 2004, V. 52, 3065.19. Del Mar Caja M., Preston C., Kempf M., Schreier P., Journal of Agricultural and

Food Chemistry, 2007, V. 55, 6700.20. Mueller C., Intorp M., Russow R., Application of isotope mass-spectrometry to

study fate and behaviour of ingredients applied on tobacco, 2006, CORESTA Congress. Smoke science / Product Technology, Paris, Oral Presentation SS16.

21. Mueller C., Intorp M., Fate of tobacco ingredients: Application of isotope mass-spectrometry to study the behaviour of humectants, geranyl acetate and tetramethyl pyrazine during combustion» 2009, CORESTA Congress. Smokey science / Product Tecnology, Aix-en-Provence, Oral Presentation SSPT22.


6

Using isotope ratio mass spectrometry of carbon in doping control

T. Sobolevskii, I.S. Prasolov and G.M. Rodchenkov

Anti-doping Centre of the Ministry of Sports, Tourism and Youth Policy of the Russian Federation, Moscow

Introduction

Currently, in doping control there is a need to identify the cases of use doping agents that are close analogs of hormones produced in the body person, or their precursors (testosterone, dehydroepiandrosterone, androstenedione, androstenediols, etc.). According to the results of classical methods – gas chromatography coupled with mass spectrometry – this fails because of the lack of specific metabolites, and only the so-called atypical changes in the hormonal profile of the athlete (or the change in the concentration relations of certain steroids) are noted.

The use of synthetic testosterone in the sport was banned more than 25 years ago. Previously, the only way to establish the fact of its use was to determine the ratio of the concentrations of testosterone (T) and its inactive isomer – epitestosterone (Epi) in the urine. However, this approach has some significant limitations since the T/Epi ratio in the population varies within a rather wide range (from 0.1 to 4.0 and above) [1], and in some people as a result of genetic special features the T/Epi ratio does not reach 4 even after taking testosterone.

Since the vast majority of synthetic steroids are derived from sitosterol and stigmasterol – compounds of plant origin with the carbon isotopic composition of about –30‰ and below (relative to the international standard belemnite), their application can be determined

189Using isotope ratio mass spectrometry of carbon

using isotope ratio mass spectrometry. The method is based on the measurement of the isotope ratios 13C/12C (δ13C) of endogenous steroids in the human body, which lies in the range from –17 to –26‰ and almost never reaches the values below –27‰ [2–4]. Studies have shown that the isotopic composition of steroid hormones of people living on different continents is markedly different: endogenous steroids in people in North and South America are enriched in 13C to a greater extent than in Europe [5, 6]. This is due to the fact that in the USA many crops such as corn and sugar cane use for photosynthetic CO2 consumption the so-called C4 mechanism (δ13C values vary from –6 to –19‰), whereas in Europe, the predominant mechanism in the plants is C3 (δ13C varies from –24 to –34‰).

In 2004, the World Anti-Doping Agency (WADA) published a technical paper [7], which regulates the use of the method of isotope ratio mass spectrometry, but even today the anti-doping laboratories have no single approach to the method of analysis because of its complexity and labour intensity. At the same time, of the 35 accredited laboratories in the world (the number as of January 2010), only two thirds of them have the technical ability to conduct such an analysis. In most cases they use multi-stage solid-phase extraction (SPE) [8–11] and SPE together with semipreparative liquid phase chromatography [12].

The aim of this work is the development of methods for determining the isotope ratios 13C/12C in the steroids isolated from human urine, by gas chromatography coupled with isotope ratio mass spectrometry (GC-C-IMS) and the establishment of the intralaboratory criteria for evaluation of the analysis results.

The metabolism of steroid hormones

For a better understanding of the principle of the analysis we should consider the scheme of biosynthesis of steroid hormones in the body. Their common precursor is cholesterol undergoing a series of transformations under the influence of highly specific enzymes that ensure the reactions of hydroxylation, hydrogenation, dehydrogenation, isomerization, splitting and aromatization. The final stage is characterized by the formation of conjugates with glucuronic acid. Compared with the starting materials, the conjugates are much better soluble in water and easily removed from the organism. As an example, Fig. 6.1 shows the structural formula of pregnandiol 3α-glucuronide.

Testosterone is the most important representative of androgens, which has a pronounced anabolic effect. It is synthesized by Leydig cells and


controls the development and function of the gonads, and is responsible for the formation of secondary male sex characteristics (development of muscles, hair, etc.). Testosterone is actively metabolized with the formation of 5α- and 5β-androstandiols, which in turn are converted into inactive products androsterone (A) and etiocholanolone (E). It should be noted that the concentration of testosterone in the urine is on average 20–40 ng/ml (in women it rarely exceeds 10 ng/ml), androstandiols – 20–100 ng/ml, whereas the concentration of the terminal metabolites (A and E) reaches 10 µg/ml or more. The diagram in Fig. 6.2 shows the metabolism of steroid hormones.

We should also mention the steroids that are not involved in the metabolism of androgens (in doping control they are referred to as endogenous markers, EM), such as pregnandiol (PD), pregnantriol, androstenol (16en), as well as the products of the metabolism of corticosteroids: 11β-hydroxyandrosterone, 11β-hydroxyetiocholanolone and 11-ketoetiocholanolone (Fig. 6.3). These compounds are found in the urine at concentrations 100–3000 ng/ml. Since their isotopic composition is determined only by the region of residence and the characteristics of a person’s diet, in doping control EM is used to make the decision that the athlete has used prohibited drugs. When interpreting the results of analysis, we compare the δ13C values of testosterone or its metabolites with similar values of EM, and if the difference in absolute value does not exceed 3‰, the analysis result is regarded negative, i.e. it is believed that androgens in this sample have an endogenous origin. This difference is denoted as ‘delta–delta’, Δδ. Recently, however, the anti-doping laboratories have been using more frequently the intralaboratory criteria for evaluation of the analysis results, individual for each ‘steroid–EM’ pair. These data obtained in the course of metrological certification of a particular technique in a single laboratory, which allows to consider all potential sources of systematic errors and eliminate the possibility of false

Fig. 6.1. Pregnandiol 3α-glucuronide.


Fig. 6.2. The scheme of the biosynthesis of steroid hormones: 1 – cholesterol, 2 – pregnenolone, 3 – pregnandiol, 4 – androstenol; 5 – dehydroepiandrosterone, 6 – androstenedione, 7 – testosterone, 8 – epitestosterone, 9 – 5α-androstandiol, 10 – 5β-androstandiol, 11 – androsterone, 12 – etiocholanolone, 13 – pregnantriol.

positive results. The latter method of assessing the results is called the reference range method.

Anti-doping laboratories use different compounds as endogenous markers, but mostly PD [13]. It is worth noting that its use can sometimes be inefficient, as some dietary supplements sold in the U.S. market, include pregnenolone, which is metabolized to


the PD and significantly distorts its isotopic composition [14, 15]. Other EMs are also used quite often: 11β-hydroxyandrosteron, 11β-hydroxyetiocholanolone and 11-ketoetiocholanolone [16]. However, their isotopic composition can be influenced by taking a dietary supplement containing 11-ketoandrostendion also available on the sports nutrition market. In addition, due to low volatility these compounds have ‘mediocre’ gas chromatographic properties even in the form of derivatives. In general, EM is the most appropriate androstenol, although in the urine of women its concentration is usually less than 300 ???ng/ml.


Equipment

Studies were carried out on an isotopic mass spectrometer Delta V Advantage (Thermo, Bremen) connected via an Combustion III interface to the gas chromatograph TRACE GC (Thermo, Italy). The

Fig. 6.3. The scheme of the biosynthesis of steroid hormones (continued): 14 – cortisone, 15 – cortisol, 16 – 11-ketoandrostendion, 17 – 11β-hydroxyandrostendion, 18 – 11-ketoetiocholanolone, 19 – 11β-hydroxyandrosteron, 20 –11β-hydroxyetiocholanolone.


structural identification of compounds was carried out using a mass spectrometer DSQ II (Thermo, USA), coupled with a second gas chromatograph. Liquid samples were processed with an autosampler TriPlusAS (Thermo, Italy) allowing alternate feeding of samples into injectors of the two gas chromatographs.

Target compounds (as acetates) were separated in identical capillary columns RTX-35MS with the integrated precolumn 5 m long (Restek, USA) 30 m × 0.25 mm × 0.25 µm in the same conditions of temperature programming: 120°C (2 min), heating 30°C/min up to 260°C, the heating rate of 1°C/min to 276°C, heating at a rate of 30°C/min to 310°C (2 min). The sample was introduced in the mode without dividing the stream at a temperature of 250°C. The flow rate of the carrier gas (helium 99.9999%) was 1.7 ml/min in the case of GC-MS and 2 ml/min in the case of GC-C-IRMS. The carrier gas for GC-C-IRMS was additionally dried using a thermocatalytic afterburner (Supelco, USA). GC-C-IRMS also used CO2 with a purity of 99.995% (reference gas) and O2 with a purity of 99.999%. The temperature of the oxidation reactor at the combustion interface was 940°C, and in the reduction reactor 600°C. The oxidation reactor was regenerated by saturating with oxygen for 15 min at the operating temperature for a few hours before the start of each series of tests. In the case of GC-MS data were recorded in the scanning mode from 50 to 410 amu (atomic mass units) under conditions of ionization by electron impact at 70 eV. The volume of the injected sample was 2 ml for GC-C-IRMS, and 0.5 ml for GC-MS.

Samples were prepared using high performance liquid chromatograph Agilent series 1100 (Germany), equipped with a binary pump, an autosampler, a diode-array detector and a preparative fraction collector G1364B. Separation was carried out in the SunFire C18 column (Waters, USA) 250 mm × 4.6 mm, the grain size of the sorbent 5 µm. The analytical column was protected by the precolumn 20 mm × 4.0 mm with the same sorbent. The gradient elution conditions were as follows: (1) ‘A’ (water) from 70 to 0% for 20 min, 10 min at 100% ‘B’ (acetonitrile), followed by up to 70% ‘A’ for 5 min and 5 min at 70% ‘A’, or (2) from 30 to 0% ‘A’ for 33 min, 5 min at 100% ‘B’, up to 30% ‘A’ for 5 min and 5 min at 30% ‘A’. The flow rate of the eluent through the column was 1 ml/min, the column was thermostatically controlled at 35°C. Detection was performed at 197 nm and 240 nm.

Reagents and materials

Standard samples of testosterone, 5α- and 5β-androstane-3α, 17β-diol


(5α, 5β) and pregnandiol (5β-pregnane-3α, 20S-diol) were obtained from LGC Standards (Germany), androsterone, etiocholanolone, androstenol (5α-androst-16-en-3α-ol), androstanol (5α-androstan-3β-ol), dehydropregnenolone acetate, testosterone, as well as pyridine, acetic anhydride, heptane, diethyl ether and salt for the preparation of buffer solutions were obtained from Sigma-Aldrich (USA). The diacetate of 5α-androstandiol was obtained from Research Plus Inc. (USA). β-glucuronidase from E. Coli K12 was obtained from Roche (Germany). The methanol for preparing solutions of the compounds obtained was supplied by Merck (Germany). Especially pure gases were produced by NII KM (Russia).

The eluent was deionized water (18.2 MOhm) (installation of Milli-Q Synthesis, Millipore, England), and the acetonitrile of the quality ‘for the far UV’ (JT Baker, Holland). Solid-phase extraction (SPE) was carried out using Bond Elut LRC C18 cartridges (Varian, USA), volume 10 ml, containing 500 mg of sorbent.

Sample preparation

A 10 ml urine sample (in some cases 20 or 40 ml) was passed through a pre-conditioned SPE cartridge, then washed with water and eluted with methanol (four times in 1 ml). The eluate was evaporated and the residue was dissolved in 1 ml of 0.8 M phosphate buffer solution (pH = 6.3) and extracted with 5 ml of diethyl ether. The organic layer was discarded and then 100 µl of β-glucuronidase was added to the water layer and incubated for 1 h at 57°C. After cooling, a 3 M carbonate buffer solution (pH = 10) and 5 ml diethyl ether were added and extracted on an automatic shaker for 10 min. After centrifugation and freezing of the water layer at –30°C the ether extract was transferred to a fresh tube, evaporated at 60°C and the dry residue was dissolved in 60 µl of methanol containing 50 ng/µl of dehydropregnenolone acetate (internal standard for retention time control), followed by 40 µl of water and transferred to a container (vial) with an insert of a reduced volume.

90 µl of the resultant sample was then injected into the liquid chromatograph in the conditions 1 (wavelength 197 nm). Fractions corresponding in the retention time to testosterone (I), 5α- and 5β-androstandiolam (II), androsteron and etiocholanolone (III), pregnandiol (IV) and androstenol (V) were then collected. To the fractions III–V we added a solution androstanol (300 ng/µl) in an amount equal to one tenth of the final volume of the fraction (see below), evaporated to dryness and acetylated by the action of 100

SPE[eluent – MeOH]


µl of the mixture (50:50) of pyridine and acetic anhydride for 2 h at 80°C. The reaction mixtures were evaporated, and the fractions I and II were redissolved in 60 µl of methanol containing 50 ng/µl of dehydropregnenolone acetate, followed by adding 40 µl of water, transfer to a vial and by adding 90 µl to the liquid chromatograph. The fraction, corresponding to the testosterone acetate, was collected in the conditions 1 at a wavelength of 240 nm and the fraction containing acetates of 5α- and 5β-androstanediols was collected in the conditions 2 at a wavelength of 197 nm. To control the time intervals of collection of the fractions, in each batch of samples test mixtures, containing steroids in the native form or in the form of acetates, were injected into the liquid chromatograph.

The fractions I and II were evaporated until dry, after which a solution of pentacosane in heptane (100 ng/µl) in an amount equal to one tenth of the final volume fraction was added to the fractions I–IV together with 9/10 of the volume of net heptane. The resulting I–V samples were analyzed by GC-MS and GC-C-IRMS. Figure 6.4 shows a block diagram of sample preparation.

Fig. 6.4. Block diagram of sample preparation.

SPE[eluent – MeOH]

LLE[pH ~ 7, extragent Et2O]

Aqueous phase Organic phase

Fermentative hydrolysis[β-glucuronidase 57oC, 1 h ]

LLE[pH ~ 9, extragent Et2O]

HPLC-fractionation

Derivatization[Ac2O, pyridine, 80oC, 2 h]

HPLC-fractionation for fractions I and II

GC-MS and GS-S-IRMS analysis



The main problem of determining the isotopic composition of endogenous steroids in urine is the complexity of this matrix and a significant difference in the concentration of target compounds – from tens of nanograms (testosterone) to ten or more micrograms (androsterone, etiocholanolone). Moreover, the linear range of the isotope mass spectrometer in the continuous flow mode is limited, and to achieve the right results the analytical signal must be in a fairly narrow range, which correspond to 30–100 ng of the steroid injected into the chromatographic column. All this leads to the need for sample fractionation, which is most conveniently done in the process of sample preparation by HPLC. Figure 6.5 shows the chromatograms of a test mixture of target compounds used to control the time intervals of collection of fractions and of the extract obtained from the urine.

To improve the gas chromatographic properties of steroids it is necessary to obtain more volatile derivatives. Silylation, being one of the simplest and most common methods of derivatization, is not suitable for GC-C-IC, since the oxidation of silyl derivatives leads to

Fig. 6.5. Chromatograms corresponding to the HPLC separation of a test mixture of steroids (top) and the real object (bottom). Indicated zones correspond to time intervals of collecting fractions: I – testosterone, II – 5α/5β-androstanediols, III – A/E, IV – PD, V – 16en, STD – time marker (dehydropregnenolone acetate).


the formation of silica deposits of which over time lead to decreased functionality of the oxidation reactor. Use of fluorine-containing reagents (trifluoroacetic anhydride) will cause the poisoning of the oxidative reactor due to the formation of stable fluorides of copper and nickel which would be prevent regeneration of the reactor. On the other hand, the use of acetic anhydride as a derivatizing agent does not harm the system, and the reaction products have good chromatographic properties. Change in the isotopic composition of steroids in acetylation is calculated on the basis of experimental data [17], although in the case of non-quantitative conversion or unstable conditions of the reaction it is possible to obtain non-reproducible results due to isotopic fractionation.

The effect of isotopic fractionation may be caused by both kinetic and thermodynamic factors. The cause of this effect lies in the difference in the energy of the vibrational levels of the bonds containing heavy isotopes in comparison with the bonds containing light isotopes [18]. In the case of the kinetic isotope effect, this energy difference leads to a difference in the rate of processes involving compounds of different isotopic composition (derivatization reaction). The influence of this effect is especially strong in the process in which rupture or formation of a bond is the limiting stage. The second type of isotopic fractionation effect (thermodynamic) is associated with differences in the physico-chemical properties such as the absorption of thermal radiation, molar volume, vapour pressure, melting and boiling points. This effect manifests itself most distinctively in phase transitions therefore sample injection into the evaporator (injector) of the gas chromatograph in GC-C-IRMS should be carried out in the mode without splitting the flow essential to prevent the discrimination of isotopic composition [19]. The ideal solution is to use an injector with direct introduction of samples into the column bypassing evaporation (the so-called on-column injector), but its application is subject to certain technical difficulties [20]. In this case, to minimize losses of the target compounds in the evaporator, we proposed to introduce the sample by increasing the pressure in it 2.5 times for 1 min after injection. For steroid acetates with increasing pressure in the evaporator the area of the chromatographic peaks was approximately doubled, whereas for the native steroids, as shown by our preliminary studies – it increases by almost an order of magnitude. Given the low concentrations of some of the target steroids in the urine, such losses were unacceptable. In addition, the non-quantitative mass transfer to the column of the gas chromatograph can lead to the registration of distorted values of δ13C due to isotopic fractionation, which is also unacceptable in doping control.


Another striking example of isotope fractionation is chromatographic separation: in liquid chromatography the carbon isotope composition of matter at the beginning and end of the chromatographic peak can vary by 10‰ [21] and, therefore, the use of HPLC for sample preparation for isotope mass spectrometry requires strict control of the retention times and quantitative data collection fractions. It is important to understand that the isotopic fractionation accompanies virtually every stage of the analysis and, if it cannot be fully excluded, then it should be made permanent.

Chromatographic analysis in the GC-C-IRMS system also has a number of features associated with the design of the interface between the gas chromatograph and the isotope mass spectrometer. A large number of compounds and long capillaries of different diameters leads to broadening of the chromatographic zones, including the CO2 peak formed after combustion of the sample components. To minimize this erosion, we have modified the interface of the GC-C-IRMS system by replacing the metal T piece in the thermostat of the gas chromatograph by a glass connector (‘pressfit’) with a quartz capillary through which the sample comes into the oxidation reactor, the reactor itself (in the original construction the connection is realized via a metal connector). Even after these changes the width of the peak in the GC-C-IRMS system was on average 25–40 s depending on the substance and its amount that imposes additional requirements on the efficiency of separation of the neighbouring peaks. It should be noted that to obtain correct results in the determination of the isotopic composition, the oxidation reactor should provide a 100% conversion of the analyzed substances, because otherwise, along with the formation of CO2 incomplete combustion to CO takes place. This process leads to isotopic fractionation of the conversion products of the analyzed compounds since due to kinetic isotope effects the CO is enriched with the 13C isotope and the recorded δ13C values are shifted into the negative region [21]. Overloading of the reactor with a large amount of organic matter can also lead to incomplete combustion and distortion of the measurement results. On the other hand, the residual amounts of water vapour in the carrier gas flow can protonate 12CO2 with the formation of the H12CO2

+ ion which has the same mass to charge ratio as 13CO2. This leads to the measured δ13C values shifted to the positive region.

The connection of the mass spectrometer and the combustion interface is implemented as an open flow divider and, therefore, 80% of the sample introduced into the chromatograph is lost. This leads to the need to introduce to the GC-C-IRMS a sample with a large volume compared to GC-MS, which in some cases cause an overload


of the column. It could be avoided by using a column with a larger internal diameter (0.32 mm) or by increasing the thickness of the film of the stationary phase, but in the first case, this may reduce the effectiveness of the separation of the investigated compounds with similar chemical structures (5α- and 5β-androstandiols, androsterone and etiocholanolone), while in the second case it may lead to a large increase of the duration of analysis. In this regard, it was decided that the use of the capillary column with the size 30 m × 0.25 mm × 0.25 µm is a reasonable compromise to solve the problem.

We determined the isotopic composition of the above steroids by the GC-C-IRMS method in more than 100 urine samples of athletes and volunteers. This was necessary to formulate statistically substantiated criteria for evaluating the analysis results, i.e. to determine the reference intervals.

In developing the methodology we were faced with the need to obtain δ13C values for the steroids, which are in the urine in both small and in very large quantities. To obtain correct results, it is necessary to assess in advance the concentration of the target steroids in the urine sample to ensure that the analytical signal to the isotope mass spectrometer is within its linear range. The concentrations of the steroids was determined by the preliminary analysis of samples by the existing doping control method [22], and this was followed by calculating the volume to which a specific fraction should be dissolved to the content of 20–50 ng/µl of steroid in the final extract. If the calculated volume of the fraction was less than 50 µl, then the dry residue was redissolved in 50 µl of heptane and then evaporated in a nitrogen stream to a specified volume. Through the use of narrow glass microinserts into the vials the minimum volume of the fraction in our work was only 10 µl.

Two compounds were used as an internal standard: pentacosane and androstanol. Androstanol was added prior to acetylation to the fractions III, IV and V to control the isotopic fractionation during derivatization. In the case of fractions I and II, which were subjected to

Table 6.1. Retention times of the compounds (steroids in the form of acetates, IS – the internal standard)

Compound tret, s Compound tret, s

Pentacosane (IS) 548.0 5β-Androstandiol 970.8Androstenol 606.1 5α-Androstandiol 995.55α-androstan-3β-ol (IS) 657.7 Testosterone 1127.1Etiocholanolone 893.5 Pregnandiol 1223.9Androsterone 910.4


repeated separation by the HPLC method the androstenol was not added, since this would lead to the need to collect an additional fraction.

The average δ13C value of pentacosane (–30.0 ± 0.8‰), determined from more than 300 analyzes, was used as an indicator of the state of the oxidative reactor and the system as a whole.

Table 6.1 shows the retention times of the investigated compounds in the GC-C-IMS system. Figure 6.6 shows as an example chromatograms

m/z 44, mV m/z 44, mV

m/z 44, mV m/z 44, mV

m/z 44, mV m/z 44, mV

t, s t, s

t, s t, s

t, s t, s

a b

c d

e f

Fig. 6.6. Chromatograms of a test mixture of steroids and certain fractions of the urine sample. The rectangular peaks represent pulses of CO2 with a known isotopic composition: a – a test mixture of steroids, use to check the status of the system and for component identification (name and retention times in Table 6.1), b – fraction I (testosterone), c – fraction II (androstanediols), d – fraction III (androsterone, etiocholanolone), e – fraction IV (pregnandiol), f - fraction V (androstenol).


of a test mixture of steroids and individual fractions of urine, which show that the use of semipreparative HPLC provides selective separation of the investigated steroids and minimizes the effect of interfering matrix components.

Since this technique involves a lengthy and laborious process of sample preparation (preparation of a series of six samples lasts almost 3 days), and taking into account the complexity of the GC-C-IRMS systems, each of the sample (a series of samples) should be accompanied by two quality control samples: clearly positive and clearly negative. The intentionally positive sample was obtained from a volunteer who took two capsules of Andriol drug preparation (80 mg of testosterone undecanoate), and the intentionally negative – from a volunteer not taking steroids. Both volunteers collected urine sample for within 2 days, after which the urine was stabilized sodium azide (1 g/l), filtered, poured into 10 ml vials and frozen, with preliminary determination of the concentration of the target steroids. Each sample was then analyzed several times by GC-C-IRMS to obtain information on the δ13C values of the target steroids and the intermediate precision

Table 6.3. δ13C values of the target steroids, established in the study

Compound Mean, % Interval, ‰ SD Number of measurements

Testosterone –23.4 –(21.2–25.5) 0.93 835α-Androstandiol –23.8 –(20.8–25.7) 0.91 1055β-Androstandiol –22.0 –(19.5–25.2) 0.94 127Androsterone –22.9 –(20.7–24.9) 0.78 131Etiocholanolone –22.8 –(20.8–24.6) 0.71 128Pregnandiol –21.9 –(19.9–24.1) 0.73 131Androstenol –22.6 –(21.2–24.1) 0.65 130

Table 6.2. The values of δ13C of target steroids in the knowingly positive and knowingly negative urine samples (n = 20)

CompoundPositive test Negative test

Mean, ‰ SD SDMean, ‰Testosterone −28.2 0.8 −22.65α-Androstandiol −29.4 0.7 −22.15β-Androstandiol −27.4 0.5 −21.9Androsterone −27.5 0.5 −22.8Etiocholanolone −27.3 0.4 −22.9Pregnandiol −22.2 0.5 −22.4Androstenol −22.5 0.4 −21.6


of the method and it was found that the standard deviation (SD) in terms of reproducibility is 0.4–0.8‰ depending on the compound. Table 6.2 presents data on the isotopic composition of the knowingly positive and knowingly negative samples, obtained during the certification procedure.

The statistical processing of the data yielded the values of δ13C of the steroids (Table 6.3). The fulfilment of the normal distribution

Frequency

Frequency

Test

oste

rone

, %5α

-And

rost

andi

ol, %

5β-A

ndro

stan

diol

, %

And

rost

eron

e, %

Etio

hola

nolo

n, %

Preg

nand

iol,

%16

-And

rost

enol

, %‰

‰‰

‰‰

‰

Etio

chol

anol

one,

‰

Fig.

6.7

. Gra

phs

of t

he d

ensi

ty o

f di

stri

butio

n of

pro

babi

lity

of t

he v

alue

s of

δ13

C o

f th

e ta

rget

ste

roid

s.


law was visually checked by constructing the ‘quantile–quantile’ graphs using software for statistical analysis of the data SPSS, version 17. Figure 6.7 presents the histogram for each steroid characterizing the density of distribution of the probability of the corresponding values of δ13C.

Table 6.4 shows the reference intervals calculated for two endogenous markers (pregnandiol, androstenol) and five target steroids (testosterone, 5α-androstandiol, 5β-androstandiol, androsterone, etiocholanolone). The reference intervals, as mentioned above, represent the ‘steroid–an endogenous marker’ difference averaged over all samples analyzed in the laboratory. By adding the tripled standard deviation to this value which corresponds to the 99.7% confidence level, we obtain the critical value for each pair of steroids (calculations were carried out using two significant digits after the decimal point, rounding the result only). Similarly, by constructing the ‘quantile–quantile’ graphs we have shown that the values of Δδ are distributed in accordance with the normal law.

Thus, our results suggest that when interpreting the results it is not advisable to follow a single threshold of 3‰ set by WADA, but one should use the intralaboratory criteria, for example, for testosterone and 5α-androstandiol this value is 4.8, and 5.1‰ respectively (relative to pregnandiol). If the value set in the laboratory is less than 3‰, it is recommended to use a threshold of 3‰ to avoid contradiction with the WADA technical document.

Table 6.4. Reference ranges for the investigated steroids

Pair The difference in a pair Δδ, mean ±SD, ‰ Critical value,‰

PD-T 1.5 ± 1.1 4.8PD-5α 1.9 ± 1.1 5.1PD-5β 0.1 ± 0.6 2.0PD-A 1.1 ± 0.8 3.4PD-E 0.9 ± 0.6 2.516en-T 0.9 ± 0.8 3.316en-5α 1.2 ± 0.8 3.816en-5β –0.6 ± 1.0 2.316en-A 0.4 ± 0.6 2.016en-E 0.2 ± 0.7 2.3


Literature

1. Sutras P., Saudan C., Schweizer C., Baume N., Mangin P., Saugy M., Forensic Sci., 2008, V. 174, No. 2, 166–209

2. Meldensohn D., Immelman A. R., Vogel J. C., von la Chevallerie G., Biomed. En-viron. Mass Spectrom., 1986, V. 13, No. 1, P. 21.

3. von Kuk C., Flenker U., Guntner U., Hulsemann F., Schanzer W., Proceedings of Cologne Workshop on Dope Analysis, Cologne, 2005, 219.

4. Flenker U., von Kuk C., Guntner U., Hulsemann F., Gougoulidis V., Schanzer W., Proceedings of Cologne Workshop on Dope Analysis, Cologne, 2005, 227.

5. von Kuk C., Flenker U., Guntner U., Hulsemann F., Schanzer W., Recent advances in doping analysis, 2005, V. 13, 219–225.

6. Flenker U., von Kuk C., Guntner U., Hulsemann F., Gougoulidis V., Schanzer W., Recent advances in doping analysis, 2005, V. 13, 227–233.

7. WADA Laboratory Committee, Montreal, 2004. WADA Document TD2004EAAS (Http://www.wada-ama.org/rtecontent/document/end_steroids_aug_04.pdf).

8. Aguilera R., Catlin D. H., Becchi M., Phillips A., Wang C., Swerdloff R. S., Pope H. G., Hatton C.K., J. Chromatogr., 1999, V. 727, No.1, 95.

9. Aguilera R., Catlin D. H., Chapman T.H., Rapid Commun., Mass Spectrom., 2000, V. 14, No. 23, 2294.

10. Aguilera R., Catlin D. H., Chapman T.H., Hatton C.K., Starcevic B., Clin. Chem. 2001, V. 47, No. 2, 292.

11. Saudan C., Emery C., Marclay F., Strahm E., Mangin P., Saugy M., J. Chro-matogr., B, 2009, V. 877, No. 23, 2321.

12. Piper T., Mareck U., Geyer H., Flenker U., Thevis M., Platen P., Schanzer W., Rapid Commun. Mass Spectrom., 2008, V. 22, No. 14, 2161.

13. Becchi M., Aguilera R., Farizon Y., Flament M., Casabianca H., James P., GC-C-IRMS analysis of urinary steroids to detect misuse of testosterone in sport, Rapid Commun. Mass Spectrom., 1994, V. 8, 304–308.

14. Ferry M., Mathurin J.-C., Becchi M., de Ceaurriz J., Influence of pregnenolone administration on IRMS analysis, Recent advances in doping analysis, 2000, V. 8, 209–213.

15. Saudan C., Baume N., Mangin P., Urinary analysis of 16 (5α)-androsten-3α-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis, J. Chromatogr., B., 2004, V. 810, 157–164.

16. Trout G. J., Rogerson J. H., Cawley A. T., Alma C. W., Developments in sports drug testing, Aust. J. Chem., 2003, V. 56, 175–180.

17. de Groot P. A., Handbook of stable isotope analytical techniques, Bristol: Elsevier, 2004, 857.

18. Meier-Augenstein W., Applied GC-C-IRMS, J. Chromatogr., A, 1999, V. 842, 351–371.

19. Thermo Finnigan. GC Combustion Interface Operating Manual, 2002, 71.20. High-performance gas chromatography / ed. K. Khaiver, Moscow: Mir, 1993, 288.21. Horning S., Nolteernsting E., Geyer H., Flenker U., Schanzer W., Recent advances

in doping analysis, 1999, V. 6, 243–256.22. Mareck U., Geyer H., Opfermann G., Thevis M., Schänzer W., J. Mass Spectrom.,

2008, V. 43, No. 7, 877.

205Isolation methods in isotope geochemistry of noble gases

7

Isolation methods in isotope geochemistry of noble gases

A.I. BuikinV.I. Vernadsky Institute of Geochemistry and

Analytical Chemistry, Russian Academy of Sciences, Moscow

Introduction

The study of the abundance of the noble gases and their isotopes in a variety of natural objects is of great importance as a tool for understanding patterns of evolution of matter, from the earliest stages of formation of the solar system and ending with current processes in the Earth crust, which are otherwise cannot be studied. For example, the presence of solar helium and neon in the Earth mantle shows that our planet captured Solar gases in the process of accretion, either directly from the solar nebula, or as a result of implantation of solar wind. Excesses of radiogenic nuclides (40Ar, formed from 40K, 4He and 21Ne from U and Th, 129Xe from short-lived 129I and xenon of fission of 238U and short-lived 244Pu) indicate a large-scale degassing of the mantle, which occurred in the early stages of development of the Earth.The isotopes of noble gases carry important information about the early evolution of the Earth atmosphere. Finally, differences in the isotopic composition of noble gases of the mantle-source of MORB (basalts of mid-ocean ridges) and the source of mantle plumes are closely related to the structure and evolution of Earth’s mantle. This contributes to the discussion of the problem origin of mantle plumes – whether they are related to less depleted and less degassed (compared to the top mantle) deep reservoirs and whether their existence requires consideration of models of layered mantle convection or whether it permits full convection of the mantle. The answer to these important questions depends greatly on a correct evaluation of the isotopic composition of


the noble gas of the Earth mantle: both the primary isotope composition and the excess of radiogenic nuclides of rare gas formed from different (with different lifetime) of the parent nuclei.

Radiogenic isotopes of noble gases are a source of information on a variety of physico-chemical, geochemical and geological phenomena occurring in the lithosphere, hydrosphere and atmosphere. Knowing the concentration of radiogenic noble gases and their parent chemical elements in the relevant objects we can determine the age of minerals and rocks, natural waters and the atmosphere and estimate the time and the temperature of secondary geological phenomena.

The width of the range of application of the isotopes of noble gases has been the reason for the intensive development of experimental techniques to study the elemental and isotopic ratios of noble gases in rocks and minerals. The sensitivity of mass spectrometric methods of analysis of trace amounts of noble gases has increased over the past 50 years by several orders of magnitude. Accordingly, it was possible to carry out all more detailed investigations using various techniques.

In this paper, we propose to consider one of the most important components of the experimental studies, namely the methods of separation of the noble gases from the rocks and minerals.

Stepwise annealing

For several decades the isotopic composition of noble gases in different objects has been studied mainly by the method of stepwise annealing. In stepwise annealing the sample is heated slowly, with holding for a certain period of time (typically from 10 to 60 min) at each temperature level. This allows partial or complete separation of the components of the gases, which are characterized by different activation energies and/or located in different mineral phases. Before beginning stepwise annealing, the weighed samples packed in Al- or Ni-foil are placed in a glass manifold with magnetic pushers and evacuated. To remove adsorbed atmospheric gases the sample is heated and pumped out at a temperature of 150°C to achieve high vacuum (about n·10–8 mbar); the duration of heating is determined by the degree of weathering of the sample and by the amount of secondary minerals. Prior to analysis the furnace is held at the maximum temperature to reduce gas emission from crucible. Monitoring is carried out by performing a blank experiment with a foil charge (blank). Typically, a series of blank experiments at different temperatures is carried out for the construction of the curve of the dependence of the amount of emitted gas on temperature and further integration of the blank in the analysis.


The samples can be analyzed when the amount of gas in the form is reduced to an acceptable level. A sample is placed in a cold crucible to prevent ‘boiling’, pumping of the crucible is then stopped and temperature is increased. The gases, generated by heating the samples, travel into the cleaning system (Fig. 7.1), which consists of two consecutive stages. Each stage contains a ‘finger’ with activated charcoal to collect the sorbed gas in the housing with Ti- and Zr–Al-getters (Fig. 7.1) for the purification of gases from chemically active impurities (CO2, H2O, N2, etc.). After purification and separation the evolved gases are supplied into a mass spectrometer. The cleaning and separation procedure of gases is repeated for the next temperature step. When using a resistance furnace, the furnace temperature between the step is usually not reduced. When using an induction furnace (Figs. 7.2 and 7.3), in which the background of the blank experiment at which the amount of gas accumulated in the furnace without heating the sample is determined is much higher, the holding time of the sample at the given temperature is reduced (10–15 min) and the heating of the crucible between the steps is switched off.

The total content of the noble gas in terrestrial samples (and almost always in space samples) is often the sum of the components of different origins occupying physically different positions in the substance of the sample. In general, these components are characterized by different curves of gas evolution, i.e. the gases collected in the different temperature levels will have different ratios of components. Partial separation is a great advantage of stepwise annealing: it allows us to estimate the content and compositions of these components or at least demonstrate their very existence [1, 2].

Fig. 7.1. The system for isolation and purification of noble gases. In the background the induction furnace is visible (see also Figs. 7.2 and 7.3).

Ti- and Zr-Al getters Induction furnace


Fig. 7.3. Testing induction furnace at high temperature (~1700°C).

Fig. 7.2. Induction furnace before heating. The molybdenum crucible inserted in a quartz vessel which, in turn, is soldered into a glass flask with the water cooling circuit.

Molybdenum crucible in a quartz vessel


Fig. 7.4. Hole in a nickel holder with a sample, melting under the influence of the laser beam.

Release of gases by heating with a laser beam

Most minerals contain mineral and/or melt inclusions, which can (sometimes significantly) distort the resulting isotope–geochemical or geochronological information for the host mineral. Such inclusions can be mechanically inseparable from the mineral. In such cases, the method using a laser can be applied. For the first time the laser method of release of gases from the solids was applied by Megrue [3] to study the abundance of helium, neon and argon in the lunar rocks from the Sea of Tranquility. Since then, this method has become widely used for dating of lunar and meteoritic breccias containing a number of fragments of different ages. The method consists in the fact that the melting of a substance occurs in a very small volume due to the impact of the laser beam (Fig. 7.4). The latter is focused on the surface of the sample placed on the table (if a flat polished section is examined) or poured into a hole in table of the vacuum chamber. Depending on the type of laser it is possible to burn immediately the entire sample (weighing usually from a fraction to a few milligrams), or analysis can be performed at the point constructing profiles along the grains and exploring in such a manner changes in the isotopic composition or age from the centre to the edges of mineral grains. The emitted gas, as in the case of stepwise annealing, is cleaned and supplied into the chamber of the mass spectrometer.

Gas emission from small volumes of solids with lasers has several advantages over conventional methods: 1) the amount of gas in the blank experiment (blank) decreases and this can significantly reduce


the sample charge, which becomes very important in the study of such rare specimens as meteorites, diamonds, zircons; 2) we can effectively explore the rocks, containing minerals of different ages and/or genesis of such polymict breccia, with no mechanical separation of minerals, which is sometimes very difficult or even impossible; 3) we can also study variations in the isotopic composition or age from the edges to the centre of individual crystals learn the thermal history of the sample.

The main factor limiting the use of the laser is the amount of emitted gas. Limitations of the laser method may also include the possibility that the laser beam can cause the release of gases from other minerals, if the spot from the ray captures them. Experiments, however, showed that less than 1% of gas is released from an area located at a distance of more than 10 µm from the boundary of the spot [4]. Another limitation of the laser method is that the results obtained with this method correspond to the data obtained in the of ‘complete separation’ method and the use of these data, for example, in K-Ar-dating leads to an underestimation of the age of samples which lost argon after crystallization, or, conversely, to overstating the age, if the sample captured excess argon during formation [5].

The stratified oxidation method

This method can be used for the extraction of noble gases from diamonds [6]. It is based on burning the diamond in an oxygen environment. The oxidation system consists of a reactor into which samples are transferred through a gate, a second reactor with copper oxide, and two vacuum gauges. The reactors are made of a nickel-based alloy. The temperature in the reactor with the sample can rise up to 1200°C. The heating of the reactor with copper oxide up to 870°C provides a pressure of oxygen in the oxidation system at 15 torr. The required temperature regime in the CuO bulk and in the oxidation volume is provided with the automatic temperature control and stabilization system. Control of the oxidation–reduction process and quantification of amount of CO2 formed in combustion of diamond are carried out by the vacuum gauges connected with the oxidation volume.

One of the advantages of this method is the possibility of applying a combined scheme of separation of noble gases from the diamond. First, the samples can be heated up to 1200°C without oxygen thus removing the atmospheric adsorbed gases and gases from the impurity mineral phases. This is followed by the oxidation of the diamond, with possible stratified oxidation of diamonds, as the oxidation reaction of the diamond depends on the temperature and pressure of produced CO2.


The use of the stratified oxidation method of diamonds is promising for studying the zonal heterogeneity of the isotopic composition of noble gases in the radial direction of diamond crystals.

Stepwise fragmentation

In recent years, there has been a growing interest in the study of fluid inclusions, in particular in the mantle minerals. In order to separate the mantle fluid component of noble gases from the radiogenic component or possible cosmogenic components (for example, 3He, which is formed from the lithium nuclei under the effect of cosmic rays) located in the crystal lattice, the gas is released by stepwise fragmentation [7, 8]. To do this, the prepared samples are placed in metallic (non-magnetic) cylinders (Fig. 7.5), which are then connected to a high-vacuum system for separation and cleaning. The entire system is subjected to preliminary annealing 150°C and evacuated to high vacuum (n·10–8 mbar) for 48–72 hours to remove surface-adsorbed atmospheric gases. Gas is released by mechanical crushing of the samples with metal balls within a cylinder. The balls are driven manually with a permanent magnet (in some laboratories, this process is automated using

Copper interlayer

Fixing bolt

Pipe made of non-magnetic

material

Metallic sphere

Sample

Permanent magnet

Fig. 7.5. Schematic representation of the evacuated non-magnetic stainless steel tube and the crushing process. The sample is placed inside (up to 3 g in one tube), on top of the sample there is a metal ball, driven by a permanent magnet.

Copper gasket


electromagnets). With each step (fraction) of crushing the number of impacts increases and, therefore, the degree of grinding of the sample also increases. Thus, during crushing the gas is extracted from smaller and smaller bubbles. The gas isolated from the sample, as in stepwise annealing, is collected, cleaned to removed reactive gases and analyzed.

The method of stepwise crushing as a way of separating the noble gases from the gas–liquid inclusions in minerals is, in our opinion, more promising than the existing attempts to open inclusions with a laser. On the one hand, the laser has an advantage because it allows to open single inclusions by choice, on the other hand, there is a shortage of emitted gas and/or capture of the matrix material.

Conclusion

This chapter briefly describes the methods for isolation of noble gases from the rocks and minerals. In conclusion, it should be noted that for each object and objectives of the study we select the individual scheme selection of noble gases – both the method of isolation and the number and size of temperature steps or crushing steps. In some cases, it is reasonable to combine different separation methods, such as the method of stepwise crushing and stepwise annealing, which will allow to obtain a more complete picture of the evolution object [9].

Literature

1. Shukolyukov Yu. A, Levski L. K., Geochemistry and cosmochemistry of noble gases, Moscow: Atomizdat, 1972, 336.

2. Ozima M., Podosek F., Geochemistry of noble gases, Leningrad: Nedra, 1987, 343.3. Megrue G. H., J. Geophys. Res., 1971, V. 76, 4956–4968.4. Schaeffer O.A., In: L.A. Currie, ed., Nuclear and chemical dating techniques,

139–148, Amer. Chem. Soc., 1982. Symp. Ser. 176, 516.5. Dalrymple G. B., Lanphere M. A., Earth Planet. Sci. Lett., 1971, V. 12, No. 3,

300–308.6. Pleshakov A. M., Dissertation, Moscow, V.I. Vernadskii Institute of Geochemistry

and Analytical Chemistry, 1998, 184.7. Hopp J., Trieloff M., Altherr R., Earth Planet. Sci. Lett., 2004, V. 219, 61–76.8. Buikin A. I., Trieloff M., Hopp J., Althaus T., Korochantseva E. V., Schwarz W.

H., Altherr R., Earth Planet. Sci. Lett., 2005, V. 230, 143–162.9. Trieloff M., Weber H. W., Kurat G., Jessberger E. K., Janicke J., Geochim. Cos-

mochim. Acta, 1997, V. 61, 5065–5088.

213Using laser spectroscopy for measuring ratios

8

Using laser spectroscopy for measuring the ratios of stable isotopes

V.S. Sevastyanov

V.I. Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences,

Moscow

Introduction

Studies that address the global climate change require very precise measurements of the content and isotopic composition of greenhouse gases (mainly CO2 and CH4) to develop improved models of the carbon cycle. It is known that the use of fossil fuels leads to an increase in the concentration of carbon dioxide in the atmosphere at about 3·10–4 vol.% per year and this leads to global changes in the Earth’s climate. Terrestrial ecosystems act as both natural sources and sinks for atmospheric carbon but the mechanisms of this process have not been studied sufficiently. Despite the fact that isotope ratio mass spectrometry (IRMS) is a well developed isotope method, it is preferred to determine the isotopic composition of atmospheric greenhouse gases by absorption infrared spectroscopy which has recently made great strides.

In the infrared spectra associated with the vibrational–rotational motion of molecules, there are molecular absorption lines with different isotopic composition. From these lines we can determine the concentration of isotopologues, such 12CO2 and 13CO2, and calculate isotopic ratios of light elements in the compound.

For water isotopic ratios can be calculated by the following formulas:

R R18 218

216

16

216O

H O

H O D

HD O

H O=

=

, ,


where [H218O], [H2

16O], [HD16O], [H216O] are the concentrations of the

corresponding isotopologues. The isotopic composition of elements in the compound is expressed by the formula

δ = stand

RR

−1 1000

⋅ ,

where δ is given in thousandths of a fraction (‰). The international standard is the water Vienna Standard Mean Ocean Water (VSMOW) with the isotopic composition of δ18O = δD = 0‰.

For the first time infrared spectroscopy was used to measure the deuterium content in water in 1950 [1]. A non-dispersive infrared spectrometer with a photoacoustic detector for the determination of the isotopic composition of carbon in CO2 was developed in 1951 [2]. So far the accuracy of determining the isotopic composition of light elements is inferior to the accuracy of the IRMS method. However, the optical isotope analyzers are significantly more compact, cheaper and easier to manage. In infrared spectroscopy of isotope ratios the sensitivity is less important than the large linear range and a high signal to noise ratio.

Thus, absorption spectroscopy for determining the isotopic composition of light gas-forming elements is an alternative method in relation to IRMS.

Currently, special attention is given to the development of laser infrared spectroscopy, since lasers can operate at room temperature and have a narrow emission line width of 1–10 MHz. For lasers, emitting in the wavelength region near λ = 3 µm, it is about 10–3–10–2 Å. Widely used are diode lasers which are small, easy to operate and have single-mode structure of radiation in the infrared region (Table 8.1) [3] and, at the same time, are easily tunable in a wide spectral range. The diode lasers can operate in continuous and pulsed modes. The spectral generation region extends from near UV to the far-infrared.

Great prospects are characteristics of the recently developed single-mode quantum-cascade lasers (QCL) (Alpes Laser, Neuchatel, Switzerland), emitting at wavelengths from 5 to 12 µm and having a narrow lasing line with a width of 1.3 MHz. They are multi-layered heterostructures of semiconductor materials such as InGaAs–InAlAs, in which lasing takes place by the cascade of intraband transitions. The lasing wavelength is determined by the thickness of the semiconductor layers of the heterostructure and can therefore be specified in the manufacture of the laser. The quantum-cascade lasers have characteristics similar to tunable diode lasers. They can


be tuned over a wide spectral range by changing the temperature and pump current, and their lasing power can be tens of milliwatts. It is essential to note that developers hope in the near future to realize these characteristics at room temperatures [4].

Absorption spectroscopy

In absorption spectroscopy, the absorption coefficient associated with the concentration of the compound in the sample is given by

α ν ν ν( ) ( ) ,= Sf nl− 0

where S is the power factor, which depends on the properties of the isotopologue and temperature, f(ν – ν0) is the normalized line shape function, ν is the frequency of the light source, ν0 is the frequency at the centre of the peak, n is the density of the gas, l is the effective optical path length.

In [5] it was shown that by using a diode laser with a wavelength of 4.7 µm for well-resolved lines of the rotational spectrum 12C16O and 12C18O it was possible to obtain a 3‰ reproducibility of the determination of δ18O for pure CO at a pressure of 3 mbar. A double-beam spectrometer with two unequal paths of radiation transmission to align the transmission of two cells was used. The isotopic composition of hydrogen and oxygen of water was determined using a dye laser with

Table 8.1. Semiconductor diode lasers

Semiconductor Chemical compounds Wavelength, μmLasers with electron excitation

Zinc sulphide ZnS 0.33Gallium selenide GaSe 0.6Gallium arsenide GaAs 0.85Lead sulphide PbS 4.3Indium antimonide InSb 5.3Lead selenide PbSe 8.5

Injection lasersGallium arsenide GaAs 0.85Indium arsenide InAs 3.2Lead telluride PbTe 6.5Lead selenide PbSe 8.5

Lasers with optical excitationCadmium sulphide CdS 0.5Indium arsenide InAs 3.2Indium antimonide InSb 5.3Lead telluride (Pb + Sn) Te 6.5–16.5


a wavelength of 2.73 µm [6]. A water sample with a volume of 5–10 µl was injected into a container with a volume of 1 liter. Measurements of δD were taken in the range from –428 to +15000‰, δ18O – in the range from –55 to +200‰, δ17O – in the range from –100 to 300‰. The accuracy of measurement δD was 1‰, and δ18O 0.5‰. The measurement time of three isotopic ratios was 20 min.

The sensitivity of absorption spectroscopy can be significantly improved by using frequency modulation of the laser light with phase-sensitive detection. Samples of atmospheric methane were analyzed. The resulting accuracy of measurement of δ13C was 0.6‰ (the minimum methane concentration 2 µM) and 5‰ for δD (minimum concentration of methane 80 µM). The duration of analysis was 15 min [7]. The use of two diode lasers allowed to carry out the isotopic analysis of water with the reproducibility of 0.2‰ for δ18O, 0.5‰ for δ17O and δD. The reproducibility of determination of δ13C in the isotopic analysis of methane was 0.3‰ [8].

In [9] attention was given to the prospects of using a tunable diode laser on lead salts in absorption spectroscopy for the determination of the isotope composition of the carbon of CO2 in air to study the nature of the carbon exchange between the soil and the atmosphere. Equipment was constructed on the basis of the gas analyzer TGA 100 (Cambell Scientific Inc., Logan, UT, USA). The standard deviation of the results of determination of δ13C was 0.35‰ (n = 82), and the deviation from the results for the isotopic ratios, obtained using a Finnigan MAT 252 mass spectrometer (Finnigan MAT, San Jose, CA, USA), was 1.77‰. The experiments showed that the values of δ13C of CO2 at a distance of 1 and 60 cm from the soil surface changed overnight from –29.1 ± 0.4 to –22.7 ± 0.8‰. Absorption lines with a wavelength of 2308.225 (12CO2) and 2308.171 cm–1 (13CO2) were selected for analysis. The intensity of the 12CO2 line was half the intensity of the 13CO2 line. The narrow molecular lines are formed at a reduced pressure in the cell (2.1 kPa) and with increasing pressure they expand.

The use of infrared spectroscopy with a krypton laser (wavelength 0.647 µm) allows high accuracy while determine the isotopic ratios 2H/1H, 17O/16O, and 18O/16O of water. Reproducibility of determination is 0.7‰ for δD, 0.5‰ for δ17O and δ18O. The pressure of water vapour in the cell with the addition of 10 ml of water using a chromatographic syringe was 13 mbar [6].

Infrared spectroscopy with Fourier transform

The resolution of the infrared spectrometer with Fourier transform can


reach 0.01 cm–1. This is sufficient to determine the carbon isotopic composition with an accuracy of 0.1‰ in atmospheric CO2 and the air exhaled by persons. The analyzer can measure δ17O and δ18O in CO2 and CO, and also δ15N in N2O [10].

Non-dispersive absorption spectroscopy

The existing non-dispersive infrared (NDIR) analyzers for monitoring atmospheric CO2 do not yet fully meet requirements of these measurements for several reasons: they have non-linear calibration curves, are sensitive to water vapor, there is a drift of the measured results with time and the results depend on temperature. In order to obtain accurate results, it is necessary to frequently (hourly) calibrate the device using an expensive standard gas. NDIR-analyzers have a double-beam configuration with two cuvettes. In one cuvette there is the sample and in the other one a standard or an inert gas. The analyzer has a low cost and simple design. The analyzer with a photoacoustic detector can be used to determine the carbon isotopic composition δ13C of CO2 in the exhaled air (the concentration of 3–5%) with an accuracy of 0.3‰ [11]. Isotopic analysis of a sample of CO2 with a volume of 50 ml and a concentration of 0.5–5% was carried out using a commercial device based on the NDIR IRIS 2TM (Wagner Analysen Technik, Bremen, Germany). It was possible to achieve reproducible results of determination of δ13C equal to 0.3‰.

Laser cavity ring down spectroscopy

The basic principles of the new technology, called Cavity Ring Down Spectroscopy (CRDS), were developed in 1988 [12]. CRDS is based on measuring the decay time of the light intensity in a cell (cuvette) with three high-reflection mirrors with multiple passage of light between them. Ultrahigh precision of measurement and low drift of its characteristics make it ideal for atmospheric research. CRDS has three distinctive features. First, CRDS provides a very large path length of the laser beam in an optical cuvette. This increases the sensitivity compared to conventional absorption methods, such as infrared spectroscopy with Fourier transform (FTIR), spectroscopy and non-dispersive infrared spectroscopy (NDIR). In the analyzer based on the CRDS, having a ring-shaped optical cuvette with three mirrors, the laser beam traverses a path of ~12 km with a decay time of ~40 µs. The second distinguishing feature of the analyzer is the measurement of the decay time, and not the weakening of light intensity, as occurs in


conventional optical absorption spectroscopy. Therefore, the results of measuring the concentration of the absorbing gas do not depend on fluctuations in the intensity of the light source, which is especially important when using pulsed lasers. The third advantageous feature of the analyzer, based on CRDS is its high resolution. The resolution of the analyzer based on CRDS is 0.0001 cm–1, whereas the FTIR analyzers have a resolution of ~ 0 and 5 cm–1 (the resolution is raised about 5000 times). The low gas pressure (50–150 torr) in the analytical cuvette leads to a narrowing of the spectral lines compared to their width at atmospheric pressure. This decreases the probability of overlap of neighbouring lines and of spectral lines of interfering compounds. Consequently, the sensitivity and accuracy spectral analysis increase.

The basis of the CRDS method is the determination of the decay time of the intensity of radiation in the flow optical cuvette filled with the analyzed gas (Fig. 8.1). Figure 8.2 shows the curve of changes of the light intensity in the cuvette of the CRDS-based analyzer as a

Fig. 8.1. Diagram of the optical analyzer based on the CRDS.

Cuvette

Input of sample in flow of carrier gas

Pressure gaugeThermometer

Output of sample in flow of carrier gas

Adjustable diode laser Wave

monitorOptical system Photodetector

Laser control

unitComputer for collecting

and processing data

Fig. 8.2. Variation of the intensity of light in the optical cell based on the CRDS analyzer before and after switching off the laser.

Det

ecto

r sig

nal

With sampleWithout sample

Laser switched off

Time, ms

With sampleWithout sample

Laser switched off Time, ms

Det

ecto

r si

gnal


function of time. When the laser frequency coincides with the resonance frequency of the analyzed gas energy builds up in the cuvette. After switching off the laser the radiation intensity (I), accumulated in the cuvette, decreases exponentially as a result of optical losses with a decay time (τ)

I t I e t( ) ,/= 0− τ

where I0 is the intensity at the time of switching off the laser. Assuming that absorption in the cell is governed by Beer’s law, we can write

τ να ν

( )( )

,=+[ ]d

c R1−

where ν is the radiation frequency, c is the velocity of light, d is the path length of radiation in the cuvette, R is the reflectivity mirrors, α(ν) = σ(ν)nd is the absorption coefficient of the substance in the cuvette, n is the density of gas

σ ντ τ

( ) ,nc

=

1 1 1

0

−

τ0 is the decay time in an empty cuvette. Since the laser beam does not enter the cuvette during the decay time τ of radiation in the cuvette with the sample, this leads to a decrease in the oscillations of the baseline and improvement of the accuracy of analysis by 4‰.

If the radiation frequency is not resonant for the analyzed gas, the decay time is determined by the optical losses upon reflection from the mirrors. When using the resonant frequency the radiation is absorbed by the sample in the cuvette, leading to a decrease in decay time depending on the concentration of the sample. A special feature of the CRDS-based analyzer is that the decay time of light intensity can be measured with greater accuracy than the absolute or relative intensity of light. The analyzer uses the precision wave monitor to measure and control radiation wavelengths. The designed wave monitor provides a detailed scan of the optical spectra, which can not be done through the analyzer based on NDIR. This avoids interference from the compounds of the gas mixture and removes one of the main reasons for re-calibration of the analyzer necessary for most devices of this type.

The analyzer has a digital signal processing system for determining the rate of decay of radiation (optical loss) in dependence on the wavelength. The measurements are carried out for about ~6 ms, which allows to obtain a typical spectrum, consisting of 10–100 points. Precise


control of temperature and pressure is critical for obtaining accurate and reliable data in the automatic mode. The temperature in the cuvette is maintained with an accuracy of 3·10–6 and the pressure with an accuracy of 2·10–3 [13].

Currently, analyzers capable of continuous, real-time measurement of isotopic ratios in the atmospheric gases, such as CO2 and water vapor, are being developed. For example, on the basis the change of the carbon isotopic composition of CO2 we can study the carbon cycle of the ecosystem, as all plants somewhat less effectively absorb CO2, containing a heavy isotope of carbon (13C). The measurement of changes in the isotopic composition of the oxygen and hydrogen of water also allows us to study the water cycle. Figures 8.3 and 8.4 are examples of spectra used in the measurement of isotopic ratios [14]. The extremely

Fig. 8.3. Isotopic high-resolution spectrum of CO2 obtained with an CRDS-based optical analyzer.

Ads

orpt

ion

loss

es, p

pm/c

m

Shift of optical frequency, cm–1

1.1

1.0

0.9

0.8–0.4 –0.2 0 0.2 0.4

Fig. 8.4. The isotopic high-resolution spectrum of water obtained with the aid of an CRDS-based optical analyzer.

Shift of optical frequency, cm–1–0.4 –0.2 0 0.2 0.4

Ads

orpt

ion

loss

es, p

pm/c

m

5.04.5

4.03.53.0

2.52.01.5


high spectral resolution of the analyzer allows to measure closely spaced spectral lines corresponding to different gas isotopologues.

Field trials on the CRDS-based analyzer showed that the analyzer is able work for many months under different environmental conditions, without requiring frequent calibration, providing the necessary linearity in a wide range, precision and accuracy of the measurement results. The analyzed samples require little or no preparation.

To measure the δ13C in CO2, the Picarro Company developed a CRDS-based Picarro G1101-i analyzer. Due to its small size the analyzer can be easily moved and measurements can be taken several minutes after switching on the analyzer. The samples do not require any preparation or removal of moisture. The linear measurement range of CO2 concentration is 0–2000 ppmv, the reproducibility of determination of δ13C (CO2 in air) is 0.3‰ [14].

Test runs for the determination of the CO2 content in 45 days with analyzers based on CRDS and NDIR showed that the mean difference between the results was 180 ppbv. The observed drift for the Piccaro G 1101-i analyzer was 0.8 ppbv per day. It means that the daily drift is 0.0002%, and in 45 days 0.045%. Only one calibration of the CRDS-based analyzer based on the standards of CO2 and water was required during the investigations. In contrast, the NDIR-based analyzer was calibrated on the basis of the CO2 standard every four hours and it was necessary to remove water vapor from the gas stream. The reproducibility of the measurement results of concentration for the CRDS-based analyzer was 50 ppbv for CO2 and 100 ppmv for H2O with the full measurement time of 3 s [14].

To measure the δD and δ18O in water, the Picarro company developed a CRDS-based analyzer Picarro L1102-i. The structure of the analyzer includes an autosampler which can be used for multiple introduction into the analyzer of a liquid sample with a volume of up to a few microliters. Typically, a water sample with a volume of 1 ml is analyzed. Table 8.2 gives the analytical characteristics of the isotope water analyzer Picarro L1102-I [13].

This analyzer is capable of working in the field and laboratory conditions. The memory effect of the previous sample disappears after three injections of the sample. The volume of the optical cuvette is 35 cm3. The temperature in the cuvette is maintained with an accuracy of 80 ± 0.02°C, and the pressure with an accuracy of 35 ± 0.1 torr [15].The wavelength of the tunable diode laser is 1392 nm. The time of a single measurement is 30 s, full analysis of one sample takes 9 min. Determination of the isotopic composition of oxygen and hydrogen is carried out in a range of water concentration from 6000 to 26 000


ppmv. Trials of the analyzer have shown that the shift of the results of measurement of δD is 2‰ within 4 weeks of the experiment [14]. The weight of the analyzer is 26.3 kg and the dimensions 43 × 25 × 59 cm (width × height × depth).

The evaporator shown in Fig. 8.5 [15] is used to convert water into steam, homogenize it and supply into the analyzer. It consists of a cylinder with a volume of 150 cm3, a valve to vent the carrier gas, a valve for the fore-vacuum pumping of the cylinder, the unit for supplying water samples and a three-way valve for injection of the sample or dry air into the analyzer. The carrier gas is nitrogen or dry air. The input unit allows to enter the sample by a chromatographic syringe through a rubber gasket. The evaporator temperature is 140°C. The evaporator can be switched from the measurement of water vapour in the air to measurement of liquid water.

In [16] the results of isotopic analysis of water using an CRDS-based optical analyzer Picarro L1102-i and the isotope ratio mass spectrometer

Table 8.2. The analytical characteristics of the water isotope analyzer Picarro L1102-i

Sample Isotopic composition

Repeata-bility, ‰

Memory, per-centage of the

preli minary sample,%

Drift (24 h),

‰

Injection of water with the aid of autosampler

δ18OδD

0.10.5

26.5

±0.3±0.9

Injection of water using a syringe

δ18OδD

26.5

±0.3±0.9

Injection of water vapour

δ18OδD

±0.3±0.9

Fig. 8.5. The scheme of the water evaporator connected to the optical isotope analyzer.

Syringe

Pumping To analyzer

Sample introduction

section

Vacuum valve

Carrier gasCylinder

Analyzed gas

Inlet valve

Three-way valve


Delta Plus XP with a TC/EA pyrolyzer (Thermo Fisher, Bremen, Germany) are compared. The reproducibility of the measurements of δD and δ18O of clean water for the two methods was similar and amounted to ±0.22 and ±0.5‰ respectively. The average shift of the results of measurements of mass-spectrometric and spectroscopic data was 0.15‰ for δD and 0.03‰ for δ18O (Table 8.3). A linear dependence of the results of determination of δD and δ18O when entering the water samples in the range of 0.5–l µl was found.

When using dry air as a carrier gas, the shift between the analysis results obtained in two methods increased to 1–2‰ for δ18O and 5‰ for δD.

Laser off-axis integrated cavity output spectroscopy

The Los Gatos Research company (LGR) [17] developed three optical isotope analyzer: for water – Liquid Water Isotope Analyzer (LWIA-24d), for carbon dioxide – CO2 Isotope Analyzer, and for methane – Methane Carbon Isotope Analyzer. Their work is based on the measurement of the radiation damping time in a cell (cuvette) with two high-reflection mirrors with multiple light passes between them. This method of analysis is called off-axis integrated cavity output spectroscopy (OA-ICOS) [17-19]. The isotopic analytical characteristics of the OA-ICOS and CRDS methods are similar (Tables 8.2–8.4). There is no need to conduct daily calibration of the analyzers, the isotopic ratios D/H, 18O/16O are measured simultaneously in single input of samples, and the errors caused by conversion of H2O molecules to H2 and CO/CO2, as is the case in isotope ratio mass spectrometry, are eliminated. The design of

Table 8.3. Results of isotopic analysis of water by the TC/EA-IRMS method (Delta Plus XP with attached TC/EA) and comparison with results obtained by the CRDS method

Sample δD. ‰ Standard deviation, ‰

The difference ΔD (TC/EA-IRMS–CRDS), ‰

R1 −101.19 0.15 −0.63R3 −4.87 0.51 0.15R5 114.77 0.11 0.60R6 418.59 0.10 0.42

Sample δ18O, ‰ Standard deviation. ‰

The difference Δ18O (TC/EA-IRMS–CRDS). ‰

R1 −12.56 0.00 0.00R3 −3.10 0.01 0.11R5 −3.27 0.10 −0.08R6 −3.41 0.09 −0.15


the analyzers based on OA-ICOS and CRDS is very similar, they use similar diode lasers with tunable wavelength and the optical cuvettes differ only in the number of reflecting mirrors. The composition of the CRDS-based analyzer also includes an additional wave monitor (see Fig. 8.1). The path length of light in the optical cuvette of the analyzer based on OA-ICOS is approximately 3 km. Analyzers are easy to operate and are small, allowing them to be used in the field conditions. The price of the analyzers is low, but the OA-ICOS-based analyzers are somewhat cheaper. Isotopic LGR analyzers allow to determine the concentration of CO2 in the range 0–20000 ppmv, the concentration of CH4 in the range 10–10000 ppmv.

The analyzers based on OA-ICOS for the determination of the isotopic composition of water have an autosampler for 200 liquid samples which can be used to automatically supply with a syringe 0.2–1 µl of water into the injector of the analyzer at a temperature of 70°C. The experiments [19] showed that the reproducibility of the isotopic analysis results of clean water for 11 hours of work (160 injections) was ±0.29‰ for δD and ±0.16‰ for δ18O, and the magnitude of the instrument shift was 0.6 and 0.4‰ for δD and δ18O, respectively. The memory effect of the input of previous samples on the results of isotopic analysis of water with other isotopic composition is removed after three sample inputs. Another reason for the error of the isotopic analysis of water for the OA-ICOS-based analyzers (also for the CRDS-based analyzers) is the linear dependence on the volume of the sample introduced into the analyzer in the range of 0.2–1 µl. With the increase in the sample

Table 8.4. Averaged results (n = 3) of isotopic analysis of water by the OA-ICOS method

International standard water

δD, ‰Difference ΔD. ‰

Standard value Measured valueOH 5 −2.1 −2.5 −0.4OH 6 −39.1 −38.3 0.8OH 7 −77.8 −77.7 −0.1OH 8 −122.0 −120.7 1.3

International standard water

δ18O, ‰ Difference Δ18O, ‰Standard value Measured value

OH 5 −0.26 −0.38 −0.12OH 6 −4.22 −4.19 0.03OH 7 −10.72 −10.87 −0.15OH 8 −16.27 −16.21 0.06


volume size the values δD become more positive by 11‰ and the δ18O values more negative in the range from –17.56‰ to –17.80‰.

Table 8.4 [19] shows the deviation of the results of measurements of the isotopic composition of the waters of international standards through the OA-ICOS-based analyzer from the defined values. The average deviation of the measurement results for δD ranged from –0.4 to 1.1‰ at the value of standard deviation ±0.8‰, and for the δ18O values the deviations ranged from 0.03 to –0.15‰ with a standard deviation of ±0.11‰.

The analytical characteristics of optical methods for measuring isotope ratios are presented in Table 8.5.

New methods and results

To determine the isotopic composition of carbon in solid samples, the LiaisonTM interface has been designed to connect sample preparation systems (reactors for burning samples) Picarro Combustion Module, Costec elemental analyzer, AutoMate FX DIC to the isotopic analyzer. The reproducibility of determining the values of δ13C is 0.4‰, and the minimum detectable concentration of CO2 200 ppbv (for molecules with the isotope 12C) and 10 ppbv (for molecules with 13C).

To demonstrate the capabilities of the i-TOC-CRDS optical isotope analyzer (Picarro and IO Analytical), experiments were carried to determine the isotopic composition of carbon in the olive oil from different countries (Table 8.6). The results of isotopic analysis were compared with the data obtained in the isotope ratio mass spectrometer. To do this, 2 µl of oil was injected with a by chromatographic syringe into a quartz crucible which was then moved to the oxidation reactor. The resulting CO2 was automatically transferred into the cuvette of the i-TOC-CRDS optical analyzer. The analysis time was 5 min. Table 8.6 shows that the data obtained by the i-TOC-CRDS optical isotope analyzer are correlated with the data of the isotope ratio mass spectrometer. Thus, the i-TOC-CRDS isotope analyzer can be used for rapid screening of olive oil to identify fakes and dilution by cheap corn oil [20].

Study [21] described a device for determining the isotopic composition of carbon in a mixture of volatile organic compounds based on a Picarro analyzer, coupled with an HP 5890 Series 2 gas chromatograph (Agilent), and a burning reactor (1 Pt and 3 Ni wires, 1000–1150°C). The analytical characteristics of the device were investigated using CH4, C2H6, C3H8. The gas sample of the mixture was introduced into the chromatograph and divided in the column


Table 8.5. The analytical characteristics of optical methods for measuring isotope ratios

The method Laser/wavelength

Gas sample/concentration

Reproduci-bility, % Literature

Absorption spectroscopy

Laser on Pb salts /3.3 µm Pure CH4 / 2 µmol 0.6 (δ13C) [22]

Diode laser/1.66 µm Pure CH4/100 µmol 0.3 (δ13C) [23]

Laser on Pb salts/3.3 µm Pure CH4/80 µmol 5 (δ13C) [22]

Laser on Pb salts / 4.7 µm Pure CO/50 µmol 2.5 (δ18O) [5]

Laser on Pb salts / 4.3 µm Pure CO2/10 µmol 4 (δ13C) [24]

Laser on Pb salts / 4.3 µm

Air/350–700 ppmv CO2

0.25 (δ13C) [9]

Diode laser / 1.66 µm

Exhaled air / 5% CO2

10 (δ13C) [25]

Laser on Pb salts / 4,3 µm

Exhaled air/380 ppmv CO2

0.2 (δ13C) [26]

Laser on Pb salts / 4.3 µm Pure CO2 / 2 µmol 2 (δ13C) [27]

Diode laser / 1.39 µm Water 0.2 (δ18O)

0.5 (δ17O)0.5 (δD)

0.4 (δ18O)

[28]

Laser on Pb salts / 8.2 µm N2O [29]

Infrared spectroscopy with Fourier transform

Resolution of 1 cm–1 Air/350 ppmv CO2 0.15 (δ13C) [30]

Resolution of 1 cm–1

Exhaled air / 5% CO2

0.1 (δ13C) [30]

Resolution of 8 cm–1 Water 3 (δD) [31]

Resolution 0.012 cm–1 N2O/6–9 µmol 2.5–4 (δ18O)

1–2 (δ15N) [10]

Non-dispersive infrared spectroscopy Photoacoustic Exhaled air/3–5 %

CO20.3 (δ13C) [11]

Cavity ring down spectroscopy (CRDS) Diode laser

Air/50 ppbv CO2 Water Water vapor in air

0.3 (δ13C) 0.1 (δ18O) 0.5 (δD)

0.2 (δ18O) 1 (δD)

[13]

Cavity ring down spectroscopy (CRDS) Diode laser

CH4 CO2 Water

1 (δ13C) 0.25 (δ13C)

0.3 (δD) 0.1 (δ18O)

[19, 20]


(HP-PLOT Q, 30 m × 0.53 mm) into components according to the retention time. The separated components are oxidized in the burning reactor. The produced CO2 with other oxidation products penetrated into the stream of nitrogen, which passed through the optical cuvette of the isotope analyzer. The temperature in the optical cuvette was 45°C, pressure 140 torr. The reproducibility of the results to determine the isotopic composition of the carbon of ethane and propane was 0.95‰ and 0.67‰, respectively. The accuracy of determination of δ13C was not worse than 3‰. Table 8.7 shows the results of comparative determination of the isotopic composition of the carbon of hydrocarbon gases using the CRDS-based optical analyzer and the isotope ratio mass spectrometer. Despite the low reproducibility and accuracy of determining the isotopic composition of carbon in the hydrocarbon gases using the optical analyzer, the developed device has a great potential for use. Further modification of the device will improve its analytical characteristics.

Table 8.6. Comparison of the results of the determination of carbon isotope composition of olive oil by different methods

Country δ13C (Pizarro CDRS), ‰

Standard deviation

(n = 3), ‰

δ13C (IRMS), ‰

Spain –28.95 0.18 –28.94Italy –28.98 0.05 –29.27Greece –29.29 0.02 –29.21Turkey –30.34 0.11 –30.32Lebanon –29.11 0.23 –28.87Australia –31.23 0.01 –31.19

Table 8.7. Comparison of δ13C values of methane, ethane and propane, measured by the isotope ratio mass spectrometer connected to the chromatograph, (the GC-C-IRMS method, the sample volume 2.5 µl, dilution 1:30), and the optical analyzer, connected to the chromatograph (GC-C-CDRS method, sample volume 25 µl, without dilution), the number of measurements for each value of 5

Compound

GC-C-IRMS δ13C (‰), individual

compounds

GC-C-CRDS δ13C (‰), the

individual compounds

GC-C-CRDS δ13C (‰) in a

mixture

Methane −44.07 ± 0.46 −47.16 ± 0.68 −45.76 ± 0.61Ethan −37.68 ± 0.41 −39.63 ± 0.89 −36.15 ± 0.86Propane −39.57 ± 0.20 −39.57 ± 0.82 −38.75 ± 1.04


Conclusions

Currently, the most developed isotope optical devices are the laser isotope analyzers based on CRDS and OA-ICOS. They allow one to accurately determine the carbon isotopic composition of CO2, hydrogen and oxygen in H2O and water vapour. As regards the analytical characteristics, the optical analyzers are comparable to those of the isotope ratio mass spectrometer, but they are much easier to manage and a lot cheaper. To determine the isotopic composition of light elements in isotope ratio mass spectrometry, it is necessary to carry out the chemical conversion of compounds or isotopic equilibration of water with H2 and CO2. Thanks to highly accurate control of temperature and pressure in the optical cuvette and also strict control of the wavelength of the tunable laser, the drift of the system can be significantly reduced. This allows calibration of the optical analyzer once within a few days. The isotopic optical analyzers are low-cost, portable and can be applied in the field conditions for environmental monitoring.

Literature

1. Thornton V., Condon F. E., Infrared spectrometric determination of deuterium oxide in water, Anal. Chem, 1950, V. 22, 680–681.

2. Milatz J. M., Kluyver J. C., Hardebol J., Determination of isotope ratios, e.g. in tracer work, by infrared absorption method, J. Chem. Phys., 1950, V. 19, 887–888.

3. Fedorov B. F., Lasers. Fundamentals of devices and applications, Moscow: DOSAAF, 1988, 190.

4. Stepanov E. V., Tr. Inst. Obshch. Fiz. im A.M. Prokhorova, RAN, 2005, V. 61, 5–47.

5. Lee P. S., Majkowski R. F., High resolution infrared diode laser spectroscope for isotope analysis – measurement of isotopic carbon monoxide, Appl. Phys. Lett., 1986, V. 48, 619–621.

6. Kerstel E. R. T., van Trigt R., Dam N., Reuss J., Meijer H. A. J., Simultaneous determination of the 2H/1H, 17O/16O, and 18O/16O isotope abundance ratios in water by means of laser spectrometry, Anal. Chem., 1999, V. 71, 5297–5303.

7. White J. U., Very long optical paths in air, J. Opt. Soc. Am., 1976, V. 66, 411–416.8. Gianfrani L., Gagliardi G., van Burgel M., Kerstel E., Isotope analysis of water

by means of near-infrared dual-wavelength diode laser spectroscopy, Opt. Express, V. 11, 1566–1576.

9. Bowling D. R., Sargent S.D., Tanner B.D., Ehleringer J. R., Tunable diode laser absorption spectroscopy for stable isotope studies of ecosystem–atmosphere CO2 exchange, Agricultural and Forest Meteorology, 2003, V. 118, 1–19.

10. Turatti F., Griffith D.W. T., Wilson S. R., Esler M. B., Positionally dependent 15N fractionation factors in the UV photolysis of N2O determined by high resolution FTIR spectroscopy, Geophys. Res. Lett., 2000, V. 27, 2489–2492.

11. Haisch M., Hering P., Fabinski W., Zochbauer M., Isotopenselective kozentra-tionsmessungen an atemgasen mit einem NDIR-spectrometer, Technisches Messen,


1996, V. 63, 322–328.12. O‘Keefe A., Deacon D. A. G., Cavity ring-down optical spectrometer for absorption

measurements using pulsed laser sources, Rev. Sci. Instr., 1988, V. 59, 2544–2554.13. http://www.picarro.com.14. Van Pelt A., Real-time atmospheric monitoring of stable isotopes and trace green-

house gases, http://www.picarro.com.15. Gupta P., Noone D., Galewsky J., Sweeney C., Vaughn B. H., Demonstration of

high-precision continuous measurements of water vapor isotopologues in laboratory and remote field deployments using wavelength-scanned cavity ring-down spec-troscopy (WS-CRDS) technology, Rapid Communications in mass spectrometry, 2009, V. 23, 2534–2542.

16. Brand W.A., Geilmann H., Crosson R., Rella C.W., Cavity ring-down spectroscopy versus high-temperature conversion isotope ratio mass spectrometry; a case study on δ2H and δ18O of pure water samples and alcohol/water mixture, Rapid Com-munications in Mass Spectrometry, 2009, V. 23, 1879–1884.

17. http://www.lgrinc.com.18. Nikolaev I. V., et al., Absorption Spectroscopy attenuation of light with multiple

heart non-axial cuvettes, Preprint 4, Lebedev Physical Institute (FIAN), Moscow, 2005, pp. 1–12.

19. Lis G., Wassenaar L. I., Hendry M. J., High-precision laser spectroscopy D/H and 18O/16O measurements of microliter natural water samples, Anal. Chem., 2008, V. 80, 287–293.

20. Automated measurement of δ13C for identifying and classifying edible oils, http://www.picarro.com.

21. Zare R. N., Kuramoto D. S., Haase C., Tan S.M., Crosson E. R., Saad N. M., High-precision optical measurements of 13S/12S isotope ratios in organic compounds at natural abundance, PNAS, 2009, V. 106, pp/ 10928–10932.

22. Bergamaschi P., Schupp M., Harris G.W., High-precision direct measurements of 13SH4/

12SH4 and 12SH3D/12SH4 ratios in atmospheric methane sources by means of a long-path tunable diode laser absorption spectrometer, Applied Optics, 1994, V. 33, 7704–7716.

23. Uehara K., Yamamoto K., Kikugawa T., Yoshida N., Isotope analysis of environ-mental substances by a new laser-spectroscopic method utilizing different path lengths, Sensors and Actuators, 2001, V. B74, pp .173–178.

24. Becker J. F., Sauke T. B., Loewenstein M., Stable isotope analysis using diodes laser spectroscopy, Appl. Opt, 1992, V. 31, pp. 1921–1927.

25. Cooper D. E., Martinelli U., Carlisle C. B., Measurement of 12SO2: 13SO2 ratios for

medical diagnostics with 1.6-μm distributed feedback semiconductor diode lasers, Appl. Opt., 1993, V. 32, 6727–6731.

26. McManus J. B., Zahniser M. S., Nelson D. D., Williams L. R., Kolb C. E., Infrared laser spectrometer with balanced absorption for measurement of isotopic ratios of carbon gases, Spectrochim. Acta, 2002, V. A58, pp. 2465–2480.

27. Sauke T. B., Becker J. F., Stable isotope laser spectrometer for exploration of Mars, Planet. Space Sci., 1998, V. 46, pp. 805–812.

28. Kerstel E. R. T., Gagliardi G., Gianfrani L., Meijer H. A. J., van Trigt R., Ramaker R., Determination of the 2H/1H, 17O/16O, and 18O/16O isotope ratios in water by means of tunable diode laser spectroscopy at 1.39 μm, Spectrochim. Acta, 2002, V. A58, 2389–2396.

29. Wahlen M., Yoshinari T., Oxygen isotope ratios in N2O from different environ-ments, Nature, 1985, V. 313, pp. 780–782.

30. Esler M. B., Griffith D. W. T., Wilson S. R., Steele L. P., Precision trace gas


analysis by FT-IR spectroscopy. 2. The 13S/12S isotope ratio of CO2, Anal. Chem., 2000, V. 72, 216–221.

31. Fusch C., Spririg N., Moeller H., Fourier transform infrared spectroscope measures 1H/2H ratios of native water with a precision comparable to that of isotope ratio mass spectrometry, Eur. J. Clin Chem. Clin. Biochem., 1993, V. 31, 639–644.

ISBN-13: 978-1-4665-9407-4

9 781466 594074

9 0 0 0 0

K20665

C H E M I S T R Y

Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements explores different methods of isotope analysis, including spark, secondary ion, laser, glow discharge, and isotope ratio mass spectrometry. It explains how to evaluate the isotopic composition of light elements (H, C, N, O) in solid, liquid, and gaseous samples of organic and inorganic substances, as well as:

• Presents a universal, economical, simple, and rapid technique for sample preparation of organic substances to measure the isotopic composition of carbon• Describes how to determine microbial mineralization of organic matter in soil and the effect of exogenous substrates on environmental sustainability• Examines use of the isotopic composition of n-alkanes from continental vegetation to study the paleoclimate and plant physiology• Proposes a systematic approach to identifying tobacco areas of origin and tobacco products based on data from the isotopic composition of light elements• Discusses ways to detect doping drugs and suggests results assessment criteria based on determining reference intervals for endogenous markers• Reviews methods of release of gases from inclusions of rocks and minerals for further implementation of isotope mass spectrometric analysis • Considers use of optical isotope analyzers for determining the isotopic composition of carbon in CO2 and of hydrogen and oxygen in water

Providing a complete picture of the latest advancements in the field, Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements aids readers from a variety of disciplines in identifying the fundamental processes in biological, ecological, and geological systems and in revealing the subtle features of many physicochemical processes and chemical transformations.

Isotope Ratio Mass Spectrometry of Light Gas-Forming ElementsEdited by V. S. Sevastyanov

Isotope Ratio Mass Spectrom

etry of Light Gas-Forming Elem

entsSevastyanov

K20665_Cover.indd All Pages 6/11/14 3:11 PM

Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements

Documents

Transcript of Isotope Ratio Mass Spectrometry of Light Gas-Forming Elements